Redox-Bohr and other cooperativity effects in the nine-heme cytochrome C from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies

The nine-heme cytochrome c is a monomeric multiheme cytochrome found in Desulfovibrio desulfuricans ATCC 27774. The polypeptide chain comprises 296 residues and wraps around nine hemes of type c. It is believed to take part in the periplasmic assembly of proteins involved in the mechanism of hydrogen cycling, receiving electrons from the tetraheme cytochrome c3. With the purpose of understanding the molecular basis of electron transfer processes in this cytochrome, we have determined the crystal structures of its oxidized and reduced forms at pH 7.5 and performed theoretical calculations of the binding equilibrium of protons and electrons in these structures. This integrated study allowed us to observe that the reduction process induced relevant conformational changes in several residues, as well as protonation changes in some protonatable residues. In particular, the surroundings of hemes I and IV constitute two areas of special interest. In addition, we were able to ascertain the groups involved in the redox-Bohr effect present in this cytochrome and the conformational changes that may underlie the redox-cooperativity effects on different hemes. Furthermore, the thermodynamic simulations provide evidence that the N- and C-terminal domains function in an independent manner, with the hemes belonging to the N-terminal domain showing, in general, a lower redox potential than those found in the C-terminal domain. In this way, electrons captured by the N-terminal domain could easily flow to the C-terminal domain, allowing the former to capture more electrons. A notable exception is heme IX, which has low redox potential and could serve as the exit path for electrons toward other proteins in the electron transfer pathway.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.     

The Tmc complex from Desulfovibrio vulgaris hildenborough is involved in transmembrane electron transfer from periplasmic hydrogen oxidation

Three membrane-bound redox complexes have been reported in Desulfovibrio spp., whose genes are not found in the genomes of other sulfate reducers such as Desulfotalea psycrophila and Archaeoglobus fulgidus. These complexes contain a periplasmic cytochrome c subunit of the cytochrome c(3) family, and their presence in these organisms probably correlates with the presence of a pool of periplasmic cytochromes c(3), also absent in the two other sulfate reducers. In this work we report the isolation and characterization of the first of such complexes, Tmc from D. vulgaris Hildenborough, which is associated with the tetraheme type II cytochrome c(3). The isolated Tmc complex contains four subunits, including the TpIIc(3) (TmcA), an integral membrane cytochrome b (TmcC), and two cytoplasmically predicted proteins, an iron-sulfur protein (TmcB) and a tryptophan-rich protein (TmcD). Spectroscopic studies indicate the presence of eight hemes c and two hemes b in the complex pointing to an alpha(2)betagammadelta composition (TmcA(2)BCD). EPR analysis reveals the presence of a [4Fe4S](3+) center and up to three other iron-sulfur centers in the cytoplasmic subunit. Nearly full reduction of the redox centers in the Tmc complex could be obtained upon incubation with hydrogenase/TpIc(3), supporting the role of this complex in transmembrane transfer of electrons resulting from periplasmic oxidation of hydrogen.     

Crystal structures at atomic resolution reveal the novel concept of "electron-harvesting" as a role for the small tetraheme cytochrome c

The genus Shewanella produces a unique small tetraheme cytochrome c that is implicated in the iron oxide respiration pathway. It is similar in heme content and redox potential to the well known cytochromes c(3) but related in structure to the cytochrome c domain of soluble fumarate reductases from Shewanella sp. We report the crystal structure of the small tetraheme cytochrome c from Shewanella oneidensis MR-1 in two crystal forms and two redox states. The overall fold and heme core are surprisingly different from the soluble fumarate reductase structures. The high resolution obtained for an oxidized orthorhombic crystal (0.97 A) revealed several flexible regions. Comparison of the six monomers in the oxidized monoclinic space group (1.55 A) indicates flexibility in the C-terminal region containing heme IV. The reduced orthorhombic crystal structure (1.02 A) revealed subtle differences in the position of several residues, resulting in decreased solvent accessibility of hemes and the withdrawal of a positive charge from the molecular surface. The packing between monomers indicates that intermolecular electron transfer between any heme pair is possible. This suggests there is no unique site of electron transfer on the surface of the protein and that electron transfer partners may interact with any of the hemes, a process termed "electron-harvesting." This optimizes the efficiency of intermolecular electron transfer by maximizing chances of productive collision with redox partners.     

