JOURNAL OF MOLECULAR RECOGNITION, VOL. 4,27-33 (1991)

Ferredoxin Electron Transfer Site on Cytochrome c3. Structural Hypothesis of an Intramolecular Electron Transfer Pathway within a Tetra-heme Cytochrome? A. Dolla, F. Guerlesquin and M. Bruschi* Laboratoire de Chirnie Bacterienne, Centre National de la Recherche Scientifique, BP 71, 13277 Marseille Cedex 9, France

R. Haser Laboratoire de Cristallisation et Cristallographie des Macromolecules Biologiques (URA 1296), Centre National de la Recherche Scientifique, Faculte de Medecine, Secteur Nord, Universite d'Aix-Marseille I1 13326 Marseille Cedex 15, France

To specify electron exchanges involving Desulfovibrio desulfuricuns Norway tetra-heme cytochrome c3, the chemical modification of arginine 73 residue, was performed. Biochemical and biophysical studies have shown that the modified cytochrome retains its ability to both interact and act as an electron carrier with its redox partners, ferredoxin and hydrogenase. Moreover, the chemical modification effects on the cytochrome c3 'H NMR spectrum were similar to that induced by the presence of ferredoxin. This suggests that arginine 73 is localized on the cytochrome c3 ferredoxin interacting site. The identification of heme 4, the closest heme to arginine 73, as the ferredoxin interacting heme helps us to hypothesize about the role of the three other hemes in the molecule. A structural hypothesis for an intramolecular electron transfer pathway, involving hemes 4 , 3 and 1, is proposed on the basis of the crystal structures of D. vulgaris Miyazaki and D. desulfuricans Norway cytochromes c3. The unique role of some structural features (ahelix, aromatic residues) intervening between the heme groups, is proposed.

INTRODUCTION

Cytochrome c is one of the most investigated electroncarrier proteins, in understanding the factors that govern the rate and specificity of proteidprotein electron transfer (Pielak et a f . , 1987). Complex formation between cytochrome c and both physiological and non physiological partners have been extensively studied: cytochrome c/cytochrome c peroxidase (Hazzard et al., 1988), cytochrome c/cytochrome b, (Wendolowski et af., 1987), cytochrome clflavodoxin (Simondsen et a f . , 1982) and cytochrome c/photosynthetic center (Bosshard el a f . , 1987). In multiheme c-type cytochrome like di-heme cytochrome c4 (Lietch et a f . , 1985), threeheme cytochrome c, (Ambler, 1971) and tetraheme cytochrome ci (Bruschi, 198l), the functional meaning of hemes exhibiting various redox potentials in the same molecule is a matter of current research. In an attempt to investigate the structure/function relationships in multiheme cytochrome c3,we have investigated the electron transfer mechanism between Desuffuvibriu desuffuricans Norway cytochrome c3 and ferredoxin I as a model for the study of electron transfer reactions involving hemes and non heme iron clusters. D. desulfuricans Norway cytochrome c3 (Mr 13 OOO) contains four low redox potential hemes ( - 165 mV, -305mV, -365mV and -400mV) (Bianco and t This work was supported by the Centre National de la Recherche Scientifique and by Grants from M.R.E.S. Aide 86. C. 0688. * Author to whom correspondence should be addressed. 0952-3499/91/010027-07 $05.00

01991 by John Wiley & Sons, Ltd.

