Eur J Biochem. 141, 207-307 (1984) ( kbBS 1984

NMR studies of a dihaem cytochrome from Pseudomonas perfectomarinus (ATCC 14405)

Isabel MOURA, Ming Cheh LIU, Jean LeGALL, Harry D. PECK Jr, William J. PAYNE, Antbnio V. XAVIER, and Jose J. G. MOURA Centro de Quimica Estrutural, Universidade Nova de Lisboa; and Department o f Biochemistry and o f Microbiology, University o f Georgia, Athens

(Received December 6; 1983/January 24, 1984) -. EJB 83 130Y

Pseu~lornonasperfectomarinus (ATCC 14405) dihaem cytochrome cs52 was studied by 300-MHz proton magnetic resonance. Some of the haem resonances were assigned in the fully reduced and fully oxidized states. No evidence was found for methionine haem axial coordination. The oxidation-reduction equilibrium was studied in detail. Due to the large difference in mid-point redox potential between the two haems (+ 174mV, for haem I1 and -180mV for haem I) an intermediate oxidatioii state could be obtained containing reduced haem I and oxidized haem 11. In this way the total paramagnetic shift at different oxidation levels could be decomposed in the intrinsic and extrinsic contributions. It was found that the two haems inleracl. The rate of electron cxchange is slow on the NMR time scale. Thc rcdox equilibria arc discussed for four possible redox species in solution.

Pseutkonzonas pe~fectomarinus (ATCC 14405) is a marine denitrifier [I]. Several c-type cytochromes have been isolated from these bacteria and seem to be implicated in the de- nitrification process [2, 31.

Recently, a dihaem cytochrome was isolated from this organism [4]. Tt is a c-type cytochronie with an a-band absorption maximum at 551.7 nm. It contains two covalciitly bound haem groups and it is isolated as a monomer ( M , = 25 800). The midpoint oxidation-reduction potentials of the two haems are far appart. One is ascorbate-reducible and the other only dithionite-reducible. The high-potential haem (haem 11) has a formal midpoint oxidation-reduction potential of 174 & 37mV and that of the low-potential haem (haem I) was estimated to be approximately - 180 mV [4].

EPR studies at 9 K revealed two distinct ferric low-spin signals arising from two non-equivalent haem species [4]. The high-potential haem (haem 11) has g,,, = 3.25, while the low- potential haein (haem I) has gmax = 2.88 and g,,, = 2.22. The sixth ligand of haem I was proposed to be a histidine due to the characteristic haem g-valucs [4]. The high-potential c-type hacms gencrally usc a methionine residue as the sixth ligand; however, the visible absorption spectrum in the oxidized state lacks the 695-nin absorption band, so characteristic of me- thionine ligation [5].

The amino acid analysis indicates the presence of only three cysteinyl residues, suggesting that one of the haems is only bound by a thioether linkage to the polypeptide chain.

The physiological significance of P. pevfectomavi~ius di- haem cytochrome c552 remains unclear but in some pseudo- monads it was suggested that dihaem cytochromes are related to the nitrite reductase system during the denitrification process [b-91. The fact that neither cytochrome cSs2 nor the nitrite reductase cytochrome cd are synthesized when P. perfecto-

Ahhrc~viuticiu. LPR, electron paramagnetic resonance

inarinus is grown aerobically is an indication that cytochrome r S s 2 is linked to the nitrate respiration process [lo].

Our interest i n cytochrome c S s 2 results from its interesting redox and haein axial ligation properties. In addition, this cytochromc represents a simplitied model for multihaem cytochroincs which we have been extensively probing by nuclear magnetic resonance spectroscopy, i.e. cytochromc c, (three haems) [1 I] and cytochrome c3 (four haenis) [12,13]. The spatial arrangement of the two haems inside the same polypep- tide chain represents a unique system where the following qucstioiis may he contemplated: (a) electron transfer between two redox cciitcrs in a fixed geommetry; (b) evaluation of extrinsic and intrinsic contributions for the haem paramagnetic shifts; (c) thc tcsting of a redox model useful for the calculation of populations or redox species in solution for multiredox centre proteins.

