Electron harvesting bacteria are key targets to develop microbial electrosynthesis technologies, which are valid alternatives for the production of value-added compounds without utilization of fossil fuels. Geobacter sulfurreducens, that is capable of donating and accepting electrons from electrodes, is one of the most promising electroactive bacteria. Its electron transfer mechanisms to electrodes have been progressively elucidated, however the electron harvesting pathways are still poorly understood. Previous studies showed that the periplasmic cytochromes PccH and GSU2515 are overexpressed in current-consuming G. sulfurreducens biofilms. PccH was characterized, though no putative partners have been identified. In this work, GSU2515 was characterized by complementary biophysical techniques and in silico simulations using the AlphaFold neural network. GSU2515 is a low-spin monoheme cytochrome with a disordered N-terminal region and an α-helical C-terminal domain harboring the heme group. The cytochrome undergoes a redox-linked heme axial ligand switch, with Met91 and His94 as distal axial ligand in the reduced and oxidized state, respectively. The reduction potential of the cytochrome is negative and is modulated by the pH in the physiological range: -78 mV at pH 6 and -113 mV at pH 7. Such pH-dependence coupled to the redox-linked switch of the axial ligand allows the cytochrome to drive a proton-coupled electron transfer step that is crucial to confer directionality to the respiratory chain. Biomolecular interactions and electron transfer experiments indicated that GSU2515 and PccH form a redox complex. Overall, the data obtained highlights for the first time how periplasmic proteins bridge the electron transfer between the outer and inner membrane in the electron harvesting pathways of G. sulfurreducens.

Geobacter sulfurreducens is a widely applied microorganism for the reduction of toxic metal salts, as an electron source for bioelectrochemical devices, and as a reagent for the synthesis of nanoparticles. In order to understand the influence of metal salts, and of electron transporting, multiheme c-cytochromes on the electron flux during respiration of G. sulfurreducens, the reduction kinetic of Fe³⁺, Co³⁺, V⁵⁺, Cr⁶⁺, and Mn⁷⁺ containing complexes were measured. Starting from the resting phase, each G. sulfurreducens cell produced an electron flux of 3.7 × 10⁵ electrons per second during the respiration process. Reduction rates were within ± 30% the same for the 6 different metal salts, and reaction kinetics were of zero order. Decrease of c-cytochrome concentrations by downregulation and mutation demonstrated that c-cytochromes stabilized respiration rates by variation of their redox states. Increasing Fe²⁺/heme levels increased electron flux rates, and induced respiration flexibility. The kinetic effects parallel electrochemical results of G. sulfurreducens biofilms on electrodes, and might help to optimize bioelectrochemical devices.

{Cytochromes are electron transfer proteins essential in various biological systems, playing crucial roles in the respiratory chains of bacteria. These proteins are particularly abundant in electrogenic microorganisms and are responsible for the efficient delivery of electrons to the cells’ exterior. The capability of sending electron outside the cells open new avenues to be explored for emerging biotechnological applications in bioremediation, microbial electrosynthesis and bioenergy fields. To develop these applications, it is critical to identify the different redox partners and elucidate the stepwise electron transfer along the respiratory paths. However, investigating direct electron transfer events between proteins with identical features in nearly all spectroscopic techniques is extremely challenging. NMR spectroscopy offers the possibility to overcome this difficulty by analysing the alterations of the spectral signatures of each protein caused by electron exchange events. The uncrowded NMR spectral regions containing the heme resonances of the cytochromes display unique and distinct signatures in the reduced and oxidized states, which can be explored to monitor electron transfer within the redox complex. In this study, we present a strategy for a fast and straightforward monitorization of electron transfer between c-type cytochromes, using as model a triheme periplasmic cytochrome (PpcA) and a membrane associated monoheme cytochrome (OmcF) from the electrogenic bacterium Geobacter sulfurreducens. The comparison between the 1D 1H NMR spectra obtained for samples containing the two cytochromes and for samples containing the individual proteins clearly demonstrated a unidirectional electron transfer within the redox complex. This strategy provides a simple and straightforward means to elucidate complex biologic respiratory electron transfer chains.}

