Role of the surface charges D72 and K8 in the function and structural stability of the...

Role of the surface charges D72 and K8 in the functionand structural stability of the cytochrome c6 from Nostocsp. PCC 7119Christian Lange1, Irene Luque2, Manuel Hervas1, Javier Ruiz-Sanz2, Pedro L. Mateo2 andMiguel A. De la Rosa1

1 Instituto de Bioquımica Vegetal y Fotosıntesis, Centro de Investigaciones Cientıficas Isla de la Cartuja, Seville, Spain

2 Departamento de Quımica Fısica e Instituto de Biotecnologıa, Universidad de Granada, Spain

In the oxygenic photosynthesis of cyanobacteria and

many unicellular algae, cytochrome c6 acts as a soluble

electron carrier between the cytochrome bf complex and

photosystem I (PS I) [1,2]. In recent years, considerable

efforts have been undertaken to gain insight into the

structure–function relationships of this small heme

protein [3–7]. The structures of the cytochromes c6 from

various organisms have been determined [8–14]. Study

of the mutant series has contributed to our understand-

ing of the functional roles of important individual

amino acid residues within the protein [15–17]. NMR

studies with mutant bacterial c-type cytochromes [18–

20] have provided important insights into the role of

heme–protein interactions for the structural stability of

this class of proteins.

The mutant D72K of cytochrome c6 from Nostoc

(formerly Anabaena) sp. PCC 7119 (cyt c6) [21], was

found to be a more reactive electron donor towards

PS I than the wild-type [16]. However, D72K was

shown to have considerably reduced stability against

Keywords

cytochrome c6; electron transfer;

electrostatic interactions; protein folding;

protein stability

Correspondence

Christian Lange, Martin-Luther-Universitat

Halle ⁄Wittenberg, Institut fur

Biotechnologie, Kurt-Mothes-Str. 3,

06120 Halle (Saale), Germany

Fax: +49 345 552 7013

Tel: +49 345 552 4948

E-mail: christian.lange@biochemtech.

uni-halle.de

(Received 10 February 2005, revised 21

April 2005, accepted 3 May 2005)

doi:10.1111/j.1742-4658.2005.04747.x

We investigated the role of electrostatic charges at positions D72 and K8

in the function and structural stability of cytochrome c6 from Nostoc sp.

PCC 7119 (cyt c6). A series of mutant forms was generated to span the

possible combinations of charge neutralization (by mutation to alanine)

and charge inversion (by mutation to lysine and aspartate, respectively) in

these positions. All forms of cyt c6 were functionally characterized by laser

flash absorption spectroscopy, and their stability was probed by urea-

induced folding equilibrium relaxation experiments and differential scan-

ning calorimetry. Neutralization or inversion of the positive charge at

position K8 reduced the efficiency of electron transfer to photosystem I.

This effect could not be reversed by compensating for the change in global

charge that had been introduced by the mutation, indicating a specific role

for K8 in the formation of the electron transfer complex between cyt c6and photosystem I. Replacement of D72 by asparagine or lysine increased

the efficiency of electron transfer to photosystem I, but destabilized the

protein. D72 apparently participates in electrostatic interactions that stabil-

ize the structure of cyt c6. The destabilizing effect was reduced when aspar-

tate was replaced by the small amino acid alanine. Complementing the

mutation D72A with a charge neutralization or inversion at position K8

led to mutant forms of cyt c6 that were more stable than the wild-type

under all tested conditions.

Abbreviations

cyt c6, cytochrome c6 from Nostoc sp. PCC 7119; DSC, differential scanning microcalorimetry; EmpH 7, midpoint redox potentials at pH 7 and

at 25 �C; DGint, interaction Gibbs energies; kbim, bimolecular reaction rate constant; N, native folded state; PS I, photosystem I; U, ensemble

of unfolded states; [urea]50, transition midpoint for urea-induced unfolding.

FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS 3317

unfolding by urea [22], and preliminary experiments

indicated that its thermal stability was affected in a

similar way. Electrostatic interactions play a critical

role in guiding and stabilizing functional protein–pro-

tein interactions [6], as well as in the structural stability

of proteins [23–25].

Inspection of a preliminary structural model of

cyt c6, based on the known structure of cytochrome c6from Synechococcus elongatus [11] and NMR data,

revealed a spatial proximity between the side chain of

D72, which is located in the C-terminal a helix, and a

positively charged residue in the protein’s N-terminal

helix, K8. In S. elongatus cytochrome c6, the conserved

residues K8 and D72 are located near the crossing

region of the helices, and the Nx atom of K8 is found

at a distance of 6.2 A from the nearest side-chain oxy-

gen of D72.

Taking the functionally and structurally interesting

mutant D72K as a material starting point, we aimed

to study the contribution of the electrostatic charges at

positions D72 and K8 to the interaction of cyt c6 with

PS I, as well as to its structural stability. For this pur-

pose, a series of mutant forms of cyt c6 was generated

that spans the possible combinations of charge neutral-

ization (by mutation to alanine) and charge inversion

(by mutation to lysine and aspartate, respectively) at

positions D72 and K8. After functional characteriza-

tion by laser-flash absorption spectroscopy, urea-

induced folding equilibrium relaxation experiments

and differential scanning microcalorimetry (DSC) were

used to assess the changes in the stability against

denaturant-induced folding ⁄unfolding as well as against

thermal unfolding.

