A New Paradigm of Multiheme Cytochrome Evolution by Grafting and Pruning Protein Modules.

Multiheme cytochromes play key roles in diverse biogeochemical cycles, but understanding the origin and evolution of these proteins is a challenge due to their ancient origin and complex structure. Up until now, the evolution of multiheme cytochromes composed by multiple redox modules in a single polypeptide chain was proposed to occur by gene fusion events. In this context, the pentaheme nitrite reductase NrfA and the tetraheme cytochrome c554 were previously proposed to be at the origin of the extant octa- and nonaheme cytochrome c involved in metabolic pathways that contribute to the nitrogen, sulfur, and iron biogeochemical cycles by a gene fusion event. Here, we combine structural and character-based phylogenetic analysis with an unbiased root placement method to refine the evolutionary relationships between these multiheme cytochromes. The evidence show that NrfA and cytochrome c554 belong to different clades, which suggests that these two multiheme cytochromes are products of truncation of ancestral octaheme cytochromes related to extant octaheme nitrite reductase and MccA, respectively. From our phylogenetic analysis, the last common ancestor is predicted to be an octaheme cytochrome with nitrite reduction ability. Evolution from this octaheme framework led to the great diversity of extant multiheme cytochromes analyzed here by pruning and grafting of protein modules and hemes. By shedding light into the evolution of multiheme cytochromes that intervene in different biogeochemical cycles, this work contributes to our understanding about the interplay between biology and geochemistry across large time scales in the history of Earth.

Electron transfer in Gram-positive bacteria: enhancement strategies for bioelectrochemical applications.

To this day, bioelectrochemical systems are still perceived as one of the rising technologies due to their versatile applications in electricity production, bioremediation, biosensors, and production of value-added products. While the majority of bioelectrochemical applications utilize Gram-negative bacteria, Gram-positive bacteria has not received sufficient attention. The lack of adequate knowledge about their electron transfer pathways along with the presence of a thick non-conductive cell wall are among the reasons behind their limited use. In this review, the electroactivity of Gram-positive bacteria will be covered describing the different pathways of electron transfer among different electroactive Gram-positive strains. Special emphasis will be given to the role of multiheme cytochromes, quorum sensing molecules, peptide-based signalling, and pili in the extracellular electron transfer. This review will also provide an overview of possible approaches for enhancement strategies of electron transfer such as enhancing biofilm formation, biocomposites and cell perforation. Understanding the fundamentals is critical for improving the use of Gram-positive bacteria in bioelectrochemical systems and may lead to the discovery of new applications.

Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role.

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.

Interaction studies between periplasmic cytochromes provide insights into extracellular electron transfer pathways of Geobacter sulfurreducens.

Geobacter bacteria usually prevail among other microorganisms in soils and sediments where Fe(III) reduction has a central role. This reduction is achieved by extracellular electron transfer (EET), where the electrons are exported from the interior of the cell to the surrounding environment. Periplasmic cytochromes play an important role in establishing an interface between inner and outer membrane electron transfer components. In addition, periplasmic cytochromes, in particular nanowire cytochromes that contain at least 12 haem groups, have been proposed to play a role in electron storage in conditions of an environmental lack of electron acceptors. Up to date, no redox partners have been identified in Geobacter sulfurreducens, and concomitantly, the EET and electron storage mechanisms remain unclear. In this work, NMR chemical shift perturbation measurements were used to probe for an interaction between the most abundant periplasmic cytochrome PpcA and the dodecahaem cytochrome GSU1996, one of the proposed nanowire cytochromes in G. sulfurreducens The perturbations on the haem methyl signals of GSU1996 and PpcA showed that the proteins form a transient redox complex in an interface that involves haem groups from two different domains located at the C-terminal of GSU1996. Overall, the present study provides for the first time a clear evidence for an interaction between periplasmic cytochromes that might be relevant for the EET and electron storage pathways in G. sulfurreducens.