Modeling electron transfer thermodynamics in protein complexes: interaction between two cytochromes c(3)

Redox protein complexes between type I and type II tetraheme cytochromes c(3) from Desulfovibrio vulgaris Hildenborough are here analyzed using theoretical methodologies. Various complexes were generated using rigid-body docking techniques, and the two lowest energy complexes (1 and 2) were relaxed using molecular dynamics simulations with explicit solvent and subjected to further characterization. Complex 1 corresponds to an interaction between hemes I from both cytochromes c(3). Complex 2 corresponds to an interaction between the heme IV from type I and the heme I from type II cytochrome c(3). Binding free energy calculations using molecular mechanics, Poisson-Boltzmann, and surface accessibility methods show that complex 2 is more stable than complex 1. Thermodynamic calculations on complex 2 show that complex formation induces changes in the reduction potential of both cytochromes c(3), but the changes are larger in the type I cytochrome c(3) (the largest one occurring on heme IV, of approximately 80 mV). These changes are sufficient to invert the global titration curves of both cytochromes, generating directionally in electron transfer from type I to type II cytochrome c(3), a phenomenon of obvious thermodynamic origin and consequences, but also with kinetic implications. The existence of processes like this occurring at complex formation may constitute a natural design of efficient redox chains.     

Spectroscopic investigation and determination of reactivity and structure of the tetraheme cytochrome c3 from Desulfovibrio desulfuricans Essex 6.

Cytochrome c3, a small (14-kDa) soluble tetraheme protein was isolated from the periplasmic fraction of Desulfovibrio desulfuricans strain Essex 6. Its major physiological function appears to be that of an electron carrier for the periplasmic hydrogenase. It has been also shown to interact with the high-molecular-mass cytochrome complex in the cytoplasmic membrane, which eventually feeds electrons into the membraneous quinone pool, as well as with the membrane-associated dissimilatory sulfite reductase. The EPR spectra show features of four different low-spin Fe(III) hemes. Orthorhombic crystals of cytochrome c3 were obtained and X-ray diffraction data were collected to below 2 A resolution. The structure was solved by molecular replacement using cytochrome c3 from D. desulfuricans ATCC 27774 as a search model.     

A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.     

Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site.

Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9%. The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

Crystal structure of the oxidised and reduced acidic cytochrome c3from Desulfovibrio africanus.

Unique among sulphate-reducing bacteria, Desulfovibrio africanus has two periplasmic tetraheme cytochromes c3, one with an acidic isoelectric point which exhibits an unusually low reactivity towards hydrogenase, and another with a basic isoelectric point which shows the usual cytochrome c3reactivity. The crystal structure of the oxidised acidic cytochrome c3of Desulfovibrio africanus (Dva.a) was solved by the multiple anomalous diffraction (MAD) method and refined to 1.6 A resolution. Its structure clearly belongs to the same family as the other known cytochromes c3, but with weak parentage with those of the Desulfovibrio genus and slightly closer to the cytochromes c3of Desulfomicrobium norvegicum. In Dva.a, one edge of heme I is completely exposed to the solvent and surrounded by a negatively charged protein surface. Heme I thus seems to play an important role in electron exchange, in addition to heme III or heme IV which are the electron exchange ports in the other cytochromes c3. The function of Dva.a and the nature of its redox partners in the cell are thus very likely different.By alignment of the seven known 3D structures including Dva.a, it is shown that the structure which is most conserved in all cytochromes c3is the four-heme cluster itself. There is no conserved continuous protein structure which could explain the remarkable invariance of the four-heme cluster. On the contrary, the proximity of the heme edges is such that they interact directly by hydrophobic and van der Waals contacts. This direct interaction, which always involves a pyrrole CA-CB side-chain and its bound protein cysteine Sgammaatom, is probably the main origin of the four-heme cluster stability. The same kind of interaction is found in the chaining of the hemes in other multihemic redox proteins.The crystal structure of reduced Dva. a was solved at 1.9 A resolution. The comparison of the oxidised and reduced structures reveals changes in the positions of water molecules and polar residues which probably result from changes in the protonation state of amino acids and heme propionates. Water molecules are found closer to the hemes and to the iron atoms in the reduced than in the oxidised state. A global movement of a chain fragment in the vicinity of hemes III and IV is observed which result very likely from the electrostatic reorganization of the polypeptide chain induced by reduction.     

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c3 from Desulfovibrio vulgaris Hildenborough: kinetic, microcalorimetric, EPR and electrochemical studies

The complex formation between the tetraheme cytochrome c3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c3 and Hmc demonstrates a redox-shuttling role of cytochrome c3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with cytochrome c3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.     

Nonaheme cytochrome c, a new physiological electron acceptor for [Ni,Fe] hydrogenase in the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex: primary sequence, molecular parameters, and redox properties

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.     