Haladjian, 1981) localized in non equivalent protein environments as described by EPR (Gayda et al., 1985), NMR (Guerlesquin et a f . , 1985a) and X-ray crystallographic studies (Haser et a f . , 1979; Pierrot et a f . , 1982). To date, the three dimensional structure of two cytochromes c3 isolated from D. desuffuricans Norway and D. vulgaris Miyazaki, have been determined at 2.5A and 1.8A resolution, respectively (Haser et al., 1979; Higuchi et a f . , 1984). It should be pointed out that despite the poor degree of sequence homology among D. desuffuricans Norway and D. vulgaris Miyazaki cytochromes (28% strict homology), the redox cores are remarkably conserved in terms of relative positions and orientations of the porphyrins: the iron/iron separations differed only by 0 . 3 A on average. D. desuffuricans Norway ferredoxin I is a (4Fe-4s) cluster ferredoxin (Mr 6000) exhibiting a redox potential of - 374 mV (Guerlesquin et a f . , 1982; Bruschi et a f . , 1985). Rapid kinetic studies of the electron exchange reaction between cytochrome c3 and ferredoxin I (Capeillere-Blandin et a f ., 1986) have shown the formation of an intermediate complex followed by a bidirectional electron transfer between heme and ferredoxin cluster, as established by in uitru experiments (Fig. 1) (Akagi, 1967; Suh and Akagi, 1969). The complex formation between cytochrome cj and ferredoxin I has been studied by biophysical techniques and computer graphics modelling. From microcalorimetric measurements, a stoichiometry of one cytochrome c3 molecule per ferredoxin subunit and an association Received 22 March I990 Accepted I.? March 1991

28

A. DOLLA E T A L.

Figure 1. Electron transport chain from Desulfovibrio. I: The phosphoroclastic reaction; II: The sulfite reduction.

constant of K,= 1.3 X lo6M-' (285 K, 10 mM Tris buffer, pH 7.7) were determined. Although an electrostatic effect is an important driving force in the interaction, a hydrophobic process is also involved in the complex formation (Guerlesquin et a f . ,1987). 'H NMR experiments (Guerlesquin et a f . , 1985b) gave the same stoichiometric ratio, and they also found that the resonance of the methyl group in the two highest potential hemes of the cytochrome ( - 165 mV and - 305 mV) were affected by the complex formation. Using the known crystal structure of cytochrome c3 and a ferredoxin I model, based on the X-ray structure of the Peptococcus aerogenes ferredoxin, a three dimensional hypothetical complex between these two proteins has been generated using interactive computer graphics methods (Cambillau et a f . , 1988). Among the four different models investigated, in which the iron/sulfur cluster faces each heme successively, the most favorable model involves heme 4 (sequential heme numbering from amino-terminus of the amino-acid sequence), in terms of charge interactions and surface topology complementarity. It should be pointed out that a covalent cross-linked cytochrome cJferredoxin I complex has been obtained (Dolla and Bruschi, 1988). Peptide mapping of this covalent complex has allowed to specify the ferredoxin interacting site on cytochrome c3. It has been shown that this site involves basic residues localized around heme 4 crevice (Dolla et al., 1991). Moreover this analysis confirms the hypothetical structure of the complex previously proposed by graphics computer modelling (Cambillau et al., 1988). The first step in the understanding of the electron exchange in a multiheme protein such as cytochrome c3, when its redox properties and three dimensional structure are known, is to correlate the two pieces of information. One approach is to generate site specific structural modifications using genetic or chemical engineering. Our methodology has been based on chemical modification of several amino acids close to the heme groups. Using this approach, we have chemically modified arginine residue 73 of D. desulfuricans Norway cytochrome c3. In this paper, we report the effects of this modification on the biophysical properties of cytochrome c3 and discuss a functional role of the four hemes.

EXPERIMENTAL Chemical modification. Cytochrome c3 and ferredoxin I were purified from Desulfovibrio desulfuricans Norway as previously described (Bruschi et al., 1977). The arginine chemical modification was performed using