Extracellular electron transfer is a key metabolic process of many organismsthat enables them to exchange electrons with extracellular electrondonors/acceptors. The discovery of organisms with these abilities and theunderstanding of their electron transfer processes has become a priority for thescientific and industrial community, given the growing interest on the use ofthese organisms in sustainable biotechnological processes. For example, inbioelectrochemical systems electrochemical active organisms can exchangeelectrons with an electrode, allowing the production of energy and added-valuecompounds, among other processes. In these systems, electrochemical activeorganisms exchange electrons with an electrode through direct or indirectmechanisms, using, in most cases, multiheme cytochromes. In numerouselectroactive organisms, these proteins form a conductive pathway that allowselectrons produced from cellular metabolism to be transferred across the cellsurface for the reduction of an electrode, or vice-versa. Here, the mechanisms bywhich the most promising electroactive bacteria perform extracellular electrontransfer will be reviewed, emphasizing the proteins involved in these pathways.The ability of some of the organisms to perform bidirectional electron transferand the pathways used will also be highlighted.

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 ( 42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C ( 11 kDa/three hemes) and D ( 10 kDa/three hemes), as well as for bi-domain CD ( 21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes.

Abstract Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from −150 to −358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.

The recent reclassification of the strict anaerobe Geobacter sulfurreducens bacterium as aerotolerant brought attention for oxidative stress protection pathways. Although the electron transfer pathways for oxygen detoxification are not well established, evidence was obtained for the formation of a redox complex between the periplasmic triheme cytochrome PpcA and the diheme cytochrome peroxidase MacA. In the latter, the reduction of the high-potential heme triggers a conformational change that displaces the axial histidine of the low-potential heme with peroxidase activity. More recently, a possible involvement of the triheme periplasmic cytochrome family (PpcA-E) in the protection from oxidative stress in G. sulfurreducens was suggested. To evaluate this hypothesis, we investigated the electron transfer reaction and the biomolecular interaction between each PpcA-E cytochrome and MacA. Using a newly developed method that relies on the different NMR spectral signatures of the heme proteins, we directly monitored the electron transfer reaction from reduced PpcA-E cytochromes to oxidized MacA. The results obtained showed a complete electron transfer from the cytochromes to the high-potential heme of MacA. This highlights PpcA-E cytochromes’ efficient role in providing the necessary reducing power to mitigate oxidative stress situations, hence contributing to a better knowledge of oxidative stress protection pathways in G. sulfurreducens.

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the extracellular environment, freely diffusing cytochromes or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons, while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.

Abstract Multiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA-E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra-promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.

Abstract Decades of research describe myriad redox enzymes that contain cofactors arranged in tightly packed chains facilitating rapid and controlled intra-protein electron transfer. Many such enzymes participate in extracellular electron transfer (EET), a process which allows microorganisms to conserve energy in anoxic environments by exploiting mineral oxides and other extracellular substrates as terminal electron acceptors. In this work, we describe the properties of the triheme cytochrome PgcA from Geobacter sulfurreducens. PgcA has been shown to play an important role in EET but is unusual in containing three CXXCH heme binding motifs that are separated by repeated (PT)x motifs, suggested to enhance binding to mineral surfaces. Using a combination of structural, electrochemical, and biophysical techniques, we experimentally demonstrate that PgcA adopts numerous conformations stretching as far as 180 Å between the ends of domains I and III, without a tightly packed cofactor chain. Furthermore, we demonstrate a distinct role for its domain III as a mineral reductase that is recharged by domains I and II. These findings show PgcA to be the first of a new class of electron transfer proteins, with redox centers separated by some nanometers but tethered together by flexible linkers, facilitating electron transfer through a tethered diffusion mechanism rather than a fixed, closely packed electron transfer chain.