Unraveling the electron transfer processes of a nanowire protein from Geobacter sulfurreducens.

The extracellular electron transfer metabolism of Geobacter sulfurreducens is sustained by several multiheme c-type cytochromes. One of these is the dodecaheme cytochrome GSU1996 that belongs to a new sub-class of c-type cytochromes. GSU1996 is composed by four similar triheme domains (A­D). The C-terminal half of the molecule encompasses the domains C and D, which are connected by a small linker and the N-terminal half of the protein contains two domains (A and B) that form one structural unit. It was proposed that this protein works as an electrically conductive device in G. sulfurreducens, transferring electrons within the periplasm or to outer-membrane cytochromes. In this work, a novel strategy was applied to characterize in detail the thermodynamic and kinetic properties of the hexaheme fragment CD of GSU1996. This characterization revealed the electron transfer process of GSU1996 for the first time, showing that a heme at the edge of the C-terminal of the protein is thermodynamic and kinetically competent to receive electrons from physiological redox partners. This information contributes towards understanding how this new sub-class of cytochromes functions as nanowires, and also increases the current knowledge of the extracellular electron transfer mechanisms in G. sulfurreducens.

Modulation of the reactivity of multiheme cytochromes by site-directed mutagenesis: moving towards the optimization of microbial electrochemical technologies.

Dissimilatory metal-reducing bacteria perform extracellular electron transfer, a metabolic trait that is at the core of a wide range of biotechnological applications. To better understand how these microorganisms transfer electrons from their metabolism to an extracellular electron acceptor, it is necessary to characterize in detail the key players in this process, the multiheme c-type cytochromes. Shewanella oneidensis MR-1 is a model organism for studying extracellular electron transfer, where the heme protein referred to as small tetraheme cytochrome is one of the most abundant multiheme cytochromes found in the periplasmic space of this bacterium. The small tetraheme cytochrome is responsible for the delivery of electrons to the porin-cytochrome supercomplexes that permeate the outer-membrane and reduce metallic minerals or electrodes. In this work, well-established thermodynamic and kinetic models that discriminate the electron transfer activity of the four individual hemes were employed to characterize a set of single amino-acid mutants of the small tetraheme cytochrome and their interaction with small inorganic electron donors and acceptors. The results show that electrostatics play an important role in the reactivity of the small tetraheme cytochrome with small inorganic electron partners, in particularly in the kinetics of the electron transfer processes. This thorough exploration using site-directed mutants provides key mechanistic insights to guide the rational manipulation of the proteins that are key players in extracellular electron transfer processes, towards the improvement of microbial electrochemical applications using dissimilatory metal-reducing bacteria.

Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella.

Many enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c3 of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron-two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella.

Unveiling the details of electron transfer in multicenter redox proteins.

Metalloproteins modulate the intrinsic properties of transition metals to achieve controlled catalysis, electron transfer, or structural stabilization. Those performing electron transport, redox proteins, are a diverse class of proteins with central roles in numerous metabolic and signaling pathways, including respiration and photosynthesis. Many redox proteins have applications in industry, especially biotechnology, making them the focus of intense research. Redox proteins may contain one or multiple redox centers of the same or a different type. The complexity of proteins with multiple redox centers makes it difficult to establish a detailed molecular mechanism for their activity. Thermodynamic and kinetic information can be interpreted using the molecular structure to elucidate the protein's functional mechanism. This Account reviews experimental strategies developed in recent years to determine the detailed thermodynamic properties of multicenter redox proteins and their kinetic properties during interactions with redox partners. These strategies allow the discrimination of thermodynamic and kinetic properties of each individual redox center. The thermodynamic characterization of the redox transitions results from the combined analysis of data from NMR and UV-visible spectroscopy. Meanwhile, the kinetic characterization of intermolecular electron transfer comes from stopped-flow spectrophotometry. We illustrate an application of these strategies to a particular redox protein, the small tetraheme cytochrome from the periplasmic space of Shewanella oneidensis MR-1. This protein is a convenient prototype for developing methods for the detailed analysis of multicenter electron transfer proteins because hemes have strong UV-visible absorption bands and because heme resonances have exquisite discrimination in NMR spectra. Nonetheless, the methods are fully generalizable. Ultimately, this Account highlights the relevance of detailed characterization of the thermodynamic and kinetic properties of redox proteins. These properties are responsible for the directionality and specificity of the electron transfer process in bioenergetic pathways; a more thorough characterization of these properties should allow better-designed proteins for industrial applications.