the following procedure: cytochrome c3 (1.6 x lo-, M) was incubated for 4 h at 37 "C under N2 with cyclohexane 1-2 dione (2x 1 0 - 2 ~ )in 0 . 2 ~borate buffer (pH 8.5). The reaction mixture was then chromatographed on a Sephadex G-25 column (100 cm X 1cm) equilibrated in the same buffer, to separate modified cytochrome c3 from the reagents. The extent of the modification was checked up by acid hydrolysis at 110 "C for 20 h in 6 N HCl and 30 pL thioglycollate. Thioglycollate was added to prevent arginine regeneration during hydrolysis. The decrease of arginine amount, deduced from the amino acid composition, corresponds to the modification yield. From our experiments, 100% of arginyl residues were modified, with no modification of lysine residues. In vitro enzyme assays. Manometric assays using Warburg systems were carried out to determine both native and modified cytochromes c3 activities in two in vitro reactions: the phosphoroclastic reaction and the sulfite reduction, respectively: reaction 1 (phosphoroclastic reaction): the main compartment of each manometric vessel contained: phosphate buffer (pH 7) 150 pmol, CoA 4pmol, TPP 5pmol, MgC12 20pmo1, pmercaptoethanol 10 pmol, native or modified cytochrome 50nmol and crude extract devoided of cytochrome c3 (2.8 mg protein) in a final volume of 3.0 ml. The center well contained 0.1 mL NaOH 10 M. 30 pmol of natrium pyruvate was added from the side arm after incubation of the flask for 30 min at 37 "C under N2. Reaction 2 (sulfite reduction): phosphate buffer (pH 7) 150pmol native or 70nmol modified cytochrome c3, and crude extract (9mg protein) devoided of cytochrome c3, were added in a 3.0 mL final volume to the main compartment of the flask. Like the first reaction, the center well contained 0.1 mL NaOH 10 M.The flask was incubated for 30min at 37°C under H2 before adding natrium sulfite (4pmol) from the side arm. The crude extract devoided of cytochrome c3 was obtained by chromatography on a silica gel column equilibrated in 0.01 M Tris-HCl buffer (pH 7.6). The activities of both modified and native cytochromes were measured in reactions 1 and 2, respectively, in terms of production or use of H2. 'H N M R measurements. The cytochrome c3 samples were prepared in 0.1 M borate buffer by D 2 0exchanging, for 3 h at 45 "C and by successive lyophilizations. Ferredoxin I was concentrated in D 2 0 on a centricon microconcentrator Amicon (Beverley, MA, USA). The p2H values of the protein solutions were adjusted by addition of minute amounts of Na02H and/or 2HC1. The titration of the different oxidoreduction states was obtained by addition of small amounts of solid disodium dithionite. 'H NMR spectra were recorded in the Fourier mode on a Bruker AM200 spectrometer (Wissembourg, France). Chemical shifts (Si)are in parts per million (ppm) from internal CCI, tetramethyl silane (TMS). The titration of cytochrome c3/ferredoxin I complex formation was obtained as previously described (Guerlesquin et al., 1985b).

Crystallographic data. In the present work, the atomic

coordinates from D. desulfuricans Norway cytochrome c3 (Haser et af.,1979; Pierrot et al., 1982) and from D. vulgaris Miyazaki cytochrome c3 (Higuchi et of., 1984)

Plate 2. Stereo drawing of the (I carbon chain of D. desulfuricans Norway cytochrome cg.The hemes are labelled from 1H to4H. The side chain of Arginine 73 is indicated.

Plate 5. Stereo drawing of the heme cluster in D. desulfuricans Norway cytochrome cg. Intervening amino acids thought to play an electron transfer mediator role (histidine ligands, Phe 34, Phe88) are shown. When an electron enters the system via 4H it is suggested (see text) that it is further transferred t o 3H and afterwards to 1H. The iron-iron distances are given in Table 3.

Plate 3. Stereographic view of the important residues in the binding site model. The ligand haloperidol (purple) is centrally located and forms a salt-bridge between the protonated piperidinyl nitrogen of the ligand and ASP(H:lOOa) (red). In addition there is a hydrogen bond between the keto group and the phenolic hydroxy of TYR (L:96) and ring stacking between the fluorophenyl ring and TRP (H:50) (green).

