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Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR‐1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell‐cell click chemistry strategy. By introducing alkyne‐ or azide‐modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C‐type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell‐cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications. [ABSTRACT FROM AUTHOR]

Cupriavidus necator, despite lacking direct electron transfer capabilities, demonstrates efficient reduction of various redox mediators in oxygen‐free cultivation within bioelectrochemical systems. This study investigates the reduction site of ferricyanide through inhibition and expression rate analysis of oxygen and nitrate respiration chain complexes, comparing aerobic cultivation conditions with fructose as carbon and electron donor to autotrophic (CO2/H2/O2) and anodic cultivation conditions (fructose/anode). Azide inhibition identified cytochrome c oxidase as the primary complex facilitating electron transfer to ferricyanide, with a secondary role proposed for nitrite reductase NirS, demonstrating a 3.9±1.1‐fold higher expression when exposed to anodic conditions. The 2.9±0.6‐fold increase in the expression of the natural porin OmpA under anodic conditions implies its potential involvement in ferricyanide uptake. Additionally, chemically permeabilizing cell membranes with cetyltrimethylammonium bromide doubles ferricyanide reduction rates without an anode present, offering insights for optimizing redox mediation in C. necator based bioelectrochemical systems. This study opens up new possibilities for the targeted optimization of mediated electron transfer in C. necator and other organisms. [ABSTRACT FROM AUTHOR]

Anaerobic digestion is a valuable strategy for environmentally-friendly and sustainable disposal of kitchen waste. However, anaerobic digestion of kitchen waste (KWAD) is prone to acidification, which can cause poor reaction outcomes. Utilising conductive materials can ameliorate hydrogen, acid and ammonia inhibition during KWAD, ultimately enhancing methane production. This technique is widely applied to increase methane yield from anaerobic digestion. An analysis of conductive materials to promote methanogenesis in KWAD indicates that applying such materials can enhance methane production, alleviate acid and ammonia inhibition, enrich functional microorganisms, and replace conductive pili and c-type cytochromes for direct interspecies electron transfer. Additionally, metagenomics has shown that conductive materials can increase the expression of functional genes on microbial metabolism, ultimately improving the activity of key enzymes in the methanogenic process. Finally, it is recommended that the combination of macrogenomics, macrotranscriptomics and macroproteomics should be used to investigate KWAD's mechanism and to anticipate future engineering applications that reduce treatment expenses and ensure environmentally responsible disposal. [ABSTRACT FROM AUTHOR]

Geobacter sulfurreducens mediates extracellular electron transfer (EET) reactions with different substrates, such as solid-phase Fe(III)-containing minerals, anodes and the cells of Geobacter metallireducens. To compare their roles in EET, the pilA-N, omcE, omcS, omcT and omcZ genes of G. sulfurreducens were systematically deleted. All mutants showed impaired and varied ability to form biofilms on nonconductive surface. Deletion of omcE also impaired bacterial ability to reduce ferrihydrite, but its impacts on the ability for anode reduction and the co-culture of G. metallireducens-G. sulfurreducens were minimal. The mutant without omcS showed diminished ability to reduce ferrihydrite and to form the co-culture, but was able to regain its ability to reduce anodes. Deletion of omcT, omcZ or pilA-N alone impaired bacterial ability to reduce ferrihydrite and anodes and to form the co-culture. Deletion of all tested genes abolished bacterial ability to reduce ferrihydrite and anodes. Triple-deletion of all omcS, omcT and omcZ abolished the ability of G. sulfurreducens to co-culture with G. metallireducens. However, deletion of only omcZ or pilA-N or both omcS and omcT abolished the ability of G. sulfurreducens without hydrogenase gene hybL to co-culture with G. metallireducens, which show their indispensable roles in direct electron transfer from G. metallireducens to G. sulfurreducens. Thus, the roles of pilA-N, omcE, omcS, omcT and omcZ for G. sulfurreducens in EET vary substantially, which also suggest that possession of PilA-N and multiple cytochromes of different structures enables G. sulfurreducens to mediate EET reactions efficiently with substrates of different properties. [ABSTRACT FROM AUTHOR]

