Review Article Cytochrome c: A Multifunctional Protein ...Cytochrome has served as a model system...

Review ArticleCytochrome c A Multifunctional Protein CombiningConformational Rigidity with Flexibility

Reinhard Schweitzer-Stenner

Department of Chemistry Drexel University 3141 Chestnut Street Philadelphia PA 19104 USA

Correspondence should be addressed to Reinhard Schweitzer-Stenner rschweitzer-stennerdrexeledu

Received 26 February 2014 Accepted 3 June 2014 Published 22 July 2014

Academic Editor Hamid Mobasheri

Copyright copy 2014 Reinhard Schweitzer-StennerThis is an open access article distributed under the Creative CommonsAttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Cytochrome has served as a model system for studying redox reactions protein folding and more recently peroxidase activityinduced by partial unfolding on membranes This review illuminates some important aspects of the research on this biomoleculeThe first part summarizes the results of structural analyses of its active site Owing to heme-protein interactions the heme groupis subject to both in-plane and out-of-plane deformations The unfolding of the protein as discussed in detail in the second partof this review can be induced by changes of pH and temperature and most prominently by the addition of denaturing agentsBoth the kinetic and thermodynamic folding and unfolding involve intermediate states with regard to all unfolding conditions Ifallowed to sit at alkaline pH (115) for a week the protein does not return to its folding state when the solvent is switched back toneutral pH It rather adopts a misfolded state that is prone to aggregation via domain swapping On the surface of cardiolipincontaining liposomes the protein can adopt a variety of partially unfolded states Apparently ferricytochrome c can performbiological functions even if it is only partially folded

1 Introduction

Among the multitude of biomolecules the family of hemeproteins has played a peculiar and prominent role in bio-physical research over many decades [1] All members ofthis family contain at least one sometimes multiple hemegroup as active sites Heme groups are iron-protoporphyrinderivatives Figure 1 exhibits heme c the prosthetic group ofthe electron carrier cytochrome cThe simplest hemeproteinsare monomers with a variety of secondary and tertiarystructures the former being dominated by helical segmentsin most cases (Figure 2) However for some of these proteinsthe monomers assemble to form a quaternary structure Themost prominent example is hemoglobin which contains fourglobular heme proteins as subunitsThis quaternary structureis pivotal for the proteinrsquos biological function in that it enablesa cooperative communication between the four binding siteswhich allows for an effective uptake and release of oxygen inthe lungs and the tissue respectively

All heme proteins contain a heme group which functionsas their active site for performing a diverse set of tasks suchas ligand binding and transfer (myoglobin and hemoglobin)

[2] electron transfer (cytochrome reductase and oxidasecytochrome c) [3 4] facilitation of enzymatic reactions cat-alyzing peroxidase (horseradish peroxidase) [5]monooxyge-nase (cytochrome P450) [6] cyclase (guanylate cyclase) [7]and catalase reactions [8] and signaling process involvingthe binding and release of nitric oxide (nitrophorin) [9] Theindividual functions of these heme groups are regulated bythe axial ligands of their heme iron multiple noncovalentinteractions between the heme (porphyrin) macrocycle andthe protein environment and in the case of members of thecytochrome c family even covalent bonds between peripheralsubstituents of the heme and protein side chains (Figure 1)[4] Thus the protein creates a rather asymmetric and chiralenvironment which affects the symmetry of the macrocycleand also the electronic structure of the central metal [10 11]All heme groups exhibit a rather strong absorption in thevisible region reminiscent of color centers in crystals

This review provides an overview on work performedin our laboratories over the last twenty years (Universityof Bremen University of Puerto Rico and Drexel Univer-sity) which was aimed at exploring cytochrome c functiondynamics and folding In spite of nearly 60 years of research

Hindawi Publishing CorporationNew Journal of ScienceVolume 2014 Article ID 484538 28 pageshttpdxdoiorg1011552014484538

2 New Journal of Science

HS

N

N N

N

Fe

SH

HO OO OH

Figure 1 Structure of heme c the prosthetic group in cytochromec

cytochrome c is still of great interest to biophysicists andbiochemists alike owing to its multifunctionality it serves asan electron carrier between major membrane proteins in theinner membrane of the mitochondria [4] it functions as ascavenger for H

2O2as peroxidase [12ndash15] and it functions

as an initiator of a biochemical cascade which finally causesmitochondrial apoptosis [15ndash17] The protein has also beenutilized as a model system for protein folding studies [18]The research in our laboratories has mostly been focused onelucidating heme-protein interactions and asymmetric defor-mations of the heme macrocycle [11 19] but lately we usedspectroscopic means to probe partially unfolded states of theprotein [20] The second chapter of this review describeshow heme-protein interactions affecting the prosthetic hemegroup of cytochrome can be probed by resonance Ramanvisible circular dichroism and low temperature absorptionspectroscopyThe third chapter reviews the studies of foldingand unfolding triggered by pH and temperature change Thisincludes the recent discovery of a partially unfolded statewhich remains stable at conditions that normally favor anative fully folded state (neutral pH room temperature) Inthe short fourth section we briefly review recent results of ourstudies on cytochrome c-liposome interactions Cardiolipin-containing liposomes serve as model systems for the innermembrane mitochondria in these studies [21]

2 Heme-Protein Interactions in Cytochrome c

21 Secondary and Tertiary Structure Figure 3 shows thecrystal structure of horse heart ferri-cytochrome c Thecoloring of the different structural segments follows a codeintroduced by Englander and coworkers [18 23] Its sig-nificance for the understanding of the proteinrsquos folding isexplained below Cytochrome c is a globular protein with an120572-helical fraction of ca 40 [25 26]That value is lower thanwhat one observes for the classical heme proteins myoglobinand hemoglobin [2]The twomain helical sections are the so-called N- and C-terminal helices Noncovalent interactions

Figure 2 Visualization of the secondary and tertiary structures offerro-horse heart cytochrome c obtained from pdb 2FRCThe figurehas been produced with VMD software [22]

105

10090

80

70

60 50

40

30

20

10

Foldon 3

HEME

Foldon 3

C-helixFo

ldon

4

Foldon 2

Foldon 5-lo

op

60rsquos helix

Foldon 1-N-helix

NE

E

EEN ADT

TFYGP

AQGT

KRGF

LG

HL

NP

GT

KH

KG

GK

EV

CHT

NKKKWT GI

L

LTM

Y

PKKYI

IFAGI

PGTKM

ATNE XGDVEKGKKIFVQKCA

Q

KKLYA

KKKTE

EDLI

R

Figure 3 Structure of oxidized horse heart cytochrome c presentedwith a color scheme that illustrates the different foldons identifiedby Krishna and coworkers [23] These foldons and the respectivecolor code are explained in detail in the text The outside circleprovides the primary amino acid sequence of the different proteinsegments The figure has been taken from Soffer [24] The originalfigure exhibiting the different foldons can be found in [23]

between these two helices are pivotal for the stabilization ofthe native state [18 27ndash29] A third helix (the so-called 60rsquoshelix) is much shorter than the C- and N-helices The restof the protein comprises rather long loop segments Two ofthem are of particular importance Firstly the loop startingfrom the N-terminal helix contains H18 the imidazole sidechain of which provides the proximal ligand of the heme ironTheCxyCmotif with the two cysteins covalently linked to theheme via thioether bridges is located between H18 and theN-terminal helix The notation xy indicates different amino

New Journal of Science 3

acid residues in different cytochrome c derivatives In horseheart cytochrome c the segment reads as CAQC Secondlythe red loop region in Figure 3 theΩ-loop contains theM80the sulfur atom of which provides the second axial ligand forthe heme iron

22 Asymmetric Deformations of the Functional Heme GroupIn spite of a rather asymmetric heme environment and strongheme-protein interactions via the two axial ligands [10 3031] the thioether bridges [32] the internal electric field atthe heme [33ndash38] and to a lesser extent substituent-hydrogenbonding [25 26 39ndash41] and multiple van der Waals contacts[42] spectroscopists have for a long period of time consideredthe heme as exhibiting an ideal D

4h-symmetry This was(and in part still is) particularly true for the interpretationof optical absorption and resonance Raman data [43ndash49]This view ignores very early EPR data for many paramagneticheme proteins including ferricytochrome c that revealed arhombic deformation of the heme ironrsquos ligand field whichreflects deformations of the heme group assignable to theirreducible representation 119861

1119892of the D

4h-point group [10 3050] Such a deformation lowers the overall symmetry of theheme to C

2v Chemical shift distributions in for example 1HNMR spectra of methyl groups revealed the nonequivalenceof the methyl substituents of the heme and thus a hemesymmetry much lower than D

4h [30 51 52] Senn et alreported NMR-data that revealed a rather asymmetric spindensity distribution of the heme macrocycle in a cytochromec derivative [53] However none of these studies involved asystematic investigation of heme deformationsThis task wasfirst carried out by resonance Raman dispersion spectroscopy(RRDS) the results of which are described in detail in thefollowing

Already in the seventies Collins et al [54] reported thatthe depolarization ratios of structure sensitive bands in theresonance Raman spectrum of horse heart ferrocytochromec show a rather strong dispersion in the 119876-band regionof the optical spectrum exhibited in Figure 4 Thus theyclearly deviate from their D

4h-expectation values which donot depend on the choice of the excitation wavelength [43]Shelnutt as well as Zgierski and Pawlikowski explained thesedispersions in terms of electronic and vibronic perturba-tions which will be explained below [55 56] A systematicinvestigation of Raman depolarization ratio dispersions andresonance excitation profiles of various heme proteins wassubsequently in our research group at the University of Bre-men [57ndash60] For ferrocytochrome c we reported dispersionand excitation data over a rather broad range of excitationwavelengths that encompasses the entire119876-band region [60]As an example Figure 5 shows the depolarization dispersionand resonance excitation profile of the oxidation marker ]

4

It is the dominant band in spectra taken with 119861-(Soret) bandexcitation while it is comparatively weak in spectra takenwith 119876-band excitation (cf the optical spectrum in Figure 4)[61] The eigenvector of the corresponding normal mode isshown in Figure 6 It involves C

120572-C120573and C

120572-N stretching

modes which make it sensitive to axial ligands induced

100

50

0

Abso

rptio

n (

)

300 400 500 600

Wavelength (nm)

B0

BV

Q0

QV

Figure 4 Visible absorption spectra of cytochrome c ferrocy-tochrome c (blue) and ferricytochrome c (red) Taken from [64] andmodified

asymmetric deformations of the heme [11] In an ideal D4h-

symmetry the depolarization ratio of this band is 0125indicative of the 119860

1119892-symmetry of its eigenvector (Figure 6)

[43] Raman excitation of this band is generally assumedto be exclusively governed by Franck-Condon type vibroniccoupling describable byAlbrechtrsquos A-term contribution to theRaman tensor [43 45 48 49] If this was the case the bandwould be practically undetectable in the entire preresonanceand resonance regions of the Q-band contrary to what theexcitation profile in Figure 5 suggests In reality the ]

4-

band gains considerable Raman activity fromHerzberg-Tellercoupling [19 60 62 63]

The underlying theory utilized to analyze the data inFigure 5 is rather complex and has been described in severalpapers and comprehensive review articles [11 19] Since thispaper focuses on structural and related functional propertiesof cytochrome c we consider only the structural informationthat emerged from the analysis of the Raman data If oneneglects multimode mixing and stays in the regime of weakcoupling limit the Raman cross section will be proportionalto the squares of the following vibronic coupling matrixelements

119888Γ119903

119890119904= ⟨119890

100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (1)

where 119890119897is the electronic Hamiltonian which also contains

the Coulomb interactions between the nuclei 119876Γ11990301

is transi-tion matrix element of the vibrational 0 rarr 1 transition forthe normal coordinate 119876Γ119903 of the 119903th Raman active vibra-tion exhibiting the symmetry Γ

119903in an ideal D

4h-symmetryEquation (1) describes the vibronic coupling between excitedelectronic states |119904⟩ and |119890⟩ which for porphyrins can beequated with |119861

119909⟩ |119861119910⟩ |119876119909⟩ and |119876

119910⟩ In D

4h the 119861- and119876-states are twofold degenerates that is 119864

119861119909= 119864119861119910

and119864119876119909

= 119864119876119910 Since 119876- and 119861-states exhibit 119864

119906-symmetry


17 19 21 23 25 27

Excitation wavenumber (103 cmminus1)

A1g minus 1363 cmminus1

100

10

1

01

Tota

l exc

itatio

n

(a)

Q B 00 1001 11

00 1001 11

1

01

Dep

olar

isatio

n ra

tio

17 19 21 23 25 27


(b)

Figure 5 Depolarization ratio dispersion (left) and resonance excitation profile of the ]4-band of horse heart ferrocytochrome c in solution

at pH 7The solid line results from amultimode fitting procedure described by Schweitzer-Stenner et al [60] fromwhere the figure was takenwith permissionThe data in the Soret band region of the resonance excitation profile have been measured earlier by Champion and Albrecht[49]

Figure 6 Normal mode pattern of the ]4-mode of Ni (II)-

octaethylporphyrin a canonical model system for the vibrationalanalysis of metal porphyrins The analysis was carried out by DrEskoUnger in the authorrsquos former research group at theUniversity ofBremen and has not been published The theoretical approach usedfor this analysis has been described by Unger et al [62]

only vibrations of 1198601119892 1198611119892 1198612119892 and 119860

2119892can contribute

to vibronic coupling Thus only modes exhibiting one ofthese symmetries can be resonance Raman active They canbe distinguished by their excitation wavelength independent

depolarization ratios of 0125 075 075 andinfin [43 44 65]1198611119892- 1198612119892- and 119860

2119892-modes are predominantly though not

exclusivelyHerzberg-Teller active this type of couplingmixescomponents of different electronic states (119876

119909 119876119910with 119861

119909

119861119910 in our case) Generally modes of different symmetry can

be selectively enhancedwith different excitationwavelengthsThis is illustrated by the resonanceRaman spectra of yeast fer-rocytochrome c in Figure 7 The (polarized) spectrum takenwith 442 nm excitation is dominated by Franck-Condon(1198601119892) and to a lesser extent Jahn-Teller type (119861

1119892) coupling

to the 119861-band transitions Raman bands in the 521 nmspectrum (119876V excitation) are mostly depolarized and areassignable to both 119861

1119892and 119861

2119892modes which both gain their

Raman excitation mostly from Herzberg-Teller couplingThe 531 nm excitation lies between 119876

0and 119876V resonances

and is dominated by inverse polarized bands assignable to1198602119892-modes In this spectral region Herzberg-Teller coupling

in this region exhibits constructive interference for 1198602119892-

modes and destructive interference for 1198601119892- 1198611119892- and 119861

2119892-

modes Excitation with 568 nm coincides with the 119876119900-band

region Here constructive interferences between Jahn-Tellerand Herzberg-Teller coupling make bands assignable to 119861

1119892-

modes dominate the spectrum [62]As indicated above the real symmetry of a heme group is

not D4hThis implies that a perturbation potential119881 has to be

added to 119890119897that is produced by the peripheral substituents

and the protein environment [19 66]

119888

Γ1199031015840

119890119904 = ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (2)


where the electronic perturbation 119881Γ119895 is classified in terms

of irreducible representations Γ119895of the D

4h point groupThis approach which was first introduced by Shelnutt [55]assumes that the perturbation of the heme group is not largeenough to eliminate all resemblancewith theD

4h point groupWhile 119881Γ119895 does not depend on the chosen normal vibrationthe derivative of this potential in (2) is different for eachRaman active vibration Following Zgierski and Pawlikowskiwe call this contribution vibronic perturbation [56]

Based on the assumption that 119881 ≪ 119867119890119897 we expressed

the perturbation potential in terms of so-called normalcoordinate deformations 120575119876Γ119895 [11]

119881Γ119895

=

120597119890119897

120597119876Γ119895

120575119876Γ119895

(3)

As later shown by Jentzen et al each possible distortionfrom D

4h is describable as a superposition of deformationsalong normal coordinates of the porphyrin macrocycle [67]For energetic reasons coordinates of modes exhibiting thelowest frequency for a given symmetry Γ

119895contribute pre-

dominantly to the overall heme deformation The patterns ofsuch deformations which can be subdivided into in-planeand out-of-plane deformations are shown in Figure 8 In-plane deformations exhibit 119860

1119892- 1198611119892- 1198612119892- 1198602119892- and 119864

119906-

symmetry In what follows we will focus on 1198611119892- and 119861

2119892-

type deformationsThe former involves the pyrrole nitrogensand produces a rhombic deformation of the ironrsquos crystalfield 119861

2119892-deformation affects the meso-carbons the formed

deformation can be described as triclinic Out-of-planedeformations transform like 119860

1119906 1198611119906 1198612119906 1198602119906

and 119864119892

which Jentzen et al termed propellering ruffling saddlingdoming andwaving In what follows wewill focus on rufflingand saddling which both affect the relative orientation of thepyrroles rather than the metal position [32 67]

The relationship between119881 and 120575119876 is that between causeand effect A perturbing potential exerted by substituentsand the protein environment forces the heme group into anew equilibrium structure that can be constructed from thepure D

4h-conformation by subjecting the latter to a linearcombination of 120575119876119904

Apparently the occurrence of in-plane (ip) and out-of-plane (oop) deformations mixes contributions of differentsymmetries into the vibronic coupling matrix elementsWhile the first term in (1) transforms like the irreduciblerepresentation of 119876Γ119903 in D