Plate 4. En face view of the modelled mAb185 with haloperidol in the binding site (same orientation as Plate 3). The CDR loops are colored light blue (Ll), red (L2), yellow (L3), green (Hl), pink (H2) and dark blue (H3). Haloperidol (Hal) is colored white (carbons), green (hydrogens), red (oxygens) and blue (fluorine and chlorine). The framework areas of the Fv are colored grey.

29

INTERACTING SITE BETWEEN CYTOCHROME ~j AND FERREDOXIN

were used. The views were done with the TOM/FRODO program (Cambillau and Horjales, 1987) implemented on a Silicon Graphics (Jory en Yosas, France) 4D 70GT Station. The heme groups are numbered sequentially from the amino-terminus.

RESULTS

The presence of a single arginine residue in D. desulfuricans Norway cytochrome c3 is very convenient for chemical modification. Moreover arginine 73 is not buried in the molecule and then is accessible to chemical reagents (Plate 2). Under the previous specified conditions, cyclohexane 1-2 dione reacts with the arginine 73 residue of cytochrome c3, as it was checked by acid hydrolysis and amino acid analysis. The arginine was then converted to a DHCH-arginine (N7-Ns-(l,2dihydroxycyclohex- 1,2-xylene)-arginine) derivative, only stable in borate buffer. The conditions, described in the Experimental, generate a homogenous population of cytochrome c3 molecules in which arginine 73 is fully converted to a DHCH-arginine derivative. To understand the effects of chemical modification, in terms of redox potentials and interactions with the oxidoreduction partners, it was essential to evaluate the conformational changes of the molecule after such a modification. Biochemical studies The shape of the optical spectrum of the modified cytochrome, in both oxidized and reduced states, was identical to the native one. This suggests that the chemical modification did not strongly disturb the heme chromophores. Moreover, no cytochrome polymerization was observed by SDS-PAGE, after treatment with cyclohexane 1-2 dione. To test the effect of the chemical modification on the cytochrome cj activities, two in uitro enzyme assays were performed as previously described (Bruschi et al., 1977). These enzyme assays restore the two oxidoreduction reactions in which cytochrome c3 has been reported to act (Akagi, 1967; Suh and Akagi, 1969) Fig. 1). The activities, in terms of evolved or used hydrogen, are reported in Table 1. These results show that the electron transfer between cytochrome c3and its redox partners (i.e., ferredoxin and hydrogenase) were not abolished by the chemical modification, although a slight decrease could be noticed. The decrease of the activities might be explained by an alteration of the proteidprotein affinity.

Table 1. Activity measurements of both native and arginine modified cytochromes c3 in the phosphoroclastic reaction and the sulfite reduction

Native cytochrome c, Arginine modified cytochrome c3

Phosphoroclastic reaction Hz evolved (prnollrnin)

Sulfite reduction Hz used (prnollrnin)

0.255 0.181 (71%)

0.212 0.143 (67%)

N M R spectroscopy To further analyze subtle effects of chemical modification on the cytochrome c3 three-dimensional structure, NMR spectroscopy studies were performed. The 'H NMR spectrum of modified ferricytochrome c3 (Fig. 3) shows that arginine modification does not significantly affect the linewidth of ring methyl lines (30-10ppm). Thus, the reaction conditions and the chemical modification are adequate enough to further characterize the structural properties of the modified cytochrome. 'H NMR spectra obtained at different oxidoreduction states of D. desulfuricans Norway cytochrome c3 have enabled the assignment of the heme methyl resonances in four groups corresponding to the four hemes, respectively (Guerlesquin et al., 1985a). The same experiments, carried out on native and modified cytochrome in borate buffer, have shown that the chemical modification gave an important shift of two ring methyl lines of heme I (-165mV) and the four ring methyl lines of heme I1 ( - 305 mV), but no significant change for the heme methyl lines of heme I11 (-365mV) and heme IV (-400mV) resolved in between 30 to 10ppm. These effects on the NMR spectra of modified cytochrome c3 were compared to changes observed on titrations of the ferricytochrome c3/ferredoxin complex formation (Guerlesquin et al., 1985b). In both experiments the two highest redox potential hemes ( - 165 and - 305 mV) were affected. We concluded that arginine 73 might be localized on the cytochrome c3/ferredoxin interacting domain. This result agrees with the model building of the complex (Cambillau et al., 1988) in which arginine 73 is involved in the interacting site. To determine the role of arginine 73 in the protein association process, two comparative titrations of complex formatin of native and modified cytochrome c3 with ferredoxin were performed as pre-