Electroactive microbial cells have evolved unique extracellular electron transfer to conduct the reactions via redox outer-membrane (OM) proteins. However, the electron transfer mechanism at the interface of OM proteins and nanomaterial remains unclear. In this study, the mechanism for the electron transfer at biological/inorganic interface is investigated by integrating molecular modeling with electrochemical and spectroscopic measurements. For this purpose, a model system composed of OmcA, a typical OM protein, and the hexagonal tungsten trioxide (h-WO3) with good biocompatibility is selected. The interfacial electron transfer is dependent mainly on the special molecular configuration of OmcA and the microenvironment of the solvent exposed active center. Also, the apparent electron transfer rate can be tuned by site-directed mutagenesis at the axial ligand of the active center. Furthermore, the equilibrium state of the OmcA/h-WO3 systems suggests that their attachment is attributed to the limited number of residues. The electrochemical analysis of OmcA and its variants reveals that the wild type exhibits the fastest electron transfer rate, and the transient absorption spectroscopy further shows that the axial histidine plays an important role in the interfacial electron transfer process. This study provides a useful approach to promote the site-directed mutagenesis and nanomaterial design for bioelectrocatalytic applications. [ABSTRACT FROM AUTHOR]

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments. [ABSTRACT FROM AUTHOR]

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

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used. Anaerobic methanotrophic archaea play crucial roles in the methane cycle. Here, Zhang et al. provide experimental evidence supporting that multi-heme cytochromes mediate extracellular electron transfer for the reduction of metals and electrodes in these microorganisms. [ABSTRACT FROM AUTHOR]

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer. [ABSTRACT FROM AUTHOR]

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

Photosynthesis converts light energy to chemical energy to fuel life on earth. Light energy is harvested by antenna pigments and transferred to reaction centers (RCs) to drive the electron transfer (ET) reactions. Here, we present cryo-electron microscopy (cryo-EM) structures of two forms of the RC from the microaerophilic Chloracidobacterium thermophilum (CabRC): one containing 10 subunits, including two different cytochromes; and the other possessing two additional subunits, PscB and PscZ. The larger form contained 2 Zn-bacteriochlorophylls, 16 bacteriochlorophylls, 10 chlorophylls, 2 lycopenes, 2 hemes, 3 Fe4S4 clusters, 12 lipids, 2 Ca2+ ions and 6 water molecules, revealing a type I RC with an ET chain involving two hemes and a hybrid antenna containing bacteriochlorophylls and chlorophylls. Our results provide a structural basis for understanding the excitation energy and ET within the CabRC and offer evolutionary insights into the origin and adaptation of photosynthetic RCs. During photosynthesis, light energy is harvested by an antenna systems and delivered to the reaction centers (RCs) for charge separation and electron transfer (ET). The authors report cryo-EM structures of two forms of RC from the microaerophilic Chloracidobacterium thermophilum (CabRC), providing a structural basis for ET within the CabRC. [ABSTRACT FROM AUTHOR]

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR. [ABSTRACT FROM AUTHOR]

