Electron transfer in an acidophilic bacterium: interaction between a diheme cytochrome and a cupredoxin.

Acidithiobacillus ferrooxidans, a chemolithoautotrophic Gram-negative bacterium, has a remarkable ability to obtain energy from ferrous iron oxidation at pH 2. Several metalloproteins have been described as being involved in this respiratory chain coupling iron oxidation with oxygen reduction. However, their properties and physiological functions remain largely unknown, preventing a clear understanding of the global mechanism. In this work, we focus on two metalloproteins of this respiratory pathway, a diheme cytochrome c4 (Cyt c4) and a green copper protein (AcoP) of unknown function. We first demonstrate the formation of a complex between these two purified proteins, which allows homogeneous intermolecular electron-transfer in solution. We then mimic the physiological interaction between the two partners by replacing one at a time with electrodes displaying different chemical functionalities. From the electrochemical behavior of individual proteins, we show that, while electron transfer on AcoP requires weak electrostatic interaction, electron transfer on Cyt c4 tolerates different charge and hydrophobicity conditions, suggesting a pivotal role of this protein in the metabolic chain. The electrochemical study of the proteins incubated together demonstrates an intermolecular electron transfer involving the protein complex, in which AcoP is reduced through the high potential heme of Cyt c4. Modelling of the electrochemical signals at different scan rates allows us to estimate the rate constant of this intermolecular electron transfer in the range of a few s-1. Possible routes for electron transfer in the acidophilic bacterium are deduced.

The effect of pH on the dynamics of natural membranes.

Pure phospholipids and membrane fragments from bacterial cells living under various conditions were studied against the influence of the surrounding acidity on the internal dynamics. For that we compared mean square displacements extracted from elastic incoherent neutron scattering data, measured both at low and at neutral pH, of the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine and of samples from neutralophilic and acidophilic micro-organisms (some being hyperthermophilic and others mesophilic). The lipids showed a slight shift in the phase transition temperature of about 4 degrees under pH variation and became slightly more mobile at lower pH. The membrane fragments not used to extreme acidic conditions were significantly more sensitive to variations in the pH values, whereas the acidophilic and -tolerant samples were much less influenced by this parameter. They presented the higher softness at low pH, which was closer to their native condition. Such findings might be a hint for adaptation mechanisms to different acidity conditions.

Reconstitution of supramolecular organization involved in energy metabolism at electrochemical interfaces for biosensing and bioenergy production.

How the redox proteins and enzymes involved in bioenergetic pathways are organized is a relevant fundamental question, but our understanding of this is still incomplete. This review provides a critical examination of the electrochemical tools developed in recent years to obtain knowledge of the intramolecular and intermolecular electron transfer processes involved in metabolic pathways. Furthermore, better understanding of the electron transfer processes associated with energy metabolism will provide the basis for the rational design of biotechnological devices such as electrochemical biosensors, enzymatic and microbial fuel cells, and hydrogen production factories. Starting from the redox complexes involved in two relevant bacterial chains, i.e., from the hyperthermophile Aquifex aeolicus and the acidophile Acidithiobacillus ferrooxidans, examination of protein-protein interactions using electrochemistry is first reviewed, with a focus on the orientation of a protein on an electrochemical interface mimic of a physiological interaction between two partners. Special attention is paid to current research in the electrochemistry of essential membrane proteins, which is one mandatory step toward the understanding of energy metabolic pathways. The complex and challenging architectures built to reconstitute a membrane-like environment at an electrode are especially considered. The role played by electrochemistry in the attempt to consider full bacterial metabolism is finally emphasized through the study of whole cells immobilized at electrodes as suspensions or biofilms. Before the performances of biotechnological devices can be further improved to make them really attractive, questions remain to be addressed in this particular field of research. We discuss the bottlenecks that need to be overcome in the future.

Dynamics measured by neutron scattering correlates with the organization of bioenergetics complexes in natural membranes from hyperthermophile and mesophile bacteria.