Results and Discussion

Functional characterization of wild-type cyt c6and its mutants

Wild-type cyt c6 and its mutant forms were success-

fully expressed in Escherichia coli strain GM119 and

purified to homogeneity. The UV ⁄Vis spectra of wild-

type and mutants were virtually identical (not shown).

Their midpoint redox potentials at pH 7 (EmpH 7) were

very similar throughout (Table 1), as expected for the

mutation of surface residues located far from the heme

moiety. Thus, the thermodynamic driving force for the

reaction of cyt c6 with PS I was essentially unaltered

by the introduction of the mutations. Eventual differ-

ences in reactivity, therefore, have to be ascribed to

structural factors that influence the formation and

productivity of the encounter complex. Indeed, such

differences were observed. All forms of cyt c6 were

highly active electron donors to PS I (Fig. 1). The

determined values for the bimolecular rate constant

(kbim) for wild-type cyt c6 and the mutant D72K were

in excellent agreement with the values that had been

reported previously [16]. Overall, a good correlation

(R ¼ 0.83) between the proteins’ net charge and their

reactivity towards PS I was observed (Fig. 1). This

confirms the importance of long-range charge interac-

tions for the electron carrier function of cyt c6. How-

ever, closer inspection of the data revealed interesting

deviations from the general trend.

A reversal of the charge effect of the mutation K8D

in the mutant D72K ⁄K8D did not lead to recovery of

wild-type reactivity. The same holds for the mutant

pair K8A and D72A ⁄K8A. All three variants carrying

the mutation K8D showed very similar kbim values,

and the same was observed for all variants carrying

Table 1. Redox potentials of wild-type cyt c6 and its mutant forms.

EmpH 7 (mV)

Wild-type 335

D72A 336

D72K 339

D72N 340

K8A 341

K8D 336

D72K ⁄ K8D 338

D72K ⁄ K8A 338

D72A ⁄ K8D 331

D72A ⁄ K8A 342

-2 -1 0 1 20

5

10

15

20

25

30

WT

K8D

D72K

D72N

D72A

KA

KD

AA

AD

k bim

(10

7 M-1

s-1)

relative nominal charge

K8A

Fig. 1. Bimolecular rate constants, kbim, for the reduction of PS I.

The kbim values were determined as described in the text. Data

points are marked with the name of the corresponding mutants.

Error bars represent errors from the fit to determine kbim. The solid

line represents a linear fit to the data points, dashed lines mark the

95% confidence intervals. Double mutants are abbreviated with the

first letter representing the residue at position 72 and the second

letter the one at position 8.

Surface charge mutants of cytochrome c6 C. Lange et al.

3318 FEBS Journal 272 (2005) 3317–3327 ª 2005 FEBS

the mutation K8A. This clearly indicates a specific

functional significance of the charge in amino acid

position 8 for the formation of a productive encounter

complex during the electron transfer from cyt c6 to

PS I. K8 is located at the edge of the contact surface

of cyt c6 that directly interacts with PS I [26], and a

positive charge in this position might be expected to

play an important role by establishing specific electro-

static interactions during complex formation with PS I.

The variant carrying the single mutation D72N

showed an increased kbim value compared with cyt c6wild-type, whereas for the mutant D72A, which has

the same net charge, no change in kbim was observed.

This result implies that changes in amino acid position

72 have additional specific effects on the interaction of

cyt c6 with PS I, independent of the influence of global

net charge.

Urea-induced folding ⁄unfolding of cyt c6:

equilibrium stability

Preliminary experiments had shown that cyt c6 has

maximum stability against unfolding by heat and

denaturants around pH 5.5, in its physiological work-

ing range [27]. Oxidized cyt c6 could not be fully unfol-

ded by urea at pH 5 and 30 �C. Therefore, we chose

to observe the urea-induced folding ⁄unfolding of oxi-

dized cyt c6 and its mutant forms at pH 7. The study

was carried out by performing folding equilibrium

relaxation experiments. In Fig. 2, the results of typical

experiments with wild-type cyt c6 and the mutant

forms D72K and D72A ⁄K8D are shown as examples,

along with the derived equilibrium unfolding curves

(Fig. 2D). Analysis of all performed experiments

yielded a set of parameters for comparison of the sta-

bilities of wild-type and mutants (Table 2).

