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Multicentre redox proteins participate in diverse metabolic processes, such as redox shuttling, multielectron catalysis, or long-distance electron conduction. The detail in which these processes can be analysed depends on the capacity of experimental methods to discriminate the multiple microstates that can be populated while the protein changes from the fully reduced to the fully oxidized state. The population of each state depends on the redox potential of the individual centres and on the magnitude of the interactions between the individual redox centres and their neighbours. It also depends on the interactions with binding sites for other ligands, such as protons, giving origin to the redox-Bohr effect. Modelling strategies that match the capacity of experimental methods to discriminate the contributions of individual centres are presented. These models provide thermodynamic and kinetic characterization of multicentre redox proteins. The current state of the art in the characterization of multicentre redox proteins is illustrated using the case of multiheme cytochromes involved in the process of extracellular electron transfer. In this new frontier of biological electron transfer, which can extend over distances that exceed the size of the individual multicentre redox proteins by orders of magnitude, current experimental data are still unable, in most cases, to provide discrimination between incoherent conduction by heme orbitals and coherent band conduction., (© 2025 The Author(s).)

Electro-bioremediation of exemplary water pollutants such as nitrogenous, phosphorous, and sulphurous compounds, hydrocarbons, metals and azo dyes has already been studied at a macro-scale level using mixed cultures. The technology has been generally established as a proof of concept at the technology readiness level (TRL) of 3, and there are already specific cases where the technology reached TRL 5. However, this technology is less utilized compared to traditional approaches. Although, mixed cultures result in high electro-biodegradation efficiency, their use hinders process' mechanistic insights which are better determined through pure cultures studies. This knowledge can lead to improved technologies. Therefore, this manuscript focuses on the specific pollutants' electro-biodegradation by pure cultures, assessing the availability of information regarding genes, enzymes, proteins and metabolites involved. Furthermore, studies characterizing the dominant genera or species are assessed, in which the available information at molecular level is evaluated. In total, less than 40 studies were found which were predominantly focused on the electro-biodegradation potential rather than the mechanistic insights. This highlights a gap in the field featuring a motivation to transitioning the focus on the study of pure cultures to unravel the mechanistic insights. Therefore, specific actions are suggested. Characterization of the mixed cultures followed by microorganisms' isolation is crucial. Thus, electroactive and biodegradation characteristics will be revealed using omics, genome annotation and transcriptional kinetics. This can lead to optimization at the microbiological level through genetic engineering, synthetic biology, mathematical modelling and strategic building of co-cultures. This research focus offers new avenues for sustainable wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 1 H, 13 C and 15 N 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., (© 2024. The Author(s).)

Background: Multiheme cytochromes c (MHC) provide prokaryotes with a broad metabolic versatility that contributes to their role in the biogeochemical cycling of the elements and in energy production in bioelectrochemical systems. However, MHC have only been isolated and studied in detail from a limited number of species. Among these, Desulfuromonadia spp. are particularly MHC-rich. To obtain a broad view of the diversity of MHC, we employed bioinformatic tools to study the cytochromome encoded in the genomes of the Desulfuromonadia class., Results: We found that the distribution of the MHC families follows a different pattern between the two orders of the Desulfuromonadia class and that there is great diversity in the number of heme-binding motifs in MHC. However, the vast majority of MHC have up to 12 heme-binding motifs. MHC predicted to be extracellular are the least conserved and show high diversity, whereas inner membrane MHC are well conserved and show lower diversity. Although the most prevalent MHC have homologues already characterized, nearly half of the MHC families in the Desulforomonadia class have no known characterized homologues. AlphaFold2 was employed to predict their 3D structures. This provides an atlas of novel MHC, including examples with high beta-sheet content and nanowire MHC with unprecedented high numbers of putative heme cofactors per polypeptide., Conclusions: This work illuminates for the first time the universe of experimentally uncharacterized cytochromes that are likely to contribute to the metabolic versatility and to the fitness of Desulfuromonadia in diverse environmental conditions and to drive biotechnological applications of these organisms., (© 2024. The Author(s).)

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., (© 2023 The Protein Society.)

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 CX 8 C 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 CX 8 C were isolated. These isoforms produced increased concentrations of H 2 O 2 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.

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.

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.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Madjarov, Soares, Paquete and Louro.)

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., Competing Interests: The authors declare no conflict of interest. CMP is serving as the guest editor and editorial board member of this journal. ROL is serving as the guest editor of this journal. We declare that CMP and ROL had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to RA., (© 2022 The Author(s). Published by IMR Press.)

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.

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., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added-value compounds from renewable and carbon-neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real-world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram-positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram-negative bacteria. In our study, three multiheme c -type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferriacetica , were cloned and over-expressed in E. coli . Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the peptidoglycan wall. However, based on the homology with the outer-membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola .

The growing interest on sustainable biotechnological processes for the production of energy and industrial relevant organic compounds have increased the discovery of electroactive organisms (i.e. organisms that are able to exchange electrons with an electrode) and the characterization of their extracellular electron transfer mechanisms. While most of the knowledge on extracellular electron transfer processes came from studies on Gram-negative bacteria, less is known about the processes performed by Gram-positive bacteria. In contrast to Gram-negative bacteria, Gram-positive bacteria lack an outer-membrane and contain a thick cell wall, which were thought to prevent extracellular electron transfer. However, in the last decade, an increased number of Gram-positive bacteria have been found to perform extracellular electron transfer, and exchange electrons with an electrode. In this mini-review the current knowledge on the extracellular electron transfer processes performed by Gram-positive bacteria is introduced, emphasising their electroactive role in bioelectrochemical systems. Also, the existent information of the molecular processes by which these bacteria exchange electrons with an electrode is highlighted. This understanding is fundamental to advance the implementation of these organisms in sustainable biotechnological processes, either through modification of the systems or through genetic engineering, where the organisms can be optimized to become better catalysts., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Microbial electrochemical technologies (METs) have emerged in recent years as a promising alternative green source of energy, with microbes consuming organic matter to produce energy or valuable byproducts. It is the ability of performing extracellular electron transfer that allows these microbes to exchange electrons with an electrode in these systems. The low levels of current achieved have been the limiting factor for the large-scale application of METs. Shewanella oneidensis MR-1 is one of the most studied electroactive organisms regarding extracellular electron transfer, and it has been shown that biofilm formation is a key factor for current generation. The transcription factor bolA has been identified as a central player in biofilm formation in other organisms, with its overexpression leading to increased biofilm. In this work we explore the effect of this gene in biofilm formation and current production by S. oneidensis MR-1. Our results demonstrate that an increased biofilm formation and consequent current generation was achieved by the overexpression of this gene. This information is crucial to optimize electroactive organisms toward their practical application in METs., (Copyright © 2020 Silva, Edel, Gescher and Paquete.)

Heme containing proteins are involved in a broad range of cellular functions, from oxygen sensing and transport to catalyzing oxidoreductive reactions. The two major types of cytochrome (b-type and c-type) only differ in their mechanism of heme attachment, but this has major implications for their cellular roles in both localization and mechanism. The b-type cytochromes are commonly cytoplasmic, or are within the cytoplasmic membrane, while c-type cytochromes are always found outside of the cytoplasm. The mechanism of heme attachment allows for complex c-type multiheme complexes, having the capacity to hold multiple electrons, to be assembled. These are increasingly being identified as secreted into the extracellular environment. For organisms that respire using extracellular substrates, these large multiheme cytochromes allow for electron transfer networks from the cytoplasmic membrane to the cell exterior for the reduction of extracellular electron acceptors. In this review the structures and functions of these networks and the mechanisms by which electrons are transferred to extracellular substrates is described., (© 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA) could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis. IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes., (Copyright © 2019 Costa et al.)

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 Δ bfe 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 Shewanella IMPORTANCE Shewanella 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., (Copyright © 2019 American Society for Microbiology.)

Multihaem cytochromes c are widespread in nature where they perform numerous roles in diverse anaerobic metabolic pathways. This is achieved in two ways: multihaem cytochromes c display a remarkable diversity of ways to organize multiple hemes within the protein frame; and the hemes possess an intrinsic reactive versatility derived from diverse spin, redox and coordination states. Here we provide a brief survey of multihaem cytochromes c that have been characterized in the context of their metabolic role. The contribution of multihaem cytochromes c to dissimilatory pathways handling metallic minerals, nitrogen compounds, sulfur compounds, organic compounds and phototrophism are described. This aims to set the stage for the further exploration of the vast unknown "cytochromome" that can be anticipated from genomic databases., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Anodic electroactive biofilms (EABs) need to overcome low current densities for applications such as microbial fuel cells or biosensors. EABs can store charge in self-produced redox proteins when temporarily left in open circuit, and discharge them once the electrode is appropriately repolarized, thus behaving as pseudocapacitors. Here we investigated the effect of such periodic polarization on the intrinsic nature of the EABs during their entire growth (i.e. starting from inoculation and for 10 days) on glassy carbon electrodes. An optimal periodic polarization (half-period of 10 s) greatly increased the maximum steady-state current density delivered by the Geobacter-dominated EABs (up to 1.10 ± 0.02 mA cm -2 , n = 3 electrodes) when compared to continuously polarized EABs (0.41 ± 0.04 mA cm -2 ); and increased the amount of electric charges produced per hour by 69 ± 17% even taking into account the half-periods of open circuit. This enhancement was highly correlated with a substantial increase in charge carriers concentration (10.6 ± 0.5 mM e- vs. 2.9 ± 0.6 mM e- ), allowing higher charge storage capacity and higher electron mobility across the EABs. Our results suggest that appropriate periodic polarizations may upregulate the expression of heme-containing redox proteins associated with the matrix, such as c-type cytochromes. The EABs grown under periodic polarization presented mushroom-like structures on their top layers, while EABs grown under continuous polarization were flat., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Electroactive microorganisms have attracted significant interest for the development of novel biotechnological systems of low ecological footprint. These can be used for the sustainable production of energy, bioremediation of metal-contaminated environments and production of added-value products. Currently, almost 100 microorganisms from the Bacterial and Archaeal domains are considered electroactive, given their ability to efficiently interact with electrodes in microbial electrochemical technologies. Cell-surface exposed conductive proteins are key players in the electron transfer between cells and electrodes. Interestingly, it seems that among the electroactive organisms identified so far, these cell-surface proteins fall into one of four groups. In this review, the different types of cell-surface conductive proteins found in electroactive organisms will be overviewed, focusing on their structural and functional properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 ., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