Mind the gap: cytochrome interactions reveal electron pathways across the periplasm of Shewanella oneidensis MR-1.

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.

Resonance assignments of cytochrome MtoD from the extracellular electron uptake pathway of sideroxydans lithotrophicus ES-1.

The contribution of Fe(II)-oxidizing bacteria to iron cycling in freshwater, groundwater, and marine environments has been widely recognized in recent years. These organisms perform extracellular electron transfer (EET), which constitutes the foundations of bioelectrochemical systems for the production of biofuels and bioenergy. It was proposed that the Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes soluble ferrous Fe(II) at the surface of the cell and performs EET through the Mto redox pathway. This pathway is composed by the periplasmic monoheme cytochrome MtoD that is proposed to bridge electron transfer between the cell exterior and the cytoplasm. This makes its functional and structural characterization, as well as evaluating the interaction process with its physiological partners, essential for understanding the mechanisms underlying EET. Here, we report the complete assignment of the heme proton and carbon signals together with a near-complete assignment of 1H, 13C and 15N backbone and side chain resonances for the reduced, diamagnetic form of the protein. These data pave the way to identify and structurally map the molecular interaction regions between the cytochrome MtoD and its physiological redox partners, to explore the EET processes of S. lithotrophicus ES-1.

Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation.

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.

A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions.

Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.

The quest to achieve the detailed structural and functional characterization of CymA.

Shewanella oneidensis MR-1 is a sediment organism capable of dissimilatory reduction of insoluble metal compounds such as those of Fe(II) and Mn(IV). This bacterium has been used as a model organism for potential applications in bioremediation of contaminated environments and in the production of energy in microbial fuel cells. The capacity of Shewanella to perform extracellular reduction of metals is linked to the action of several multihaem cytochromes that may be periplasmic or can be associated with the inner or outer membrane. One of these cytochromes is CymA, a membrane-bound tetrahaem cytochrome localized in the periplasm that mediates the electron transfer between the quinone pool in the cytoplasmic membrane and several periplasmic proteins. Although CymA has the capacity to regulate multiple anaerobic respiratory pathways, little is known about the structure and functional mechanisms of this focal protein. Understanding the structure and function of membrane proteins is hampered by inherent difficulties associated with their purification since the choice of the detergents play a critical role in the protein structure and stability. In the present mini-review, we detail the current state of the art in the characterization of CymA, and add recent information on haem structural behaviour for CymA solubilized in different detergents. These structural differences are deduced from NMR spectroscopy data that provide information on the geometry of the haem axial ligands. At least two different conformational forms of CymA are observed for different detergents, which seem to be related to the micelle size. These results provide guidance for the discovery of the most promising detergent that mimics the native lipid bilayer and is compatible with biochemical and structural studies.

Molecular Mechanisms of Microbial Extracellular Electron Transfer: The Importance of Multiheme Cytochromes.