Geobacter’s unique ability to perform extracellular electron transfer (EET) to electrodes in Microbial Fuel Cells (MFCs) has sparked the implementation of sustainable production of electrical energy. However, the electrochemical performance of Geobacter’s biofilms in MFCs remains challenging to implement industrially. Multiple approaches are being investigated to enhance MFC technologies. Protein engineering of multihaem cytochromes, key components of Geobacter’s EET pathways, can, conceivably, be pursued to improve the EET chain. The periplasmic cytochrome PpcA bridges ET from the inner to the outer membrane and its deletion impairs this crucial step. The functional characterisation of PpcA homologs from G. sulfurreducens (Gs) and G. metallireducens (Gm) revealed a significantly different redox behaviour even though they only differ by thirteen amino acids. In a previous study, we found that the single replacement of a tryptophan residue by methionine (W45M) in PpcAGm shifted the reduction potential value 33% towards that of PpcAGs. In this work, we expanded our investigation to include other non-conserved residues by conducting five mutation rounds. We identified the most relevant residues controlling the redox properties of PpcAGm. With just four mutations (K19, G25, N26, W45) the reduction potential value of PpcAGm was shifted 71% toward that of PpcAGs. Additionally, in the quadruple mutant, it was possible to replicate the haem oxidation order and the functional mechanisms of PpcAGs, which differ from those in PpcAGm. Overall, the mutants exhibit diverse redox and functional mechanisms that could be explored as a library for the future design of minimal, synthetic, ET chains in Geobacter.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s−1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105 s−1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.

Electroactive bacteria mediate electron exchange with external compounds through a process known as extracellular electron transfer (EET). A key step in EET is the transfer of electrons from the menaquinone pool to inner membrane-associated quinol-cytochrome c oxidoreductase complexes, which subsequently relay electrons to periplasmic redox partners. Gene-knockout and proteomic analyses have identified several critical components involved in EET in Geobacter sulfurreducens, including six inner membrane oxidoreductase gene clusters. Of these, three – CbcL, ImcH, and CbcBA - have been linked to specific respiratory pathways depending on the redox potential of the terminal electron acceptor. Cbc4 is one of the other inner membrane oxidoreductase complexes and is composed by three domains: a membrane-anchored tetraheme c-type cytochrome (CbcS), an iron–sulfur protein containing four [4Fe4S] clusters (CbcT), and an integral membrane subunit (CbcU). In this study, the sequence and AlphaFold model of CbcS were analyzed and its cytochrome domain was produced, and structurally and functionally characterized using Nuclear Magnetic Resonance spectroscopy. CbcS has four bis-histidine low-spin hemes and the structure of its hemecore is homologous to CymA and NrfH from Shewanella and Desulfovibrio species, respectively, despite differences on its axial ligands. Potentiometric titrations showed that the redox active window of CbcS overlaps with those of its putative redox partners of the triheme periplasmic cytochrome family (PpcA-E). However, NMR-monitored electron transfer experiments revealed that CbcS can transfer electrons to PpcA through the heme group closer to the C-terminal (heme IV). Together, these findings provide insights on a putative new respiratory pathway in G. sulfurreducens.

The bacterium Geotalea uraniireducens, commonly found in uranium-contaminated environments, plays a key role in bioremediation strategies by converting the soluble hexavalent form of uranium (UVI) into less soluble forms (e.g. UIV.). While most of the reduction and concomitant precipitation of uranium occur outside the cells, there have been reports of important reduction processes taking place in the periplasm. In any case, the triheme periplasmic cytochromes are crucial players, either by ensuring an effective interface between the cell´s interior and exterior or by directly participating in the reduction of the metal. Therefore, understanding the functional mechanism of the highly abundant G. uraniireducens’ triheme cytochromes is crucial to assist the elucidation on the respiratory pathways in this bacterium. In this work, a detailed functional characterization of the triheme cytochromes PpcA and PpcB from G. uraniireducens was conducted using NMR and visible spectroscopy techniques. Despite sharing high amino acid sequence and structural homology with their counterparts from G. sulfurreducens, the results obtained showed that the heme reduction potential values are less negative, the order of oxidation of the hemes is distinct, and the redox and redox-Bohr network of interactions revealed unprecedented functional mechanisms of the G. uraniireducens cytochromes. In these cytochromes, the reduction potential values of the three heme groups are much more similar, hence covering a narrow range of values, features that facilitate the directional electron flow from the inner membrane, thereby favouring the optimal reduction of uranium.