As previously reported [22], the transition midpoint

for urea-induced unfolding ([urea]50) of the mutant

D72K was shifted to significantly lower urea concen-

trations ([urea]), and its Gibbs energy of unfolding at

0 [urea] (DGU0) was significantly reduced (D[urea]50 ¼

)2.4 ± 0.2 m, DDGU0 ¼ )2.7 ± 1.6 kJÆmol)1). The

mutant protein D72N was similarly affected (D[urea]50 ¼)1.9 ± 0.1 m, DDGU0 ¼ )3.1 ± 2.9 kJÆmol)1). All

other single mutations, including D72A, had a

comparably minor effect. Charge neutralization at

position K8 has no significant effect on the structural

0

1

2

3

4

[ure

a]fin

al[u

rea]

final

[ure

a]fin

al

wild typeA

B

C

D

fluo.

sig

nal (

V)

0

1

2

3

AD

D72Kflu

o. s

igna

l (V

)

0.0 0.3 0.6 0.9 1.2 1.5

1

2

3

4

fluo.

sig

nal (

V)

time (s)

0 2 4 6 8 10

0

20

40

60

80

100

% u

nfol

ded

[urea] (M)

Fig. 2. Folding equilibrium relaxation experiments. The represented

experiments were performed with wild-type cyt c6 (A), D72K (B)

and D72A ⁄ K8D (AD) (C). Arrows on the right indicate increasing

[urea]final (0–9.8 M). Percentages of unfolded protein at equilibrium

as determined from the experiments shown in (A) to (C) for wild-

type cyt c6 (squares), D72K (circles) and AD (triangles) are plotted

as a function of [urea] in (D). The lines represent fits of two state

transitions to the data for wild-type cyt c6 (solid), D72K (dashed)

and AD (dotted). Double mutants are abbreviated with the first let-

ter representing the residue at position 72 and the second letter

the one at position 8.

C. Lange et al. Surface charge mutants of cytochrome c6


stability of the protein, whereas inversion of the charge

at position 72 considerably reduces the stability against

urea-induced unfolding, i.e. electrostatic interactions of

the negative charge at position D72 seem to play an

important role for the stability of cyt c6. It is interesting

to note that charge neutralization with a hydrogen-

bond-forming asparagine residue leads to destabiliza-

tion of the protein, whereas neutralization with a small

hydrophobic alanine residue does not.

Partial or total neutralization of the effect of the

mutation D72K on the protein’s global charge was not

sufficient to fully reverse the negative effect on the

stability of cyt c6. The apparent stability of the mu-

tants D72K ⁄K8A (D[urea]50 ¼ )1.7 ± 0.1 m, DDGU0 ¼)1.4 ± 2.1 kJÆmol)1) and D72K ⁄K8D (KD) (D[urea]50 ¼)1.4 ± 0.1 m, DDGU0 ¼ +0.2 ± 2.8 kJÆmol)1) against

urea-induced unfolding was still reduced when com-

pared with wild-type cyt c6. This indicates that the elec-

trostatic interactions of the side-chain of D72 that

contribute to the stability of cyt c6 are at least partially

local, and that the adverse effects of the mutations

D72K and D72N cannot be entirely ascribed to global

electrostatic repulsion within the positively charged

protein.

However, although mutation D72A did not lead to

a significant change in stability, the additional elimin-

ation of the positive charge K8 in the double mutants

D72A ⁄K8D and D72A ⁄K8A resulted in an increased

apparent stability against urea-induced unfolding, as

well as an increased Gibbs energy of unfolding of

these mutants (D[urea]50 ¼ +0.2 ± 0.1 m, DDGU0 ¼+3.5 ± 2.4 kJÆmol)1 and D[urea]50 ¼ +0.2 ± 0.1 m,

DDGU0 ¼ +0.5 ± 1.7 kJÆmol)1, respectively).

The equilibrium urea interaction parameter (meq),

which represents the steepness of the unfolding

transition with respect to [urea] and may be interpreted

as a measure of the change in solvent-accessible area

upon unfolding [28], was affected by changes at position

D72, but not at position K8. It was significantly

increased for all mutants in which position 72 had been

modified, with the exception of D72A ⁄K8A. This find-

ing might be partially explained by a prevalence of more

extended conformations in the unfolded ensemble of

states due to local electrostatic repulsion in the vicinity

of the mutated position 72.

Urea-induced folding ⁄unfolding of cyt c6: kinetic

parameters

The performed folding relaxation experiments also

allowed for the determination of kinetic parameters

(Table 2). The most thermodynamically unstable

mutants, D72N and D72K, were found to have the

highest unfolding rate constant in absence of denatu-

rant (kU0 ¼ 0.075 and 0.080 s)1, respectively), while

the mutant with the highest DGU0 value, D72A ⁄K8D,

was found to show the lowest unfolding rate (kU0 ¼0.023 s)1). The value for wild-type cyt c6 lay between

these extremes (kU0 ¼ 0.035 s)1) (Table 2). In general,

a good correlation (R ¼ –0.73) was found between log

kU0 and the Gibbs energy of unfolding at 0 [urea]

(DGU0). In the mutant series, the overall height of the

energy barrier for the rate-determining step of the fold-

ing ⁄unfolding transition is mainly determined by the

overall difference in Gibbs energy between the native

state (N) and the ensemble of unfolded states (U), in

agreement with a single transition state with a disor-

dered (unfolded-like) structure [28]. This indicates that

the folding ⁄unfolding transition proceeds along a sim-

ilar pathway for wild-type cyt c6 and for all mutants.

When the folding ⁄unfolding traces were analysed

individually, and the apparent rate constants (k) for

the individual traces were plotted against [urea] in

Chevron plots, pronounced deviations from linearity

Table 2. Evaluation of urea-induced folding equilibrium relaxation experiments. Data represent means ± SD from 2–4 experiments for each

protein.