4h the second one adds productrepresentations Γ

119903otimes Γ119895 This mixing of symmetries changes

the depolarization ratio of the Raman mode Combined withsome interference between Franck-Condon Jahn-Teller andHerzberg-Teller coupling this causes a dispersion of thedepolarization ratio as shown in Figure 5 for the 119860

1119892-mode

]4[60]The depolarization dispersion of ]

4can be used for

a qualitative assessment of the deformations of the hemesymmetry in ferrocytochrome c In D

4h the depolarizationratio is 0125 The experimental data suggest that it is largerat all excitation wavelengths Any in-plane deformation of1198611119892- 1198612119892- or 119860

2119892-symmetry increases the depolarization

ratio Only1198602119892-deformations can increase it above 075 what

15 ClO3minus 22 21 4 1119

568nm

531nm

521nm

442nm

times103

20

15

10

5

0

20

15

10

5

0

60

40

20

0

100

50

0

Ram

an in

tens

ityRa

man

inte

nsity

Ram

an in

tens

ityRa

man

inte

nsity

400 600 800 1000 1200 1400 1600

Wavenumber (cmminus1)

Figure 7 Polarized resonance Raman spectra of horse heartferrocytochrome c in aqueous solution taken at the indicated exci-tation wavelengths Spectra recorded with 119909-polarization (parallelpolarized to the exciting laser beam) are displayed in black andthose with 119910-polarization (perpendicular polarized to the excitinglaser beam) are displayed in gray 119861-band excitation was achievedwith 442 nm excitation Excitation wavelengths 521 531 and 568 nmcorrespond to119876V-band the region between1198760 and119876V band and1198760band excitation respectively Taken from [61] with permission

indeed happens for ]4(cf Figure 5) in the region between

the 1198760and the 119876V resonances However the contribution

of antisymmetric vibronic coupling is negligible outside ofthe 119876-band region [19 43] Thus the dispersion between119876- and 119861-band region must result from 119861

1119892- and 119861

2119892-type

deformations While the depolarization of the ]4-mode does

not allow for distinguishing between 1198611119892

and 1198612119892 this task

can be accomplished by measuring the depolarization ratiosof 1198611119892- and 119861

2119892-modes (]

10]11) [41 59] Respective data

clearly indicate that both types of deformations affect theheme group of ferro- and ferricytochrome c [41]

Thus far we have interpreted deviations of the depolar-ization ratio from its D

4h value solely in terms of in-plane


SADB1u

WAX

RUFB1u

WAY

DOMA2u

PROA1uEg119909 Eg119910

(a)

MSTB2g

NSTB1g

TRX TRY

BREA1g

ROTA2g

Eu119909 Eu119910

(b)

Figure 8 Representations of (a) in-plane and (b) out-of-plane deformations of the porphyrinmacrocycle classified in terms of the irreduciblerepresentations of the D

4h point group Each of these deformations reflects the normal coordinate pattern of the lowest frequency mode ofthe respective symmetry Taken form [67] and modified

deformations However for cytochrome c this would lead tothe conclusion that antisymmetric119860

2119892-type deformations are

dominant This is at odds with two observations Firstly thelowest frequency of 119860

2119892-modes of the porphyrin macrocycle

lies well above respective values for 1198611119892

and 1198612119892

[65] whichmakes it much more difficult to deform the heme along theformer than along the latter Secondly a decomposition ofthe deviation of the cytochrome heme group in the crystalstructure of the protein from its idealD

4h-symmetry into nor-mal coordinate deformations (called normal mode structuraldecomposition NSD) revealed very weak 119860

2119892-deformations

[67] The reason for the observed large depolarization ratioof the ]

4-band lies in the existence of rather significant

oop-deformations of the heme group of cytochrome c [3267] These deformations have escaped the attention of crys-tallographers for decades but they were finally discoveredand analyzed by concerted efforts of Jentzen Shelnutt andcoworkers [32 67ndash70] These researchers developed NSDas a tool of structural exploration of heme chromophoresof a large number of heme proteins [67] For ferro- andferricytochrome c they inferred a larger ruffling from thecrystallographic data This is followed by saddling whichexhibits 119861

2119906-symmetry Figure 9 visualizes the result of the

NSD analysis of the heme group in horse heart cytochromec Together they lower the symmetry from D

4h to 1198784 If bothof these deformations are present they can affect vibroniccoupling via the second order term [63 71]

119881Γlowast

=

1205972119890119897

12059711987611986111199061205971198761198612119906

1205751198761198611119906

1205751198761198612119906

(4)

where 1205751198761198611119906 and 1205751198761198612119906 denote ruffling and saddling normaldeformationsThe electronic part of (4) transforms like 119861

1119906otimes

1198612119906

= 1198602119892 which explains the strong 119860

2119892-type contribution

to vibronic coupling of ]4 Strong antisymmetric contribu-

tions to the Raman tensor of symmetric1198601119892-typemodes have

been observed for highly nonplanar metal porphyrins witha symmetric arrangement of peripheral substituents [63 71]thus confirming the above interpretation

The qualitative group theoretical analysis presented thusfar and the more detailed analysis described in our earlierpapers [60] reveal a significant presence of in-plane 119861

1119892-

and 1198612119892-type deformations as well as the combined effect

of ruffling (1198611119906) and saddling (119861

2119906) for ferrocytochrome

c Raman dispersion data for ferricytochrome c are morelimited in scope but they are diagnostic of an above theaverage rhombic 119861

1119892-deformation which leads to rather large

depolarization values for the ]4-band in the Soret band region

(120588 gt 02) [72] The depolarization ratio of 1198601119892-modes is

rather insensitive to 1198611119892- and 119861

2119892-type deformations in this

spectral region owing to the dominance of 1198601119892-type Franck-

Condon coupling in the Raman tensor [19 59] A similarobservation has been made earlier for myoglobin cyanide[59] which as ferricytochrome c exhibits a hexacoordinatedlow spin state A large rhombic distortion in heme proteinswith this spin and ligation state is likely to result fromstatic Jahn-Teller distortions which are facilitated by the 119864

119892-

ground state of the heme iron The existence of a ratherstrong rhombic deformation in ferric cytochrome c speciesis confirmed by EPR data which allowed for the discoveryof two types of ferric cytochrome c proteins type I with anegligible and type II with a very strong rhombic splitting ofthe 119889119909119911119910119911

energies [30] Apparently horse heart cytochromec belongs to the type II category

The NSD analysis of Shelnutt and coworkers allows acomparison of the magnitude of out-of-plane and in-plane


Pero

xida

seYeast

Manganese

A ramosus

Displacement (A)minus12 minus10 minus08 minus06 minus04 minus02 00 02 04 06

(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450

A)minus06 minus04 minus02 00 02 04 06 08 10 12

(b)

Rice

Horse

Tuna

Displacement (A)minus06 minus04 minus02 00 02 04 06 08 10 12

cyt-c

(c)

Alcaligenes sp NCIB

A denitrificans NCTC

R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)

Figure 9 Out-of-plane deformations of the heme group obtained from the NSD analysis of the indicated heme proteins The color code forthe different deformations is displayed at the bottom of the figure Technical details of the NSD analysis and the pdb-structures used for theanalysis can be found in [32] from where this picture was taken and modified

deformations While the former can reach 10 A (Figure 12)the latterwere found to be in the range between 005 and 02 A[32]

Another strategy to analyze out-of-plane deformations ofmetalloporphyrins has been developed in our group basedonmeasurements of Raman bands assignable to out-of-planevibrations They would all be forbidden if the porphyrin(heme) was planar since modes of ungerade symmetrycannotmix states of119864

119906-stateHowever in the presence of out-

of-plane deformations the vibronic coupling matrix elementreads as

119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D

4h-symmetry of the out-of-plane deforma-tion To illustrate the meaning of (5) consider a mode of 119861

2119906-

symmetry In the presence of a 1198612119906-deformation (ruffling)

corresponding matrix elements in (5) transform like Γ119895otimesΓ119903=

1198612119906

otimes 1198612119906

= 1198601119892 indicating that the out-of-plane mode

becomes Franck-Condon active with B-band excitationThisis exactly what one obtains We reported a thorough analysisof the intensities and depolarization ratios of several out-of-plane modes of ferrocytochrome c derivatives (horse heartchicken and yeast) and found major differences between thedegrees of heme ruffling in these three cytochromes [73] Acomparison with out-of-plane deformation derived from X-ray data revealed stronger doming and propellering defor-mations for the respective proteins in solution Interestinglythis could be reproduced bymolecular dynamics simulationsThis suggests some subtle differences between the structuresof heme groups of proteins in solution and in single crystals

Thus far we have based our discussion on the assump-tion that the depolarization ratio dispersion solely reflectsdeformations of the ground state Strictly speaking this is anoversimplification As a matter of fact the matrix elements


described by (2) reflect differences between the equilibriumcoordinates of the excited state and the ground state (if 119890 =

119904) or between different excited states (119890 = 119904) However byinserting (3) and (4) into (2) we obtain

119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)

where the 120575119876 terms can be moved out of the second andthird terms of the electronic matrix element Hence wecan conclude that 119888Γ

119890119904depends on both the ground state

deformation and structural differences between the groundstate and excited states

There is now ample evidence based on the investigation ofa variety of heme proteins that heme deformations are func-tionally relevant More than twenty years ago we reportedcorrelations between pH-induced changes of the ligand affin-ity of O

2and CO binding to hemoglobin and corresponding

variations of 1198601119892- and 119861

1119892-type heme deformations [74 75]

Several experiments on variousmetal porphyrins revealed aninfluence of out-of-plane deformations particularly rufflingon ligand binding and redox potentials [76 77] Michel et alused EPR-data of ferric cytochrome c

552mutants to show a

correlation between heme ruffling (1198611119906) a destabilization of

the ironrsquos 119889119909119910-orbital and a decrease of the redox potential

119864119898-value [78 79]The destabilization of the 119889

119909119910-orbital (119861

2119892)

is a result of the capability of 1198611119906-type ruffling to mix with

the 1198602119906(120587) orbital Based on EPR and NMR investigations of

Pseudomonas aeruginosa cytochrome c551

and Nitrosomonaseuropaea cytochrome c

552 Zoppelaro et al proposed a corre-

lation between rhombic (1198611119892) deformations and ruffling (119861

1119906)

[30] As shown in a more recent paper from our group eachtype of out-of-plane deformation can indeed induce in-planedeformation of the porphyrin macrocycle described by [72]

ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)

where ΔΓ120582is the total ground state deformation of symmetry

Γ The first sum runs over all vibrations exhibiting the sym-metry Γ 120575119876Γ119894

119894denotes any normal deformation of symmetry

Γ119894of the D

4h point group Obviously the largest deformation(ruffling in the case of cytochrome c) is capable of inducingall types of in-plane deformations of the ground state

23 Probing the Electric Field of the Protein in the Heme PlaneThe deformations discussed thus far are generally assigned tononcovalent heme-protein interactions Ligand-heme inter-actions are generally asymmetric and can promote in-planeand out-of-plane deformations [11 76] In cytochrome cthe influence of peripheral substituents cannot be neglectedThe thioether bridges between heme and CxyC motif area dominant cause for ruffling which can be modified bythe axial ligands [32 68] Propionic acid substituents are

hydrogen bonded to the protein matrix a change of the lattercan induce additional deformations (119861

2119892in the case of a Y57F

mutant) as shown in one of our more recent studies [41]In addition to these contact interactions the influence ofthe electric field on the heme pocket has to be taken intoaccount Fluorescence hole burning experiments on hemeproteins with Zn-substituted heme groups revealed electricfield strengths in the range of 107cmTheoretical calculationsbased on the solution of Laplace equations yielded values inthe same order of magnitude [36] Manas et al argued ontheoretical grounds that this internal electric field can causea splitting of both the 119876- and 119861-band if the field vector doesnot exactly bisect the two transition dipole moments of thetwofold degenerate electronic transitions [35] Band splittinghas indeed been observed for the119876-band of ferrocytochromec derivatives at cryogenic temperatures [33ndash35 61 80] Itis generally not detectable in the 119861-band region owing tothe large band width of the overlapping 119861

119909- and 119861

119910-band

However absorption spectra of Zn-substituted cytochromec in which the 119861-band is narrower due to the absence ofnonradiative decay via the metal states seem to indicate sucha splitting [35]

More direct evidence for 119861-band splitting was recentlyprovided by us [37 81] based on a comparison of visible CDand absorption profiles in the Soret band region Figure 10depicts CD and absorption in the Soret band region of ferri-and ferrocytochrome c The CD spectrum of the oxidizedprotein shows a clear couplet whereas the correspondingspectrum of the reduced species is somewhat more com-plex In both cases the CD spectra do not resemble therespective absorption profile This indicates an underlyingband structure which results from band splitting due to theinfluence of an internal electric field via a quadratic Starkeffect If one assumes the field to be homogeneous (which is asomewhat crude approximation) second order perturbationtheory yields the following splitting of the 119861-state energies[37]

119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)

where119864 is the electric field strength and 120579 is the angle betweenthe electric field and one of the N-Fe-N axis of the heme 1198640

119892

is the ground state energy of the unperturbed heme 1198640119861119909

=

1198640

119861119910denote the energies of the unperturbed excited state If

120579 = 1205874 the electric fields shift the 119909- and 119910-componentsby a different amount thus causing a splitting of the 119861-bandThis splitting can be significant owing to the strong transitiondipole moment of the 119861-state transition This splitting canlead to a CD couplet if the two components carry rotationalstrength of opposite sign which is the case in both redoxstates of cytochrome c

The solid lines in Figure 10 resulted froma comprehensiveanalysis which also took vibronic perturbations into consid-eration 119861

1119892-perturbations for instance can either increase


22 24 26 28

Wavenumber (cmminus1 lowast103)22 24 26 28 22 24 26 28

Wavenumber (cmminus1 lowast103) Wavenumber (cmminus1 lowast103)

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

Horse heart Bovine Yeast8

6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28

Wavenumber (cmminus1 lowast103) Wavenumber (cmminus1 lowast103) Wavenumber (cmminus1 lowast103)

10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

Horse heart Bovine Yeast

(b)

Figure 10 Circular dichroism and absorption spectra in the Soret (119861) band region of horse heart ferri- (upper figures) and ferrocytochromec (lower figures) The solid lines in these figures result from a global vibronic analysis described by Schweitzer-Stenner [37] from where thisfigure was taken and modified


or decrease electric field induced coupling depending on thesign of the coupling matrix elements [37 59] While the Starkeffect does not alter the individual band profile of 119861

119909and 119861

119910

vibronic 1198611119892-perturbations can have a major impact in this

regard since they enter the Franck-Condon coupling terms of119861119909and119861

119910with different signs As a consequence the vibronic

progression of one component is enhanced while that ofthe other one gets substantially diminished [82] Consideringthese vibronic contributions is essential for simulating theCD-band profiles of both cytochrome c species correctlyFor myoglobin cyanide strong 119861

1119892-perturbations lead to the

dispersion of the absorption polarization of single MbCNcrystals [59]

The electric field induced and vibronic splitting of the 119861-bands of ferri- and ferrocytochrome c as obtained from theabove discussed analysis of Schweitzer-Stenner are listed inTable 1 The electric field values are in good agreement withresults from the earlier mentioned hole burning experimentsThe difference between the electric field strengths in the twooxidation states accounts for a substantial fraction of theproteinrsquos redox potential It should be noted that the electricfield strength and the difference between the oxidation statesare less in yeast cytochrome c [59]

Some common misconceptions in interpreting spectro-scopic data of heme proteins deserve to be briefly discussedhere It is well established that a substantial part of therotational strength of heme transitions is induced by dipole-dipole coupling with chromophores in the protein (aromaticside chains and protein backbone) [83 84] Another con-tribution results from nonplanar deformations which candestroy the inversion center of the heme [85] In this contextthe two components of the CD couplet displayed in the Soretregion of ferricytochrome c are interpreted as resulting fromthe coupling of the heme with different amino acid residuesin the heme pocket without wasting any thoughts about thephysical origin of the couplet A prominent example is theeffect of the reduction of the negative component of thecouplet observed in CD spectra of cytochromes in whicha single phenylalanine residue in the Ω-loop is replaced bya nonaromatic residue [86 87] In yeast cytochrome c thisresidue is at position 87 while horse heart cytochrome ccontains it at position 82 Pielak et al measured the Soret CDof yeast cytochrome c mutants in which F87 was replaced byother aromatic (Y) and nonaromatic residues (GS) [86] Ineach case the negative component of the couplet disappearedSince the negative component in the native CD spectrumis considered as an indicator of the native state its absenceis interpreted as a conformational change into a less foldedstateThis notion is corroborated by the negative componentrsquosdisappearance in ferricytochrome c spectra measured atextreme pH high temperatures [88] and high denaturantconcentrations [89] However the same effect does not implythe same cause First of all a sole inspection of the CDspectrum does not reveal all necessary facts one has tocompare CD with their corresponding absorption spectra[81 82]The absence of the negative couplet in the CD spectraof the above discussed F87 mutants of yeast ferricytochromec could just reflect the elimination of one important modeof transition dipole coupling between protein and heme

Table 1 Listing of B-band spectral parameters of ferri- andferrocytochrome derivatives inferred from a vibronic analysis of therespectiveCDand absorption band profileΔ119864119861elect electronic (Stark)splitting Δ119864119861vib vibronic splitting V

119861 wavenumber associated the

average transition energy of the split B-band and Γ119861and 120590

119861

Lorentzian and Gaussian halfwidths of the Voigtian B-band profileTaken from [37]