MATERIALS AND METHODS

Cytochrome c s 5 2 from Pseudowonas prrfwtomrcriiius was isolated as previously described 141. The concentration of the solution was about 2mM, determined using the molar ab- sorption coefficients at 551.7nm (c: = 33020 M-' cm-I). The cytochrome was dialysed against deionized water at 4 "C, and Iyophilized twice from D20. The samples were dissolved in D,O and the nominal 'pH' was adjusted with NaOD and DCl. Quoted 'pH' readings refer to direct pH measurements. Samples were reduced under argon with a slight excess of solid sodium dithionite. They were allowed to reoxidize by introduc- ing small amounts of air with a Hamilton syringe through serum caps. The chemical shifts are always referred to sodium 3-trimethyI~ilyl-(2,2,3,3-~H,) propionate, positive shifts re- ferring to down field.

The NMR spectra were recorded using a Bruker 300 CXP spectrometer, equiped with an Aspect 2000 computer where mathematical manipulations were performed.

298

LO 30 20 lo 6 ippml

Fig. 1. 300-MH: Xh4K vpi’ctru ( l o ~ c ~ / k l d / ([f P. perfectomarinus ,4 TCC 14405) c,rtoc.lrromc c,;~, in ~ e w r a l oxi~lation-rerluution s q e s ,

sturtifrg f iorn IIW fid1.s rethrcrd ( lower specrvum) arid going to fhe ititermediate oxidized protein (hoem I 1 oxidized, upper speclrum). The titration was carried o u t at 205 K and pH* 7.24

RESULTS

The bottom spectrum of Fig. 1 and the top spectrum of Fig. 2, respectively, represent details of the low-field region of the fully reduced and fully oxidized spectra of Pseudomonas pe~fi.ctornarinus dihaem cytochronie c552. In Fig. 3, the top and bottom spectra show the high-field region of the f~dly oxidized and fully reduced spectra. The most striking feature of these spectra is the presence of resonances outside the range 11 pprn to -2ppm for the fcrricytochrome and the absence of such resonances for the ferro form. The chemical shift values of these resonances are ternperaturc-dependent (see below) and arise from protons in the vicinity of the iron atom. They experience large shifts due to the paramagnetism of the ferricytochrome.

The ferrocytochrome i s diamagnetic in agreement with EPR measurements since no signal could be detected [4]. The most powerful source of secondary shifts for diamagnetic haem proteins is the ring-current of the haem group and, as a consequence of this shifts, resonances of the axial ligands are shifted upfield [14]. [lowever, only in the case of methionine ligation resonances of axial ligands have been observed beyond

~ 1 ppm [14]. Tn the case that two histidines are used as axial ligands. which is the ligation mode found, for example, in the tetrahaem cytochrome c ’ ~ , the C2 and C4 protons of histidine may be found between 1 ppm and Oppm [15]. In the fully reduced state the dihaem cytochrome shows a typical dia- magnetic haem protein spectrum, with well resolved mew- proton resonances between + 11 ppm and + 8 ppm. However, no evidence was found for methionine coordination. At high field a resonance at --2.lhppm is detected with a one-

Ml

I I I I I 1 I

LO 30 20 10 6 ippml

Fig. 2. 300-MHz IVMR spectra ( l o ~ c - / k l d ) of’ P. perfectomarinus (ATCC 14405) cytochrome cjj2 iiz several oridaiion-recitictiorl siqyes, startiiig,from the iritermediaie (huem I1 osidized, lower spectrum) atid going to thejidly oxidiredpmtein (upper spectrum). The titration was carried out at 295K and pH*7.24. Some relevant resonances are i dentitied

proton intensity. Also two broad resonances are observed at - 4.20 ppm and - 5.00 ppm. The three-proton resonance around - 3 ppm, which is characteristic of cytochromes with methionine as axial ligand, is absent.