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET) is an effective mechanism for microbial species to exchange electrons cooperatively during syntrophic metabolism. It is generally accepted that DIET is mainly mediated by electrically conductive pili and outer surface c -type cytochromes (c -Cyts). However, as an extracellular matrix is ubiquitous and abundant on the surface of microorganisms, the effect and mechanism of exopolysaccharides on DIET are still unclear. This study constructed a co-culture of exopolysaccharides-deficient Geobacter sulfurreducens with Geobacter metallireducens to explore the role of exopolysaccharides in DIET. Results revealed that the deficiency of exopolysaccharides extended the metabolic period of the co-culture by 44.4% and changed the proportions of each species in the co-culture. The exopolysaccharides-deficient co-culture failed to form large, tight spherical aggregates and the expression of c -Cyts and pili was decreased. The addition of magnetite and granular activated carbon (GAC), respectively, might compensate for the functions of c -Cyts and pili in the first generation of co-culture, but the stimulatory effect on the metabolic stable period co-culture was fairly limited. These findings demonstrate that non-conductive exopolysaccharides are an important component of DIET aggregates and an extracellular matrix for DIET-required c -Cyts. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Anaerobic methanotrophic (ANME) archaea have recently been reported to be capable of using insoluble extracellular electron acceptors via extracellular electron transfer (EET). In this study, we investigated EET by a microbial community dominated by " Candidatus Methanoperedens" archaea at the anode of a bioelectrochemical system (BES) poised at 0 V vs. standard hydrogen electrode (SHE), in this way measuring current as a direct proxy of EET by this community. After inoculation of the BES, the maximum current density was 274 mA m–2 (stable current up to 39 mA m–2). Concomitant conversion of 13CH4 into 13CO2 demonstrated that current production was methane-dependent, with 38% of the current attributed directly to methane supply. Based on the current production and methane uptake in a closed system, the Coulombic efficiency was about 17%. Polarization curves demonstrated that the current was limited by microbial activity at potentials above 0 V. The metatranscriptome of the inoculum was mined for the expression of c -type cytochromes potentially used for EET, which led to the identification of several multiheme c -type cytochrome-encoding genes among the most abundant transcripts in " Ca. Methanoperedens." Our study provides strong indications of EET in ANME archaea and describes a system in which ANME-mediated EET can be investigated under laboratory conditions, which provides new research opportunities for mechanistic studies and possibly the generation of axenic ANME cultures. [ABSTRACT FROM AUTHOR]

[Display omitted] • NCDs (C: N precursor = 1:0.5–1:3) were used to modified the biocathode. • NCDs 1:1 modification achieved highest acetate synthesis of MES (1.9 ± 0.1 mM d−1). • Pyrrolic N positively correlated with Sporomusa abundance and cytochrome expression. • NCDs 1:1 accelerated the EEU of S. ovata by upregulating cytochrome expression. • NCDs 1:1 enhanced the metabolism of S. ovata by increasing 2 H+-pumping step. This study investigated the molecular mechanism behind the highly efficient performance of nitrogen-doped carbon dots (NCDs)-assisted microbial electrosynthesis systems (MESs). The impact of NCDs (C:N precursor = 1:0.5–1:3) on acetogens was examined in the biocathode. The highest electrocatalytic performance was observed with NCDs 1:1. The maximum acetate production rate of 1.9 ± 0.1 mM d−1 was achieved in NCDs 1:1 - modified MESs, which was 26.7–216.7 % higher than other MESs (0.6–1.5 mM d−1). With NCDs 1:1 modified, the biocathode exhibited a 129.3–186.8 % increase in the abundance of Sporomusa , and 38.5–104.6 % increase in cytochrome expression (cydAB , cybH). Transcriptome confirmed that cytochromes played a crucial role in the extracellular electron uptake (EEU) of NCDs 1:1 -modified Sporomusa. NCDs 1:1 enhanced EEU efficiency, thereby increasing the two H+-pumping steps and accelerating microbial CO 2 fixation. These results provide valuable insights into increasing CO 2 fixation by maximizing EEU efficiency in acetogens. [ABSTRACT FROM AUTHOR]

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here, we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features that separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor. A comparative genomics study of anaerobic methanotrophic (ANME) archaea reveals the genetic "parts list" associated with the repeated evolutionary transition between methanogenic and methanotrophic metabolism in the archaeal domain of life. [ABSTRACT FROM AUTHOR]