M

30.0

20.0 PPR

10.0

Figure 3. 'H NMR spectra of Desulfovibrio desulfuricans Norway ferricytochrome c,. (A) native cytochrome c3 in D20 (p2H7.6). The protein concentration was 0.7 mM. (B) native cytochrome c3 in borate buffer 0.1 M (p2H7.6). The protein concentration was 0.67 mM. (C) DHCH-arginine cytochrome c, derivative in borate buffer (pZH7.6). The protein concentration was 0.92 mM. Each roman number (I-IV) corresponds to one heme methyl resonance according to the decreasing potentials; for instance, resonances I correspond to methyl resonances of the highest redox potential heme ( - 165 mV).

30

A. DOLLA E T A L .

Table2. Arginine 73 chemical modification on redox potentials and on 'H NMR spectrum of D. desulfuricans Norway cytochrome c3 hemes Chemical modificaton effect

AEo (mV)

Adi (ppm)

HEME I ( - 165 mV)

- 50

- 0.43 - 0.06

HEME II ( - 305 rnV)

- 20

HEME 111 ( - 365 mV)

- 10

+ 0.30 -0.14

- 0.28

- 0.52 - 0.65 - 0.49 +0.10 0.00 0.00 0.00

Ferredoxin binding effect Native Modified cytochrome cytochrome Adi lppm) Adi (ppm)

- 0.04 -0.14 - 0.29 - 0.29 -0.12 0.04 - 0.09 - 0.26 - 0.07

+

+ 0.08 + 0.06 + 0.06

0.00 -0.12 -0.13 - 0.20 -0.12 +0.13 - 0.03 + 0.27 0.00 + 0.04 - 0.03 - 0.03

- 20 HEME IV ( - 400 mV) A€,= Redox potential changes (from Dolla eta/., 1987). (Asi)= Chemical shift changes in the NMR spectra (6, bound or modified ferricytochrome cg ring methyl-6, free or native ferricytochrome c, ring methyl).

viously described (Guerlesquin et al., 1985b). In both cases the ferredoxin recognition which occured involved the same heme methyl resonances (Table 2), even if the association constant was altered as suggested by the decrease in physiological activity (Table 1). The microcalorimetric measurements of cytochrome c,/ferredoxin complex formation (Guerlesquin et al., 1987), crosslinking experiments (Dolla and Bruschi 1988) and model building (Cambillau et al., 1988) have shown there is only one heme interacting site namely heme 4. Computer graphics modelling of the modified arginine 73 in the cytochrome structure and analysis of this structure have shown that the arginine residue, in both native and modified forms, was closer to heme 4 than to the other three hemes (Dolla et al. , 1987). From the NMR data, two hemes were affected by both the complex formation and the chemical modification of cytochrome c3 arginine 73. It is not known which heme is involved in the interacting site and which one is a long range perturbated heme. However, on the basis of electrochemical studies of arginine modified cytochrome c3, the highest redox potential ( - 165 mV) could be associated to heme 4 (Dolla et al., 1987). This suggestion can be correlated with the NMR data previously described but does not agree with the redox potential assignment recently proposed by Guigliarelli et al. (1990), on the basis of EPR results in which the redox potential (-300mV) is assigned to heme 4. Presently, a study involving site directed mutagenesis will definitively establish the heme redox potential. In any case, the attribution of the lowest redox potential to heme 4 on the basis of the Stellwagen hypothesis correlating the heme solvent exposure and the redox potential (Stellwagen, 1987) must be discarded.