Now, if we observe the fully oxidized state spectrum. there are six three proton intensity resonances with chemical shift values larger than 10ppm and at least six one-proton intensity resonances resolved in the same region. These resonances are temperature-dependent. In Fig. 4, the chemical shifts of these resonances are plotted against 1jT. Solid lines represent the haem methyl protons. All the resonances appear to obey Curie’s law. On the basis of their low-field chemical shift values and intensity, six methyl resonances are clearly assigned to the haem methyl groups, accounting for the presence of two haems. The other two haem methyl group resonances are missing in this spectral region and must be present in a more crowded region of the spectrum. The molecular mass of the protein does not enable a Large resolution in the spectra between about 10ppm and 0. At higher field, several resonances are resolved between 0 and - 8 ppm, which also present Curie law tempera- ture dependence (see Fig. 4). There are also two very broad resonances around - 16 ppm and - 1 1 ppm, which have not yet been identified.

299

equilibria proposed, all the populations can be expressed in terms of any other two (e.g. Po and P,). With the additional

condition C Pi = 1 the resultanl second-order equation can be

solved for any value of P, with subsequent calculation of Po.

3

i = O

Pi (F) +. P" (1 + lo"*) + (P* - 1) = 0

where AE* = (Ei,-Ei)F/2.303 RT and F is the Faraday equivalent.

The calculation of all Pi is straightforward and Pi can then be plotted as functions of the solution redox potential and their values compared with the intensities of the resonances present in the NMR experiments, assuming that an equilibrium state is attained at each experimental point. The curves using the midpoint redox potentials previously estimated (EII = +174mV and El = -180mV) are shown in Fig.7A. Also included in this figure is the influence of the difference between the microscopic midpoint redox potentials of the two haems, 4 E = El , -& in the intensities of the intermediate oxidation states.

The difference of midpoint redox potentials determined for P. pecfectonznrinus [14] (LIE = 354mV) is so large that the intensities of intermediate 2 (haem TI oxidized and haem I reduced) is negligeable. So we face a very simple situation where three redox species must be expected (0, 1 and 3). The model predicts that for a ;1E = IOOmV, the maximum value of intermediate 2 is only 0.02 and from 100mV to OmV, it increases up to 0.25. The intensities of P, can also provide an indication of the redox potential between the two haems. When LIE > 200 mV only a lower limit can be determined.

-- __ -

--

I

1_~__

. __--~

L---L-__ _ _ _ _ _ _ _ ~

0 -5 -10 -15 -20 6 (pprnl

Fig. 3. 300-MHz N M R spectru (high-field) of P. perfectomarinus ( A TC'C 14405 j cytochronie c , - ~ ~ in setwul oxidation-reduction sta,ycs, stnrfing,fiorn rhe $illy reduced (lower spectrum) and going 10 the.$illjl oxidiiedyrotein (zipper spectrunz). The titration was carried out at and pH* 7.24

The pH dependence of the haem resonances was studied over a pH range (5.8 - 8.5) at 295 K (Fig. 5). In this range of pH a number of resonances have slight shifts but the nature of the ionizing groups has not been unequivocally identified.

A model ,fbr [he reoxidation of CI diliaeoz cytochrome

A detailed study of the oxidation-reduction mechanism of a dihaem cytochrome requires the design of a model which considers all the possible redox species present in solution. Such a simple model is shown in Fig. 6, together with all thc pathways by which the Sour redox species (0, fully oxidized; 1 and 2, intermediate oxidation states; 3, fully reduced) can inter-convert during the oxidation-reduction process. The haems are numbered according to their midpoint redox potentials (El more negative).

Two intermediate states can be generated and their popu- lations are critically dependent on thc difference between E, and Eli. The population fractions of the four redox species (Pi, i = 0- 3 ) can be calculated assuming that each Ei i s inde- pendent of the oxidation state of the other haem. By combining the Nernst equations that can be established for the redox

Oxi~ation-recZzlctio~ equilibria

Fig.1, 2 and 3 show in detail the 300-MHz NMR re- oxidation pattern of P. perfectoinurinus cytochrome c552 followed in the low-field (Fig. 1 and 2) and high-field (Fig. 3) regions of the spectra. The drastic modifycation observed in the NMR spectra of the dihaem cytochrome when going from the fully reduced diamagnetic species to the fully oxidized para- magnetic one provides a powerful way to determine the mechanism involved in thc electron process, as well as to probe a possible haem-haem interaction. Several redox titrations were performed at different pH and temperatures.