Natural microbes employing c‐type cytochromes (c‐Cyts) for extracellular electron transport (EET) were greatly valuable to develop redox‐based bioelectrochemical applications. However, low levels of c‐Cyt expression limited the phylogenetically diverse Gram‐positive species to be used in bioelectrochemical devices. This work reported the remarkable finding that a high level of c‐Cyts was expressed in the cells of the Gram‐positive strain Lysinibacillus varians GY32. c‐Cyts in Lysinibacillus varians GY32 cells were expressed at a level close to those in Shewanella oneidensis. These c‐Cyts were found to cluster in cytoplasmic membrane and periplasmic space, along the length of Lysinibacillus varians GY32 cell. With voltammetry and spectroelectrochemical titration, phenazine methosulfate‐mediated electron transfer between the c‐Cyts and an electrode was proved. These results expanded the accessible natural models of EET and suggested a new microbial platform for developing bioelectrochemical applications. [ABSTRACT FROM AUTHOR]

During photosynthesis, electrons are transferred between the cytochrome b 6 f complex and photosystem I. This is carried out by the protein plastocyanin in plant chloroplasts, or by either plastocyanin or cytochrome c 6 in many cyanobacteria and eukaryotic algal species. There are three further cytochrome c 6 homologs: cytochrome c 6A in plants and green algae, and cytochromes c 6B and c 6C in cyanobacteria. The function of these proteins is unknown. Here, we present a comprehensive analysis of the evolutionary relationship between the members of the cytochrome c 6 family in photosynthetic organisms. Our phylogenetic analyses show that cytochromes c 6B and c 6C are likely to be orthologs that arose from a duplication of cytochrome c 6, but that there is no evidence for separate origins for cytochromes c 6B and c 6C. We therefore propose renaming cytochrome c 6C as cytochrome c 6B. We show that cytochrome c 6A is likely to have arisen from cytochrome c 6B rather than by an independent duplication of cytochrome c 6, and present evidence for an independent origin of a protein with some of the features of cytochrome c 6A in peridinin dinoflagellates. We conclude with a new comprehensive model of the evolution of the cytochrome c 6 family which is an integral part of understanding the function of the enigmatic cytochrome c 6 homologs. [ABSTRACT FROM AUTHOR]

The electrode‐immobilized Met80Ala variant of yeast iso‐1 cytochrome c in mixed water/dimethylsulfoxide (DMSO) solutions up to 60 % v/v DMSO shows thermodynamic and kinetic parameters of electron exchange and electrocatalytic properties towards O2 reduction fully comparable to those in water. This is the result of moderate protein conformational changes thanks to immobilization that, to a certain extent, preserves protein structure, possibly due to the constraints on protein mobility/flexibility induced by the electrostatic interactions with the electrode‐coating SAM. Upon increasing the DMSO content of the mixed solution beyond 60 %, a much larger perturbation occurs that leads to the progressive loss of the electrocatalytic ability. Therefore, under these conditions, the organic solvent remarkably affects the structure and properties of the protein probably involving major conformational changes or even the replacement of the 6th axial hydroxide ligand of the heme iron with a strong protein ligand, possibly a lysine residue. [ABSTRACT FROM AUTHOR]

Extracellular electron transfer (EET) is an important biological process in microbial physiology as found in dissimilatory metal oxidation/reduction and interspecies electron transfer in syntrophy in natural environments. EET also plays a critical role in microorganisms relevant to environmental biotechnology in metal-contaminated areas, metal corrosion, bioelectrochemical systems, and anaerobic digesters. Geobacter species exist in a diversity of natural and artificial environments. One of the outstanding features of Geobacter species is the capability of direct EET with solid electron donors and acceptors, including metals, electrodes, and other cells. Therefore, Geobacter species are pivotal in environmental biogeochemical cycles and biotechnology applications. Geobacter sulfurreducens, a representative Geobacter species, has been studied for direct EET as a model microorganism. G. sulfurreducens employs electrically conductive pili (e-pili) and c-type cytochromes for the direct EET. The biological function and electronics applications of the e-pili have been reviewed recently, and this review focuses on the cytochromes. Geobacter species have an unusually large number of cytochromes encoded in their genomes. Unlike most other microorganisms, Geobacter species localize multiple cytochromes in each subcellular fraction, outer membrane, periplasm, and inner membrane, as well as in the extracellular space, and differentially utilize these cytochromes for EET with various electron donors and acceptors. Some of the cytochromes are functionally redundant. Thus, the EET in Geobacter is complicated. Geobacter coordinates the cytochromes with other cellular components in the elaborate EET system to flourish in the environment. [ABSTRACT FROM AUTHOR]