[Urea]50

(M)

DGU0

(kJÆmol)1)

meq

(kJÆmol)1ÆM)1)

log

(kU0Æs)1)

mk1

(kJÆmol)1ÆM)1)

mk2

(kJÆmol)1ÆM)2)

Wild-type 5.94 ± 0.07 21.4 ± 1.3 )3.6 ± 0.2 )1.5 ± 0.5 )1.7 ± 0.9 0.03 ± 0.06

K8A 5.8 ± 0.2 21.4 ± 0.8 )3.7 ± 0.2 )1.3 ± 0.2 )1.7 ± 0.3 0.05 ± 0.03

K8D 5.51 ± 0.02 20.6 ± 0.2 )3.74 ± 0.02 )1.26 ± 0.09 )1.6 ± 0.2 0.03 ± 0.01

D72K 3.6 ± 0.1 18.7 ± 0.3 )5.2 ± 0.2 )1.10 ± 0.05 )2.5 ± 0.2 0.11 ± 0.02

D72N 4.05 ± 0.06 18.3 ± 1.6 )4.6 ± 0.5 )1.13 ± 0.04 )2.0 ± 0.1 0.08 ± 0.01

D72A 5.41 ± 0.05 21.8 ± 1.4 )4.0 ± 0.2 )1.4 ± 0.5 )2.3 ± 0.7 0.09 ± 0.05

D72K ⁄ K8D 4.59 ± 0.02 21.6 ± 1.5 )4.7 ± 0.3 )1.3 ± 0.7 )2.8 ± 1.3 0.1 ± 0.1

D72K ⁄ K8A 4.27 ± 0.03 20.0 ± 0.8 )4.7 ± 0.2 )1.1 ± 0.1 )2.4 ± 0.4 0.11 ± 0.04

D72A ⁄ K8D 6.12 ± 0.02 24.6 ± 1.1 )4.0 ± 0.2 )1.6 ± 0.5 )2.4 ± 0.9 0.07 ± 0.07

D72A ⁄ K8A 6.07 ± 0.07 22.0 ± 0.4 )3.62 ± 0.05 )1.0 ± 0.4 )1.6 ± 0.6 0.04 ± 0.04



(‘roll over’ and ‘roll down’) were observed (not

shown). These deviations were interpreted as indicative

of a Hammond shift [29] in the structure of the trans-

ition state � with increasing denaturant concentration.

For the global analysis, this was taken into account by

introducing the parameter mk2. From the fit parame-

ters, a values were calculated (as a function of urea)

according to Eqn (3) (see Experimental procedures).

These values may be interpreted as a measure of

native-like structure in the transition state [22,28].

Whereas a ¼ 1 would indicate an all native-like trans-

ition state, a ¼ 0 would indicate an all unfolded-like

one. Wild-type cyt c6 and all mutant forms showed a

transition state shift to higher a-values with increasing

[urea] (not shown). The single mutants D72K and

D72N, as well as the double mutants D72K ⁄K8D and

D72K ⁄K8A, showed the steepest increase in a with

[urea] (i.e. a higher mk2 value), and therefore signifi-

cantly higher a-values at high denaturant concentra-

tions than wild-type cyt c6 and the other mutant

forms. Their kinetic properties were clearly more sus-

ceptible to the effect of urea and the structure of their

transition state shifted more strongly towards the

native conformation under the influence of destabiliza-

tion of the latter.

Microcalorimetry studies

In order to obtain the thermodynamic parameters of

the thermal unfolding of cyt c6 and its mutants, DSC

was performed.

At pH 7, oxidized cyt c6 was found to be redox-

unstable in the absence of oxidizing agents, whose

presence would affect the calorimetric trace of the

DSC experiment. It was also observed that the heating

of reduced protein at pH 7 gave rise to complex calori-

metric traces, probably due to an oxidative process of

the protein upon thermal unfolding. It was found that

at lower pH values ranging from 3.0 to 5.0, close to

the physiological working range of cyt c6, the oxidized

form was redox-stable in absence of oxidizing agents

during DSC experimental time. Consequently, DSC

experiments were carried out at pH 3.0, 4.0 and 5.0

for all cyt c6 variants. In all cases, heat-induced unfold-

ing was highly reversible and independent of both

the scanning rate and the protein concentration. All

calorimetric traces could be fitted very well to a two-

state equilibrium model (NfiU).

The experimental DSC curves, together with their

best fits, for the thermal unfolding of wild-type cyt c6,

and for the mutants D72K and D72A ⁄K8D at pH 5.0

are shown in Fig. 3A as typical examples. The effect

of pH on thermal unfolding of wild-type cyt c6 is

shown in Fig. 3B. The Gibbs energy of unfolding as a

function of temperature, DGU(T), could be obtained

using the thermodynamic parameter values from the

multiple fits of the DSC traces at the studied pH con-

ditions. The values of DGU(T) at 30 �C (DG 30 �CU ) for

wild-type were 54.8 kJÆmol)1 at pH 5.0, 49.3 kJÆmol)1

at pH 4.0 and 34.5 kJÆmol)1 at pH 3.0. It is clear from

these results that there is a pronounced pH effect on

the stability of cyt c6. Taking into account the value of

21.4 kJÆmol)1 obtained at pH 7.0 by the urea-induced

folding ⁄unfolding experiments (Table 2), it could be

confirmed that the maximum stability of wild-type

cyt c6 is around pH 5.0. As it is shown in Table 3, this

behaviour was common to all studied mutants.