The redox titration shown in Fig. 1 - 3 was done at pH 7.3 and 295 K. Fig. 1 presents the spectra obtained for the first part of'the redox titration (i.e. reoxidation of the haem with lowest midpoint redox potential) starting with the spectrum of fully reduced state (bottom). From the breadth and progressivc shift observed for the resonances of thc methyl groups belonging to this haem, it is evident that the intermolecular electron exchange rate for this redox steps is intermediate to slow on the NMR time scale. The linewidths of the resonances sharpen up to its final linewidth when the half-reduced state is attained (top spectrum). At this stage no resonance belonging to the other haem is yet observed in the low-field region of the NMR spectrum.

Fig. 2, starting with the spectrum of half-reduced state (bottom), follows the second part of the redox titration. As the titration proceeds, haem I1 becomes oxidized until the fully oxidized state is obtained (top spectrum). The resonances grow without change of either chemical shift or linewidth, indicating that the electron exchange rate for this last redox step is slow.

300

- E a

a Q -

Due to the large differcncc in midpoint redox potential between these two haems, an half-reduced redox state is observed whcre one of the haems is still fully reduced (haem 11) while lhe other is already fully oxidized (haem 1). Then it is possible to assign the haem methyl groups (Mi) and one proton resonances ( S i ) observed outside the main protein spectral envelope to hacm I.

2

Fig. 6. Diagram shottirig ull [he possihk~ rc4o.y species of n dihavtii cylochrome. 3, fully reduced species; 2 and I . intermediate species; 0, fully oxidized species. The open circlcs represent oxidized states. For clarity of discussion a methyl group belonging to haem I is shown

MI, Mz and M4 togcthcr with S, , S, and S, belong to the hacm with lower redox potential (- 180 mV). M3, M, and M6 together with S2 (not obscrvcd at 295 K because i t is under M4), S3 and s6 belong Lo the haem with higher redox potential ( + 174 mV).

For a dihaem cytochrome (as well as [or a multiredox system) the total paramagnetic shift 6 M i felt by each haem can have two contributions, as illustrated schematically in Fig. 8: (a) the contact plus the pseudocontact shift due to the ferric state of its own haem (intrinsic shift. jinl); and (b) the pseudocontact shift, due to the ferric hacm in the vicinity (extrinsic shift, lcJ. So the total chemical shift is 6 M i = I:,, + 'k,.

An analysis of the paramagnetic shifts in ferricytochromes shows that AinI are generally very large [14] and positive, whereas Aoll are much smaller or even negligeable and may have both signs.

Only two methyl group resonances (M, and M,) that belong to haem I (- 180 mV) experience a measurable extrinsic chemical shift when haem II (+174mV) starts to reoxidize.

301

Fig. I . ( A ) Plot of the iritensities ofthe several redox species as rr,function ofthe rc~tlo.vpotmtialspublished~irr the P. perfectomarinus (ATCC 14405) cjm+rome cXX2 (41 iE,, = + 174m V and E, = ~ IS'OmVi. i R i Plot o f the maximum iniensities of the intermediate species as a,fiirlc.tiorl ofthe tliffkrence in rer1o.c potential between the iwo hawi.v ( 1 E = E,- Elr)

Fig. 8. Schmuric representution of the chemical o j a methyl resonance belonging to the huem with lower redox potential in three .vtnte.v (IJ' o,vidation: ( a ) fully reduced; ( b ) intermediated oxidation state; (c).full j oxidized. din, and A,, , are the intrinsic and thc extrinsic chemical shifts of this haem methyl resonance

Methyl MI shows a negative I,,, (z - 0.47 ppm) and methyl M, a positive one (z 0.08 ppm). The exchange process, probed by the observation of the evolution of resonances M, and M,, results from the redox equilibrium between species 0 and 1, and it is slow on the NMR time scale. A limit exchange rate can be estimated from the chemical shift difference. The exchange rate ( k ) between 0 and 1 must be smaller than 2.4 x lo4 M-'s-' in '

order that two separate peaks could be observed for M,. The observation of extrinsic shifts for some of the haems

proves that the two haems are sufficiently close so that some of the haem proton chemical shifts of one haem are perturbed by the ferric ion of the other.