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.

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 (A–D). 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.

Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB-OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC.

Nanocarriers with a pH responsive behavior are receiving an ever growing attention due to their potential for promoting on-demand drug release and thus increase the therapeutic effectiveness of anti-tumoral pharmaceutics. However, the majority of these systems require costly, time-consuming and complex chemical modifications of materials or drugs to synthesize nanoparticles with pH triggered release. Herein, the development of dual drug loaded pH-responsive mesoporous silica nanoparticles (MSNs) with a calcium carbonate-based coating is presented as an effective alternative. This innovative approach allowed the loading of a non-steroidal anti-inflammatory drug (Ibuprofen) and Doxorubicin, with high efficiency. The resulting dual drug loaded MSNs have spherical morphology and a mean size of 171nm. Our results indicate that under acidic conditions the coating disassembles and the drugs are rapidly released, whereas at physiologic pH the release is slower and gradually increases with time. Furthermore, an improved cytotoxic effect was obtained for Doxorubicin-Ibuprofen MSNs coated with CaCO3 in comparison with non-coated particles. The cytotoxic effect of dual loaded carbonate coated particles, was similar to that of Doxorubicin+Ibuprofen free drug administration at 72h, even with the delivery of a significantly lower amount of drug by MSNs-CaCO3. These results suggest that the carbonate coating of MSNs is a promising approach to create a pH-sensitive template for a delivery system with application in cancer therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bioelectrochemical systems (BES) rely on the metabolism of sediment bacteria capable of forming electrogenic biofilms to generate electrical work. The environment across the thickness of the biofilm is variable and in order for the cells to maintain their viability they require molecular sensors that allow them to adapt their metabolism to their respective environment. The DcrA sensor from Desulfovibrio vulgaris and the GSU 0582 and 0935 sensor domains from Geobacter sulfurreducens appear to function as redox sensors. The SO2144 sensor domain from Shewanella oneidensis MR-1 and the cytochrome c″ from Methylophilus methylotrophus appear to function as NO sensors. Although M. methylotrophus is not known to colonize electrodes on BES, the characterization of cytochrome c″ serves to illustrate the general mechanism of NO sensing similar to that of the heme based sensors of sediment bacteria used in BES. In all cases, conformational changes initiated by the signal trigger the response. What appears to set these two groups of proteins apart is the poise of the heme in the sensors. In the case of redox sensors the hemes appear to be low spin iron II axially coordinated by two residues of the protein. In the case of NO sensors the heme appears to be high spin iron II with the distal coordination site vacant. Understanding of the molecular bases for signal and ligand discrimination may enable a fine control of biofilm formation in bioelectrochemical systems or the development of novel biosensors., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Non-steroidal anti-inflammatory drugs (NSAIDS) are emerging as a particularly valuable class of drugs due to their recently reported anti-tumoral activity in colorectal cancer. However, despite this tremendous potential, their bioavailability at the tumor microenvironment remains rather limited. To overcome this issue, in this work we synthesized biocompatible micellar nanocarriers composed of amphiphilic chitosan to deliver ibuprofen into breast cancer cells and evaluate its anti-tumor activity, while avoiding side-effects. Our results reveal that the formulations produced herein self-assembly into spherical micelles with suitable sizes for tumor accumulation (108-252 nm). Furthermore, by using a vortex-sonication method, ibuprofen was successfully encapsulated with high efficiency. Cell uptake studies show that ibuprofen-loaded micelles are readily internalized by tumor cells and deliver their cargo in the intracellular compartment as demonstrated by confocal microscopy images. This fact led to a remarkable reduction in cancer cell viability (<13%), at a relatively low drug dosage, illustrating the anti-tumoral activity of ibuprofen when delivered to breast cancer cells. These findings demonstrate the promising potential of chitosan micelles as carriers of cost-effective NSAIDS for application in breast cancer therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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.