A similar dependence on the pH was observed for

the Tm values obtained for all proteins (Table 3). By

contrast, the effect of pH on the unfolding enthalpies

30 50 70 90 11010

20

30

40

50

60

temperature (ºC)

temperature (ºC)30 50 70 90 110

10

20

30

40

50

60

A

B

Cp

(kJ·

K-1

·mol

-1)

Cp

(kJ·

K-1

·mol

-1)

Fig. 3. Temperature dependence of the partial molar heat capacity

of cyt c6. (A) DSC traces at pH 5.0 for wild-type (h), D72K (s) and

D72A ⁄ K8D (n). (B) DSC traces for wild-type at pH 5.0 (h), pH 4.0

(s) and pH 3.0 (n). Symbols correspond to experimental data, for

clarity every third data point is plotted. Solid lines correspond to

best fits to the two-state equilibrium model.



seemed to be more complex (Fig. 4). For many pro-

teins the dependence of DHU,m on Tm is linear, indica-

ting an entropic origin of the differences in stability

[30–32]. The slope of such a representation corres-

ponds to DCp,U. In our case, as shown in Fig. 4, this

dependence was not linear for many of the studied

forms of cyt c6. Moreover, in the mutants for which

a linear dependence was observed, e.g. K8D, the

obtained slope values were much greater than the

DCp,U values calculated from the global analysis of

the calorimetric traces. As may be observed in Fig. 4,

the enthalpic variation with pH is most pronounced

between pH 3 and 4 for the wild-type and most

mutants, with the exception of D72A, for which the

biggest pH effect was observed between pH 4 and 5.

These results indicate that the stability differences

found are not only of entropic nature but that there is

a considerable enthalpic contribution. Because the

ionic strength was fixed in all experiments, the pH

effects are exclusively due to changes in the electro-

static interactions. The enthalpic contributions of pro-

tonation ⁄deprotonation events are small compared

with the changes in enthalpy observed in these mutants

(up to 60 kJÆmol)1). So, it is likely that these ionization

events are responsible for some kind of structural rear-

rangement that is associated with enthalpic changes.

The changes in the thermodynamic parameters of

unfolding of each mutant with respect to the wild-type

protein are summarized in Fig. 5. To avoid extrapola-

tion over a large temperature range, the values of

DDGU and DDHU are reported at 75 �C, which is at

approximately the median unfolding temperature. At

all studied pH conditions, wild-type cyt c6 had a signi-

ficantly higher Gibbs energy of unfolding than the

mutants, with the exception of D72A at pH 4 and 3,

and of the double mutants D72A ⁄K8D and

Table 3. Thermal unfolding transition of wild-type cyt c6 and its mutant forms. For each protein, thermodynamic parameters were deter-

mined by global fits of the two-state equilibrium model to the DSC traces recorded at all three pH conditions. Tm values refer to the trans-

ition midpoint and DG 30 �CU values represent Gibbs energies of unfolding extrapolated to 30 �C. The reported values are estimated to be

accurate within ±0.4 �C for Tm and ±10% for DG 30 �CU . The DGU0 values for pH 7.0 from Table 2 are included for comparison.

pH 7.0 DGU0

(kJÆmol)1) Tm (�C)pH 5.0 DG 30 �C

U


U


U

(kJÆmol)1)

Wild-type 21.4 82.1 54.8 77.3 49.3 69.6 34.5

K8A 21.4 80.5 53.2 76.2 48.0 69.3 33.5

K8D 20.6 77.6 48.7 74.0 41.7 67.0 29.9

D72K 18.7 75.1 46.0 71.9 42.6 65.0 30.3

D72N 18.3 75.9 50.2 72.0 42.5 64.9 29.9

D72A 21.8 81.8 51.5 78.1 39.0 72.9 36.6

D72K ⁄ K8D 21.6 79.1 51.3 74.5 44.7 66.5 32.0

D72K ⁄ K8A 20.0 77.9 50.6 74.3 46.7 67.4 33.4

D72A ⁄ K8D 24.6 83.6 54.7 79.3 49.8 73.1 35.0

D72A ⁄ K8A 22.0 83.7 53.9 80.1 50.8 75.0 38.6

65 70 75 80 85

300

330

360

390

420

∆HU

,m (

kJ m

ol-1

)∆H

U,m

(kJ

mol

-1)

65 70 75 80 85

300

330

360

390

420

A

B

Tm (°C)

Tm (°C)

Fig. 4. Temperature dependence of DHU,m. Temperature depend-

ence of the unfolding heat effect for wild-type cyt c6 and its mutant

forms. Symbols correspond to the DHU,m and Tm values obtained

by multiple fits of the DSC traces at the three pH conditions for (A)

wild-type ( ), K8A (s), D72A ⁄ K8D (n), D72K ⁄ K8D (,) and

D72A ⁄ K8A (e), and for (B) K8D ( ), D72K (s), D72N (n), D72A (,)

and D72K ⁄ K8A (e). The lines correspond to the DHU(T) functions

obtained by multiple fits for wild-type (A) and for K8D (B) at pH 5.0

(solid line), pH 4.0 (dashed line) and pH 3.0 (dotted line).