(a) Ferricytochrome c

Horse heart (bovine) YeastΔ119864119861

elect [cmminus1] minus505 minus380

Δ119864119861

elect + Δ119864119861

vib [cmminus1] minus546 minus392

V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome

Horse heart Bovine YeastΔ119864119861

elect [cmminus1] 126 186 217

Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237

which contributes to the negative rotational strength of oneof the 119861-state transitions In this case the substitution ofF87 by a nonaromatic residue would just reduce the negativeCotton band without shifting the absorption band and thepositivemaximum If thismutation causes structural changes(eg involving a rupture of the Fe3+-M80 coordination) onewould expect a blueshift of the absorption spectrum anda reduced noncoincidence between absorption and positiveCotton band As will be described in more detail in thenext chapter that is what is generally observed for partiallyunfolded states of ferricytochrome c in solution

The above discussed Stark splitting is much less pro-nounced in the 119876-band region owing to the much weakerdipole moment of the 119876-state transition [35 37] Howeveroptical absorption measurements on ferrocytochrome c atcryogenic temperatures revealed a sim100ndash120 cmminus1 splitting ofthe 119876-band with intensities for the 119876

119909and 119876

119910components

[35 61 90] Such an intensity redistribution cannot beexplained with a quadratic Stark effect We invoked insteada 1198611119892-type electronic perturbation combined with vibronic

contributions which can be shown to indeed cause a splittingand a redistribution of oscillator strength [61]This perturba-tion (and thus the splitting) is larger in horse heart than inyeast cytochrome c (Table 1) We measured the cryogenic 119876-band spectra of a series of yeast ferrocytochrome c mutants[91] for which Blouin and Wallace had determined theenthalpic and entropic contributions to the redox potential[40] We could show that the 119861

1119892perturbations inferred

from the optical spectra correlate nicely with the square ofthe enthalpic contribution to the redox potential This againunderscores the functional relevance of heme perturbationsof cytochrome c


16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

Wavenumber (cmminus1 lowast10minus3)

Figure 11 Circular dichroism and absorption spectra in the Q-bandregion of horse heart ferricytochrome cThe solid lines resulted froma global vibronic analysis of the spectra described by Schweitzer-Stenner [37] from where this figure was taken and modified

The 119876-band region of ferricytochrome c is more difficultto analyze because the rather broad bands associated withindividual transitions into 119876

119909and 119876

119910states do not allow

a separation even at cryogenic temperatures However acomparison of CD and absorption spectra reveals a nonco-incidence at the 119876

0-position which indicates band splitting

(Figure 11) [37 81] Moreover the CD spectra show a muchbetter resolution of the vibronic119876V-bandwhich facilitates theanalysis further The 119876-band splitting in ferricytochrome c isagain assignable to an electronic 119861

1119892-type perturbation

Taken together this chapter describes a variety of exper-imental techniques which can be employed to explore in-plane and out-of-plane deformation of the heme group incytochrome c The same methods can of course be appliedto other heme proteins Out-of-plane deformations haveattracted more attention in the last fifteen years owing tothe fact that they can be easily inferred from the proteinsrsquocrystal structures However in-plane deformations loweringthe symmetry of the crystal field probed by the heme ironshould be of similar functional relevance The same can besaid about the internal electric field in the heme plane

3 Folding and Unfolding ofCytochrome c in Solution

31 Thermal and pH-Induced Unfolding of Cytochrome cOver the last twenty years cytochrome c has emerged as one ofthe most prominent model systems for studying the folding

and unfolding of proteins Most of the research has therebyfocused on ferricytochrome c because it is much less stablethan the reduced state of the protein To unfold the lattertemperatures above 100∘C or extreme pH values below 3 andabove 12 are required [92ndash96] Ferricytochrome c howeveradopts a variety of states if the pH is changed between 1 and12 and begins to thermally unfold above 50∘C [20]The reasonfor the oxidized proteinrsquos lesser stability lies (a) in the reducedstrength of the Fe3+-M80 coordination [97] which is pivotalformaintaining the proteinrsquos native structure [98] and (b) to aminor extent in larger conformational flexibility [99 100] andsolvent accessibility of the proteinrsquos structure in the oxidizedstate [101 102]

pH-induced unfolding of ferricytochrome c has a longtradition of research It was investigated even prior to theclassical folding unfoldingrefolding experiment of Anfinsen[104] Nearly 70 years ago Theorell and Akesson discoveredfive different titration states which they denoted by theRoman numerals I to V [105] Since then numerous studieshave been performed to characterize these states the resultof which is summarized by the scheme shown in Figure 12States I and II are known to be partially unfolded and arepopulated at acidic pH [94 106ndash108] The actual state of Idepends heavily on ionic strength It is highly disorderedwithreduced secondary structure content at low ionic strengthbut gains secondary structure upon addition of NaCl owingto the shielding of repulsive Coulomb interactions betweenpositively charged groups by chloride ions [109 110] ThisA-state unfolds cooperatively with increasing temperatureDifferent spin states of the heme iron can coexist in state IIdepending on solution conditions [110] State III is generallyconsidered as the most folded state often referred to as theldquonativerdquo state States IV and V are the alkaline states in whichM80 is replaced by lysine residues andhydroxyl ions [111ndash113]Each of them can be divided into substates with different axialligands

TheTheorell-Akesson schemewas recently augmented byus when we reported the existence of a state IIIlowast which is anintermediate between III and IV [103] It is similar to III inthat it still contains a (most likely weakened) Fe-M80 bondIt is detectable only at low ionic strength explaining why it isoften missed in spectroscopic experiments Figure 13 showsthe integrated intensities of 695 nm subbands (their originwill be discussed below) as a function of pHThe data clearlyreveal two different phases which merge into a single oneat high phosphate ion concentration Cherney and Bowlerassigned the state to the deprotonation of one of the propionicacid substituents of the heme group which is known toexhibit a very high pK-value [114] A similar state called III

35

was observed from site specific IR-experiments of Weinkamet al which as IIIlowast is populated at pH 9 [115] These authorsargued that III

35does not exhibit a 695 nmband in the optical

spectrum which is indicative of the integrity of the Fe-M80linkage Our data however show that a weaker 695 nm bandis still present at pH 9 and low ionic strength This contra-diction could be resolved by assuming the coexistence of twodifferent intermediates IIIlowast one with and one without M80ligation At low ionic strength the former is more populated


pH 2 4 6 8 10 12 14

State(s) I II III IIIlowast

IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla

Figure 12 Schematic representation of the different protonation states of ferricytochrome c with the corresponding ligation states of theheme iron In addition to the canonical Theorell-Akesson states I II III IV and V the scheme displays a recently discovered intermediateIIIlowast and the different isomer of states II IV and V Produced by Dr Jonathan Soffer in the authorrsquos research group

800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


Figure 13 Integrated intensities of two subbands of the 695 nm charge transfer band of horse heart ferricytochrome c plotted as a functionof pH The decomposition of the absorption and CD band is shown in the left The solid lines in the plots resulted from the fit of a titrationmodel outlined by Verbaro et al [103] from where we took the figure with permission


while the latter is predominant at high ionic strength atwhich the experiments of Weinkam et al were performed

The transitions III rArr IV and III rArr II can be consideredas the prime pH-induced unfolding transitions It is thereforeno surprise that they were particularly intensively studiedIII rArr IV is called the alkaline transition which involvesthe above intermediates IIIlowast andor III

35 The disappearance

of the 695 nm band in IV indicated early that M80 isreplaced by another ligand in this state [116] Optical andresonance Raman studies on yeast cytochrome c mutants byMauck Hildebrandt and their respective coworkers revealedthe coexistence of two state IV isomers with K73 and K79(Figure 12) [112] Since these isomers are in thermodynamicequilibrium they cannot be responsible for the two phasesof the III rArr IV transition observed by Verbaro et al [103]and Weinkam et al [115] In horse heart cytochrome c theexistence of another lysine (K72) complicates the picturefurther in that it can also bind to the heme [117] In allthese histidine-lysine ligand complexes of the heme thereduction potential is substantially reduced thus stabilizingthe oxidized state [113 118]

One would be tempted to assign the III rArr IIIlowast rArr IVtransition to lysine deprotonation However the intrinsic pKof lysine is at 115 which is way too high to explain theobserved titrations Very early Davis et al based on kineticexperiments showed that the alkaline transition involvesa kinetic intermediate the protonation process is followedby a conformational change which lowers the effective pK-value(s) of the transition [116] Our analysis showed that bothIII hArr IIIlowast and IIIlowast hArr IV equilibria are describable by aHill-type equation which indicates the involvement of morethan a single proton As indicated above Cherney and Bowlersuggested that one of the propionic acid groups of the hemeis a candidate [114]

The effective pK-value of the alkaline transition dependson the ionic strength of the solution Battistuzzi et al foundthat the pK-value increasesmonotonicallywith ionic strength[118 119] However our data reveal that this is true only forthe pK-value of the III hArr IIIlowast the pK-value of IIIlowast hArr

IV decreases with increasing ionic strength [103] It is thiscombined effect which obfuscates the identification of IIIlowast athigh ionic strength According to Battistuzzi et al an increasein the temperature decreases the effective pK of the alkalinetransition [119]

Hoang et al used HD exchange and kinetic studies tofurther characterize the III hArr IV transition [120]They foundstrong evidence for this transition to involve the unfolding ofthe Ω-loop foldons Unfolding means here a misligation ofthe heme In order to allow ligation by one of the lysines theΩ-loop has to carry out something like a translational movewith respect to the heme group This view is in agreementwith the NMR-based structure that Assfalg et al reported oftwo yeast cytochrome c mutants K73A and K79A [121] Themeasurements were carried out at pH 11 and moderate ionicstrength (50mM phosphate buffer) It is not clear whetherthe protein is exclusively in state IV at these conditions Theuse of the two mutants avoids complications which wouldarise from the coexistence of the two state IV isomers TheNMR structure reveals that themost dominant changes occur

Figure 14 Comparison of the structure of alkaline (state IVVred) and native (grey) yeast ferricytochrome c derived from two-dimensional NMR experiments The figure was taken from [121]with permission

indeed in the Ω-loop region Figure 14 shows a comparisonof the structure of the native and the state IV (V) proteinreported by Assfalg et al [121]

Unfortunately state V has not yet attracted as muchattention of researchers as state IV The Raman study ofDopner et al revealed the existence of two V-state isomers adominant V

119886 and a less populated V

119887state [112]The authors

managed to extract the Raman spectra of the latter from theirdata in which the position of spin marker bands is consistentwith OHminus being the sixth ligand The ligand of V

119886might

still be a lysine CD and absorption spectra to be presentedbelow provide further evidence that states IV and V areindeed distinguishable particularly with respect to thermalunfolding The acid unfolding of horse heart cytochrome cwas first investigated thoroughly byMyer [123]Their data ledthem to propose a scheme III hArr III

119886hArr II hArr I where III

119886

was described as a III-like intermediate very similar to theIIIlowast state detected by Verbaro et al [103] The respective pK-values were reported as 36 27 and 12 at low ionic strengthThey barely change in the presence of salt The authorssuggested that state II is hexacoordinated high spin and stateI is pentacoordinated high spin A somewhat different pictureemerged from the kinetic studies of Dyson and Beattle onthe same cytochrome c derivative [106] They found that theprotein has lost both axial ligands in state I which is highlydisordered Two coexisting states were proposed for stateII one with low and the other with a high spin heme Theauthors proposed that the former should still exhibit M80as ligand However this seems to be very unlikely since the695 nm band is absent in the pH range at which state II ispopulated Work of Jeng and Englander as well as of Goto etal revealed a strong influence of salt on the conformational


mixture at low pH [107 124] The protein is predominantlymolten globule (ie with an intact secondary structure) instate II and a statistical coil in state I at low ionic strengthAt high ionic strength the statistical coil state I converts to amolten globule which was later termedA-state [109 110 125]More recently Battistuzzi et al used visible absorption andmagnetic circular dichroism spectra to probe conformationaltransition of yeast iso-1 ferricytochrome c and three mutants(M80A M80AY67H and M80AY67A) in the acidic regionand a low ion concentration [126]They found that state III ofthe wild type first converts into a hexacoordinated high spinstate most likely with water as sixth ligand Below pH 26a new hexacoordinated high spin state is formed for whichthe protein is mostly unfolded and the heme is ligated bytwo water molecules The replacement of M80 by an alanineproduces a hexacoordinated low spin state with OHminus as sixthligand reminiscent of state V

119887[112] Lowering the pH causes

the population of different high spin states with H2O as sixth

ligandwhich differ in terms of the protonation state of one theheme grouprsquos propionic acids and the nature of the proximalligand

In our laboratory we recently measured the absorptionand visible CD spectra as function of pH in the absence ofsalt and at the lowest possible ionic strength [127] Figure 15shows the Kuhn anisotropy Δ120576120576-value measured at thewavelength position of the negative maximum of the nativestate spectrum as a function of pH These data clearlyallow the identification of all protonation states The datareproduced the finding of Verbaro et al [103] in that theydistinguish between states IIIlowast and IV but gave no indicationof the existence of the III

119886-state reported by Myer [123 128]

States IV and V are clearly distinguishableThe thermal unfolding of ferricytochrome has been

studied for quite some time Earlier UVCD measurementsby Myer and Pande clearly indicate two phases of thermalunfolding at physiological conditions an observation imply-ing the existence of an unfolding intermediate [128] Theexistence of the latter followed also from the observation thatthe 695 nm band disappears at temperatures below the onsetof protein unfolding Taler et al based on the results of NMRexperiments suggested a pK-shift of the alkaline transitionwith increasing temperature as the source of this intermediate[129] However this notion was questioned by Battistuzziet al who from their analysis of the temperature dependenceof the alkaline transition found a nonnative state populatedat high temperatures which is clearly distinct from state III[119]

The thermal unfolding of ferricytochrome c was recentlyinvestigated further by us using spectroscopic means Firstwe measured the 695 nm band profile as a function oftemperature at pH 6 and 7 [130] We found that the bandprofile itself is changing at high temperature This wasrelated to the slightly different partial unfolding of differentsubconformation of the protein We had shown earlier thatthe 695 nm band can be decomposed into different subbandsthat should be related to different substates of the Fe-M80linkage [81] Our analysis was later confirmed by the resultsof low temperature CDmeasurements [131] As recognized by

pH2 4 6 8 10 12

I II III IIIlowast IV V

Δ120576120576

times10minus4

24

20

16

12

08

04

Figure 15 Kuhn anisotropy (Δ120576120576) of 05mM oxidized cyt-119888measured at 405 nm as a function of pH in 01mM MOPS bufferat 298K (25∘C) Taken from [122]

100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)

Figure 16 Oscillator strengths of the three subbands of the 695 nmabsorption band of bovine heart ferricytochrome c as function oftemperature Filled circles 1198783 triangles 1198784 open circles 1198782 Thesolid lines result from a thermodynamic analysis described in thetext and by Schweitzer-Stenner et al [20] from where this figurewas taken and modified

Frauenfelder and coworkers proteins in different subconfor-mations perform the same function but with different rates[132]

Figure 16 shows the temperature dependence of extinc-tion coefficients measured at three different wavelengths andpH 7 which according to Dragomir et al correspond to theabove subbands [81] The transition curves are clearly bipha-sic one of the curves indicates even the existence of anotherlow temperature phase which becomes more apparent atacidic pH These data will be discussed again below whenwe present a thermodynamic analysis of different data setsHowever just a visual inspection of the data reveals thatan intermediate state is populated at temperatures around320K and that the Fe3+-M80 ligation is still intact in thisintermediate It is thus much more reminiscent of the stateIIIlowast reported by Verbaro et al [103]

To explore thermal unfolding further we measured thetemperature dependence of the Soret absorption and CDspectra at different pH values in the neutral and alkalineregions [88] The selected pH values correspond to popu-lations of the states III IIIlowast IV and V We used bovinerather than horse heart ferricytochrome c because the former


22000 23000 24000 25000 26000 27000 28000Wavenumber (cmminus1)

20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0

Figure 17 CD and absorption spectra of the 119861-band region of oxi-dized bovine heart cytochrome c dissolved in a 1mMMOPS buffer(pH 7 at room temperature) measured as a function of temperaturebetween 5∘ and 90∘C Arrows indicate changes in temperatureTaken from [20] and modified The original experimental data arefrom Hagarman et al [88]

is less prone to aggregation at high temperatures than thelatter Figure 17 shows the visible CD and absorption 119861-band spectra measured at pH 7 The CD couplet convertsinto a positive Cotton band with increasing temperaturethe corresponding absorption band shifts to the blue thusminimizing the noncoincidence between absorption andCDThis measurement was deliberately performed with a Tris-HCl buffer which exhibits a pH shift with increasing temper-atureThus the authors avoided any overlap with the alkalinetransition Subsequently they performed the same measure-ments with aMOPS buffer which is very thermostableThesetwo protocols yielded practically identical results Figure 18shows Δ120576 values measured at the position of the negativeand positive maximums of the native protein as a functionof temperature In addition the temperature dependence ofa Δ120576 of the corresponding UVCD spectrum is displayed Thetransition curve obtained from the latter is clearly biphasica closer inspection reveals the same for the former Figure 19shows the visible Soret CD and absorption profiles measuredat pH 105 and 115 at different temperatures At the ionicstrength conditions used for these measurements (01M) thepH values correspond to states IV and V The CD spectrashow a positive Cotton band even at room temperature therotational strength increases with increasing temperature

