Extracellular electron transfer is the key metabolic trait that enables some bacteria to play a significant role in the biogeochemical cycling of metals and in bioelectrochemical devices such as microbial fuel cells. In Shewanella oneidensis MR-1, electrons generated in the cytoplasm by catabolic processes must cross the periplasmic space to reach terminal oxidoreductases found at the cell surface. Lack of knowledge on how these electrons flow across the periplasmic space is one of the unresolved issues related with extracellular electron transfer. Using NMR to probe protein-protein interactions, kinetic measurements of electron transfer and electrostatic calculations, we were able to identify protein partners and their docking sites, and determine the dissociation constants. The results showed that both STC (small tetrahaem cytochrome c) and FccA (flavocytochrome c) interact with their redox partners, CymA and MtrA, through a single haem, avoiding the establishment of stable redox complexes capable of spanning the periplasmic space. Furthermore, we verified that the most abundant periplasmic cytochromes STC, FccA and ScyA (monohaem cytochrome c5) do not interact with each other and this is likely to be the consequence of negative surface charges in these proteins. This reveals the co-existence of two non-mixing redox pathways that lead to extracellular electron transfer in S. oneidensis MR-1 established through transient protein interactions.

Microorganisms show an astonishing versatility in energy metabolism. They can use a variety of different catabolic electron acceptors, but they use them according to a thermodynamic hierarchy, which is determined by the redox potential of the available electron acceptors. This hierarchy is reflected by a regulatory machinery that leads to the production of respiratory chains in dependence of the availability of the corresponding electron acceptors. In this study, we showed that the c-proteobacterium Shewanella oneidensis produces several functional electron transfer chains simultaneously. Furthermore, these chains are interconnected, most likely with the aid of c-type cytochromes. The cytochrome pool of a single S. oneidensis cell consists of ca. 700 000 hemes, which are reduced in the absence on an electron acceptor, but can be reoxidized in the presence of a variety of electron acceptors, irrespective of prior growth conditions. The small tetraheme cytochrome (STC) and the soluble heme and flavin containing fumarate reductase FccA have overlapping activity and appear to be important for this electron transfer network. Double deletion mutants showed either delayed growth or no growth with ferric iron, nitrate, dimethyl sulfoxide or fumarate as electron acceptor. We propose that an electron transfer machinery that is produced irrespective of a thermodynamic hierarchy not only enables the organism to quickly release catabolic electrons to a variety of environmental electron acceptors, but also offers a fitness benefit in redox-stratified environments.

Bacteria of the genus Shewanella contain an abundant small tetraheme cytochrome in their periplasm when growing anaerobically. Data collected for the protein isolated from S. oneidensis MR-1 and S. frigidimarina indicate differences in the order of oxidation of the hemes. A detailed thermodynamic characterization of the cytochrome from S. oneidensis MR-1 in the physiological pH range was performed, with data collected in the pH range 5.5-9.0 from NMR experiments using partially oxidized samples and from redox titrations followed by visible spectroscopy. These data allow the parsing of the redox and redox-protonation interactions that occur during the titration of hemes. The results show that electrostatic effects dominate the heme-heme interactions, in agreement with modest redox-linked structural modifications, and protonation has a considerable influence on the redox properties of the hemes in the physiological pH range. Theoretical calculations using the oxidized and reduced structures of this protein reveal that the bulk redox-Bohr effect arises from the aggregate fractional titration of several of the heme propionates. This detailed characterization of the thermodynamic properties of the cytochrome shows that only a few of the multiple microscopic redox states that the protein can access are significantly populated at physiological pH. On this basis a functional pathway for the redox activity of the small tetraheme cytochrome from S. oneidensis MR-1 is proposed, where reduction and protonation are thermodynamically coupled in the physiological range. The differences between the small tetraheme cytochromes from the two organisms are discussed in the context of their biological role.

Sulphate-reducing bacteria are important players in the global sulphur and carbon cycles, with considerable economical and ecological impact. However, the process of sulphate respiration is still incompletely understood. Several mechanisms of energy conservation have been proposed, but it is unclear how the different strategies contribute to the overall process. In order to obtain a deeper insight into the energy metabolism of sulphate-reducers whole-genome microarrays were used to compare

A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely.