D72A ⁄K8A at all three pH values (Fig. 5A). This ten-

dency was also observed when comparing the Tm val-

ues (Fig. 5B). These results are in good agreement

with the apparent order of stabilities obtained from

the urea-induced unfolding experiments (Table 3). It is

interesting to note that the stabilizing effect of substi-

tuting D72 to alanine (in single and double mutants)

was neither observed when the position 72 was

mutated to a charged lysine residue, nor when it was

changed to the neutral and hydrogen bond forming

asparagine.

With respect to DDHU75 �C values (Fig. 5C) two

groups of mutants could be distinguished. The point

mutations at position 8, for which no significant

enthalpic difference was observed, and the rest (where

position 72 is mutated), which presented lower enthal-

pies of unfolding than the wild-type protein.

Inspection of the values obtained for the Gibbs ener-

gies of unfolding of the mutant forms of cyt c6revealed significant nonadditive effects in the stabilities

of the double mutants when compared with the stabili-

ties of the respective single mutants. The interaction

Gibbs energies (DGint) at 30 �C for the different double

mutants were estimated by double-mutant cycle analy-

sis [33,34] and are summarized in Table 4. At pH 5.0,

the DGint values obtained when K8 was substituted by

aspartate were � 5 kJÆmol)1 higher than when K8 was

substituted by alanine, independent of the nature of

the substitution at amino acid position 72. This addi-

tional interaction energy was reduced to 2 kJÆmol)1

(for the double mutants of D72K ⁄K8D and

D72K ⁄K8A) or even abolished (for the double

mutants D72A ⁄K8D and D72A ⁄K8A) at pH 3.0.

Assuming a standard pKa value for the carboxyl

group, the aspartate residue introduced at position 8

would be almost completely neutralized at pH 3.0.

Thus, replacement of K8 with a negatively charged

residue resulted in stronger destabilizing interactions

than a neutral residue, when the negative charge at

position 72 was removed.

The influence of the nature of the introduced muta-

tions and the complex pH dependence of the DGint

values for the different mutant pairs strongly suggest

that much more complex effects (multiple interactions

and ⁄or structural rearrangements) than a direct elec-

trostatic interaction between the residues at amino acid

positions 8 and 72 play a role. A refined model for the

structure of cyt c6, incorporating NMR data, has

become available during revision of this work [35]. In

this model (Fig. 6), K8 is located relatively far from

other charged residues, and its side chain is highly

solvent exposed. It forms backbone hydrogen bonds

AA

AD

KA

KD

D72A

D72N

D72K

K8A

K8D

10 5 0 -5

∆∆GU

75 ºC (kJ mol-1) ∆∆HU

75 ºC (kJ mol-1)∆Tm

(K)

AA

AD

KA

KD

D72A

D72N

D72K

K8A

K8D

100 50 0 -50

AA

AD

KA

KD

D72A

D72N

D72K

K8A

K8D

8 4 0 -4

A B C

Fig. 5. Effect of mutations on the thermodynamic parameters of unfolding. Difference of the thermodynamic parameters DDG 75 �CU (A), Tm

(B) and DDHU75 �C between wild-type and each mutant. Black bars correspond to pH 5.0, grey bars to pH 4.0 and light grey bars to pH 3.0.

Double mutants are abbreviated with the first letter representing the residue at position 72 and the second letter the one at position 8.

Table 4. Double-mutant cycle analysis. Interaction Gibbs energies

at 30 �C (DGint) were calculated from the DG 30 �CU -values reported

in Table 3 according to DGint (D72X ⁄ K8Y) ¼ DG 30 �CU (wild-

type) + DG 30 �CU (D72X ⁄ K8Y) – DG 30 �C

U (D72X) – DG 30 �CU (K8Y).

DGint (kJÆmol)1)

pH 5.0 pH 3.0

DGintpH5

– DGintpH3

(kJÆmol)1)

D72K ⁄ K8D 11.4 6.3 5.1

D72K ⁄ K8A 6.2 4.1 2.1

D72A ⁄ K8D 9.3 3.0 6.3

D72A ⁄ K8A 4.0 3.0 1.0



along the N-terminal a helix involving the amino acids

V4 ⁄N5, and S11 ⁄A12. The side chain of D72 is part of

an acidic patch, together with the residues D2 and

E68. It forms backbone hydrogen bonds along the

C-terminal a helix to E68 and Y76. Interestingly, one

of the side chain oxygens of D72 appears to be hydro-

gen-bonded to the backbone N atom of G6, bridging

the crossing between the N- and C-terminal a helicesof cyt c6 (Fig. 6). Any mutation leading to charge

neutralization or inversion of a residue within this

helix-crossing region may cause rearrangements and a

reorganization of the local hydrogen-bond network. It

is, for example, conceivable that upon replacement of

D72 by alanine one of the residues E68 or Q69 ‘takes

over’ in forming a stabilizing hydrogen bond to an

amino acid within a reoriented N-terminal a helix.However, any such interpretation of the experimental

data remains speculative until reliable structural infor-

mation for cyt c6 and its mutant forms becomes avail-

able.