280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV

Figure 18 Δ120576 versus temperature of oxidized bovine horse heartcytochrome c between 278 and 363K Upper panel Δ120576 obtainedfrom the CD spectra in Figure 17 at 24876 cmminus1 (triangles) and24010 cmminus1 (filled circles) Lower panel Δ120576 obtained from thecorresponding CD spectra at 44964 cmminus1 The experimental datawere taken from [88] The solid lines result from fits described inthe text The figure was taken from [20] and modified

The room temperature spectra for states IV and V are clearlydistinct which is diagnostic of different heme environmentsFor state V the increase of the Cotton band with increasingtemperature is particularly large In addition one observesa rather surprising redshift of the spectrum At room tem-perature the CD spectrum is slightly blueshifted comparedwith the absorption bandThis is indicative of somemoderateband splitting The noncoincidence is practically absent athigh temperatures The temperature dependences of Δ120576max(ie the dichroism measured at the wavelength at which theroom temperature CD is maximal) are depicted in Figure 20A two-phase transition is clearly on display for state V onlya closer inspection of the data reveals the population of anintermediate state for the ldquounfoldingrdquo of state IV

We subjected these data to a global fitting based on thefollowing equation [20]

Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576

119906are the dichroism values in the native

(119899) intermediate (119894) and (119906) unfolded state 119866119894and 119866

119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)

Figure 19 CD and absorption in the 119861-band region of oxidized bovine cytochrome c measured as a function of temperature between 5∘ and90∘ Left the measurements were performed with a pH of 105 with a 01M Tris buffer Right the measurements were performed with a pHof 115 with a 01M Tris buffer The arrows in the left figure indicate the changes with increasing temperature for both data sets The originaldata were obtained from Hagarman et al [88] the figure was taken from [20] and modified

the Gibbs energies of states 119894 and 119906 with respect to 119899 whichare written as follows

119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867

119906indicate enthalpies of the intermediate and

unfolded state 119879119894and 119879

119906are the corresponding transition

temperatures 119878119894and 119878

119906are the entropies and 120575119888

119901is the

change of the heat capacity associated with the 119894 rArr 119906

transition This formalism was used to fit the data in Figures18 and 20 The obtained thermodynamic parameters areshown in Table 2 We used a modified formalism to globallyfit the 695 nm data in Figure 16 [20] which led to the solidlines shown therein

Our analysis revealed a rather complex picture ofcytochrome c thermal and pH-induced unfoldingTheir dataclearly revealed the existence of three nonnative alkalinestates termed IIIlowast IV and V Synchrotron UVCD spectraof these states clearly show that the secondary structureis mostly maintained and the structural changes are pre-dominant on the tertiary level Each of these states shows

Table 2 Gibbs free energy of hydrogen exchange obtained forthe indicated protein segments of horse heart ferricytochrome creported by Krishna et al [23]

Foldon Identity Δ119866HX[kJmol]

Blue bihelix N- and C-terminal120572-helices 535

Green helix and green loop 60rsquos 120572-helix and 19ndash36Ω-loop 4018

Yellow 120573-sheet 37ndash39 58ndash61 antiparallel120573-strands 3071

Red loop 71ndash85 Ω-loop 2614Infrared loop 40ndash57Ω-loop 158

a two-phase thermal unfolding (not shown for IIIlowast butreported by Hagarman et al) The respective visible CDsignals suggest that all these thermally unfolded states arestructurally distinct For III

119906 IIIlowast119906 and IV

119906the absorption and

visible CD spectra still suggest a low spin hexacoordinatedstate of the heme iron The thermally unfolded state V

119906

is somewhat mysterious The rather large redshift of boththe absorption and the Cotton band at high temperaturesseems to be indicative of a ferrous pentacoordinated complex


280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)

Figure 20 Δ120576 versus temperature of oxidized bovine horse heartcytochrome c between 278 and 363K The Δ120576 values were obtainedfrom theCD spectra in Figures 18 (state IV filled circles) and 19 (stateV open circles) at the position of the Cotton band maximum Theexperimental data were taken from Hagarman et al [88] The solidlines result from fits described in the text and in [20] from wherethe figure was taken and modified

If this is indeed the case the thermal unfolding would beaccompanied by a change of the proteinrsquos redox state

Like many biochemical interactions the foldingunfold-ing of proteins is governed by enthalpy-entropy compensa-tion [133ndash137] The compensation is exact at the folding tem-peratureThe comparability of different folding processes canbe judged based on the similarity of their foldingunfoldingtemperature A closer look on the values listed in Table 2reveals that in particular the values for 119879

119906are not very

different indicating that very similar forces are involved in therespective unfolding processes In this case Δ119867 and Δ119878 showa high degree of correlation which can be described by [133]

Δ119867 = 119879119888Δ119878 (11)

If all folding temperatures of the considered processes areidentical 119879

119888equals these folding temperatures and the corre-

lation coefficient for a fit of (11) to the experimental data willyield 1 A distribution of still similar transition temperaturesleads to a representative 119879

119888-value and correlation coefficients

in the 097ndash099 regime [127]It is interesting to compare the thermodynamic parame-

ters listed in Table 2 with Gibbs energies of the HD exchangeof cytochrome c unfolding reported byKrishna et al (Table 3)[23] The values of Δ119866

119894are by a factor of 3 lower than the

Δ119866HD-values these authors reported for the infraredΩ-loopwhich is the least stable foldon whereas the values obtainedfor Δ119866

119906are comparable with the Δ119866HD values reported for

the green helixloop and the blue bihelix foldons Remember-ing that the alkaline transition at room temperature involvesthe unfolding of the Ω-foldons we suspect that the 119899 rArr

119894 transition involves the destabilization of different foldonsat different pH while the 119894 rArr 119906 transition involves thehigher lying foldons The thermal unfolding reported by usin Hagarman et al [88] and Schweitzer-Stenner et al [20] istherefore somewhat different from the recently investigated

thermal unfolding at low ionic strength [127] Here we foundthat even the 119894 rArr 119906 transition does not involve completeunfolding and leaves the blue foldon mostly intact over arather large pH range from 1 to 12 This shows that thepresence of anions in solution does destabilize rather thanstabilize cytochrome c

32 Oligomerization of Cytochrome c We have mentionedabove that a misfolded state of the protein is temporarilypopulated during cytochrome c folding at neutral pH androom temperature [89 138ndash140] The nonnative states of theprotein adopted at alkaline and acidic pH can be consideredmisfolded as well because they bind the wrong ligand butshow a high degree of folding otherwise It is well docu-mented thatmisfolded as well as unfolded proteins are proneto aggregation Dobson even argued that the amyloidogenicstate of proteins in which the unfolded protein has formedrather longer fibrils of parallel or antiparallel 120573-sheets is themost stable state for all proteins [141] This would imply thatthe respective native states are in fact metastable nonequilib-rium states With regard to cytochrome c thermal unfoldingleads to protein aggregation into 120573-sheet structures andsubsequent precipitation at temperatures above 350ndash360K[142] Acidic states I and II are more prone to aggregationthan the alkaline state State II can be converted into solubleaggregates even at moderate temperatures [139]

Besides aggregation into soluble and insoluble fibrilscytochrome c exhibits another mode of aggregation whichis much more interesting It is well known for more thanforty years that cytochrome c can form nonamyloidogenicoligomers after dissolving the protein in ethanol [143] How-ever only recently Hirota et al reported the crystal structureof the formed oligomers [144] They found that dimerstrimers and even tetramers of cytochrome c are formed bydomain swapping that is the C-terminal becomes displacedfrom its original position and links a proteinwith another onevia nonbonding interactions (Figure 21) Here monomersand oligomers are in equilibrium The involved structuralchanges of the protein involve the dissociation of the Fe3+-M80 linkage and the ligation of the heme iron with OHminuswhich makes them reminiscent of state V

119887as characterized

by Dopner et al [112]This phenomenon of domain swappingwas recently obtained for other proteins such a serpin [145]Subsequent investigations by Hayashi et al revealed similardomain swapping for other cytochrome c species [29] Mostrecently they found that dimer formation via domain swap-ping occurs during the folding of the protein from a dena-tured state in solutions withmoderate GuHCl concentrations[140]Thus the formed cytochromeoligomers showenhancedperoxidase activities which made researchers wonderwhether cytochrome c oligomers might be involved in suchactivities on the inner membrane of the mitochondria [146]

A rather peculiar strategy for obtaining cytochrome coligomers with an intact secondary structure was recentlyreported by us [147] We oxidized cytochrome c at pH 115(state V) and allowed incubation times up to a week atthis condition After switching the solution back to moreneutral pH the protein did not refold We could show that


Table 3Thermodynamic parameters obtained from fitting the data in Figures 16 18 and 20 reflecting the two-step thermal unfolding of theprotein Taken from [20]

pH 7 MOPS pH 7 Tris-HCl pH 105 pH 115Δ1198670

119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355

119888119901[kJmolsdotK] minus29 minus29 minus28 minus28

C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)

Figure 21 Crystal structures of oxidized monomeric and oligomeric cyt-c (a) Structure of monomeric cyt-c (PDB ID code 1HRC) (b)Structure of dimeric cyt-c (red and blue) (c) Structure of trimeric cyt-c (red blue and green) The hemes are shown as gray stick modelsSide-chain atoms of M80 are displayed as yellow stick models The N and C termini and the N- and C-terminal helices are labeled as N C120572N and 120572C respectively The T79-A83 residues (hinge loop of the monomer) are depicted in pale colors Taken from [144] with permission


25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)

Figure 22 Charge transfer band region of the absorption spectrumof 05mM ferricytochrome c (equine) measured between 13500and 17000 cmminus1 in 01mM potassium phosphate buffer Prior tothe measurement the oxidized protein was exposed to alkalineconditions at pH 115 for one weekThe label CT1 exhibits the regionof the so-called 695 nm charge transfer band which is practicallyabsent in the displayed spectra CT2 is assignable to a heme rarr irontransition in high and quantum mixed heme irons The spectrumwas taken from [147] with permission

this was not the result of chemical modifications Moreoverwe found that the protein predominantly stays monomericat 50 120583M concentration while substantial aggregation intodimers and trimers was obtained at 05mM Below pH 7 anew nonnative species is formed which was probed by theoccurrence of a weak absorption band at 625 nm Figure 22shows the spectral region between 600 nm and 750 nm ofthe absorption spectrum of oxidized cytochrome c measuredat the indicated pH after one-week oxidation at pH 115The Fe3+-M80 indicator at 695 nm is absent indicating anonnative state The band at 625 nm appears below pH 6and gains intensity with decreasing pH A titration curveplotting the integrated intensity of the 625 nm band versuspH is plotted in Figure 23 An analysis of these data yieldedapparent pK-values of 47 and 64

The 625 nm band would normally be interpreted asindicative of a high spin hexa- or pentacoordinated statesof the heme group [148ndash150] However resonance Ramandata and the position of the respective absorption and CDbands in the Soret band region are inconsistent with such aninterpretationThe only commondenominator of all data setsis a quantum mixed state of the heme iron as found in typeC peroxidases such as horseradish peroxidase [151ndash154] Aquantum mixed state can be formed if the lowest iron stateis high spin with an intermediate spin state not lying higherthan 300 cmminus1 [154] In such a case the two statesmix via spin-orbit coupling Such a quantum mixed state yields low spin-like Raman spectra and a charge transfer band in the regionbetween 620 and 630 nm as obtained [155] If the quantummixed state is pentacoordinated the Soret absorption peaksbetween respective low and high spin band positions againin agreement with our absorption spectra [147]

160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2

Figure 23 Integrated intensity of the CT2 band in the optical spec-trum of horse heart ferricytochrome c incubated under oxidizingconditions at pH 115 for seven days plotted as a function of pHThesolid curve results from a fit explained in the text The picture wastaken from [147] and explained in the text

300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10

Figure 24 Resonance Raman spectra of 05mM ferricytochromec measured at the indicated pH after the protein was subjected tooxidizing conditions at pH 115 for 7 days The spectra were takenwith 442 nmnear119861-band excitationThefigurewas taken from [147]with permission

The UVCD spectra of this nonnative apparently mis-folded protein indicate rather intact secondary structurewhich rules out amyloid formation The oligomers obtainedwith 05mM concentration should therefore be assignedto domain swapping Interestingly these oligomers do notremain in the oxidized state Resonance Raman (Figure 24)spectra clearly reveal coexisting oxidized and reduced statesthe ratio being pH dependentThe population of the reducedstate becomes maximal between pH 8 and 9 and decreasessubstantially towards acidic and alkaline pH This is indica-tive of a yet undiscovered proton-redox linkage which iscurrently under investigation


The work described above reveals that a pronouncedfrustration occurs along the alkaline pathway provided thatthe protein is allowed to stay longer at pH 115 in state V A lesseffective frustration was earlier reported for acid unfolding[156] Frustration means that the protein occupies an inter-mediate state during the folding process which is separated bya large barrier from the native state Since resonance Ramandata obtainedwith 05mMprotein concentration exhibit spinand oxidation marker lines close to positions found in OHminusligated hemes Soffer et al conjectured that the mixture of V

119886

and V119887changes over time in that the predominant fraction

of V119886converts into V

119887over time If this is true it becomes

understandable that state V refolds without any problems ifthe protein stays at the corresponding alkaline pH only for ashort period of time On the contrary refolding of V

119887might

bemuchmore frustrating that is misfolded nonnative statesbecome occupied very early on in its downhill motion in thefolding funnel Since the respective UVCD spectra indicatea lot of helical content it can be assumed that at least theN- and C-helices are fully folded Since they do not formthe native contacts the former is free for domain swappingthus producing dimers trimers and even tetramers with arather regular and ordered quaternary structureThis is likelyto stabilize the misfolded state Zheng et al used the funnel-theory to investigate the formation of multidomain aggre-gates during the folding process [157] Their results indeedsuggest that such oligomers might be more stable than thenative state and that protein are prone to misfolding at mildlyunfolding conditions That is exactly what we obtained [147]

4 Unfolding of Cytochrome c on Surfaces

All folding studies discussed in the preceding paragraph dealtwith cytochrome c in solution While of general interestfor the protein folding community in general and the hemeprotein community in particular the obtained results mightbe considered of limited relevance for an understandingof the proteinrsquos function owing to the fact that its naturalenvironment is the inner membrane of the mitochondria towhich a fraction of the cytochromes in the intermembranespace is bound [158] Investigating the protein in vivo andin vitro is rather challenging even though various attemptshave been made [158 159] Over the last ten years two typesof surfaces have emerged as suitable model system to studystructure and function of cytochrome c in a more realisticenvironment functionalized self-assembled monolayers andanionic phospholipid liposomes In what follows experi-ments with these two types of surfaces are briefly discussed

41 Unfolding of Cytochrome c on Electrode Surfaces Mur-diga Hildebrandt and their respective coworkers usedspectroscopic and electrochemical techniques to studycytochrome immobilized on gold and silver electrodes coatedwith self-assembled monolayers of for example pyridineterminated alkanethiols [160 161] They found a potentialdependent equilibrium between two coordination statesnamely a pentacoordinated high spin and a hexacoordinatedlow spin state of the heme iron In the latter the M80 ligand

was found to be replaced by a pyridinyl residue whichobviously requires that the respective structures are differentfrom the native one The two states were found to differwith respect to their redox potentials In another approachWackerbarth and Hildebrandt coated electrodes with anionsof appropriate acids which produced an array of negativecharges on metal surfaces [162] This way they mimicked tosome extent the anionic surface of the mitochondrial innermembrane Cytochrome c can easily bind to such a surfacevia the positive patches on its surfaceThe authors observed apotential dependent equilibrium between two states termed1198611 and 1198612 The former is the authorsrsquo notation for the nativestate III and the latter is partially unfolded and involves avariety of hexacoordinated and pentacoordinated subspecieswith the former exhibiting bis-histidine complexes Corre-spondingly their redox potential was found to vary betweenminus0425 and minus0385 V

More recently Monari et al used surface enhanced res-onance Raman spectroscopy and electrochemical measure-ments to investigate the influence of urea on surface immobi-lized yeast cytochrome c and its K72AK73AK79A [163]Theyfound that above a urea concentration of ca 6M the nativeM80 ligand is replaced by a histidine ligand reminiscent ofwhat one finds in solution This structural change was foundto cause a 04 V shift of the redox potential towards negativevaluesThey found further that the transition to the nonnativestate requires slightly less urea for the triple mutant whichthey interpreted as indicating a role of the three replacedlysines with regard to the stabilization of the native structureon the surface Interestingly these authors found a nearlyperfect enthalpy-entropy compensation with regard to theGibbs energy contribution to the redox potential whichthe authors measured for different urea concentrations In asubsequent study Ranieri et al showed that yeast cytochromeimmobilized on the type of electrode used by Monari etal became what the authors termed a pseudoperoxidase inthe presence of urea [164] The proteinrsquos activity involved atwo-electron reduction of water The Michaelis-Menten-likeparameters used to describe the process were shown to beaffected by the above replacements of the lysine residues byalanine

42 Unfolding of Cytochrome c on Anionic Liposome SurfacesApoptosis is essential for maintaining the functioning of thehuman body Damaged genetically modified or otherwiseunwanted cells are eliminated by a regulated chain of bio-chemical reactions In an average human adult apoptosiscauses the death of between 50 and 70 billion cells eachday [16] While apoptosis is generally beneficial defectiveapoptotic processes can lead to an extensive variety ofdiseases Excessive apoptosis causes atrophy whereas aninsufficient amount results in uncontrolled cell proliferationsuch as cancer Therefore gaining a complete and thoroughunderstanding of the parameters governing the apoptosispathway is of great importance for any therapeutic approachaimed at battling such defective behavior