DISCUSSION From chemical modification of cytochrome c3 arginine 73, the interacting site for ferredoxin is proposed to be heme 4. The proposal of a high redox potential for this

heme leads us to consider the electron transfer process in terms of thermodynamic factors. Rapid kinetic experiments have shown that electron exchange in the cytochrome c3/ferredoxin I complex occurs by a bimolecular complex formation, in a bidirectional electron flow (Capeillere-Blandin et al. , 1986). If the reduction of the interacting heme ( - 165 mV or - 305 mV) by ferredoxin I ( - 374 mV) is compatible with our model, the reverse reaction is difficult to understand in terms of energy levels. In this latter process, we assume that the three other hemes have a crucial role in the modulation of a macroscopic potential of the cytochrome c3. In light of this, cytochrome c3 may be considered as a mono-heme cytochrome, in which heme 4 is the interacting site of the molecule and the three other hemes are involved in modulating the redox potential, thus allowing a bidirectional electron transfer. This hypothesis may be linked to the electron storage properties of cytochrome c3 as suggested by Dobson et al. (1974).

Are there one or several interacting heme groups in cytochrome c3? The presence of four hemes in cytochrome c3 has been associated with the large diversity of its redox partners: iron-sulfur proteins such as hydrogenases or ferredoxins and flavoproteins like flavodoxins. Each cytochrome c3 heme is a specific reactive group for one of the physiological partners. Our results show that the D. desulfuricam Norway cytochrome c3 heme 4, the heme with the highest redox potential interacts with the ferredoxin (4Fe-4s) cluster in the cytochrome c3/ferredoxin complex. Moreover, two other models of electron transfer complexes, have been recently proposed (Stewart et al., 1988; 1989), which are based on the three-dimensional structure of D. vulgaris Miyazaki cytochrome c3 and those of both flavodoxin and rubredoxin isolated from D. vulgaris Hildenborough. Although these complexes were obtained by computer graphics modelling and still require the support of biochemical evidence, heme 4 does appear to interact with the three kinds of redox group: the ferredoxin

31

INTERACTING SITE BETWEEN CYTOCHROME c3 AND FERREDOXIN

(4Fe-4s) cluster, the flavodoxin flavin and the rubredoxin iron center. This analysis suggests that heme 4 is the interacting heme of cytochrome c3. Moreover, in both known cytochrome c3 structures, two distinct heme groups, namely hemes 1 and 4, are thought to be reponsible for the fast intermolecular electron transfer (Haser, 1981; Haser and MossC, 1987), which in turn may explain the high conductivity of these proteins (Kimura et al., 1979). In fact the molecular packing in the crystal brings heme 1 of one molecule close and nearly parallel to the ring of heme 4 of a symmetry-related molecule (Pierrot et al., 1982). This structural feature confers an important role to heme 4 in the cytochrome c3intermolecular electron exchange. The role of the three other hemes would be associated either to a specificity for various redox partners, to a redox potential modulation or to an electron storage property. Whatever the functional meaning of the various hemes, an intramolecular electron transfer within the tetra-heme cytochrome is expected.

Table3. Conservation of the heme core in multihemic cytochromes: distances (A) and angles between iron atoms in cytochromes c3 and cytochrome c, D. desulfuricas Norway tetra-heme cytochrome c3