Various models on membrane structure and organization of proteins and complexes in natural membranes emerged during the last years. However, the lack of systematic dynamical studies to complement structural investigations hindered the establishment of a more complete picture of these systems. Elastic incoherent neutron scattering gives access to the dynamics on a molecular level and was applied to natural membranes extracted from the hyperthermophile Aquifex aeolicus and the mesophile Wolinella succinogenes bacteria. The results permitted to extract a hierarchy of dynamic flexibility and atomic resilience within the samples, which correlated with the organization of proteins in bioenergetics complexes and the functionality of the membranes.

Biocatalysts for fuel cells: efficient hydrogenase orientation for H2 oxidation at electrodes modified with carbon nanotubes.

We report the modification of gold and graphite electrodes with commercially available carbon nanotubes for immobilization of Desulfovibrio fructosovorans [NiFe] hydrogenase, for hydrogen evolution or consumption. Multiwalled carbon nanotubes, single-walled carbon nanotubes (SWCNs), and amine-modified and carboxyl-functionalized SWCNs were used and compared throughout. Two separate methods were performed: covalent attachment of oriented hydrogenase by controlled architecture of carbon nanotubes at gold electrodes, and adsorption of hydrogenase at carbon-nanotube-coated pyrolytic graphite electrodes. In the case of self-assembled carbon nanotubes at gold electrodes, hydrogenase orientation based on electrostatic interaction with the electrode surface was found to control the electrocatalytic process for H(2) oxidation. In the case of carbon nanotube coatings on pyrolytic graphite electrodes, catalysis was controlled more by the geometry of the nanotubes than by the orientation of the enzyme. Noticeably, shortened SWCNs were demonstrated to allow direct electron transfer and generate high and quite stable current densities for H(2) oxidation via adsorbed hydrogenase, despite having many carboxylic surface functions that could yield unfavorable hydrogenase orientation for direct electron transfer. This result is attributable to the high degree of oxygenated surface functions in addition to the length of shortened SWCNs that yields highly divided materials.

Hydrogen metabolism in the hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus is a microaerophilic, hydrogen-oxidizing, hyperthermophilic bacterium containing three [NiFe] hydrogenases. Two of these three enzymes (one membrane-bound and one soluble) have been purified and characterized. The Aquifex hydrogenases are thermostable and tolerant to oxygen. A cellular function for the three hydrogenases has been proposed. The two membrane-bound periplasmic hydrogenases may function in energy conservation, whereas the soluble cytoplasmic hydrogenase is probably involved in the CO(2) fixation pathway.

Hydrogenases in sulfate-reducing bacteria function as chromium reductase.

The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H(2)S produced by the bacteria, and enzymatically, by polyhemic cytochromes c(3). We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K(m) towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.

Electron transfer and binding of the c-type cytochrome TorC to the trimethylamine N-oxide reductase in Escherichia coli.

Reduction of trimethylamine N-oxide (E'(0(TMAO/TMA)) = +130 mV) in Escherichia coli is carried out by the Tor system, an electron transfer chain encoded by the torCAD operon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemic c-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamine N-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapC c-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorC(N)) to the cytochromes of this family, but TorC contains a C-terminal extension (TorC(C)) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorC(N) and TorC(C), were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (-177 and -98 mV) localized in the tetrahemic TorC(N) and one positive midpoint potential (+120 mV) in the monohemic TorC(C). In agreement with these values, the in vitro reconstitution of electron transfer between TorC, TorC(N), or TorC(C) and TorA showed that only TorC and TorC(C) were capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorC(N) strongly bind TorA. Therefore, TorC(C) directly transfers electrons to TorA, whereas TorC(N), which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorC(C) and the binding to TorA.

Characterization of a new dihemic c(4)-type cytochrome isolated from Thiobacillus ferrooxidans.

