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.

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.

Shewanella oneidensis is the best understood model organism for the study of dissimilatory iron reduction. This review focuses on the current state of our knowledge regarding this extracellular respiratory process and highlights its physiologic, regulatory and biochemical requirements. It seems that we have widely understood how respiratory electrons can reach the cell surface and what the minimal set of electron transport proteins to the cell surface is. Nevertheless, even after decades of work in different research groups around the globe there are still several important questions that were not answered yet. In particular, the physiology of this organism, the possible evolutionary benefit of some responses to anoxic conditions, as well as the exact mechanism of electron transfer onto solid electron acceptors are yet to be addressed. The elucidation of these questions will be a great challenge for future work and important for the application of extracellular respiration in biotechnological processes.

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Dissimilatory metal reducing organisms play key roles in the biogeochemical cycle of metals as well as in the durability of submerged and buried metallic structures. The molecular mechanisms that support electron transfer across the microbe-metal interface in these organisms remain poorly explored. It is known that outer membrane proteins, in particular multiheme cytochromes, are essential for this type of metabolism, being responsible for direct and indirect, via electron shuttles, interaction with the insoluble electron acceptors. Soluble electron shuttles such as flavins, phenazines, and humic acids are known to enhance extracellular electron transfer. In this work, this phenomenon was explored. All known outer membrane decaheme cytochromes from Shewanella oneidensis MR-1 with known metal terminal reductase activity and a undecaheme cytochrome from Shewanella sp. HRCR-6 were expressed and purified. Their interactions with soluble electron shuttles were studied using stopped-flow kinetics, NMR spectroscopy, and molecular simulations. The results show that despite the structural similarities, expected from the available structural data and sequence homology, the detailed characteristics of their interactions with soluble electron shuttles are different. MtrC and OmcA appear to interact with a variety of different electron shuttles in the close vicinity of some of their hemes, and with affinities that are biologically relevant for the concentrations typical found in the medium for this type of compounds. All data support a view of a distant interaction between the hemes of MtrF and the electron shuttles. For UndA a clear structural characterization was achieved for the interaction with AQDS a humic acid analog. These results provide guidance for future work of the manipulation of these proteins toward modulation of their role in metal attachment and reduction.