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.

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.

Protein modified- and membrane electrodes: strategies for the development of biomolecular sensors.

Different procedures used for constructing protein/enzyme-modified electrodes are examined, in particular adsorption, covalent attachment and film deposition. The performances of such modified electrodes with electroactive proteins or enzymes attached to their active surface are examined, especially in the case of c-type cytochromes, hydrogenases and glucose oxidase. Another strategy presented in this review consists of the use of membrane electrodes with an electroactive protein imprisoned between a dialysis membrane and the electrode surface. The versatility and other advantages of such a procedure are underlined. Applications of membrane electrodes to the bioremediation of soils and effluents and as models for investigating interactions between proteins and soils are described.

Ultrahigh-resolution study on Pyrococcus abyssi rubredoxin: II. Introduction of an O-H...Sgamma-Fe hydrogen bond increased the reduction potential by 65 mV.

The effect of D-H...S(gamma)-Fe hydrogen bonding on the reduction potential of rubredoxin was investigated by the introduction of an O-H...S(gamma)-Fe hydrogen bond on the surface of Pyrococcus abyssi rubredoxin. The formation of a weak hydrogen bond between Ser44-O(gamma) and Cys42-S(gamma) in mutant W4L/R5S/A44S increased the reduction potential by 56 mV. When side effects of the mutation were taken into account, the contribution of the additional cluster hydrogen bond to the reduction potential was estimated to be +65 mV. The structural analysis was based on ultrahigh-resolution structures of oxidized P. abyssi rubredoxin W4L/R5S and W4L/R5S/A44S refined to 0.69 and 0.86 A, respectively.

Ultrahigh-resolution study on Pyrococcus abyssi rubredoxin. I. 0.69 A X-ray structure of mutant W4L/R5S.

The crystal structure of Pyrococcus abyssi rubredoxin mutant W4L/R5S was solved by direct methods. The model of the air-oxidized protein was refined by partially restrained full-matrix least-squares refinement against intensity data to 0.69 A resolution. This first ultrahigh-resolution structure of a rubredoxin provides very detailed and precise information about the Fe(SCys)(4) centre and its environment, the peptide-backbone stereochemistry, H atoms and hydrogen bonds, static and dynamic disorder, the solvent structure and the electron-density distribution. P. abyssi rubredoxin W4L/R5S is the first of a series of mutants studied by atomic and ultrahigh-resolution X-ray crystallography which are expected to contribute to the understanding of structure-function relationships in iron-sulfur proteins.

Multiple Rieske genes in prokaryotes: exchangeable Rieske subunits in the cytochrome bc-complex of Rubrivivax gelatinosus.

Bacterial cytochrome bc1-complex encoded by the petABC operon consists of three subunits, the Rieske iron-sulphur protein, the b-type cytochrome, and the c1-type cytochrome. Disruption of the petA gene of Rubrivivax gelatinosus is not lethal under photosynthetic growth conditions. However, deletion of both petA and petB results in a photosynthesis-deficient strain, suggesting the presence of a second gene encoding a Rieske protein and rescuing a functional cytochrome bc1-complex in the PETA1 mutant. The corresponding petA2 gene was identified and the PETA2 mutant could also grow under photosynthetic conditions. The double mutant PETA12, however, was unable to grow photosynthetically. The presence of a photo-induced cyclic electron transfer was tested by monitoring the kinetics of cytochrome photo-oxidation on intact cells; the data confirm the capacity of petA2 to replace petA1 in the bc1-complex to support photosynthesis. Soluble forms of both PetA1 and PetA2 Rieske proteins were purified from Escherichia coli and found to contain correctly inserted [2Fe-2S] clusters. Electron paramagnetic resonance (EPR) spectroscopy and midpoint potential measurements showed typical [2Fe-2S] signals and E(m) values of +275 mV for both Rieske proteins. The high amino acid sequence similarity and the obtained midpoint potential values argue for a functional role of these proteins in the cytochrome bc1-complex. The presence of duplicated Rieske genes is not restricted to R. gelatinosus. Phylogenetic trees of Rieske genes from Rubrivivax and other proteobacteria as well as from cyanobacteria were reconstructed. On the basis of the phylogenetic analyses, differing evolutionary origins of duplicated Rieske genes in proteo- and cyanobacteria are proposed.