A new soluble c-type cytochrome has been purified to homogeneity from the acidophilic proteobacterium Thiobacillus ferrooxidans BRGM. It is characterized by an alpha-peak wavelength of 552 nm, a molecular mass of 26 567 Da (as determined by mass spectroscopy) and a pI value of 8. Optical redox titrations at pH 4.0 revealed the presence of two distinguishable redox species with an E(m) of 510 mV and an E(m) of 430 +/- 20 mV. EPR spectra recorded for this heme protein demonstrated the presence of stoichiometric amounts of two low-spin hemes with a g(z)() of 3.08 (510 mV species) and a g(z)() of 3.22 (430 mV species). Modifications of the physicochemical properties of the cytochrome were observed on complex formation with the blue copper protein rusticyanin, another soluble electron carrier in the genus Thiobacillus. N-Terminal sequencing yielded the polypeptide sequence up to the 50th residue. The determined sequence was found to be present (at 100% amino acid identity) in the (unfinished) genome of T. ferrooxidans ATCC 23270, and the corresponding full-length protein turned out to be surprisingly similar (34.5% amino acid identity) to another c(4)-type diheme protein from T. ferrooxidans BRGM [Cavazza, C., et al. (1996) Eur. J. Biochem. 242, 308-314], the gene of which is also present (at 97% amino acid identity) in the T. ferrooxidans ATCC 23270 genome. The physicochemical properties and sequence characteristics of both c(4) cytochromes present in the same bacteria are compared, and the functional role of this new diheme protein in the iron(II)-oxidizing electron transport chain in the genus Thiobacillus is discussed.

A sequential electron transfer from hydrogenases to cytochromes in sulfate-reducing bacteria.

A central step in the energy metabolism of sulfate-reducing bacteria is the oxidation of molecular hydrogen, catalyzed by a periplasmic hydrogenase. The resulting electrons are then transferred to various electron transport chains and used for cytoplasmic sulfate reduction. The complex formation between [NiFeSe] hydrogenase and the soluble periplasmic polyheme cytochromes from Desulfomicrobium norvegicum was characterized by cross-linking experiments, BIAcore and kinetics analysis. Analysis of electron transfer between [NiFeSe] hydrogenase and octaheme cytochrome c(3) (M(r) 26¿ omitted¿000) pointed out that this cytochrome is reduced faster in the presence of catalytic amounts of tetraheme cytochrome c(3) (M(r) 13¿ omitted¿000) isolated from the same organism. The activation of the hydrogenase-dependent reduction of polyheme cytochromes by cytochrome c(3) (M(r) 13¿ omitted¿000), which is now described in both Desulfovibrio and Desulfomicrobium, is proposed as a general mechanism. During this process, cytochrome c(3) (M(r) 13¿ omitted¿000) would act as an electron shuttle in between hydrogenase and the polyheme cytochromes and its conductivity appears to be an important factor.

A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.

Interaction-induced redox switch in the electron transfer complex rusticyanin-cytochrome c(4).

The blue copper protein rusticyanin isolated from the acidophilic proteobacterium Thiobacillus ferrooxidans displays a pH-dependent redox midpoint potential with a pK value of 7 on the oxidized form of the protein. The nature of the alterations of optical and EPR spectra observed above the pK value indicated that the redox-linked deprotonation occurs on the epsilon-nitrogen of the histidine ligands to the copper ion. Complex formation between rusticyanin and its probable electron transfer partner, cytochrome c(4), induced a decrease of rusticyanin's redox midpoint potential by more than 100 mV together with spectral changes similar to those observed above the pK value of the free form. Complex formation thus substantially modifies the pK value of the surface-exposed histidine ligand to the copper ion and thereby tunes the redox midpoint potential of the copper site. Comparisons with reports on other blue copper proteins suggest that the surface-exposed histidine ligand is employed as a redox tuning device by many members of this group of soluble electron carriers.

First evidence for the presence of a hydrogenase in the sulfur-reducing bacterium Desulfuromonas acetoxidans.

Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from Desulfuromonas acetoxidans. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium D. acetoxidans, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of D. acetoxidans, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium.

Key role of phenylalanine 20 in cytochrome c3: structure, stability, and function studies.

Aromatic residues in c-type cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme cytochrome c3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L cytochrome c3 molecules.

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 BLAcore 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 x 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.

Characterization and expression of the co-transcribed cyc1 and cyc2 genes encoding the cytochrome c4 (c552) and a high-molecular-mass cytochrome c from Thiobacillus ferrooxidans ATCC 33020.