Extracellular electron transfer is a key metabolic process of many organisms that enables them to exchange electrons with extracellular electron donors/acceptors. The discovery of organisms with these abilities and the understanding of their electron transfer processes has become a priority for the scientific and industrial community, given the growing interest on the use of these organisms in sustainable biotechnological processes. For example, in bioelectrochemical systems electrochemical active organisms can exchange electrons with an electrode, allowing the production of energy and added-value compounds, among other processes. In these systems, electrochemical active organisms exchange electrons with an electrode through direct or indirect mechanisms, using, in most cases, multiheme cytochromes. In numerous electroactive organisms, these proteins form a conductive pathway that allows electrons produced from cellular metabolism to be transferred across the cell surface for the reduction of an electrode, or vice-versa. Here, the mechanisms by which the most promising electroactive bacteria perform extracellular electron transfer will be reviewed, emphasizing the proteins involved in these pathways. The ability of some of the organisms to perform bidirectional electron transfer and the pathways used will also be highlighted.

Sporomusa ovata as Catalyst for Bioelectrochemical Carbon Dioxide Reduction: A Review Across Disciplines From Microbiology to Process Engineering.

Sporomusa ovata is a bacterium that can accept electrons from cathodes to drive microbial electrosynthesis (MES) of acetate from carbon dioxide. It is the biocatalyst with the highest acetate production rate described. Here we review the research on S. ovata across different disciplines, including microbiology, biochemistry, engineering, and materials science, to summarize and assess the state-of-the-art. The improvement of the biocatalytic capacity of S. ovata in the last 10 years, using different optimization strategies is described and discussed. In addition, we propose possible electron uptake routes derived from genetic and experimental data described in the literature and point out the possibilities to understand and improve the performance of S. ovata through genetic engineering. Finally, we identify current knowledge gaps guiding further research efforts to explore this promising organism for the MES field.

Deciphering Molecular Factors That Affect Electron Transfer at the Cell Surface of Electroactive Bacteria: The Case of OmcA from Shewanella oneidensis MR-1.

Multiheme cytochromes play a central role in extracellular electron transfer, a process that allows microorganisms to sustain their metabolism with external electron acceptors or donors. In Shewanella oneidensis MR-1, the decaheme cytochromes OmcA and MtrC show functional specificity for interaction with soluble and insoluble redox partners. In this work, the capacity of extracellular electron transfer by mutant variants of S. oneidensis MR-1 OmcA was investigated. The results show that amino acid mutations can affect protein stability and alter the redox properties of the protein, without affecting the ability to perform extracellular electron transfer to methyl orange dye or a poised electrode. The results also show that there is a good correlation between the reduction of the dye and the current generated at the electrode for most but not all mutants. This observation opens the door for investigations of the molecular mechanisms of interaction with different electron acceptors to tailor these surface exposed cytochromes towards specific bio-based applications.

Investigation of the Molecular Mechanisms of the Eukaryotic Cytochrome-c Maturation System.

Cytochromes-c are ubiquitous heme proteins with enormous impact at the cellular level, being key players in metabolic processes such as electron transfer chains and apoptosis. The assembly of these proteins requires maturation systems that catalyse the formation of the covalent thioether bond between two cysteine residues and the vinyl groups of the heme. System III is the maturation system present in Eukaryotes, designated CcHL or HCCS. This System requires a specific amino acid sequence in the apocytochrome to be recognized as a substrate and for heme insertion. To explore the recognition mechanisms of CcHL, the bacterial tetraheme cytochrome STC from Shewanella oneidensis MR-1, which is not a native substrate for System III, was mutated to be identified as a substrate. The results obtained show that it is possible to convert a bacterial cytochrome as a substrate by CcHL, but the presence of the recognition sequence is not the only factor that induces the maturation of a holocytochrome by System III. The location of this sequence in the polypeptide also plays a role in the maturation of the c-type cytochrome. Furthermore, CcHL appears to be able to catalyse the binding of only one heme per polypeptide chain, being unable to assemble multiheme cytochromes c, in contrast with bacterial maturation systems.

Let's chat: Communication between electroactive microorganisms.