DISCUSSION

The redox pattern of a dihaem eytochrome as followed by proton magnetic resonance can be very complex due to: (a) the different midpoint redox potentials of the haems; (b) the influence of the oxidation state of one haem on the redox potential of the other (interacting potential); (c) the contri- bution of both inter and intramolecular electron exchange mechanisms: and (d) the relative values of intrinsic and extrinsic paramagnetic chemical shifts [I 21. However, the analysis may be very simplified, as in the present example, when

the two haems have a very large difference in midpoint redox potentials. In this case, the solution equilibria analysis is restricted to three redox species and it is irrelevant to consider interacting potentials. This is not the general case and, for example, in the tetrahaem cytochromes isolated from Desulfovihrin vulgaris and Desulfovibrio gigas, a more complex analysis was undertaken considering the 16 possible redox species in solution [12, 131.

The redox model previously discussed for a dihaem cyto- chrome, taking into account all the redox species, can be useful for the following reasons. (a) From the intensity measurements of intermediate 1, a lower limit for the difference in midpoint redox potential of the two haems can be obtained. (b) In a situation where LIE < 200 mV, the value of LIE may be measured directly from the analysis of the intensities of the intermediate, P , and P2, if slow exchange conditions are observed.

The first reoxidation step probes essentially the redox equilibrium 3/1, i.e. it measures the intermolecular electron exchange process for haem I. By analysis of the linewidths during the first reoxidation step, a value of 2.7 x lo5 M-'s- I

( T = 295 K for 2 m M protein) for the rate constant of the redox equilibrium was obtained. Since the oxidation state 2 is not formed, the LIE value between the two haems is larger than 200 mV (in agreement with the redox potential previously estimated [4]) and the intramolecular exchange process does not need to be considered. The smaller extrinsic chemical shifts observed during the second haem reoxidation process gives an upper limit for the velocity constant of the exchange process between 0/1 of the order of 2.4 x lo4 M-'s- l ( T = 295 K for 2 mM protein). Our data analysis implies that the equilibrium for step 3jl is faster than this value.

We should emphasize that the analysis of the redox pattern of this dihaem cytochrome unequivocally demonstrates that both extrinsic and intrinsic contributions are determinant for the total paramagnetic chemical shift of the hacni resonances. This is the first example where both contributions could be separated by direct analysis of the redox pattern. As indicated before, the values of the extrinsic chemical shifts are small when compared with the intrinsic ones. This supports the analysis of the D. gigas cytochrome model [I 31 where it was assumed that, to a first approximation, the extrinsic contribution is negligible to the total chemical shift value.

The present NMR study excludes a methionine residue as an axial ligand for haem 11. Indeed, a high-field-shifted methyl group resonance, which is a fingerprint for an axial methionine, is absent in the fully reduced N M R spectrum, in agreement wilh the suggestion made by analysis of the visible spectrum [4].

302

The E P R studies [4] also support this observation. Low-spin ferric haems normally give rhombic EPR spectra with g-values that are sensitive to the typc of axial ligands coordinated, particularly the value of g,,, (g,). For niethioiiine in a c-type situation gz values betwccn 3.0 and 3.2 arc normally found [16] though slightly hisher values have been observed, as is the case of Pseudonioiiiis uerogiriosii cytochrome cs51 (gz = 3.24) [I 71. Higher gz values have been found in other c-type situations, e.g. the c-moiety haem of Thiohacillus tienitrjficans cytochrome cd (nitrite reductase) where g, = 3.60. In this case the possibility of niethionine coordination was disregarded 1181. Also hemi- cromes with gz = 3.5 values have been observed for cyto- chromes c at high pH. These high-pH forms were suggested to have a lysine r:-amino group in the axial position replacing the methionine residue 1161. The EPR g-values reported for dihaem cytochrome css2 at low temperature clearly indicate the exis- tence of two low-spin haems, in agreement with the N M R data taken at room temperatures. Haem I g-values (g,,, = 2.88) suggest the presence of a histidine in the sixth coordination position [I 91. However. thc high-potential hacm (haem 11) shows a g-value (g,a, = 3.25) that is a t the limit for a c-type mcthioninc coordiiiation. Cptochromes c3 isolated from dif- ferent Desulfbvihrio strains and Dum. aceta.xidnns cytochrome c7 show EPR spectra with g,,,-values between 3.36 and 2.80 [20, 221 (and our unpublished results). In these cytochromes there is crystallographic 123, 241 and NMR evidcnce [I 1 - 131 that all the haems have a histidine-histidine type ligation. However, the redox potcntials of thesc haems are always negative [22, 251. The ,q$,,,-valuc of the high-potential haem (haem 11) in cytocht-omc c 5 5 2 is in the range observed for c j and c7 but the redox potenlial is very different. Thus, the exclusion of a mcthioninc residue as ii haem axial ligand for haem I1 can not be made by considering only the EPR data.