The sequence of the cyc1 gene encoding the Thiobacillus ferrooxidans ATCC 33020 c552 cytochrome, shows that this cytochrome is a 21-kDa periplasmic c4-type cytochrome containing two similar monohaem domains. The kinetics of reduction and the fact that cytochromes c4 are considered to be physiological electron donors of cytochrome oxidases suggest that the last steps of the iron respiratory chain are: rusticyanin-->cytochrome c4-->cytochrome oxidase. In Thiobacillus ferrooxidans, cyc1 is co-transcribed with the cyc2 gene, encoding a high-molecular-mass monohaem cytochrome c. This suggests that the cytochromes encoded by these genes belong to the same electron transfer chain.

Structural and kinetic studies of the Y73E mutant of octaheme cytochrome c3 (Mr = 26 000) from Desulfovibrio desulfuricans Norway.

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.

Characterisation of a soluble cytochrome c4 isolated from Thiobacillus ferrooxidans.

A soluble c-type cytochrome was purified to homogeneity from Thiobacillus ferrooxidans. This cytochrome is characterised by an alpha-peak wavelength of 552 nm, a molecular mass of 21 193 Da (as determined by mass spectroscopy), and a pI value of 9. N-terminal sequencing yielded the polypeptide sequence up to the 50th residue. The iron content of 1.9 Fe/molecule and the heme/molecule ratio of 2.15 identified this cytochrome as a diheme protein. Optical redox titrations at pH 3.0 revealed the presence of two distinguishable redox species with Em = 385 mV +/- 20 mV and Em = 480 mV +/- 20 mV. EPR spectra recorded on this heme protein showed the presence of two distinct spectral species with gz = 3.1 and gz = 3.35. The gz = 3.35 heme corresponds to the higher potential redox species. In line with the differences in Em values, the two heme species were oxidised by O2 with significantly differing half-times. All the above mentioned properties demonstrate that this heme protein belongs to the c4 family of diheme cytochromes. The characteristics and functional role of the studied heme protein are discussed with reference to other c-type cytochromes described in Thiobacilli. Its properties are furthermore compared to other members of the cytochrome c4 family.

The modulation of enzyme reaction rates within multi-enzyme complexes. 1. Statistical thermodynamics of information transfer through multi-enzyme complexes.

There is now experimental evidence that association of different enzymes as a multi-enzyme complex may result in an alteration of the catalytic properties of the enzymes present in this complex. This effect is not related to the channelling of reaction intermediates between different active sites. It appears as a consequence of an information transfer that occurs within the multi-enzyme complex. A theory, based on statistical thermodynamics, has been developed which provides an understanding, on a physical basis, for how isologous as well as heterologous interactions between identical, or different, enzymes of the complex may modulate the catalytic properties of an oligomeric enzyme of that complex. The theory predicts three possible types of effects: an alteration, through heterologous interactions, of an already existing co-operativity of the oligomeric enzyme within the complex; a co-operativity, generated by heterologous interactions in the complex that could not occur if the oligomeric enzyme were isolated from the rest of the complex; a Michaelis-Menten character of the oligomeric enzyme within the complex, but with altered values of Vm and Km relative to what would have been observed with the naked enzyme. All these effects appear as a consequence of a transfer of information between different enzymes of the same multi-protein complex. The following paper in this journal shows how one can demonstrate and characterize experimentally these effects in a multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

The modulation of enzyme reaction rates within multi-enzyme complexes. 2. Information transfer within a chloroplast multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Octameric ribulose bisphosphate carboxylase-oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6-phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase-oxygenase is inserted in the five-enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase-oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein-protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase-oxygenase. This alteration may be expressed in terms of stabilization-destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi-protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co-ordinate of -1.68 kJ/mol and a constant stabilization energy of -3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi-enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase-oxygenase, as well as to increase the catalytic rate constant. The binding of the substrate analog 6-phosphogluconate to free ribulose bisphosphate carboxylase-oxygenase is non-co-operative. It becomes positively co-operative if this enzyme is inserted in the multi-protein complex. Under these conditions, only one type of enzyme-inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme-inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is -1.03 kJ/mol. Neither free nor associated ribulose bisphosphate carboxylase-oxygenase display any co-operativity relative to substrate binding. However, in the presence of the substrate analog 6-phosphogluconate, this enzyme displays positive co-operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is < or = 2. As 6-phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co-operativity might be of functional importance in vivo.