In the inner membrane space of mitochondria cyto-chrome c exhibits a delicate balance between membranebound and free cytochromes Cortese et al by performing


a conformational analysis of cytochrome c in mitochondriareported that the protein can adopt nonnative states whenbound to the IMM [158] A characteristic of the IMM isits high cardiolipin (CL Figure 25) content the biphosphatehead group is the main target for cytochrome c bindingStudies carried out over the last 15 years have revealed a rathercomplex and yet incomplete picture of cytochrome c bindingto CL-containing membranes which was mostly obtainedby using liposomes as model systems The most importantresults are briefly reviewed below

The very comprehensive work of Rytomaa and Kinnunenindicates the existence of two protein binding sites of horseheart cytochrome c termed A and C [165] Site A bindingis purely electrostatic and can therefore be inhibited by salt(NaCl) It is formed by the positively charged residues (mostlyK) on the proteinrsquos surface Site C involves hydrogen bondingand requires a slightly acidic pH and might also involve thepenetration of a lipid chain into the interior of the protein(extended lipid anchorage) Kalanxhi and Wallace providedsupport for the notion that another mode of lipid anchorageis actually associated with the A-site binding [166] The latternotion is supported by Rytomaa and Kinnunen who foundthat while the presence of salt could prevent A-site bindingbound cytochrome did not dissociate from the membrane[165 167] Bregstrom et al by investigating the bindingfor ferricytochrome c to single-giant unilamellar vesiclesfound strong evidence for pore formation by cytochromec-CL complexes [168] The picture of cytochrome-liposomeinteractions became evenmore complex recently due to workof Kawai et al who provided evidence for a third binding siteL nearly opposite to A which as site C is more effective atacidic pH [169]

Pletneva and coworkers have recently applied their FRETtechnique to study ferricytochrome c on liposomes withdifferent CL contents [170ndash173] As observed for unfoldedor partially unfolded cytochrome in solution they identifiedan equilibrium between compact and extended states Thefraction of the latter was found to increase with CL contentuntil it saturated at a fraction of 05 [172] The authors alsoinvestigated the influence of NaCl and found the extendedstate to be depleted at high ionic strength Interestingly theyfound no indication for the C-sites and the lipid anchoragereported by Rytomaa and Kinnunen [165] Sinibaldi et alused visible circular dichroism to study the binding offerricytochrome to 100 CL liposomes [174] They identifiedtwo binding sites with rather different binding affinities Bothrespond to the addition of NaCl which seems to suggest thatthe binding is electrostatic in nature In this case neither oneof these binding sites can be associated with site C proposedbyKinnunen and coworkers since it would not be susceptibleto Na+ induced inhibition

The picture which emerges from all these differentbinding and protein conformation studies is far from beingconsistent A couple of issues deserve to be mentioned Firstnone of the above studies probed the state of the unboundprotein hence it was not checkedwhether the conformationaltransition of the protein is really reversible Very recentlyPandiscia and Schweitzer-Stenner found that this is not thecase in the absence of salt [175] Instead a major fraction of

OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus

Figure 25 Schematic representations of cardiolipin The figure hasbeen taken from open sources

the protein stays in a semiunfolded state exhibiting sub-stantial tryptophan fluorescence However the addition ofsalt not only dissociates the protein but also converts mostof the protein to the native or a native-like state Theseresults show that cytochrome c binding to anionic liposomescannot be described in terms of rather simple equilibriumlaws Second it is noteworthy that the concentration rangesof the experiments conducted by the different groups arerather different Rytomaa and coworkers observed their A-state binding by titrating cytochrome c in the submicromolarrange to an access of lipids [165 167] Their binding reachessaturation at a cytochrome c concentration well below thetotal accessible CL content of the mixture (sim09 120583M) Thebinding studies performed by the Pletneva Santucci andSchweitzer-Stenner groups were carried out by adding anincreasing number of lipids (ie liposomes) to a largeramount of cytochrome c (3120583M was reported by Hanskeet al [173] 10 120583M was used by Sinibaldi et al [174] and5 120583M was employed in our study (Pandiscia and Schweitzer-Stenner [159]) The binding affinities reflected by theirisotherms are much lower than that for Kinnunenrsquos A-siteFurther experiments which involve an analysis of unboundproteins are apparently necessary for the development of acomprehensive binding model These are currently on theirway in our laboratory

5 Summary and Outlook

Cytochrome c in its oxidized state is a very flexible moleculecontrary to its reduced counterpart The reason for thisenormous difference in stability is the reduced strength ofthe Fe-M80 linkage in the oxidized state It requires lessenergy to break and thus the pK-values of acid and alkalinetransitions as well as the thermal transition temperatureare much lower than in the reduced state Ferricytochromec adopts a variety of different states at acidic and alkalinepH and at high temperatures with tertiary structures whichdeviate from the native one while most of the secondarystructure remains rather intact With the exception of stateI which we did not discuss in detail the H18-Fe3+ linkageis maintained The covalent linkage of the heme to theCAQC region is always intact which prevents a segmentof the protein from unfolding We found a nonnative statewhich appears stable at neutral pH and room temperatureafter the protein was incubated at alkaline pH for a week[147] We interpreted this as the population of a frustrated


metastable state in the proteinrsquos folding funnel but onemight wonder whether this state is even energetically lowerthan the native state which is just separated from it by alarge enough barrier to avoid spontaneous unfolding in theoxidized state We are planning a systematic investigation ofthe determinants of the processes underlying the formationof this misfiled state and its propensity for aggregation ofnonamyloidogenic aggregates via domain swappingThe pH-window from which the formation of this state becomespossible has still to be identified Currently we are doingsome long-term experiments to probe the kinetics of theproteinrsquos recovery frommisfolding Preliminary data indicatethat this might take longer than a year The results of theseinvestigations will substantially enrich our understanding ofprotein folding They will prompt searches for conditions atwhich other proteins show a similar behavior

Cytochrome c has now been researched for over 70 yearsOne might think that this is enough and that researchersshould turn to newer pastures This view is belied by therealities The results of Soffer et al show that the energylandscape of the protein is yet not fully understood Exploringit further is likely to reveal important general insights aboutthe relationship between folded and misfolded states of pro-teins We wonder whether cytochrome c oligomers formedby domain swapping might have exciting switchable redoxproperties With regard to the proteinrsquos function in mito-chondria it still needs to be explored how the environmentalconditions maintain a balance between intact (necessary forelectron transfer) and partially unfolded proteins (necessaryfor gaining peroxidase activity and for other functions of theprotein not discussed in this review) Studying this relation-ship should transcend the realm of cytochrome c by sheddinglight on how membrane surfaces can unfold proteins thustuning them to another function In this context the role ofthe strong electric field at the membrane surface needs to beexplored furtherTheoretical and computational studies haverevealed that strong electric fields can facilitate the unfold-ing of ferricytochrome c Needless to say that any resultsregarding this issue would be of most general relevance forunderstanding protein structure on membranes

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Coworkers and collaborators have over time contributed tothe research of the group which is discussed in this reviewDuring the authorrsquos time at the University of Bremen thesewereDr Ulrich Bobinger and ProfessorWolfgangDreybrodtAt the authorrsquos current institution Drexel University DrQing Huang Dr Andrew Hagarman Isabelle DragomirRonak Shah Daniel Verbaro Emma Fradkin Dr JonathanB Soffer and Leah Pandiscia contributed to the reportedresearch The author enjoyed collaborations with ProfessorCarmichael Wallace from Dalhousie University in Halifax

Canada with Professor Antonio Cupane andDrMatteo Lev-antino from the University of Palermo in Italy and with thelate Dr Monique Laberge from the St-Jean Military Collegein Richelain Canada The author thanks Leah Pandiscia forthe critical reading of the paper

References

[1] M Paoli J Marles-Wright and A Smith ldquoStructure-functionrelationships in heme-proteinsrdquo DNA and Cell Biology vol 21no 4 pp 271ndash280 2002

[2] E Antonini and M Brunori Hemoglobin and Myoglobin inTheir Reaction with Ligands Elsevier Amsterdam The Nether-lands 1970

[3] C Weber B Michel and H R Bosshard ldquoSpectroscopicanalysis of the cytochrome c oxidase-cytochrome c complexcircular dichroism and magnetic circular dichroism measure-ments reveal change of cytochrome c heme geometry imposedby complex formationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 84 no 19 pp 6687ndash6691 1987

[4] E T Adman ldquoComparison of the structures of electron transferproteinsrdquo Biochimica et Biophysica Acta vol 549 no 2 pp 107ndash144 1979

[5] N C Veitch ldquoHorseradish peroxidase a modern view of aclassic enzymerdquo Phytochemistry vol 65 no 3 pp 249ndash2592004

[6] M J Coon ldquoCytochrome P450 naturersquos most versatile biolog-ical catalystrdquo Annual Review of Pharmacology and Toxicologyvol 45 pp 1ndash25 2005

[7] E R Derbyshire and M A Marletta ldquoStructure and regulationof soluble guanylate cyclaserdquo Annual Review of Biochemistryvol 81 pp 533ndash559 2012

[8] H N Kirkman and G F Gaetani ldquoCatalase a tetramericenzyme with four tightly bound molecules of NADPHrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 81 no 14 I pp 4343ndash4347 1984

[9] A M Amoia andW R Montfort ldquoApo-nitrophorin 4 at atomicresolutionrdquo Protein Science vol 16 no 9 pp 2076ndash2081 2007

[10] F A Walker ldquoMagnetic spectroscopic (EPR ESEEM Moss-bauer MCD and NMR) studies of low-spin ferriheme centersand their corresponding heme proteinsrdquo Coordination Chem-istry Reviews vol 185-186 pp 471ndash534 1999

[11] R Schweitzer-Stenner ldquoAllosteric linkage-induced distortionsof the prosthetic group in haem proteins as derived by the the-oretical interpretation of the depolarization ratio in resonanceRaman scatteringrdquo Quarterly Reviews of Biophysics vol 22 no4 pp 381ndash479 1989

[12] NA Belikova Y AVladimirov ANOsipov et al ldquoPeroxidaseactivity and structural transitions of cytochrome C bound tocardiolipin-containing membranesrdquo Biochemistry vol 45 no15 pp 4998ndash5009 2006

[13] G G Borisenko A A Kapralov V A Tyurin A Maeda DA Stoyanovsky and V E Kagan ldquoMolecular design of newinhibitors of peroxidase activity of cytochrome ccardiolipincomplexes fluorescent oxadiazole-derivatized cardiolipinrdquo Bio-chemistry vol 47 no 51 pp 13699ndash13710 2008

[14] V E Kagan V A Tyurin J Jiang et al ldquoCytochrome c actsas a cardiolipin oxygenase required for release of proapoptoticfactorsrdquo Nature Chemical Biology vol 1 no 4 pp 223ndash2322005


[15] A A Kapralov I V Kurnikov I I Vlasova et al ldquoThe hier-archy of structural transitions induced in cytochrome c byanionic phospholipids determines its peroxidase activation andselective peroxidation during apoptosis in cellsrdquo Biochemistryvol 46 no 49 pp 14232ndash14244 2007

[16] X Jiang and X Wang ldquoCytochrome C-mediated apoptosisrdquoAnnual Review of Biochemistry vol 73 pp 87ndash106 2004

[17] M J Burkitt and P Wardman ldquoCytochrome c is a potentcatalyst of dichlorofluorescin oxidation Implications for therole of reactive oxygen species in apoptosisrdquo Biochemical andBiophysical Research Communications vol 282 no 1 pp 329ndash333 2001

[18] SW Englander T R Sosnick L CMayneM Shtilerman P XQi and Y Bai ldquoFast and slow folding in cytochrome crdquoAccountsof Chemical Research vol 31 no 11 pp 737ndash744 1998

[19] R Schweitzer-Stenner ldquoPolarized resonance Raman dispersionspectroscopy on metalporphyrinsrdquo Journal of Porphyrins andPhthalocyanines vol 5 no 3 pp 198ndash224 2001

[20] R Schweitzer-Stenner A Hagarman D Verbaro and J BSoffer ldquoConformational stability of cytochrome C probed byoptical spectroscopyrdquo Methods in enzymology vol 466 pp109ndash153 2009

[21] E K J Tuominen C J A Wallace and P K J KinnunenldquoPhospholipid-cytochrome c interactionrdquoThe Journal of Biolog-ical Chemistry vol 277 pp 8822ndash8826 2002

[22] W Humphrey A Dalke and K Schulten ldquoVMD visualmolecular dynamicsrdquo Journal of Molecular Graphics vol 14 no1 pp 33ndash38 1996

[23] M M G Krishna H Maity J N Rumbley Y Lin and S WEnglander ldquoOrder of steps in the cytochrome c folding pathwayevidence for a sequential stabilization mechanismrdquo Journal ofMolecular Biology vol 359 no 5 pp 1410ndash1419 2006

[24] J B SofferTheFolded Partially FoldedMisfolded andUnfoldedConformations of Cytochrome c Probed by Optical SpectroscopyDepartment of Chemistry Drexel University Philadelphia PaUSA 2013

[25] G V Louie and G D Brayer ldquoHigh-resolution refinement ofyeast iso-1-cytochrome c and comparisons with other eukary-otic cytochromes crdquo Journal of Molecular Biology vol 214 no2 pp 527ndash555 1990

[26] G W Bushnell G V Louie and G D Brayer ldquoHigh-resolutionthree-dimensional structure of horse heart cytochrome crdquoJournal of Molecular Biology vol 214 no 2 pp 585ndash595 1990

[27] P Weinkam E V Pletneva H B Gray J R Winkler and PG Wolynes ldquoElectrostatic effects on funneled landscapes andstructural diversity in denatured protein ensemblesrdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 106 no 6 pp 1796ndash1801 2009

[28] G J Pielak D S Auld J R Beasley et al ldquoProtein thermaldenaturation side-chain models and evolution amino acidsubstitutions at a conserved helix-helix interfacerdquo Biochemistryvol 34 no 10 pp 3268ndash3276 1995

[29] Y Hayashi S Nagao H Osuka H Komori Y Higuchi and SHirota ldquoDomain swapping of the heme and N-terminal 120572-helixin Hydrogenobacter thermophilus cytochrome c

552dimerrdquo

Biochemistry vol 51 no 43 pp 8608ndash8616 2012[30] G Zoppellaro E Harbitz R Kaur A A Ensign K L Bren and

K K Andersson ldquoModulation of the ligand-field anisotropy ina series of ferric low-spin cytochrome c mutants derived fromPseudomonas aeruginosa cytochrome c-551 and Nitrosomonaseuropaea cytochrome c-552 A Nuclear Magnetic Resonance

and Electron Paramagnetic Resonance studyrdquo Journal of theAmerican Chemical Society vol 130 no 46 pp 15348ndash153602008

[31] J M Friedman ldquoStructure dynamics and reactivity inhemoglobinrdquo Science vol 228 no 4705 pp 1273ndash1280 1985

[32] W Jentzen J Ma and J A Shelnutt ldquoConservation of theconformation of the porphyrin macrocycle in hemoproteinsrdquoBiophysical Journal vol 74 no 2 pp 753ndash763 1998

[33] I Rasnik K A Sharp J A Fee and J M Vanderkooi ldquoSpectralanalysis of cytochrome c effect of heme conformation axialligand peripheral substituents and local electric fieldsrdquo Journalof Physical Chemistry B vol 105 no 1 pp 282ndash286 2001

[34] N V Prabhu S D Dalosto K A SharpWWWright and JMVanderkooi ldquoOptical spectra of Fe(II) cytochrome c interpretedusing molecular dynamics simulations and quantum mechan-ical calculationsrdquo Journal of Physical Chemistry B vol 106 no21 pp 5561ndash5571 2002

[35] E S Manas W W Wright K A Sharp J Friedrich and J MVanderkooi ldquoThe influence of protein environment on the lowtemperature electronic spectroscopy of Zn-substituted cyto-chromerdquo Journal of Physical Chemistry B vol 104 no 29 pp6932ndash6941 2000

[36] M Laberge M Kohler J M Vanderkooi and J FriedrichldquoSampling field heterogeneity at the heme of c-type cyto-chromes by spectral hole burning spectroscopy and electrostaticcalculationsrdquo Biophysical Journal vol 77 no 6 pp 3293ndash33041999

[37] R Schweitzer-Stenner ldquoInternal electric field in cytochrome Cexplored by visible electronic circular dichroism spectroscopyrdquoJournal of Physical Chemistry B vol 112 no 33 pp 10358ndash103662008

[38] A K Churg and A Warshel ldquoControl of the redox potentialof cytochrome c and microscopic dielectric effects in proteinsrdquoBiochemistry vol 25 no 7 pp 1675ndash1681 1986

[39] A M Berghuis J G Guillemette M Smith and G D BrayerldquoMutation of tyrosine-67 to phenylalanine in cytochrome csignificantly alters the local heme environmentrdquo Journal ofMolecular Biology vol 235 no 4 pp 1326ndash1341 1994

[40] C Blouin and C J A Wallace ldquoProtein matrix and dielectriceffect in cytochrome crdquoThe Journal of Biological Chemistry vol276 no 31 pp 28814ndash28818 2001

[41] R Schweitzer-Stenner Q Huang A Hagarman M Labergeand C J A Wallace ldquoStatic normal coordinate deformations ofthe heme group in mutants of ferrocytochrome c from Saccha-romyces cerevisiae probed by resonance Raman spectroscopyrdquoJournal of Physical Chemistry B vol 111 no 23 pp 6527ndash65332007