The fastest-growing microbe Vibrio natriegens is an excellent platform for bioproduction processes. Until now, this marine bacterium has not been examined for bioremediation applications, where the production of substantial amounts of biomass would be beneficial. V. natriegens can perform extracellular electron transfer (EET) to Fe(III) via a single porin-cytochrome circuit conserved in Vibrionaceae. Electroactive microbes capable of EET to Fe(III) usually also reduce toxic metals such as carcinogenic Cr(VI), which is converted to Cr(III), thus decreasing its toxicity and mobility. Here, the performance of V. natriegens was explored for the bioremediation of Cr(VI). At a density of 100 mg/mL, V. natriegens removed 5–20 mg/L Cr(VI) within 30 s and 100 mg/L Cr(VI) within 10 min. In comparison, the model bacterium Escherichia coli grown to a comparable cell density removed Cr(VI) 36 times slower. To eliminate Cr(VI), V. natriegens had to be metabolically active, and functional outer-membrane c -type cytochromes were required. At the end of the Cr(VI) removal process, V. natriegens had reduced all of it into Cr(III) while adsorbing more than half of the metallic ions. These results demonstrate that V. natriegens , with its fast metabolism, is a viable option for the rapid treatment of aqueous pollution with Cr. [Display omitted] • The fast-growing microbe Vibrio natriegens can reduce and adsorb toxic chromium. • High-density V. natriegens removed 5–20 mg/L Cr(VI) within 30 s. • V. natriegens at the same density also removed 100 mg/L Cr(VI) within minutes. • Cr(VI) was reduced extracellularly and Cr(III) was adsorbed by the microbes. • A porin-cytochrome complex is likely involved in Cr(VI) conversion. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel hybrid system based on AGS to enhance methanogenesis. • Improve functional genes of methanogenic (mcrA) and electric transfer (omcZ). • Regulated the dense structure of EPS in AGS. • (Fe(III)/Fe(II)) as an intermediate station of electron transfer. • Promoted resistance to membrane contamination. Biotic-abiotic hybrid systems are promising technologies to enhance methane production in anaerobic wastewater treatment. However, the dense structure of the extracellular polymeric substances (EPS) present in anaerobic granular sludge (AGS) poses challenges with respect to the implementation of hybrid systems and efficient interspecies electron transfer. In this study, the use of AGS with a Ni/Fe layered double hydroxide@activated carbon (Ni/Fe LDH@C-AGS) was investigated in an anaerobic membrane bioreactor (AnMBR). The hybrid system showed a significant increase of 82% in methane production. Further research revealed that Ni/Fe LDH@C regulated the dense structure of EPS, stimulated the production of cytochromes, and facilitated the decomposition of nonconductive substances. Surprisingly, the hybrid system also promoted resistance to membrane fouling and extended membrane life by 81%. This study provides insights into the operation of a biotic-abiotic hybrid system by regulating the dense structure of EPS ultimately resulting in an enhanced methane production. [ABSTRACT FROM AUTHOR]