An important diffcrcncc bctwcen thc two haems is their midpoint rcdox potential. Indccd, a largc gap in redox potential values is observed. A wide range of redox potentials is generally covered by haem proteins [XI. This reflects the influence of the haem ligation, the important role of the polypeptide chain and the exposure of the haem group t o the environment [27], controlling in a fine-tuning mode the electron distribution in the haem centre.

In general, bis-histidine ligation is associated with low- potential values a, observed for the three and four haem- containing proteins. cytochromes c7 and cj, respectively [22, 251. A high redox potential is generally associated with a niethioiiinc-histidine ligation. It has also been suggested that a hydrophobic protein encapsulation of the haem increases its midpoint redox potential [27]. Our analysis of the electron transfcr rates also correlates with this suggestion since a higher electron exchange rate was observed for hacm I. The hacm with lower redox potential (haem I) should be the haem more exposed to solvent.

The nuniber ot’ cysteinyl residues reported per molecule of M , = 28 500 (three residues) [4] is insufficient to covalently bind two r-type haems in a normal situation. This result could indi- cate that only one thioether linkage was used for one of the haems. A similar situation was reported for Chrithitlia ocron- pclti cytochrome c 5 s 7 [28].

The physiological role of this dihaein cytochrome remains unclear. However. this protein is prcscnt in other pscudo- monads, likc Psriirlomomc sfufievi (ATCC 17591). Thc anal- ogies observed between these two dihaem proteins (our un- published results) are evident, indicating the presence of two low-spin fcrric haems. However, another dihaem protein isolated from P. srutxri (ATCC 11607) presents a different

spin-state situation [29]. This protein has a low-spin haem (high-potential ascorbate-reducible) which presents a normal methionine : haem iron: histidine coordination, and a high-spin haem (low-potential) which undergoes a spin transition on cooling below 7 7 K . This protein presents a cytochrome c-peroxidase activity and the general EPR properties are similar to those reported for Pseudonionns nevoginosn cyto- chrome c peroxidase [30, 311. Pse~irkoniotius pe~fecfomarinzis dihaein cytochromc cs52 also shows a residual cytochrome c pcroxidase activity ( 103-fold less active than the P. stutzeri, ATCC 11607, dihaem protein) (our unpublished results). This residual activity is possibly due t o the displacement of one of the axial ligands. The haem coordination (histidine-histidine) may be determincd for this type of activity. The possibility of reaching spin equilibrium states by displacement of the axial ligand may play a regulatory role for this type of enzymatic activity .

We thank Instifulo h‘acionnl de Investigci~~cio Ciciztifiiu and Junta Nacioncrl de Inve.cfigap?o Cient[fica e Tecno/(igicn (Portupal : .4VX and JJGM) National Science Foundation grant OCE-7919550 (WJP) and the Solar Energy Research Institute under contract XD-1-1155-1 (J.LeG.). We thank Ms M. Marlinez for carerul preparation of the manuscript, and Ms I . Carvalho for skilful technical help.

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I. Moura, A. V. Xavier, and J . J. G. Moura, Centro de Quimica Estrutural, Complexo Interdisciplinar, Instituto Superior Tecnico, Avenida Kovisco Pais, P-1000 Lisboa, Portugal

M. C. Liu, J. LeGall, and H. D. Peck, Jr, Department of Biochemistry, University of Georgia, Boyd Graduate Studies Research Center, Athens, Georgia, USA 30602 W. J . Payne, Department of Microbiology, University of Georgia, Boyd Graduate Studies Research Center, Athens, Georgia, USA 30602