Electroactive microorganisms can exchange electrons with other cells or conductive interfaces in their extracellular environment. This property opens the way to a broad range of practical biotechnological applications, from manufacturing sustainable chemicals via electrosynthesis, to bioenergy, bioelectronics or improved, low-energy demanding wastewater treatments. Besides, electroactive microorganisms play key roles in environmental bioremediation, significantly impacting process efficiencies. This review highlights our present knowledge on microbial interactions promoting the communication between electroactive microorganisms in a biofilm on an electrode in bioelectrochemical systems (BES). Furthermore, the immediate knowledge gaps that must be closed to develop novel technologies will also be acknowledged.

Evidence for Quinol Oxidation Activity of ImoA, a Novel NapC/NirT Family Protein from the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1.

Sideroxydans species are important chemolithoautotrophic Fe(II)-oxidizing bacteria in freshwater environments and play a role in biogeochemical cycling of multiple elements. Due to difficulties in laboratory cultivation and genetic intractability, the electron transport proteins required for the growth and survival of this organism remain understudied. In Sideroxydans lithotrophicus ES-1, it is proposed that the Mto pathway transfers electrons from extracellular Fe(II) oxidation across the periplasm to an inner membrane NapC/NirT family protein encoded by Slit_2495 to reduce the quinone pool. Based on sequence similarity, Slit_2495 has been putatively called CymA, a NapC/NirT family protein which in Shewanella oneidensis MR-1 oxidizes the quinol pool during anaerobic respiration of a wide range of substrates. However, our phylogenetic analysis using the alignment of different NapC/NirT family proteins shows that Slit_2495 clusters closer to NirT sequences than to CymA. We propose the name ImoA (inner membrane oxidoreductase) for Slit_2495. Our data demonstrate that ImoA can oxidize quinol pools in the inner membrane and is able to functionally replace CymA in S. oneidensis. The ability of ImoA to oxidize quinol in vivo as opposed to its proposed function of reducing quinone raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus. IMPORTANCE Fe(II)-oxidizing bacteria play an important role in biogeochemical cycles. At circumneutral pH, these organisms perform extracellular electron transfer, taking up electrons from Fe(II) outside the cell, potentially through a porin-cytochrome complex in the outer membrane encoded by the Mto pathway. Electrons from Fe(II) oxidation would then be transported to the quinone pool in the inner membrane via periplasmic and inner membrane electron transfer proteins. Directly demonstrating the functionality of genes in neutrophilic iron oxidizers is challenging due to the absence of robust genetic methods. Here, we heterologously expressed a NapC/NirT family tetraheme cytochrome ImoA, encoded by Slit_2495, an inner membrane protein from the Gram-negative Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1, proposed to be involved in extracellular electron transfer to reduce the quinone pool. ImoA functionally replaced the inner membrane c-type cytochrome CymA in the Fe(III)-reducing bacterium Shewanella oneidensis. We suggest that ImoA may function primarily to oxidize quinol in S. lithotrophicus.

Mapping the iron binding site(s) on the small tetraheme cytochrome of Shewanella oneidensis MR-1.

In the model microbe Shewanella oneidensis, multi-heme proteins are utilized for respiratory metabolism where metals serve as the terminal electron acceptor. Among those is the periplasm-localized small tetraheme cytochrome (STC). STC has been extensively characterized structurally and electrochemically to which electron flow in and out of the protein has been modeled. However, until the present work, no kinetic studies have been performed to probe the route of electron flow or to determine the iron-binding site on STC. Using iron chelated by EDTA, NTA, or citrate, we have used chemical modification, site-directed mutagenesis along with isothermal titration calorimetry (ITC), and stopped-flow measurements to identify the iron binding site of STC. Chemical modifications of STC revealed that carboxyl groups on STC are involved in binding of EDTA-Fe(3+). Scanning mutagenesis was performed on Asp and Glu to probe the putative iron-binding site on STC. Two STC mutants (D21N; D80N) showed ∼70% decrease in observed electron transfer rate constant with EDTA-Fe(3+) from transient-state kinetic measurements. The impaired reactivity of STC (D80N/D21N) with EDTA-Fe(3+) was further confirmed by a significant decrease (>10-fold) in iron binding affinity.