Conclusions

The results of this study stress the importance of elec-

trostatic interactions for the function, as well as for

the stability, of cyt c6. Neutralization or inversion of

the positive charge at position K8 significantly reduced

the efficiency of electron transfer to PS I. This effect

was not reversed when the global charge of the protein

was restored by additional mutations at position 72.

This implies that residue K8 plays a role in the forma-

tion of the electron transfer complex between cyt c6and PS I. However, replacement of the negative charge

at position D72 by asparagine or lysine increased the

efficiency of electron transfer to PS I, at least partly,

by favouring long-range electrostatic attraction between

the reaction partners. These mutations, however, were

found to destabilize the protein significantly. Again,

this effect of the mutations could not be fully reversed

by restoring the global charge balance. The negative

charge at position D72 apparently participates in elec-

trostatic interactions that stabilize the structure of

cyt c6, as indicated by the reduced enthalpy of unfold-

ing of the corresponding mutants. Interestingly, the

negative effect of its deletion was reduced, due to

entropic contributions, when aspartate was not

replaced by the isosteric asparagine, or by lysine, but

by the small amino acid alanine. For the variants

carrying the mutation D72A, the positive change in

entropy upon unfolding was reduced with respect to

other variants of cyt c6. This smaller unfolding entropy

could be due to the effect of the mutation on the

native state (higher flexibility and ⁄or lower exposure of

hydrophobic area to the solvent in the mutant D72A)

and ⁄or on the unfolded state (lower flexibility and ⁄orhigher solvation of hydrophobic surface area). Com-

plementing the mutation D72A with a charge neutral-

ization or inversion at position K8 led to mutant

forms of cyt c6 that were more stable than the wild-

type under all tested conditions. Of all studied forms

of cyt c6, AD was the thermodynamically most stable

at pH 7, whereas AA was the most stable at pH 4 and

3. The price for this stabilization, however, was a

reduction in catalytic efficiency by >50%.

Experimental procedures

Protein expression and purification

Expression plasmids pEACwt and pEACD72K were kindly

provided by F.P. Molina-Heredia [16,36]. Expression plas-

mids for the other mutants were generated by site-directed

mutagenesis according to the QuikChange method (Strata-

gene, La Jolla, CA) using pEACwt as template. Cyt c6wild-type and its mutant forms were expressed under aero-

bic conditions in E. coli GM119 cells that had been

cotransfected with the plasmid pEC86 [37] (kindly provided

by L. Thony-Meyer, ETH Zurich) and were purified as des-

cribed previously [16], with the exception of the mutants

K8D, K8A and D72A ⁄K8D (AD). These proteins were

Fig. 6. Structural model of cyt c6, showing secondary structure ele-

ments. The side chains of amino acids K8 (blue), E68 (light red),

Q69 (light green) and D72 (red), as well as the heme moiety

(orange), are depicted as stick models. The H-bond from the back-

bone amide group of G6 to the carboxyl oxygen of D72 is shown

as dotted green line. The image was generated with PYMOL 0.97

(DeLano Scientific, San Carlos, CA, USA) and is based on the struc-

tural model from [35].



purified by a combination of anion-exchange chromatogra-

phy and gel filtration. In brief, the periplasmic extracts con-

taining the mutants K8D, K8A or AD were dialysed

against 0.5 mm sodium phosphate buffer pH 7, oxidized by

the addition of 50 lm potassium ferricyanide, and loaded

onto a DEAE cellulose column (50 mL) that had been

equilibrated with a 1 mm sodium phosphate buffer, pH 7.

The mutant proteins were found in the flow-through. After

concentration under nitrogen pressure in an Amicon 8050

ultrafiltration device fitted with YM 3 membranes (Milli-

pore, Bedford, MA), the fractions containing the mutant

forms of cyt c6 were applied to a Superdex 75 XK16 ⁄ 60FPLC column. The running buffer for the gel filtration was

10 mm Tricine ⁄KOH pH 7.5, 100 mm NaCl. After chroma-

tography, the purified proteins were washed with 10 mm

Tricine ⁄KOH pH 7.5 in the ultrafiltration device, concen-

trated to 1–2 mm, and stored at )80 �C. The purity of all

preparations was >95% as judged from the optical

absorbance ratio at 554 and 280 nm, and from Coomassie

Brilliant Blue-stained SDS ⁄PAGE. Protein concentrations

were determined based on an absorptivity e553 of

26 200 m)1 cm)1 [36] for fully reduced cyt c6.

Redox potentials

Redox potentials were determined by potentiometric titra-

tion with sodium dithionite and potassium ferricyanide as

described previously [38]. The determinations were carried

out in 50 mm sodium phosphate buffer pH 7 and at 25 �C.

Laser-flash absorption spectroscopy

Experiments were carried out as described previously [3].

