Complex formation between flavodoxin and cytochrome c. Cross-linking studies

Complex formation between Azotobacter vinelandii flavodoxin and horse cytochrome c has been demonstrated through cross-linking studies with dimethyl suberimidate, dimethyl adipimidate, 1-ethyl-3-(3-di-methylaminopropyl)carbodiimide, and dimethyl-3,3'-dithiobispropionimidate. Essentially quantitative cross-linking of cytochrome c and flavodoxin was observed at low ionic strengths with the carbodiimide cross-linking reagent. An association constant of 4 X 10(4) M-1 was obtained between cytochrome c and flavodoxin at 88 mM ionic strength from analysis of the cross-linking studies. This value is similar to the association constant determined kinetically during the electron transfer reaction between cytochrome c and flavodoxin (Simondsen, R.P., Weber, P.C., Salemme, F.R., and Tollin, G. (1982) Biochemistry 21, 6366-6375), and suggests that the cross-linked complex may be similar to the precursor complex identified kinetically. A structural model for the flavodoxin-cytochrome c complex proposed by these workers is shown to be compatible with the present cross-linking results.     

Preliminary X-ray studies of the tetra-heme cytochrome c3 and the octa-heme cytochrome c3 from Desulfovibrio gigas

A tetra-heme and an octa-heme cytochrome c3 from the sulfate bacterium Desulfovibrio gigas have been crystallized. Diffraction quality crystals of the tetra-heme cytochrome are obtained from solution by the addition of polyethylene glycol at pH 6.5. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell parameters a = 42.27 A, b = 52.54 A and c = 52.83 A. The octa-heme cytochrome crystals develop from low ionic strength solutions of phosphate or Tris-Cl in the pH range 6.2-7.6. The crystals belong to the trigonal system, space group P3(1) or the enantiomorph P3(2), with unit cell parameters a = b = 57.4 A, c = 97.3 A, gamma = 120 degrees. Single crystal diffraction studies of the structures of these two low-potential cytochromes are in progress.     

Active site structure and dynamics of cytochrome c3 from Desulfovibrio gigas immobilized on electrodes

Cytochrome c3 from Desulfovibrio gigas is electrostatically adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid. The redox equilibria and electron transfer dynamics of the adsorbed four-heme protein are studied by surface enhanced resonance Raman spectroscopy. Immobilization on the coated electrodes does not cause any structural changes in the redox sites. The potential-dependent stationary experiments distinguish the redox potential of heme IV (-0.19 V versus normal hydrogen electrode) from those of the other hemes for which an average value of -0.3 V is determined. Taking into account the interfacial potential drops, these values are in good agreement with the redox potentials of the protein in solution. The heterogenous electron transfer between the electrode and heme IV of the adsorbed cytochrome c3 is analyzed on the basis of time-resolved experiments, leading to a formal electron transfer rate constant of 15 s(-1), which is a factor of 3 smaller than that of the monoheme protein cytochrome c.     

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.     

Kinetic study of the reduction mechanism for Desulfovibrio gigas cytochrome c3.

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J.J. & Xavier, A.V. (1991) Eur. J. Biochem. 202, 1101-1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network.     

A thermodynamic model for the cooperative functional properties of the tetraheme cytochrome c3 from Desulfovibrio gigas.

A thermodynamic model is presented to describe the redox behaviour of the tetraheme cytochrome c3 from Desulfovibrio gigas. This molecule displays different intrinsic redox potentials for the four hemes and during the redox titration process, interactions among different hemes occur, thus altering the values of redox potentials according to which of the hemes are oxidized [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A.V. (1984) Eur. J. Biochem. 141, 283-296]. This complex cooperative behaviour [Xavier, A.V. (1986) J. Inorg. Biochem. 28, 239-243] has been analyzed here using an I2H4-interaction network [Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250] coupled to a proton-linked equilibrium between two tertiary structures. Such a formalism, which requires a reduced number of parameters, is able to fully account quantitatively for the pH dependence of the NMR redox-titration curves. The 'redox-Bohr' effect is discussed in terms of the available structure and thermodynamic data and a functional mechanism is proposed.     

Kinetics and interaction studies between cytochrome c3 and Fe-only hydrogenase from Desulfovibrio vulgaris hildenborough

Hydrogenases from Desulfovibrio are found to catalyze hydrogen uptake with low potential multiheme cytochromes, such as cytochrome c₃, acting as acceptors. The production of Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough was improved with respect to the growth phase and media to determine the best large-scale bacteria growth conditions. The interaction and electron transfer from Fe-only hydrogenase to multiheme cytochrome has been studied in detail by both BIAcore and steady-state measurements. The electron transfer between [Fe] hydrogenase and cytochrome c₃ appears to be a cooperative phenomenon (h = 1.37). This behavior could be related to the conductivity properties of multihemic cytochromes. An apparent dissociation constant was determined (2 × 10^-7 M). The importance of the cooperativity for contrasting models proposed to describe the functional role of the hydrogenase/cytochrome c₃ complex is discussed. Presently, the only determined structure is from [NiFe] hydrogenase and there are no obvious similarities between [NiFe] and [Fe] hydrogenase. Furthermore, no crystallographic data are available concerning [Fe] hydrogenase. The first results on crystallization and X-ray crystallography are reported. Proteins 33:590–600, 1998. © 1998 Wiley-Liss, Inc.     

