Microbial electrosynthesis is an emerging green technology that explores the capability of a particular group of microorganisms to drive their metabolism toward the production of hydrogen or value-added chemicals from electrons supplied by electrode surfaces. The cytochrome PccH showed the largest increase in transcription when electrons are supplied to Geobacter sulfurreducens biofilms. Gene knock-out experiments have shown that the electron transfer toward G. sulfurreducens cells was completely inhibited by the deletion of the gene encoding for cytochrome PccH. This identifies a crucial role for this protein in G. sulfurreducens microbial electrosynthesis mechanisms, which are currently unknown. In this work, we present the backbone (1H, 13C and 15N) and heme assignment for PccH in the oxidized state. The data obtained paves the way to identify and structurally map the molecular interaction regions between the cytochrome PccH and its physiological redox partners.

Gene knock-out studies on Geobacter sulfurreducens have shown that the monoheme c-type cytochrome OmcF is essential for the extracellular electron transfer pathways involved in the reduction of iron and uranium oxy-hydroxides, as well as, on electricity production in microbial fuel cells. A detailed electrochemical characterization of OmcF was performed for the first time, allowing attaining kinetics and thermodynamic data. The heterogeneous electron transfer rate constant was determined at pH 7 (0.16 ± 0.01 cm s−1) indicating that the protein displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The analysis of the structures of OmcF allowed us to assign the redox-Bohr centre to the side chain of His47 residue and its pKa values in the reduced and oxidized states were determined (pKox = 6.73; pKred = 7.55). The enthalpy, entropy and Gibbs free energy associated with the redox transaction were calculated, pointing the reduced form of the cytochrome as the most favourable. The data obtained indicate that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF.

The monoheme outer membrane cytochrome F (OmcF) from Geobacter sulfurreducens plays an important role in Fe(III) reduction and electric current production. The electrochemical characterization of this cytochrome has shown that its redox potential is modulated by the solution pH (redox-Bohr effect) endowing the protein with the necessary properties to couple electron and proton transfer in the physiological range. The analysis of the OmcF structures in the reduced and oxidized states showed that with the exception of the side chain of histidine 47 (His⁴⁷), all other residues with protonatable side chains are distant from the heme iron and, therefore, are unlikely to affect the redox potential of the protein. The protonatable site at the imidazole ring of His⁴⁷ is in the close proximity to the heme and, therefore, this residue was suggested as the redox-Bohr center. In the present work, we tested this hypothesis by replacing the His⁴⁷ with non-protonatable residues (isoleucine – OmcFH47I and phenylalanine – OmcFH47F). The structure of the mutant OmcFH47I was determined by X-ray crystallography to 1.13 Å resolution and showed only minimal changes at the site of the mutation. Both mutants were ¹⁵N-labeled and their overall folding was confirmed to be the same as the wild-type by NMR spectroscopy. The pH dependence of the redox potential of the mutants was measured by cyclic voltammetry. Compared to the wild-type protein, the magnitude of the redox-Bohr effect in the mutants was smaller, but not fully abolished, confirming the role of His⁴⁷ on the pH modulation of OmcF’s redox potential. However, the pH effect on the heme substituents’ NMR chemical shifts suggested that the heme propionate P₁₃ also contributes to the overall redox-Bohr effect in OmcF. In physiological terms, the contribution of two independent acid–base centers to the observed redox-Bohr effect confers OmcF a higher versatility to environmental changes by coupling electron/proton transfer within a wider pH range.

Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of α-helical content at relatively low temperatures (Tapp (m) ~ 44 °C), followed by the disruption of both iron−sulfur clusters (Tapp (m) ~ 53−60 °C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (Tapp (m) ~ 58 °C) and a change, rather than a significant net loss, of secondary structure (Tapp (m) ~ 60 °C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron−sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc3) are rationalised by a model which involves both homotropic (e−/e−) and heterotropic (e−/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVc3 have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.

The thermodynamic parameters which govern the homotropic (e−/e−) and heterotropic (e−/H+) cooperativity in the tetrahaem cytochrome c3 isolated from Desulfovibrio vulgaris (Hildenborough) were determined, using the paramagnetic shifts of haem methyl groups in the NMR spectra of intermediate oxidized states at different pH levels. A model is put forward to explain how the network of positive and negative cooperativities between the four haems and acid/base group(s) enables the protein to achieve a proton-assisted 2e− step.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure.

Three-quarters of the carbon-13 resonances of nuclei attached to the four haems of Desulfovibrio vulgaris ferricytochrome c3 are assigned. Preliminary analysis of their Fermi contact interactions shows that the shifts are directly related to the orientation of both of the axial histidine ligands in each case and the approach can therefore be used to obtain structural information in other cytochromes with bis-histidinyl coordination. The implications for the control of redox potential in cytochromes are discussed.