Biochemical and spectroscopic characterization of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough expressed in Desulfovibrio desulfuricans G200.

The gene of high molecular weight, multiheme cytochrome c (Hmc) from the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has been overexpressed in Desulfovibrio desulfuricans G200. The recombinant protein has been purified. Its molecular weight (65,600), amino acid composition, and NH2-terminal sequence were found to be identical to those of the wild-type protein. The recombinant protein has been spectroscopically characterized (optical spectrum, EPR, circular dichroism) and compared to the wild-type protein. We have found 16 hemes per molecule by iron analysis and the pyridine hemochrome test. Both high- and low-spin features were observed in the EPR spectrum. A detailed spin quantitation analysis indicates 1 or 2 high-spin hemes and 14 or 15 low-spin hemes per molecule. The redox potentials of the hemes determined by voltammetric techniques gave an average of three different values, 0, -100, and -250 mV (versus NHE), for the wild-type and the recombinant cytochrome. The low potential values are similar to the values observed for the bis(histidinyl) coordinated hemes of cytochrome c3. A comparison of the arrangement of heme binding sites and coordinated histidines in the amino acid sequences of cytochrome c3 and Hmc has shown that the latter contains four domains, three of which are complete c3-like domains, while the fourth represents an incomplete c3-like domain which may contain His-Met coordinated hemes. These data are in agreement with the detailed study of the number and types of hemes reported in this paper.     

Structure-function relationship in type II cytochrome c 3 from Desulfovibrio africanus: a novel function in a familiar heme core

NMR and visible spectroscopy were used to characterize the type II tetraheme cytochrome c(3) isolated from the periplasmic space of Desulfovibrio africanus, a sulfate-reducing bacterium. Although structurally similar to other cytochromes c(3), this protein displays distinct functional properties. Proton NMR signals from the four hemes were assigned to the structure in the ferri- and ferrocytochromes using two-dimensional NMR experiments. The thermodynamic parameters of the hemes and of an acid-base center in the type II cytochrome c(3) were determined using NMR and visible spectroscopies. The thermodynamic features indicate that electrostatic effects dominate all of the interactions between the centers and no positive cooperativity between hemes is observed. The redox-Bohr effect in this protein is associated with the acid-base equilibrium of a propionate of heme II instead of propionate 13 of heme I as is the case for all of the type I cytochromes c(3). These novel functional properties are analyzed together with the redox-linked structural differences reported in the literature and reveal a mechanistic basis for type II cytochromes c(3) having a physiological function that is different from that of type I cytochromes c(3).     

Redox interaction of cytochrome c3 with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

Cytochrome c3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome c3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly 15N-labeled ferric cytochrome c3 except N-terminus, Pro, and Gly73 were assigned. 1H-15N HSQC spectra were recorded for 15N-labeled ferric and ferrous cytochrome c3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous cytochrome c3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome c3. To investigate the transient complex of cytochrome c3 and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome c3 in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the Km of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome c3 reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling.     

Ferredoxin electron transfer site on cytochrome c3. Structural hypothesis of an intramolecular electron transfer pathway within a tetra-heme cytochrome.

To specify electron exchanges involving Desulfovibrio desulfuricans 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 1H 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 (alpha helix, aromatic residues) intervening between the heme groups, is proposed.     

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.     

Molecular dynamics study of Desulfovibrio africanus cytochrome c3 in oxidized and reduced forms

A 5-ns molecular dynamics study of a tetraheme cytochrome in fully oxidized and reduced forms was performed using the CHARMM molecular modeling program, with explicit water molecules, Langevin dynamics thermalization, Particle Mesh Ewald long-range electrostatics, and quantum mechanical determination of heme partial charges. The simulations used, as starting points, crystallographic structures of the oxidized and reduced forms of the acidic cytochrome c(3) from Desulfovibrio africanus obtained at pH 5.6. In this paper we also report structures for the two forms obtained at pH 8. In contrast to previous cytochrome c(3) dynamics simulations, our model is stable. The simulation structures agree reasonably well with the crystallographic ones, but our models show higher flexibility and the water molecules are more labile. We have compared in detail the differences between the simulated and experimental structures of the two redox states and observe that the hydration structure is highly dependent on the redox state. We have also analyzed the interaction energy terms between the hemes, the protein residues, and water. The direct electrostatic interaction between hemes is weak and nearly insensitive to the redox state, but the remaining terms are large and contribute in a complex way to the overall potential energy differences that we see between the redox states.