Detailed thermodynamic and structural data measured in soluble monomeric multiheme cytochromes c provided the basis to investigate the functional significance of interactions between redox co-factors. The steep decay of intramolecular interactions with distance means that close proximity of the redox centers is necessary to modulate the intrinsic reduction potentials in a significant way. This ensures selection of specific populations during redox activity in addition to maintaining fast intramolecular electron transfer. Therefore, intramolecular interactions between redox co-factors play an important role in establishing the biological function of the protein by controlling how electrons flow through and are distributed among the co-factors., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The translation of non-viral gene replacement therapies for cancer into clinical application is currently hindered due to known issues associated with the effectiveness of plasmid DNA (pDNA) expression vectors and the production of gene delivery vehicles. Herein we report an integrative approach established on the synthesis of nanoparticulated carriers, in association with the supercoiled (sc) isoform purification of a p53 tumor suppressor encoding plasmid, to improve both delivery and transfection. An arginine-based chromatographic matrix with specific recognition for the different topoisoforms was used to completely isolate the biologically active sc pDNA. Our findings showed that the sc topoisoform is recovered under mild conditions with high purity and structural stability. In addition, to further enhance protection and transfection efficiency, the naked sc pDNA was encapsulated within chitosan nanoparticles by ionotropic gelation. The mild conditions for particle synthesis used in the former technique allowed the attainment of a high encapsulation efficiency for sc pDNA (>75%). Moreover, in vitro transfection experiments confirmed the reinstatement of the p53 protein expression and most importantly, the sc pDNA transfected cells exhibited the highest p53 expression levels when compared to other formulations. Overall, given the fact that sc pDNA topoisoform indeed enhances transgene expression rates this approach might have a profound impact on the development of a sustained nucleic acid-based therapy for cancer., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

Recent progress in bacterial genomic analysis has revealed a vast number of genes that encode c-type cytochromes that contain multiple heme cofactors. This high number of multiheme cytochromes in several bacteria has been correlated with their great respiratory flexibility, and in what concerns biotechnological applications, has been correlated with electricity production in Microbial Fuel Cells. Desulfuromonas acetoxidans, a member of the Geobactereaceae family, is one of these organisms for which the genome was recently made available, coding for 47 putative multiheme cytochromes. The growth of D. acetoxidans in different media allowed the identification of the cytochromes dominant in each condition. The triheme cytochrome c(7) is always present suggesting a key role in the bioenergetic metabolism of this organism, and a dodecaheme cytochrome of low homology with other proteins in the databases was also isolated. Different cytochromes are found for different growth conditions showing that their roles can be assigned to specific bioenergetic electron transfer routes.

Shewanella are facultative anaerobic bacteria of remarkable respiratory versatility that includes the dissimilatory reduction of metal ores. They contain a large number of multiheme c-type cytochromes that play a significant role in various anaerobic respiratory processes. Of all the cytochromes found in Shewanella, only the two most abundant periplasmic cytochromes, the small tetraheme cytochrome (STC) and flavocytochrome c(3) (Fcc(3)) have been structurally characterized. For these two proteins the molecular bases for their redox properties were determined using spectroscopic methods based on paramagnetic NMR, that allow the contribution of specific hemes to be discriminated. In this perspective these results are reviewed in the context of the continuing effort to understand the molecular mechanisms of electron transfer in the respiratory chains of these organisms.

Biological macromolecules involved in electron transfer reactions display chains of closely packed redox cofactors when long distances must be bridged. This is a consequence of the need to maintain a rate of transfer compatible with metabolic activity in the framework of the exponential decay of electron tunneling with distance. In this work intermolecular electron transfer was studied in kinetic experiments performed with the small tetraheme cytochrome from Shewanella oneidensis MR-1 and from Shewanella frigidimarina NCIMB400 using non-physiological redox partners. This choice allowed the effect of specific recognition and docking to be eliminated from the measured rates. The results were analyzed with a kinetic model that uses the extensive thermodynamic characterization of these proteins reported in the literature to discriminate the kinetic contribution of each heme to the overall rate of electron transfer. This analysis shows that, in this redox chain that spans 23 A, the kinetic properties of the individual hemes establish a functional specificity for each redox center. This functional specificity combined with the thermodynamic properties of these soluble proteins ensures directional electron flow within the cytochrome even outside of the context of a functioning respiratory chain.

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.

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c(3) isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.

Type I cytochrome c(3) is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c(3). This work presents the NMR assignment of the haem substituents in type I cytochrome c(3) isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c(3) belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.