[42] B R Gelin A W Lee and M Karplus ldquoHemoglobin tertiarystructural change on ligand binding its role in the co-operativemechanismrdquo Journal of Molecular Biology vol 171 no 4 pp489ndash559 1983

[43] T G Spiro ldquoThe resonance Raman spectra of metallopor-phyrins and heme proteinsrdquo in Iron Porphyrins A B P Leverand H B Gray Eds pp 89ndash166 Addison-Wesley London UK1993

[44] T G Spiro and T C Strekas ldquoResonance Raman spectra ofheme proteins Effects of oxidation and spin staterdquo Journal ofthe American Chemical Society vol 96 no 2 pp 338ndash345 1974

[45] S Hu K M Smith and T G Spiro ldquoAssignment of protohemeResonance Raman spectrum by heme labeling in myoglobinrdquoJournal of the American Chemical Society vol 118 no 50 pp12638ndash12646 1996


[46] S Hu I K Morris J P Singh K M Smith and T G SpiroldquoComplete assignment of cytochrome c resonance Ramanspectra via enzymatic reconstitution with isotopically labeledhemesrdquo Journal of the American Chemical Society vol 115 no26 pp 12446ndash12458 1993

[47] S Choi T G Spiro K C Langry K M Smith D L Buddand G N La Mar ldquoStructural correlations and vinyl influencesin resonance raman spectra of protoheme complexes andproteinsrdquo Journal of the American Chemical Society vol 104 no16 pp 4345ndash4351 1982

[48] B R Stallard P R Callis P M Champion and A C AlbrechtldquoApplication of the transform theory to resonance Ramanexcitation profiles in the Soret region of cytochrome-crdquo TheJournal of Chemical Physics vol 80 no 1 pp 70ndash82 1984

[49] P M Champion and A C Albrecht ldquoInvestigations of Soretexcited resonance Raman excitation profiles in cytochrome crdquoThe Journal of Chemical Physics vol 71 no 3 pp 1110ndash1121 1979

[50] D L Brautigan B A Feinberg B M Hoffman E MargoliashJ Preisach and W E Blumberg ldquoMultiple low spin forms ofthe cytochrome c ferrihemochrome EPR spectra of variouseukaryotic and prokaryotic cytochromes crdquo Journal of BiologicalChemistry vol 252 no 2 pp 574ndash582 1977

[51] G N LaMar and J S Satterlee ldquoNuclear magnetic resonance ofheme proteinsrdquo in The Porphyrin Handbook K M Kadish KM Smith and R Guilard Eds Academic Press San FranciscoCailf USA 2000

[52] G N la Mar D L Budd D B Viscio K M Smith and K CLangry ldquoProton nuclear magnetic resonance characterizationof heme disorder in hemoproteinsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 75 no12 pp 5755ndash5759 1978

[53] H Senn M Billeter and K Wuthrich ldquoThe spatial structureof the axially bound methionine in solution conformations ofhorse ferrocytochrome c and Pseudomonas aeruginosa ferrocy-tochrome c 551 by 1H NMRrdquo European Biophysics Journal vol11 no 1 pp 3ndash15 1984

[54] D W Collins D D Fitchen and A Lewis ldquoResonance Ramanscattering from cytochrome cThe frequency dependence of thedepolarization ratiordquo Journal of Chemical Physics vol 59 no 10pp 5714ndash5719 1973

[55] J A Shelnutt ldquoThe Raman excitation spectra and absorptionspectrum of a metalloporphyrin in an environment of lowsymmetryrdquo The Journal of Chemical Physics vol 72 no 7 pp3948ndash3958 1980

[56] M Z Zgierski andM Pawlikowski ldquoDepolarization dispersioncurves of resonance Raman fundamentals ofmetalloporphyrinsand metallophthalocyanines subject to asymmetric perturba-tionsrdquo Chemical Physics vol 65 no 3 pp 335ndash367 1982

[57] R Schweitzer-Stenner and W Dreybrodt ldquoExcitation profilesand depolarization ratios of some prominent Raman lines inoxyhemoglobin and ferrocytochromeC in the pre-resonant andresonant region of the Q-bandrdquo Journal of Raman Spectroscopyvol 16 pp 111ndash123 1985

[58] R Schweitzer-Stenner and W Dreybrodt ldquoInvestigation ofHaem-Protein coupling and structural heterogeneity in myo-globin and haemoglobin probed by resonance raman spec-troscopyrdquo Journal of Raman Spectroscopy vol 23 pp 539ndash5501991

[59] R Schweitzer-Stenner A Cupane M Leone C Lemke JSchott and W Dreybrodt ldquoAnharmonic protein motions and

heme deformations in myoglobin cyanide probed by absorp-tion and Resonance Raman spectroscopyrdquo Journal of PhysicalChemistry B vol 104 no 19 pp 4754ndash4764 2000

[60] R Schweitzer-Stenner U Bobinger and W Dreybrodt ldquoMulti-mode analysis of depolarization ratio dispersion and excitationprofiles of seven Raman fundamentals from the haeme group inferrocytochrome crdquo Journal of Raman Spectroscopy vol 22 no2 pp 65ndash78 1991

[61] M Levantino Q Huang A CupaneM Laberge A Hagarmanand R Schweitzer-Stenner ldquoThe importance of vibronic pertur-bations in ferrocytochrome c spectra A reevaluation of spectralproperties based on low-temperature optical absorption reso-nance Raman andmolecular-dynamics simulationsrdquo Journal ofChemical Physics vol 123 no 5 Article ID 054508 2005

[62] E Unger U Bobinger W Dreybrodt and R Schweitzer-Stenner ldquoVibronic coupling in Ni(II) porphine derived fromresonant Raman excitation profilesrdquo Journal of Physical Chem-istry vol 97 no 39 pp 9956ndash9968 1993

[63] R Schweitzer-Stenner A Stichternath W Dreybrodt etal ldquoRaman dispersion spectroscopy on the highly saddlednickel(II)-octaethyltetraphenylporphyrin reveals the symmetryof nonplanar distortions and the vibronic coupling strength ofnormal modesrdquo Journal of Chemical Physics vol 107 no 6 pp1794ndash1815 1997

[64] R Schweitzer-Stenner and J B Soffer ldquoOptical spectroscopyrdquo inBiophysical Techniques for Structural Characterization ofMacro-molecules H J Dyson Ed pp 533ndash591 Oxford AcademicPress Oxford UK 2012

[65] X Li R S Czernuszewicz J R Kincaid P Stein and T GSpiro ldquoConsistent porphyrin force fieldmdash2 Nickel octaethyl-porphyrin skeletal and substituent mode assignments from15N meso-d

4 and methylene-d

16Raman and infrared isotope

shiftsrdquo Journal of Physical Chemistry vol 94 no 1 pp 47ndash611990

[66] C Lemke W Dreybrodt J A Shelnutt J M E Quirke and RSchweitzer-Stenner ldquoPolarized Raman dispersion spectroscopyprobes planar and non-planar distortions of Ni(II) porphyrinswith different peripheral substituentsrdquo Journal of Raman Spec-troscopy vol 29 no 10-11 pp 945ndash953 1998

[67] W Jentzen X Song and J A Shelnutt ldquoStructural characteri-zation of synthetic and protein-bound porphyrins in terms ofthe lowest-frequency normal coordinates of the macrocyclerdquoJournal of Physical Chemistry B vol 101 no 9 pp 1684ndash16991997

[68] J D Hobbs and J A Shelnutt ldquoConserved nonplanar hemedistortions in cytochromes crdquo Journal of Protein Chemistry vol14 no 1 pp 19ndash25 1995

[69] J A Shelnutt X Song J Ma S Jia W Jentzen and C JMedforth ldquoNonplanar porphyrins and their significance inproteinsrdquoChemical Society Reviews vol 27 no 1 pp 31ndash41 1998

[70] J A Shelnutt J D Hobbs S A Majumder et al ldquoResonanceRaman spectroscopy of non-planar nickel porphyrinsrdquo Journalof Raman Spectroscopy vol 23 p 523 1992

[71] R Schweitzer-Stenner C Lemke R Haddad et al ldquoCon-formational distortions of metalloporphyrins with electron-withdrawing NO2 substituents at different meso positions Astructural analysis by polarized resonance Raman dispersionspectroscopy andmolecular mechanics calculationsrdquo Journal ofPhysical Chemistry A vol 105 no 27 pp 6680ndash6694 2001

[72] M Alessi A M Hagarman J B Soffer and R Schweitzer-Stenner ldquoIn-plane deformations of the heme group in nativeand nonnative oxidized cytochrome c probed by resonance


Raman dispersion spectroscopyrdquo Journal of Raman Spec-troscopy vol 42 no 5 pp 917ndash925 2011

[73] A Hagarman C J Wallace M M Laberge and R Schweitzer-Stenner ldquoOut-of-plane deformations of the heme group indifferent ferrocytochrome c proteins probed by resonanceRaman spectroscopyrdquo Journal of Raman Spectroscopy vol 39no 12 pp 1848ndash1858 2008

[74] R Schweitzer-Stenner D Wedekind and W Dreybrodt ldquoCor-respondence of the pK values of oxyHb-titration states detectedby resonance Raman scattering to kinetic data of ligand disso-ciation and associationrdquo Biophysical Journal vol 49 no 5 pp1077ndash1088 1986

[75] R Schweitzer-Stenner D Wedekind and W DreybrodtldquoDetection of the heme perturbations caused by the quaternaryRrarrT transition in oxyhemoglobin trout IV by resonanceRaman scatteringrdquo Biophysical Journal vol 55 no 4 pp 703ndash712 1989

[76] S A Roberts A Weichsel Y Qiu J A Shelnutt F A Walkerand W R Montfort ldquoLigand-induced heme ruffling and bentNOgeometry in ultra-high-resolution structures of nitrophorin4rdquo Biochemistry vol 40 no 38 pp 11327ndash11337 2001

[77] M K Safo F A Walker A M Raitsimring et al ldquoAxialligand orientation in iron(III) porphyrinates effect of axial120587-acceptors Characterization of the low-spin complex[Fe(TPP)(4-CNPy)

2]C1O4rdquo Journal of the American Chemical

Society vol 116 no 17 pp 7760ndash7770 1994[78] L V Michel T Ye S E J Bowman et al ldquoHeme attachment

motif mobility tunes cytochrome c redox potentialrdquo Biochem-istry vol 46 no 42 pp 11753ndash11760 2007

[79] M D Liptak X Wen and K L Bren ldquoNMR and DFT investi-gation of heme ruffling functional implications for cytochromecrdquo Journal of the American Chemical Society vol 132 no 28 pp9753ndash9763 2010

[80] J M Friedman D L Rousseau and F Adar ldquoExcited statelifetimes in cytochromesmeasured fromRaman scattering dataevidence for iron-porphyrin interactionsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 74 no 7 pp 2607ndash2611 1977

[81] I Dragomir A Hagarman C Wallace and R Schweitzer-Stenner ldquoOptical band splitting and electronic perturbations ofthe heme chromophore in cytochrome c at room temperatureprobed by visible electronic circular dichroism spectroscopyrdquoBiophysical Journal vol 92 no 3 pp 989ndash998 2007

[82] R Schweitzer-Stenner J P Gorden and A Hagarman ldquoAsym-metric band profile of the Soret band of deoxymyoglobinis caused by electronic and vibronic perturbations of theheme group rather than by a doming deformationrdquo Journal ofChemical Physics vol 127 no 13 Article ID 135103 2007

[83] M-C Hsu and R W Woody ldquoThe origin of the heme cottoneffects in myoglobin and hemoglobinrdquo Journal of the AmericanChemical Society vol 93 no 14 pp 3515ndash3525 1971

[84] G Blauer N Sreerama and R W Woody ldquoOptical activity ofhemoproteins in the soret region Circular dichroism of theheme undecapeptide of cytochrome c in aqueous solutionrdquoBiochemistry vol 32 no 26 pp 6674ndash6679 1993

[85] C Kiefl N Sreerama R Haddad et al ldquoHeme distortions insperm-whale carbonmonoxy myoglobin correlations betweenrotational strengths and heme distortions in MD-generatedstructuresrdquo Journal of the American Chemical Society vol 124no 13 pp 3385ndash3394 2002

[86] G J Pielak K Oikawa A Grant Mauk M Smith and CM Kay ldquoElimination of the negative soret cotton effect of

cytochrome c by replacement of the invariant phenylalanineusing site-directedmutagenesisrdquo Journal of the AmericanChem-ical Society vol 108 no 10 pp 2724ndash2727 1986

[87] R Santucci and F Ascoli ldquoThe soret circular dichroism spec-trum as a probe for the heme Fe(III)- Met(80) axial bond inhorse cytochrome crdquo Journal of Inorganic Biochemistry vol 68no 3 pp 211ndash214 1997

[88] A Hagarman L Duitch and R Schweitzer-Stenner ldquoTheconformational manifold of ferricytochrome c explored byvisible and far-UV electronic circular dichroism spectroscopyrdquoBiochemistry vol 47 no 36 pp 9667ndash9677 2008

[89] C J Abel R A Goldbeck R F Latypov H Roder and D SKliger ldquoConformational equilibration time of unfolded proteinchains and the folding speed limitrdquo Biochemistry vol 46 no 13pp 4090ndash4099 2007

[90] E S Manas J M Yanderkooi and K A Sharp ldquoThe effectsof protein environment on the low temperature electronicspectroscopy of cytochrome c and microperoxidase-11rdquo Journalof Physical Chemistry B vol 103 no 30 pp 6334ndash6348 1999

[91] R Schweitzer-Stenner M Levantino A Cupane C WallaceM Laberge and Q Huang ldquoFunctionally relevant electric-field induced perturbations of the prosthetic group of yeastferrocytochrome c mutants obtained from a vibronic analysisof low-temperature absorption spectrardquo Journal of PhysicalChemistry B vol 110 no 24 pp 12155ndash12161 2006

[92] U Kubitscheck W Dreybrodt and R Schweitzer-StennerldquoDetection of heme-distortions in ferri- and ferrocyto-chromeC by resonance Raman scatteringrdquo Spectroscopy Letters vol 19no 6 pp 681ndash690 1986

[93] G R Moore and R J Williams ldquoNuclear-magnetic-resonancestudies of ferrocytochrome c pH and temperature dependencerdquoEuropean Journal of Biochemistry vol 103 no 3 pp 513ndash5211980

[94] D S Cohen and G J Pielak ldquoStability of yeast iso-1-ferricytochrome c as a function of pH and temperaturerdquo ProteinScience vol 3 no 8 pp 1253ndash1260 1994

[95] R Varhac M Antalik and M Baro ldquoEffect of temperature andguanidine hydrochloride on ferrocytochrome c at neutral pHrdquoJournal of Biological Inorganic Chemistry vol 9 pp 12ndash22 2004

[96] E Droghetti S Oellerich P Hildebrandt and G SmulevichldquoHeme coordination states of unfolded ferrous cytochrome crdquoBiophysical Journal vol 91 no 8 pp 3022ndash3031 2006

[97] A M Berghuis and G D Brayer ldquoOxidation state-dependentconformational changes in cytochrome crdquo Journal of MolecularBiology vol 223 no 4 pp 959ndash976 1992

[98] G G Silkstone C E Cooper D Svistunenko andM TWilsonldquoEPR and optical spectroscopic studies of Met80X mutantsof yeast ferricytochrome c models for intermediates in thealkaline transitionrdquo Journal of the American Chemical Societyvol 127 no 1 pp 92ndash99 2005

[99] P Baistrocchi L Banci I Bertini P Turano K L Bren andH B Gray ldquoThree-dimensional solution structure of Saccha-romyces cerevisiae reduced Iso-1-cytochrome crdquo Biochemistryvol 35 no 43 pp 13788ndash13796 1996

[100] L Banci I Bertini J G Huber G A Spyroulias and P TuranoldquoSolution structure of reduced horse heart cytochrome crdquo TheJournal of Biological Inorganic Chemistry vol 4 no 1 pp 21ndash311999

[101] YW Bai T R Sosnick L CMayne and SW Englander ldquoPro-tein folding intermediates native-state hydrogen exchangerdquoScience vol 269 no 5221 pp 192ndash197 1995


[102] L Banci I Bertini H B Gray et al ldquoSolution structure ofoxidized horse heart cytochrome crdquo Biochemistry vol 36 no32 pp 9867ndash9877 1997

[103] D Verbaro A Hagarman J Soffer and R Schweitzer-StennerldquoThe pHdependence of the 695 nm charge transfer band revealsthe population of an intermediate state of the alkaline transitionof ferricytochrome c at low ion concentrationsrdquo Biochemistryvol 48 no 13 pp 2990ndash2996 2009

[104] C B Anfinsen ldquoPrinciples that govern the folding of proteinchainsrdquo Science vol 181 no 4096 pp 223ndash230 1973

[105] H Theorell and A Akesson ldquoStudies on cytochrome c IIITitration curvesrdquo Journal of the American Chemical Society vol63 no 7 pp 1818ndash1820 1941

[106] H J Dyson and J K Beattie ldquoSpin state and unfoldingequilibria of ferricytochrome c in acidic solutionsrdquoThe Journalof Biological Chemistry vol 257 no 5 pp 2267ndash2273 1982

[107] Y Goto N Takahashi and A L Fink ldquoMechanism of acid-induced folding of proteinsrdquo Biochemistry vol 29 no 14 pp3480ndash3488 1990