Role of the tetrahemic subunit in Desulfovibrio vulgaris hildenborough formate dehydrogenase.

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH), the genome sequencing revealed the presence of three operons encoding formate dehydrogenases. fdh1 encodes an alphabetagamma trimeric enzyme containing 11 heme binding sites; fdh2 corresponds to an alphabetagamma trimeric enzyme with a tetrahemic subunit; fdh3 encodes an alphabeta dimeric enzyme. In the present work, spectroscopic measurements demonstrated that the reduction of cytochrome c(553) was obtained in the presence of the trimeric FDH2 and not with the dimeric FDH3, suggesting that the tetrahemic subunit (FDH2C) is essential for the interaction with this physiological electron transfer partner. To further study the role of the tetrahemic subunit, the fdh2C gene was cloned and expressed in Desulfovibrio desulfuricans G201. The recombinant FDH2C was purified and characterized by optical and NMR spectroscopies. The heme redox potentials measured by electrochemistry were found to be identical in the whole enzyme and in the recombinant subunit, indicating a correct folding of the recombinant protein. The mapping of the interacting site by 2D heteronuclear NMR demonstrated a similar interaction of cytochrome c(553) with the native enzyme and the recombinant subunit. The presence of hemes c in the gamma subunit of formate dehydrogenases is specific of these anaerobic sulfate-reducing bacteria and replaces heme b subunit generally found in the enzymes involved in anaerobic metabolisms.

Buildup of polyelectrolyte-protein multilayer assemblies on gold electrodes. Role of the hydrophobic effect.

The buildup of layer-by-layer assemblies onto gold surfaces from water-soluble charged polyelectrolytes and proteins is examined using quartz crystal microgravimetry (QCM) and electrochemical techniques. Polyelectrolytes such as poly(styrenesulfonate) and poly(ester sulfonic acid) (Eastman AQ-29D polymer) adsorb spontaneously onto gold, contrary to poly(ethyleneimine). From the modification of the gold surface with a thiol and specific adsorption of polymers under polarization conditions, it is concluded that the hydrophobicity of the gold surface seems to be a determining factor in the adsorption process. Alternate adsorption onto gold resonators first coated with AQ-29D polymer gives stable multilayer films in the case of positively charged lysozyme (pI = 11) or polyheme Desulfovibrio vulgaris Hildenborough cytochrome c3 (pI = 10.5). QCM frequency changes with the number of adsorption steps suggest that a linear increase in film mass occurs. Desulfomicrobium norvegicum polyheme cytochrome c3 (pI = 7), which has a null global charge at neutral pH, is shown to give also stable multilayer AQ-29D/cytochrome c3 films, suggesting that several types of interactions, especially the hydrophobic effect, are involved in the buildup process.

Multiple orientations in a physiological complex: the pyruvate-ferredoxin oxidoreductase-ferredoxin system.

Ferredoxin I from Desulfovibrio africanus (Da FdI) is a small acidic [4Fe-4S] cluster protein that exchanges electrons with pyruvate-ferredoxin oxidoreductase (PFOR), a key enzyme in the energy metabolism of anaerobes. The thermodynamic properties and the electron transfer between PFOR and either native or mutated FdI have been investigated by microcalorimetry and steady-state kinetics, respectively. The association constant of the PFOR-FdI complex is 3.85 x 10(5) M(-1), and the binding affinity has been found to be highly sensitive to ionic strength, suggesting the involvement of electrostatic forces in formation of the complex. Surprisingly, the punctual or combined neutralizations of carboxylate residues surrounding the [4Fe-4S] cluster slightly affect the PFOR-FdI interaction. Furthermore, hydrophobic residues around the cluster do not seem to be crucial for the PFOR-FdI system activity; however, some of them play an important role in the stability of the FeS cluster. NMR restrained docking associated with site-directed mutagenesis studies suggested the presence of various interacting sites on Da FdI. The modification of additional acidic residues at the interacting interface, generating a FdI pentamutant, evidenced at least two distinct FdI binding sites facing the distal [4Fe-4S] cluster of the PFOR. We also used a set of various small acidic partners to investigate the specificity of PFOR toward redox partners. The remarkable flexibility of the PFOR-FdI system supports the idea that the specificity of the physiological complex has probably been "sacrificed" to improve the turnover rate and thus the efficiency of bacterial electron transfer.