Electrochemical study of the electron exchange between cytochrome c3 and hydrogenase from Desulfovibrio desulfuricans Norway

The kinetics of the reduction of the Desulfovibrio desulfuricans Norway cytochrome c3 by its physiological partner hydrogenase, in the presence of hydrogen, was investigated by an electrochemical method; from cyclic voltammetry experiments a value of 3 X 10(7) M-1 s-1 was obtained for the second-order rate constant. Results are discussed in terms of specific interactions between physiological partner proteins.     

NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3

The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the NMR reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem-haem redox interactions. The intramolecular electron exchange is fast on the NMR time scale (larger than 10(5) s-1). The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non-interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH* = 7.2 and 9.6, shows that the haem-haem interacting potentials cover a range from -50 mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH-dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed.     

Preliminary 1H-NMR studies of the interaction between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway

The complex formation of two electron transfer proteins, cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway, has been shown by 1H-NMR spectroscopy. Presence of ferredoxin I produces ferricytochrome c3 1H-NMR spectrum modifications. The chemical shift of perturbated heme methyl resonances has been used to determine the stoichiometry of the complex. At pH 7.6 and 20 degrees C, the two proteins were found to form a complex 1:1 with an association constant, KA, of 10(4) M-1. Two of the four hemes are affected by presence of ferredoxin I and may be involved in the electron transfer sites. The heme methyl resonances are average resonances of free and bound cytochrome c3 resonances, indicating a fast exchange process on the NMR time scale.     

Redox linked conformational changes in cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774

Cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 appears to be capable of receiving two protons and two electrons from hydrogenase for transport to the membrane, and converting electronic energy into proton motive force. Detailed studies of the mechanism require control both of the redox state and of the protonation state of the protein; hence, structure determination of the protein in solution by NMR is the preferred method. This work compares the structures of the protonated protein in the fully oxidized and fully reduced states as a first step toward elucidating the pH-dependent and redox-state-dependent conformational changes that drive the energy transduction. These high-resolution structures revealed significant localized differences upon change of redox state, even though the global folds of the two families of structures are similar. There are concerted redox-linked motions within the protein that bring E61 and K75 closer to heme II in the oxidized form. This is consistent with an electrostatically driven movement that may provide an important contribution to the previously measured positive cooperativity between hemes I and II. No significant conformational changes were observed that might be related to redox?Bohr effects; the families of structures represent mainly protonated forms, and therefore, pH dependence should not play a major role in the observed structural rearrangements.     

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.     

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris.

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.     

Crystallographic study of cytochrome c553 from Desulfovibrio vulgaris Miyazaki

Cytochrome c553 from the sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki, has been crystallized. The combination of microdialysis and vapor diffusion allowed successful crystallization. The crystals were of good quality, and useful data were obtained that extended to the nominal resolution of 1.3 A. The space group is P4(3)2(1)2 with cell dimensions of a = b = 42.7 A, c = 103.4 A. More than twenty heavy-atom reagents were screened with the isomorphous replacement technique, and only the mersalyl derivative could be used for the phase determination. The single isomorphous replacement method combined with the anomalous scattering effect of the Hg-atom in mersalyl and the Fe-atom of the heme group was used for the phase determination.     

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.     

The structure of cytochrome c3 from Desulfovibrio vulgaris Miyazaki at 2.5 A resolution

The structure of tetraheme cytochrome c3 isolated from Desulfovibrio vulgaris Miyazaki has been determined at 2.5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using the native and four heavy atom derivatives, anomalous scattering measurements of the latter being considered. The mean figure of merit was 0.77. Four heme groups are exposed on the surface of the molecule. There are some short helical segments in the polypeptide chain, and hair-pin turns are often observed at glycine and alanine residues.     

EPR study of the redox interactions in cytochrome c3 from Desulfovibrio vulgaris Miyazaki

We present a new examination of the EPR redox titration data for the tetraheme cytochrome c3 from Desulfovibrio vulgaris Miyazaki. Our analysis includes the contribution of the interaction potentials between the four redox sites and is based on the model previously developed for the study of cytochrome c3 from Desulfovibrio desulfuricans Norway. We observed, as for D. desulfuricans Norway cytochrome c3, that the conformation of the heme with the lowest redox potential, heme 4, is sensitive to the redox state of the heme with the highest potential, heme 1. However in D. vulgaris Miyazaki cytochrome c3 spectral simulations show that heme 4 is present in two conformational states which interconvert partially when heme 1 is reduced. The sets of redox parameters which satisfy the fitting procedure of the titration curves are in the following domain: -250 mV less than or equal to e41 less than or equal to -220 mV, -325 mV less than or equal to e2 less than or equal to -320 mV, -335 mV less than or equal to e3 less than or equal to -330 mV, -360 mV less than or equal to e4 less than or equal to -355 mV, -5 mV less than or equal to I12 less than or equal to 20 mV, -10 mV less than or equal to I13 less than or equal to 5 mV, -15 mV less than or equal to I23 less than or equal to -5 mV, -15 mV less than or equal to I24 less than or equal to -10 mV, -25 mV, less than or equal to I34 less than or equal to -15 mV. As in D. desulfuricans Norway cytochrome c3 the interactions are moderate. Simple electrostatic considerations suggest that these moderate values could be related to the large accessibility of the hemes to the solvent. Our work does not confirm the existence of a cooperative interaction between heme 2 and heme 3 which has been proposed on the basis of electrochemical measurements.