[108] C Indiani G De Sanctis F Neri H Santos G Smulevichand M Coletta ldquoEffect of pH on axial ligand coordination ofcytochrome crdquo from Methylophilus methylotrophus and horseheart cytochrome crdquo Biochemistry vol 39 no 28 pp 8234ndash8242 2000

[109] S Zhong D L Rousseau and S-R Yeh ldquoModulation of thefolding energy landscape of cytochrome c with saltrdquo Journal ofthe American Chemical Society vol 126 no 43 pp 13934ndash139352004

[110] WColon andH Roder ldquoKinetic intermediates in the formationof the cytochrome c molten globulerdquo Nature Structural Biologyvol 3 no 12 pp 1019ndash1025 1996

[111] F I Rosell J C Ferrer and A G Mauk ldquoProton-linkedprotein conformational switching definition of the alkalineconformational transition of yeast iso-1-ferricytochrome crdquoJournal of the American Chemical Society vol 120 no 44 pp11234ndash11245 1998

[112] S Dopner P Hildebrandt F I Resell andA GMauk ldquoAlkalineconformational transitions of ferricytochrome c studied by res-onance Raman spectroscopyrdquo Journal of the American ChemicalSociety vol 120 no 44 pp 11246ndash11255 1998

[113] P D Barker and A G Mauk ldquopH-linked conformationalregulation of ametalloprotein oxidation-reduction equilibriumelectrochemical analysis of the alkaline form of cytochrome crdquoJournal of the American Chemical Society vol 114 no 10 pp3619ndash3624 1992

[114] M M Cherney and B E Bowler ldquoProtein dynamics andfunction making new strides with an old warhorse the alka-line conformational transition of cytochrome crdquo CoordinationChemistry Reviews vol 255 no 7-8 pp 664ndash677 2011

[115] P Weinkam J Zimmermann L B Sagle et al ldquoCharacteriza-tion of alkaline transitions in ferricytochrome C using carbon-deuterium infrared probesrdquo Biochemistry vol 47 no 51 pp13470ndash13480 2008

[116] L A Davis A Schejter and G P Hess ldquoAlkaline isomerizationof oxidized cytochrome crdquoThe Journal of Biological Chemistryvol 249 no 8 pp 2624ndash2632 1974

[117] C Blouin J G Guillemette and C J A Wallace ldquoResolvingthe individual components of a pH-induced conformationalchangerdquo Biophysical Journal vol 81 no 4 pp 2331ndash2338 2001

[118] G Battistuzzi M Borsari and M Sola ldquoMedium and tempera-ture effects on the redox chemistry of cytochrome crdquo European

Journal of Inorganic Chemistry vol 2001 no 12 pp 2989ndash30042001

[119] G Battistuzzi M Borsari L Loschi A Martinelli and M SolaldquoThermodynamics of the alkaline transition of cytochrome crdquoBiochemistry vol 38 no 25 pp 7900ndash7907 1999

[120] L Hoang H Maity M M G Krishna Y Lin and S WEnglander ldquoFolding units govern the cytochrome c alkalinetransitionrdquo Journal of Molecular Biology vol 331 no 1 pp 37ndash43 2003

[121] M Assfalg I Bertini A Dolfi et al ldquoStructural model for analkaline form of ferricytochrome crdquo Journal of the AmericanChemical Society vol 125 no 10 pp 2913ndash2922 2003

[122] J B SofferTheFolded Partially FoldedMisfolded andUnfoldedConformations of Cytochrome C Probed by Optical SpectroscopyDepartment of Chemistry Drexel University Philadelphia PaUSA 2013

[123] Y P Myer ldquoConformation of cytochromes III Effect of ureatemperature extrinsic ligands and pH variation on the confor-mation of horse heart ferricytochrome crdquo Biochemistry vol 7no 2 pp 765ndash776 1968

[124] M F Jeng S W Englander G A Elove H Roder and A JWand ldquoStructural description of acid-denatured cytochrome cby hydrogen exchange and 2D NMRrdquo Biochemistry vol 29 no46 pp 10433ndash10437 1990

[125] Y Goto Y Hagihara D Hamada M Hoshimo and I NishilldquoAcid-induced unfolding and refolding transitions of cyto-chrome c a three-state mechanism in H

2O and D

2Ordquo Biochem-

istry vol 32 no 44 pp 11878ndash11885 1993[126] G Battistuzzi C A Bortolotti M Bellei et al ldquoRole of

Met80 and Tyr67 in the low-pH conformational equilibria ofcytochrome crdquoBiochemistry vol 51 no 30 pp 5967ndash5978 2012

[127] J B Soffer and R Schweitzer-Stenner ldquoNear-exact enthalpy-entropy compensation governs the thermal unfolding of pro-tonation states of oxidized cytochrome Crdquo Journal of BiologicalInorganic Chemistry In press

[128] Y P Myer and A Pande ldquoCircular dichroism studies of hemo-proteins and heme modelsrdquo inThe Porphyrins D Dolphin Edpp 271ndash322 Academic Press New York NY USA 1978

[129] G Taler A Schejter G Navon I Vig and E MargoliashldquoThe nature of the thermal equilibrium affecting the ironcoordination of ferric cytochrome crdquo Biochemistry vol 34 no43 pp 14209ndash14212 1995

[130] R Schweitzer-Stenner R Shah A Hagarman and I DragomirldquoConformational substates of horse heart cytochrome c exhibitdifferent thermal unfolding of the heme cavityrdquo Journal ofPhysical Chemistry B vol 111 no 32 pp 9603ndash9607 2007

[131] A Spilotros M Levantino and A Cupane ldquoConformationalsubstates of ferricytochrome c revealed by combined opticalabsorption and electronic circular dichroism spectroscopy atcryogenic temperaturerdquo Biophysical Chemistry vol 147 no 1-2pp 8ndash12 2010

[132] H Frauenfelder PW Fenimore G Chen and B HMcMahonldquoProtein folding is slaved to solvent motionsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 42 pp 15469ndash15472 2006

[133] R Lumry and S Rajender ldquoEnthalpy-entropy compensationphenomena in water solutions of proteins and small moleculesa ubiquitous property of waterrdquo BiopolymersmdashPeptide ScienceSection vol 9 no 10 pp 1125ndash1227 1970

[134] L Liu C Yang and Q Guo ldquoA study on the enthalpy-entropycompensation in protein unfoldingrdquo Biophysical Chemistry vol84 no 3 pp 239ndash251 2000


[135] L Liu and Q Guo ldquoIsokinetic relationship isoequilibriumrelationship and enthalpy-entropy compensationrdquo ChemicalReviews vol 101 no 3 pp 673ndash695 2001

[136] J R Beasley D F Doyle L Chen D S Cohen B R Fineand G J Pielak ldquoSearching for quantitative entropy-enthalpycompensation among protein variantsrdquo Proteins StructureFunction and Genetics vol 49 no 3 pp 398ndash402 2002

[137] E B Starikov and B Norden ldquoEnthalpy-entropy compensationa phantom or something usefulrdquo Journal of Physical ChemistryB vol 111 no 51 pp 14431ndash14435 2007

[138] D J Segel D Eliezer V Uversky A L Fink K O Hodgsonand S Doniach ldquoTransient dimer in the refolding kinetics ofcytochrome c characterized by small-angle X-ray scatteringrdquoBiochemistry vol 38 no 46 pp 15352ndash15359 1999

[139] G Balakrishnan Y Hu and T G Spiro ldquoHis26 protonationin cytochrome c triggers microsecond 120573-sheet formation andheme exposure implications for apoptosisrdquo Journal of theAmerican Chemical Society vol 134 no 46 pp 19061ndash190692012

[140] P P Parui M S Deshpande S Nagao et al ldquoFormationof oligomeric cytochrome c during folding by intermolecularhydrophobic interaction betweenN- andC-terminal 120572-helicesrdquoBiochemistry vol 52 no 48 pp 8732ndash8744 2013

[141] C M Dobson ldquoProtein misfolding evolution and diseaserdquoTrends in Biochemical Sciences vol 24 no 9 pp 329ndash332 1999

[142] A Filosa andAM English ldquoProbing local thermal stabilities ofbovine horse and tuna ferricytochromes c at pH 7rdquo Journal ofBiological Inorganic Chemistry vol 5 no 4 pp 448ndash454 2000

[143] EMargoliash and J Lustgarten ldquoInterconversion of horse heartcytochrome cmonomer and polymersrdquoThe Journal of BiologicalChemistry vol 237 pp 3397ndash3405 1962

[144] S Hirota Y Hattori S Nagao et al ldquoCytochrome c polymer-ization by successive domain swapping at the C-terminal helixrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 29 pp 12854ndash12859 2010

[145] F Rousseau J W H Schymkowitz and L S Itzhaki ldquoTheunfolding story of three-dimensional domain swappingrdquo Struc-ture vol 11 no 3 pp 243ndash251 2003

[146] Z Wang T Matsuo S Nagao and S Hirota ldquoPeroxidaseactivity enhancement of horse cytochrome c by dimerizationrdquoOrganic and Biomolecular Chemistry vol 9 no 13 pp 4766ndash4769 2011

[147] J B Soffer E Fradkin L A Pandiscia and R Schweitzer-Stenner ldquoThe (not completely irreversible) population of amisfolded state of cytochrome c under folding conditionsrdquoBiochemistry vol 52 no 8 pp 1397ndash1408 2013

[148] W A Eaton and R M Hochstrasser ldquoSingle-crystal spectraof ferrimyoglobin complexes in polarized lightrdquo The Journal ofChemical Physics vol 49 no 3 pp 985ndash995 1968

[149] W A Eaton and R M Hochstrasser ldquoElectronic spectrum ofsingle crystals of ferricytochrome-crdquo The Journal of ChemicalPhysics vol 46 no 7 pp 2533ndash2539 1967

[150] S Dopner P Hildebrandt F I Rosell et al ldquoThe structuraland functional role of lysine residues in the binding domain ofcytochrome c in the electron transfer to cytochrome c oxidaserdquoEuropean Journal of Biochemistry vol 261 no 2 pp 379ndash3911999

[151] B D Howes C B Schioslashdt K GWelinder et al ldquoThe quantummixed-spin heme state of barley peroxidase a paradigm forclass III peroxidasesrdquo Biophysical Journal vol 77 no 1 pp 478ndash492 1999

[152] B D Howes A C Feis M Indiani M Morzocchi and GSmulevich ldquoFormation of two types of low spin heme inhorseradosh peroxidase isoenzyme A2 at low temperaturesrdquoJournal of Biological Inorganic Chemistry vol 276 pp 40704ndash40711 2000

[153] A Feis B D Howes C Indiani and G Smulevich ldquoResonanceRaman and electronic absorption spectra of horseradish perox-idase isozyme A2 evidence for a quantum-mixed spin speciesrdquoJournal of Raman Spectroscopy vol 29 no 10-11 pp 933ndash9381998

[154] M M Maltempo ldquoVisible absorption spectra of quantummixed spin ferric heme proteinsrdquo Biochimica et Biophysica Actavol 434 no 2 pp 513ndash518 1976

[155] Q Huang K Szigeti J Fidy and R Schweitzer-StennerldquoStructural disorder of native horseradish peroxidase C probedby resonance Raman and low-temperature optical absorptionspectroscopyrdquo Journal of Physical Chemistry B vol 107 no 12pp 2822ndash2830 2003

[156] P Weinkam F E Romesberg and P G Wolynes ldquoChemicalfrustration in the protein folding landscape grand canonicalensemble simulations of cytochrome crdquo Biochemistry vol 48no 11 pp 2394ndash2402 2009

[157] W Zheng N P Schafer and P G Wolynes ldquoFrustration inthe energy landscapes of multidomain protein misfoldingrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 110 no 5 pp 1680ndash1685 2013

[158] J D Cortese A L Voglino and C R Hackenbrock ldquoMultipleconformations of physiological membrane-bound cytochromecrdquo Biochemistry vol 37 no 18 pp 6402ndash6409 1998

[159] S Berezhna H Wohlrab and P M Champion ldquoResonanceRaman investigations of cytochrome c conformational changeupon interaction with the membranes of intact and CA2+-exposed mitochondriardquo Biochemistry vol 42 no 20 pp 6149ndash6158 2003

[160] D HMurgida P Hildebrandt JWei Y F He H Liu andD HWaldeck ldquoSurface-enhanced resonance raman spectroscopicand electrochemical study of cytochrome c bound on elec-trodes through coordination with pyridinyl-terminated self-assembledmonolayersrdquoTheJournal of Physical Chemistry B vol108 no 7 pp 2261ndash2269 2004

[161] D HMurgida and P Hildebrandt ldquoElectron-transfer processesof cytochrome c at interfacesNew insights by surface-enhancedresonanceRaman spectroscopyrdquoAccounts of Chemical Researchvol 37 no 11 pp 854ndash861 2004

[162] H Wackerbarth and P Hildebrandt ldquoRedox and conforma-tional equilibria and dynamics of Cytochrome c at high electricfieldsrdquo ChemPhysChem vol 4 no 7 pp 714ndash724 2003

[163] S Monari D Millo A Ranieri et al ldquoThe impact of urea-induced unfolding on the redox process of immobilisedcytochrome crdquo Journal of Biological Inorganic Chemistry vol 15no 8 pp 1233ndash1242 2010

[164] A Ranieri G Battistuzzi M Borsari et al ldquoA bis-histidine-ligated unfolded cytochrome c immobilized on anionic SAMshows pseudo-peroxidase activityrdquo Electrochemistry Communi-cations vol 14 no 1 pp 29ndash31 2012

[165] M Rytomaa and P K J Kinnunen ldquoEvidence for two distinctacidic phospholipid-binding sites in cytochrome crdquoThe Journalof Biological Chemistry vol 269 no 3 pp 1770ndash1774 1994

[166] E Kalanxhi and C J AWallace ldquoCytochrome c impaled inves-tigation of the extended lipid anchorage of a soluble proteinto mitochondrial membrane modelsrdquo Biochemical Journal vol407 no 2 pp 179ndash187 2007


[167] M Rytomaa P Mustonen and P K J Kinnunen ldquoReversiblenonionic and pH-dependent association of cytochrome c withcardiolipin-phosphatidylcholine liposomesrdquo Journal of Biologi-cal Chemistry vol 267 no 31 pp 22243ndash22248 1992

[168] C L Bergstrom P A Beales Y Lv T K Vanderlick and J TGroves ldquoCytochrome c causes pore formation in cardiolipin-containing membranesrdquo Proceedings of the National Academyof Sciences of the United States of America vol 110 no 16 pp6269ndash6274 2013

[169] C Kawai F M Prado G L C Nunes P Di Mascio A MCarmona-Ribeiro and I L Nantes ldquopH-dependent interactionof cytochrome c with mitochondrial mimetic membranes therole of an array of positively charged amino acidsrdquo The Journalof Biological Chemistry vol 280 no 41 pp 34709ndash34717 2005

[170] E J Snider J Muenzner J R Toffey Y J Hong and E VPletneva ldquoMultifaceted effects of ATP on cardiolipin-boundcytochrome crdquo Biochemistry vol 52 no 6 pp 993ndash995 2013

[171] E V Pletneva H B Gray and J R Winkler ldquoNature ofthe cytochrome c molten globulerdquo Journal of the AmericanChemical Society vol 127 no 44 pp 15370ndash15371 2005

[172] Y Hong J Muenzner S K Grimm and E V Pletneva ldquoOriginof the conformational heterogeneity of cardiolipin-bound cyto-chrome crdquo Journal of the American Chemical Society vol 134no 45 pp 18713ndash18723 2012

[173] J Hanske J R Toffey A M Morenz A J Bonilla K HSchiavoni and E V Pletneva ldquoConformational properties ofcardiolipin-bound cytochrome crdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 109 no1 pp 125ndash130 2012

[174] F Sinibaldi L Fiorucci A Patriarca et al ldquoInsights intocytochrome c-cardiolipin interaction Role played by ionicstrengthrdquo Biochemistry vol 47 no 26 pp 6928ndash6935 2008

[175] L A Pandiscia and R Schweitzer-Stenner ldquoSalt as a catalystin the mitochondria returning cytochrome c to its NativeState after it misfolds on the surface of cardiolipin containingmembranesrdquo Chemical Communications vol 50 no 28 pp3674ndash3676 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


HS

N

N N

N

Fe

SH

HO OO OH

Figure 1 Structure of heme c the prosthetic group in cytochromec

cytochrome c is still of great interest to biophysicists andbiochemists alike owing to its multifunctionality it serves asan electron carrier between major membrane proteins in theinner membrane of the mitochondria [4] it functions as ascavenger for H

2O2as peroxidase [12ndash15] and it functions

as an initiator of a biochemical cascade which finally causesmitochondrial apoptosis [15ndash17] The protein has also beenutilized as a model system for protein folding studies [18]The research in our laboratories has mostly been focused onelucidating heme-protein interactions and asymmetric defor-mations of the heme macrocycle [11 19] but lately we usedspectroscopic means to probe partially unfolded states of theprotein [20] The second chapter of this review describeshow heme-protein interactions affecting the prosthetic hemegroup of cytochrome can be probed by resonance Ramanvisible circular dichroism and low temperature absorptionspectroscopyThe third chapter reviews the studies of foldingand unfolding triggered by pH and temperature change Thisincludes the recent discovery of a partially unfolded statewhich remains stable at conditions that normally favor anative fully folded state (neutral pH room temperature) Inthe short fourth section we briefly review recent results of ourstudies on cytochrome c-liposome interactions Cardiolipin-containing liposomes serve as model systems for the innermembrane mitochondria in these studies [21]