The reaction mixture was buffered with 20 mm Tri-

cine ⁄KOH at pH 7.5 and contained 10 mm MgCl2. The

temperature was 25 �C. For the determination of bimolecu-

lar rate constants (kbim), the kinetics of re-reduction of PS I

from Nostoc sp. PCC 7119 were measured at increasing

concentrations of wild-type cyt c6 or of its mutant forms.

The kbim values were determined from the slope of a linear

fit of the observed rate constants (kobs), plotted against

donor concentration. For most forms of cyt c6, fast kinetic

phases were not observed, and it was possible to analyse all

kinetic according to a simple bimolecular reaction mechan-

ism without introducing significant deviations in kobs.

Stopped-flow experiments

Folding equilibrium relaxation experiments were carried

out as described previously [22]. In brief, equal concentra-

tions of cyt c6 (wild-type or mutant forms) in 20 mm

sodium phosphate pH 7.0 (buffer) and in 9.8 m urea buf-

fered with 20 mm sodium phosphate pH 7.0 (buffered urea

solution), both containing 50 lm potassium ferricyanide in

order to keep the protein in its oxidized state throughout

the folding ⁄unfolding experiment, were mixed in varying

ratios (r) in a lSFM20 stopped-flow device (BioLogic SA,

Grenoble, France) with a constant total flow rate of

5.6 mL s)1 in a constant total volume of 560 lL. Protein

folding ⁄ unfolding was monitored by tryptophan fluores-

cence. Kinetic traces were recorded in duplicate or triplicate

and averaged for analysis. The data for all mutants were

fitted globally to a two-state model assuming linear depend-

ence of the Gibbs energy of unfolding (DGU) on the urea

concentration ([urea]) according to

DGU ¼ DGU0 þmeq � ½urea� ð1Þ

with DGU at 0 [urea], DGU0, and the urea interaction

parameter meq, and assuming a second-order dependence of

the logarithm of the unfolding rate constant (kU) on [urea],

according to

ln kU ¼ ln kU0 �1

RT� ðmk1 � ½urea� þmk2 � ½urea�2Þ; ð2Þ

with kU at 0 [urea], kU0, and the first- and second-order

urea dependencies mk1 and mk2. To estimate the urea-

dependent shift in transition state structure, a values were

calculated from the fit parameters according to

a ¼ meq � ðmk1 þ 2 �mk2 � ½urea�Þmeq

: ð3Þ

The global fitting of the data was carried out with the non-

linear analysis module of origin 5.0 (Microcal, Northamp-

ton, MA, USA).

Microcalorimetry

The heat capacity of wild-type cyt c6 and its mutant forms

was measured as a function of temperature with a VP-DSC

differential scanning microcalorimeter (Microcal). Samples

were dialysed overnight against the desired buffer and sub-

sequently oxidized by addition of potassium ferricyanide to

a final concentration of 2.5 mm. Immediately before load-

ing the samples into the calorimetric cell, ferricyanide was

removed by gel filtration on a PD-10 column (Amersham

Biosciences, Little Chalfont, UK). The buffers used in the

experiments were 60 mm sodium acetate, pH 5.0, 250 mm

sodium acetate, pH 4.0, or 45 mm sodium phosphate,

pH 3.0, all giving rise to the same ionic strength (41 mm)

as the 20 mm sodium phosphate buffer, pH 7, which was

used in the urea-induced folding ⁄ unfolding experiments.

DSC experiments were performed at a heating rate of

1.5 KÆmin)1 between 5 and 100 �C, using protein concen-

trations from 10 to 50 lm (0.1–0.5 mgÆmL)1). Baseline

scans were performed with the corresponding dialysis buffer

loaded in both calorimetric cells. The partial molar heat

capacity (Cp) was calculated assuming a value of

0.73 mLÆg)1 for the partial specific volume of the proteins.



After transforming the DSC traces into partial molar heat

capacity curves, they were subjected to individual and glo-

bal curve fitting using the nonlinear analysis module of

origin 5.0 with user-defined fit functions based on the

equations corresponding to a two state model (NfiU). The

temperature dependence of the heat capacity was described

as a linear and quadratic function for the native and unfol-

ded species, respectively [39]. The corresponding parameters

were left to float during the curve fitting, with the exception

of the first- and second-order coefficients of the unfolded

heat capacity, which were fixed and evaluated from the

amino acid content as described previously [40,41]. For

each form of cyt c6, the values of the change in molar heat

capacity upon unfolding (DCp,U) were determined by global

fitting of the DSC traces recorded at all three studied pH

values.

Chemicals

Urea was SigmaUltra grade. All other chemicals were

at least analytical grade. All solutions were prepared with

MilliQ water.

Acknowledgements

This work was supported by the Spanish Ministry of

Science and Technology (MCYT grants BMC 2000-

444 and BIO2003-04274) and the Junta de Andalucıa

(PAI, CVI-198). CL received a fellowship from the

European Union’s Research Training Network pro-

gram (HRPN-CT-1999-00095). IL was supported by a

research contract from the University of Granada and

is the recipient of a Ramon y Cajal research contract

from the Spanish Ministry of Science and Technology.

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