Nuclear magnetic resonance and visible spectroscopies were used to determine the thermodynamic parameters of the four hemes in cytochrome c3 from Desulfovibrio gigas at 298 and 277 K and to investigate the mechanism of electron/proton energy transduction. Data obtained in the pH range from 5 to 9 were analyzed according to a model in which the hemes interact with each other (redox cooperativities) and with an ionizable center (redox-Bohr cooperativities). The results obtained at the two temperatures allow the deconvolution of the entropic contribution to the free energy of the four hemes, to the acid-base equilibrium of the ionizable center, and to the network of cooperativities among the five centers. The redox potentials of the hemes are modulated by the enthalpic contribution to the free energy, and evidence for the participation of the propionates of heme I in the redox-Bohr effect is presented. The network of interactions between the centers in this protein facilitates the concerted transfer of electrons and protons, in agreement with the "proton thruster" mechanism proposed for electronic to protonic energy transduction by cytochromes c3.

Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c 3 is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechanochemical coupling involved in the transfer of multiple electrons and protons in cytochrome c 3 from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.Recent developments in techniques of structural biology have opened the way for probing the mechanisms used by biological macromolecules involved in energy transduction at the molecular level. The structural analysis of bacteriorhodopsin trapped in the M photointermediate state (1), the structures in the oxidized and reduced forms of cytochromes c 3 that perform a coupled two-electron step associated with proton transfer (2, 3), and the establishment of the coupled transfer of electrons and protons to the 3Fe-4S cluster of Azotobacter vinelandii ferredoxin (4) are just a few recent examples where results from different techniques are integrated in a description at the atomic level of the energy-transducing events. The phenomenon of energy transduction relies on coupled events (5), whether they involve only electrostatic interactions or structural rearrangements of the active sites or its surroundings (mechano-chemical coupling), which may be more important than the electrostatic component of the overall coupling (6). The pumping of proton(s) at the beginning of re-reduction of cytochrome c oxidase (7) can be described using a model in which the electrostatic attraction of electrons and protons is overcome (8), a situation that requires structural changes involving charged residues. Small proteins capable of performing energy transduction provide easier access to the structural bases for the underlying mechanisms, as the recent advances in the understanding of the proton pumping by the 26-kDa bacteriorhodopsin demonstrate (1, 9).This wo...

Heteronuclear multiple quantum NMR is used to measure the paramagnetic 13C shifts of the α substituents of the hemes in five different tetraheme ferricytochromes c 3. The shifts of the 20 bis-histidine ligated hemes are assigned and then analyzed in terms of a model based on the π molecular orbitals of the heme under perturbed D 4 symmetry, which yields the orientation of the rhombic perturbation, θ, and an energy splitting parameter, ΔE. Comparison of these parameters with crystal structures provides a test of the nature and extent of the influence of axial ligand orientation on the electronic structure of the heme. Despite possible differences between structures in solution and in the crystal, a clear correlation is found between θ and the resultant of the normals to the imidazole planes, and between ΔE and the angle between the normals. A weaker dependence of ΔE upon θ is also apparent. This is analogous to the results of low-temperature EPR studies of model compounds, which have been attributed to pseudo-Jahn−Teller distortion of the porphyrin. However, the effect is also predicted by extended Hückel calculations made with undistorted geometries. This work demonstrates that the variation in the electronic structure of bis-histidinyl hemes c is dominated by the geometry of the axial ligands and that other perturbations, such as asymmetric substitution of the porphyrin or low symmetry of the surrounding protein, are relatively minor. The correlations with θ and ΔE can, therefore, be used to determine the ligand geometry with sufficient accuracy to detect differences between structures in solution and in the crystal. The analysis can also be used to locate the principal axes of the magnetic susceptibility tensors of ferrihemes as well as providing orientational constraints for the axial ligands for the calculation of solution structures of paramagnetic proteins. This is particularly important since paramagnetic relaxation may make it impossible to observe NOE effects to the imidazole protons, leaving the geometry of the heme pocket poorly defined.