Fe,-Fe3 Fel-Fe, Fe,-Fe4 Fe4-Fe, Fe,-Fe,-Fe, Fe,-Fe,-Fe, Fe3-Fel-Fe, Fe,-Fe,-Fe,

Previously spectroscopic results had suggested that the hemes of D . desulfuricans Norway cytochrome c3 interact within the redox cluster (Gayda et al., 1988). A Raman resonance study (Verma et al., 1988) has also proved that interheme interactions occur in D . vulgaris Miyazaki cytochrome c3 and that in this protein intramolecular electron exchange within the four hemes is much slower than 1 x 10'2s-'. Direct experimental support for intramolecular electron transfer within D. desulfuricans Norway cytochrome c3 has recently been observed. Cross-linked ferredoxin in the covalent complex can be reduced by hydrogenase under hydrogen atmosphere through an intramolecular electron transfer within cytochrome c3 (Dolla et a[., 1989). One of the next important questions is therefore, how is the electron transfer process in cytochrome c3 controlled by the protein molecule? As hemes 4 and 1 form the interacting redox pair within the intermolecular organization, it is tempting to assume that heme 1 is the electron exit site when an electron enters the system via heme 4. The next step therefore is to propose the best electron transfer pathway from heme 4 to heme 1, 1 A V V : AP : : :

10

20

44.9" 73.2"

Desulforomonas acetoxidans tri-heme cytochrome Q

11.0 17.8 12.0 16.4 101.4" 37.3" 41.1" 75.7"

12.5 19.3 11.6

-

106.4" 38.2" 35.1

-

within the same protein molecule. The corresponding data for D . vulgaris Miyazaki cytochrome c3 are given in brackets. Plate 5 illustrates this proposal: an electron may enter the system through direct transfer between the exposed edge of heme 4 and the (4Fe-4s) cluster of ferredoxin; it is transferred to heme 3, close to heme 4; it is propagated further to heme 1 which is very close to heme 1, the iron to iron distance being 10.9 (11.0) A (Table 3). The process is probably facilitated by the extensive pi-orbital overlap between the porphyrin rings involving an intervening phenylalanine residue Phe 34 (Phe 20); the electron could be transferred to heme 1 of another cytochrome c3 molecule. The above proposal is supported by the following observations: (i) heme 4 is significantly closer to heme 3 than to the next heme 2, the separation between the iron centrers 4 and 2 being 16.3 (16.4) A compared to 12.4 (12.0) A between the iron centers of heme 4 and heme 3 (Table 3). Moreover the porphyrin edge to edge distance is less than 5 A for the heme pair 4-3, compared to 11A between hemes 4 and 3 and should favor electron exchange. (ii) On the basis of sequence alignments (Fig. 4), the helical structure (84-101, in D. desulfuricans Norway cytochrome c3) running between hemes 4 and 3 is a conserved feature in all known cytochromes c3. Interestingly in D . vulgaris Miyazaki cytochrome c3,the corresponding helix although interrupted by a loop (Asp 71 to Lys 77), leads to an almost identical arrangement for hemes 4 and 3. Consistent with the latter suggestion, several examples of enzyme structures have been discussed with focus on some of their secondary structures ( a-helices, P-pleated

A plausible intramolecular electron transfer pathway in cytochrome cj

D.d.Norway D.d.ElAgh. D-salexigens D.v.Hilden.

10.9 17.5 12.4 16.3 96.1 39.0"

D. vulgaris Miyazaki tetra-heme cytochrome CJ

30

40

50

60

70

VISAPEGMKAK-PKGDKPGA IKApAGAKV----------mPAGAm----------Grn--------GU--------GAKIDFIAGGEKNL---

D.acetoxidans:

-DKS-AK-GYY -DKS-AK-GYY -DKS-VN-SWY Figure 4. Alignment of the amino acid sequences of cytochrome c3 from D. desulfuricans N o w a y (Bruschi, 1981), D. desulfuricansEl Agheila 2 (Ambler eta/., 19711, D. salexigens (Ambler, 1973). D. vulgaris Hildenborough (Ambler, 1968). D. vulgaris Miyazaki (Shinkai et a/., 1980). D. gigas (Ambler eta/., 19691, Desulforomonas acefoxidans (Ambler, 1971). Common residues to all proteins and heme attachment sites are enclosed in boxes.