2 Heme-Protein Interactions in Cytochrome c

21 Secondary and Tertiary Structure Figure 3 shows thecrystal structure of horse heart ferri-cytochrome c Thecoloring of the different structural segments follows a codeintroduced by Englander and coworkers [18 23] Its sig-nificance for the understanding of the proteinrsquos folding isexplained below Cytochrome c is a globular protein with an120572-helical fraction of ca 40 [25 26]That value is lower thanwhat one observes for the classical heme proteins myoglobinand hemoglobin [2]The twomain helical sections are the so-called N- and C-terminal helices Noncovalent interactions

Figure 2 Visualization of the secondary and tertiary structures offerro-horse heart cytochrome c obtained from pdb 2FRCThe figurehas been produced with VMD software [22]

105

10090

80

70

60 50

40

30

20

10

Foldon 3

HEME

Foldon 3

C-helixFo

ldon

4

Foldon 2

Foldon 5-lo

op

60rsquos helix

Foldon 1-N-helix

NE

E

EEN ADT

TFYGP

AQGT

KRGF

LG

HL

NP

GT

KH

KG

GK

EV

CHT

NKKKWT GI

L

LTM

Y

PKKYI

IFAGI

PGTKM

ATNE XGDVEKGKKIFVQKCA

Q

KKLYA

KKKTE

EDLI

R

Figure 3 Structure of oxidized horse heart cytochrome c presentedwith a color scheme that illustrates the different foldons identifiedby Krishna and coworkers [23] These foldons and the respectivecolor code are explained in detail in the text The outside circleprovides the primary amino acid sequence of the different proteinsegments The figure has been taken from Soffer [24] The originalfigure exhibiting the different foldons can be found in [23]

between these two helices are pivotal for the stabilization ofthe native state [18 27ndash29] A third helix (the so-called 60rsquoshelix) is much shorter than the C- and N-helices The restof the protein comprises rather long loop segments Two ofthem are of particular importance Firstly the loop startingfrom the N-terminal helix contains H18 the imidazole sidechain of which provides the proximal ligand of the heme ironTheCxyCmotif with the two cysteins covalently linked to theheme via thioether bridges is located between H18 and theN-terminal helix The notation xy indicates different amino





1119892of the D






4


120572-C120573and C

120572-N stretching


100

50

0

Abso

rptio

n (

)

300 400 500 600

Wavelength (nm)

B0

BV

Q0

QV






4-



119888Γ119903

119890119904= ⟨119890

100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (1)




119903in an ideal D


119909⟩ |119861119910⟩ |119876119909⟩ and |119876

119910⟩ In D


119861119909= 119864119861119910

and119864119876119909


119906-symmetry


17 19 21 23 25 27



100

10

1

01

Tota

l exc

itatio

n

(a)

Q B 00 1001 11

00 1001 11

1

01

Dep

olar

isatio

n ra

tio

17 19 21 23 25 27


(b)











119909 119876119910with 119861

119909



1119892) coupling


1119892and 119861







2119892-



1119892-





119888

Γ1199031015840

119890119904 = ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (2)








119881Γ119895

=

120597119890119897

120597119876Γ119895

120575119876Γ119895

(3)





1119892- 1198611119892- 1198612119892- 1198602119892- and 119864

119906-


2119892-




1119906 1198611119906 1198612119906 1198602119906

and 119864119892








1119892-mode


4can be used for





15 ClO3minus 22 21 4 1119

568nm

531nm

521nm

442nm

times103

20

15

10

5

0

20

15

10

5

0

60

40

20

0

100

50

0

Ram

an in

tens

ityRa

man

inte

nsity

Ram

an in

tens

ityRa

man

inte

nsity

400 600 800 1000 1200 1400 1600






1119892- and 119861

2119892-type





2119892-modes (]






SADB1u

WAX

RUFB1u

WAY

DOMA2u

PROA1uEg119909 Eg119910

(a)

MSTB2g

NSTB1g

TRX TRY

BREA1g

ROTA2g

Eu119909 Eu119910

(b)








and 1198612119892










119881Γlowast

=

1205972119890119897

12059711987611986111199061205971198761198612119906

1205751198761198611119906

1205751198761198612119906

(4)


1119906otimes

1198612119906







1119892-












119892-





Pero

xida

seYeast

Manganese

A ramosus


(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450


(b)

Rice

Horse

Tuna


cyt-c

(c)

Alcaligenes sp NCIB


R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)






119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D


2119906-



1198612119906

otimes 1198612119906







119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1119892of the D






4


120572-C120573and C

120572-N stretching


100

50

0

Abso

rptio

n (

)

300 400 500 600

Wavelength (nm)

B0

BV

Q0

QV






4-



119888Γ119903

119890119904= ⟨119890

100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (1)




119903in an ideal D


119909⟩ |119861119910⟩ |119876119909⟩ and |119876

119910⟩ In D


119861119909= 119864119861119910

and119864119876119909


119906-symmetry


17 19 21 23 25 27



100

10

1

01

Tota

l exc

itatio

n

(a)

Q B 00 1001 11

00 1001 11

1

01

Dep

olar

isatio

n ra

tio

17 19 21 23 25 27


(b)











119909 119876119910with 119861

119909



1119892) coupling


1119892and 119861







2119892-



1119892-





119888

Γ1199031015840

119890119904 = ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (2)








119881Γ119895

=

120597119890119897

120597119876Γ119895

120575119876Γ119895

(3)





1119892- 1198611119892- 1198612119892- 1198602119892- and 119864

119906-


2119892-




1119906 1198611119906 1198612119906 1198602119906

and 119864119892








1119892-mode


4can be used for





15 ClO3minus 22 21 4 1119

568nm

531nm

521nm

442nm

times103

20

15

10

5

0

20

15

10

5

0

60

40

20

0

100

50

0

Ram

an in

tens

ityRa

man

inte

nsity

Ram

an in

tens

ityRa

man

inte

nsity

400 600 800 1000 1200 1400 1600






1119892- and 119861

2119892-type





2119892-modes (]






SADB1u

WAX

RUFB1u

WAY

DOMA2u

PROA1uEg119909 Eg119910

(a)

MSTB2g

NSTB1g

TRX TRY

BREA1g

ROTA2g

Eu119909 Eu119910

(b)








and 1198612119892










119881Γlowast

=

1205972119890119897

12059711987611986111199061205971198761198612119906

1205751198761198611119906

1205751198761198612119906

(4)


1119906otimes

1198612119906







1119892-












119892-





Pero

xida

seYeast

Manganese

A ramosus


(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450


(b)

Rice

Horse

Tuna


cyt-c

(c)

Alcaligenes sp NCIB


R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)






119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D


2119906-



1198612119906

otimes 1198612119906







119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


17 19 21 23 25 27



100

10

1

01

Tota

l exc

itatio

n

(a)

Q B 00 1001 11

00 1001 11

1

01

Dep

olar

isatio

n ra

tio

17 19 21 23 25 27


(b)











119909 119876119910with 119861

119909



1119892) coupling


1119892and 119861







2119892-



1119892-





119888

Γ1199031015840

119890119904 = ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01 (2)








119881Γ119895

=

120597119890119897

120597119876Γ119895

120575119876Γ119895

(3)





1119892- 1198611119892- 1198612119892- 1198602119892- and 119864

119906-


2119892-




1119906 1198611119906 1198612119906 1198602119906

and 119864119892








1119892-mode


4can be used for





15 ClO3minus 22 21 4 1119

568nm

531nm

521nm

442nm

times103

20

15

10

5

0

20

15

10

5

0

60

40

20

0

100

50

0

Ram

an in

tens

ityRa

man

inte

nsity

Ram

an in

tens

ityRa

man

inte

nsity

400 600 800 1000 1200 1400 1600






1119892- and 119861

2119892-type





2119892-modes (]






SADB1u

WAX

RUFB1u

WAY

DOMA2u

PROA1uEg119909 Eg119910

(a)

MSTB2g

NSTB1g

TRX TRY

BREA1g

ROTA2g

Eu119909 Eu119910

(b)








and 1198612119892










119881Γlowast

=

1205972119890119897

12059711987611986111199061205971198761198612119906

1205751198761198611119906

1205751198761198612119906

(4)


1119906otimes

1198612119906







1119892-












119892-





Pero

xida

seYeast

Manganese

A ramosus


(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450


(b)

Rice

Horse

Tuna


cyt-c

(c)

Alcaligenes sp NCIB


R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)






119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D


2119906-



1198612119906

otimes 1198612119906







119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








119881Γ119895

=

120597119890119897

120597119876Γ119895

120575119876Γ119895

(3)





1119892- 1198611119892- 1198612119892- 1198602119892- and 119864

119906-


2119892-




1119906 1198611119906 1198612119906 1198602119906

and 119864119892








1119892-mode


4can be used for





15 ClO3minus 22 21 4 1119

568nm

531nm

521nm

442nm

times103

20

15

10

5

0

20

15

10

5

0

60

40

20

0

100

50

0

Ram

an in

tens

ityRa

man

inte

nsity

Ram

an in

tens

ityRa

man

inte

nsity

400 600 800 1000 1200 1400 1600






1119892- and 119861

2119892-type





2119892-modes (]






SADB1u

WAX

RUFB1u

WAY

DOMA2u

PROA1uEg119909 Eg119910

(a)

MSTB2g

NSTB1g

TRX TRY

BREA1g

ROTA2g

Eu119909 Eu119910

(b)








and 1198612119892










119881Γlowast

=

1205972119890119897

12059711987611986111199061205971198761198612119906

1205751198761198611119906

1205751198761198612119906

(4)


1119906otimes

1198612119906







1119892-












119892-





Pero

xida

seYeast

Manganese

A ramosus


(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450


(b)

Rice

Horse

Tuna


cyt-c

(c)

Alcaligenes sp NCIB


R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)






119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D


2119906-



1198612119906

otimes 1198612119906







119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SADB1u

WAX

RUFB1u

WAY

DOMA2u

PROA1uEg119909 Eg119910

(a)

MSTB2g

NSTB1g

TRX TRY

BREA1g

ROTA2g

Eu119909 Eu119910

(b)








and 1198612119892










119881Γlowast

=

1205972119890119897

12059711987611986111199061205971198761198612119906

1205751198761198611119906

1205751198761198612119906

(4)


1119906otimes

1198612119906







1119892-












119892-





Pero

xida

seYeast

Manganese

A ramosus


(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450


(b)

Rice

Horse

Tuna


cyt-c

(c)

Alcaligenes sp NCIB


R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)






119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D


2119906-



1198612119906

otimes 1198612119906







119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Pero

xida

seYeast

Manganese

A ramosus


(a)

B megaterium BM-3

Pseudomonas sp TERP

P putida CAM

Displacement (

P450


(b)

Rice

Horse

Tuna


cyt-c

(c)

Alcaligenes sp NCIB


R molischianum


cyt-c

998400(d)

SADRUFDOM

WAV(x)WAV(y)PRO

cyt-c

2

P denitrificans

R capsulatus

R rubrum


(e)






119888Γ

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

120597119881Γ119895

120597119876Γ119903

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

sum

119895

1205972119890119897

120597119876Γ119903120597119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(5)where Γ

119895is the D


2119906-



1198612119906

otimes 1198612119906







119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119888Γ119903

119890119904= ⟨119890

10038161003816100381610038161003816100381610038161003816100381610038161003816

120597119890119897

120597119876Γ119903

+sum

119895

1205972119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119895

120575119876Γ119895

+sum

119894

sum

119895

1205973119867

Γ119895

119890119897

120597119876Γ119903120597119876Γ119894120597119876Γ119895

120575119876Γ119903

120575119876Γ119895

10038161003816100381610038161003816100381610038161003816100381610038161003816

119904⟩119876Γ119903

01

(6)













119909119910-orbital (119861

2119892)







1119906)


ΔΓ

119892= sum

120582(Γ)

sum

Γ119894

sum

119894

⟨119892

100381610038161003816100381610038161205973119890119897120597119876Γ

1205821205972119876Γ119894

119894

10038161003816100381610038161003816119892⟩

(ΩΓ

120582)2

(120575119876Γ119894

119894)

2

(7)




Γ119894of the D






119909- and 119861

119910-band



119864119861119909

= 1198640

119861119909+

⟨119861119909

10038161003816100381610038161003816100381610038161003816119892⟩2

1198640

119892minus 1198640

119861119909

1198642cos2120579 (8a)

119864119861119910

= 1198640

119861119910+

⟨119861119910

10038161003816100381610038161003816100381610038161003816119892⟩

2

1198640

119892minus 1198640

119861119910

1198642sin2120579 (8b)


119892


=

1198640






22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


22 24 26 28



120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


6

4

2

0

minus2

minus4

minus6

minus8

minus10

minus12

80 times 103

60 times 103

40 times 103

20 times 103

0

(a)

22 24 26 28 22 24 26 28 22 24 26 28


10

5

0

minus5

minus1012e + 5

10e + 5

80e + 4

60e + 4

40e + 4

20e + 4

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)


(b)




119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119909and 119861

119910












119861





Δ119864119861

elect + Δ119864119861


V119861[cmminus1] 24500 24500

Γ119861[cmminus1] 600 600

120590119861[cmminus1] 100 100

(b) Ferrocytochrome



Δ119864119861

elect + Δ119864119861

vib [cmminus1] 516 311 242

V119861[cmminus1] 24300 23900 23800

Γ119861[cmminus1] 330 330 330

120590119861[cmminus1] 237 237 237



119909and 119876

119910components






16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


16 17 18 19 20

10000

8000

6000

4000

2000

0

4

3

2

1

0

minus1

minus2

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)




119909and 119876












35





pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


pH 2 4 6 8 10 12 14


IIIlowast

IV V

T

His18His18

His18His18

His18His18

His18

His18

Met80 Met80

Lys73

Lys79 Leu68U

OH

VU

Vh

IVU

IVh

IIIU

IIIh

III

IIUIU

I

Va

Vb

IVa

IVb

IIba

IIla


800

600

400

200

120576(M

minus1

cmminus1)

02

00

minus02

minus04

minus06

minus08

minus10

Δ120576

(Mminus1

cmminus1)

S3

S4

160

120

80

40

0

120

80

40

0140 145 150 155 8 9 10

pH

f(M

minus1

cmminus2)

f(M

minus1

cmminus2)


















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















119886might



119886












pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











pH2 4 6 8 10 12


Δ120576120576

times10minus4

24

20

16

12

08

04


100

80

60

40

20

0

f(M

minus1

cmminus2)

280 300 320 340

T (K)







20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



20

10

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

minus10

0



280 300 320 340 360

T (K)

25

20

15

10

5

0

minus5

minus10

minus15

minus150

minus200

minus250

minus300

minus350

minus400

Δ120576

(Mminus1

cmminus1)

Visible CD

UV




Δ120576 =

Δ120576119899+ Δ120576119894sdot 119890minus119866119894119877119879

+ Δ120576119906sdot 119890minus119866119906119877119879

1 + 119890minus119866119894119870119879

+ 119890minus119866119906119877119879

(9)

whereΔ120576119899Δ120576119894 andΔ120576



119906are



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



20

15

10

5

0

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(a)


25

20

15

10

5

0

1e + 5

8e + 4

6e + 4

4e + 4

2e + 4

0

120576(M

minus1

cmminus1)

Δ120576

(Mminus1

cmminus1)

(b)



119866119894= 119867119894minus 119879119878119894 (10a)

119866119906= 1198670

119906+ 120575119888119901sdot (119879 minus 119879

119894) + 119879 sdot (119878

119906+ 120575119888119901sdot ln( 119879

119879119906

))

(10b)

where119867119894and119867






119901is the














119906



280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


280 300 320 340 360

T (K)

24

20

16

12

8

4

Δ120576 m

ax(M

minus1

cmminus1)




119906are not very


Δ119867 = 119879119888Δ119878 (11)





















119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119894[kJmol] 100 100 100 200

Δ1198670

119906[kJmol] 300 250 200 200

119879119894[K] 315 315 320 330

119879119906[K] 355 355 350 355


C

Heme120572C

120572N

N

M80

C

Heme120572C

120572N

N

M80

(a)

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

C

C

120572C120572C

120572N

120572N

N

N

M80

M80

(b)

C

C

120572C

120572C

120572N

120572N

120572N

N

N N

C C

N

M80

M80

M80

120572C

C

C

120572C

120572C

120572N

120572N

120572N

N

N

M80

M80

M80

120572C

(c)



25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


25

20

15

10

5

0

times103

13500 14000 14500 15000 15500 16000 16500

CT1 CT2


120576(M

minus1

cmminus1)




160

140

120

100

80

60

40

20

0

Inte

grat

ed in

tens

ity (a

u)

45 50 55 60 65 70 75 80

pH

K1 K2


300

250

200

150

100

50

0

Ram

an in

tens

ity (x

-pol

ariz

ed)

1300 1400 1500 1600 1700


pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

pH 13

4 10





119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119886





119887might














OO

OO

OO

HH

H

OO

OO

OO

O

PP

OHminus +

HO

O

Ominus










Acknowledgments



References































552dimerrdquo









































4 and methylene-d






































































2O and D

2Ordquo Biochem-





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Cytochrome c: A Multifunctional Protein ...Cytochrome has served as a model system...

Documents

Transcript of Review Article Cytochrome c: A Multifunctional Protein ...Cytochrome has served as a model system...