32

A. DOLLA E T A L .

sheets) which are shown to have the possible potential of delocalizing charge, transporting protons and of being a ‘communication channel’ connecting distant sites (Korn, 1986). (iii) The strong coupling between hemes 3 and 1 is obviously enhanced by the intervening phenylalanine side-chain which contributes to direct the electron flow. Recent results clearly show that aromatic side chains could greatly facilitate the rate of electron transfer in some redox protein systems (Louie et al., 1988). In the first intramolecular electron transfer event (transfer from heme 4 to heme 3) the peculiar role of the a-helix can be underlined: the helix provides the axial histidine ligands His 89 (70) amd His 96 (83), with the N,’s of the imidazole rings bonded to the corresponding heme iron atoms. It is interesting to note that the two imidazole rings appear on the same side of the helix and that there is no intervening side-chain so that they ‘see’ each other (Plate 5). Such an arrangement may favour also the electronic coupling between the corresponding heme groups. The inter-heme helix appears therefore to have several functions: (a) it provides the rigid bridging framework for an optimal coupling between the donor and acceptor heme groups; (b) with its dipole moment, this helix may serve to modulate the redox potentials of its bound heme groups; (c) its C-terminus with its positively charged turn, Lys99-Lys100-Lys101, is involved in the presumed recognition site with a redox partner, as proposed in the computer modelling analysis of the interaction between D. desulfuricans Norway cytochrome c3 and ferredoxin (Cambillau et al., 1988). The above discussion leads us now to focus on the specific role of heme 2. Role of heme 2 The three-dimensional structure of D. desulfuricans Norway cytochrome c3shows that heme 2 is critical for the stability of the intermolecuar organization, as two propionate groups form salt bridges with two lysines of a nearby protein molecule. Apart from its structural function in the crystal, heme 2, with its two propionates pointing to the external medium, could act as an electrostatic barrier to the approach of negatively charged species or as an interacting site for positively charged partners. On the other hand, if this redox

center is involved in intramolecular electron exchange, the following structural features are therefore important: (i) heme 2 is close to heme 1, as the iron to iron distance is 12.8 (12.2) A. This proximity is a consequence of the fact that two histidine iron ligands are consecutive in the sequence and form the bonding chain Fel-His48-His49-Fe2, which is a strictly conserved segment in all known cytochromes c3; (ii) heme 2 may interact with heme 4, via the intervening aromatic side chain Phe88 (Phe76), despite a rather long distance between the two redox centers (Fe2-Fe4= 16.3 A (16.4)). Again this aromatic residue, being in the ‘line of flight’ and at 5-6 A distance from some of the atoms of the porphyrin rings may be responsible for some pi-orbital overlap between the redox sites and therefore it could act as an electron transfer mediator, like Phe 34 (Phe 20) for the heme pair 3-1. However, the coupling between hemes 2-4 is obviously weaker than that between hemes 4-3 and 3-1; (iii) heme 2 is the redox group which is lacking in the three-heme cytochrome c,, on the basis not only of sequence comparisons (Fig. 4) but of X-ray diffraction data which have led to the unambiguous location of the three iron centers (R. Haser et al., unpublished results); the observed three-iron-atom arrangement is clearly homologous to that found for the heme 1 , 3 and 4 in cytochrome c3 (Table 3). As a fast internal electron transfer can occur in cytochrome c7 (Moura et al., 1984), it is tempting to assign a privileged role to this three-heme core in controlling or directing the electronic interactions between the redox sites. Finally, since the electron transfer in the cytochrome c,/ferredoxin complex is bidirectional (CapeillereBlandin et al., 1986), which is in agreement with the two metabolic pathways, the above intramolecular electron flow is expected to be reversible to achieve the electron transfer from heme 4 to the ferredoxin (4Fe4s) cluster. However, considering another heme as a potential interacting site for another redox partner, it cannot be concluded that the proposed intramolecular electron pathway is conserved. Recently, cloning of the cytochrome c3 gene has led to a functional expression of the polyhemic cytochrome (Voordouw et al., 1990). Further understanding of cytochrome c3 structure/function relationships will be obtained by combining techniques of oligonucleotidedirected mutagenesis with protein biochemical and biophysical studies.

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