Magnetic field (MF, 200 mT) and Fe 3 O 4 (26 g/m2) are simultaneously employed to enhance acetate production from bicarbonate reduction in Serratia marcescens catalyzed cathode of microbial electrosynthesis system (MES), reaching 8.5 mM at an operational time of 9 d, higher than the controls by 1.7-time (Fe 3 O 4 only), 1.8-fold (MF only), and 2.3-time (neither Fe 3 O 4 nor MF). This confirms synergetic interaction of the MF and Fe 3 O 4 for enhanced acetate production in the MES. The presence of MF and Fe 3 O 4 induces the S. marcescens physiologically releasing more extracellular polymeric substances with a compositional diversity and containing more c -type cytochromes for facilitating extracellular electron transfer for efficient acetate production. Moreover, the favorable MF for less shedding (approximate 40%) of Fe 3 O 4 from the biocathode, also contributes to the efficient MES. This study provides a feasible strategy of the Fe 3 O 4 and MF for efficient acetate production from bicarbonate reduction in the MES. Image 1 • Synergetic interaction of magnetic field and loaded Fe 3 O 4 is observed in MES. • Magnetic field favors for less shedding of loaded Fe 3 O 4 from biocathode. • Magnetic field and Fe 3 O 4 regulate electrotrophic physiological activity. • Electrotroph releases EPS with a compositional diversity. • EPS contain c -type cytochromes for facilitating extracellular electron transfer. [ABSTRACT FROM AUTHOR]

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c -type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments. [ABSTRACT FROM AUTHOR]

Anaerobic microorganisms of the Geobacter genus are effective electron sources for the synthesis of nanoparticles, for bioremediation of polluted water, and for the production of electricity in fuel cells. In multistep reactions, electrons are transferred via iron/heme cofactors of c‐type cytochromes from the inner cell membrane to extracellular metal ions, which are bound to outer membrane cytochromes. We measured electron production and electron flux rates to 5×105 e s−1 per G. sulfurreducens. Remarkably, these rates are independent of the oxidants, and follow zero order kinetics. It turned out that the microorganisms regulate electron flux rates by increasing their Fe2+/Fe3+ ratios in the multiheme cytochromes whenever the activity of the extracellular metal oxidants is diminished. By this mechanism the respiration remains constant even when oxidizing conditions are changing. This homeostasis is a vital condition for living systems, and makes G. sulfurreducens a versatile electron source. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO), the Cu A, heme a , heme a 3 , Cu B enzyme of respiratory chain, converts the free energy released by aerobic cytochrome c oxidation into a membrane electrochemical proton gradient (ΔμH+). ΔμH+ derives from the membrane anisotropic arrangement of dioxygen reduction to two water molecules and transmembrane proton pumping from a negative (N) space to a positive (P) space separated by the membrane. Spectroscopic, potentiometric, and X-ray crystallographic analyses characterize allosteric cooperativity of dioxygen binding and reduction with protonmotive conformational states of CcO. These studies show that allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane ΔμH+. (i) Dioxygen binding to Fe a 3 at BNC selects a CcO state in which the conformation of the subunit I-helix X, next in sequence to the hemes a and a 3 histidine axial ligands, opens access of pumped protons from the N space to a Mg2+-storage site at the opposite (P) membrane side. (ii) Sequential electron transfer at Cu A and heme a is associated with additional CcO conformational change in which protons are translocated from the Mg2+-site to the propionate(s) of heme a. From there, protons are expelled in the P space on heme a oxidation by electrostatic repulsion along a hydrogen bond network of amino acid residues and/or structured water molecules. Image 1 • Cytochrome c oxidase converts the energy released in cytochrome c oxidation into a transmembrane DmH+. • The molecular mechanism of ΔμH+ generation in cytochrome c oxidase is a topic of advanced functional and structural studies. • Allosteric cooperativity in A1-type CcO plays a key role in proton energy conversion. • A scheme of the catalytic cycle in cytochrome c oxidase, which highlights the role of allosteric cooperativity, is proposed. [ABSTRACT FROM AUTHOR]

The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of direct interspecies electron transfer (DIET) in the environment. Here we tested eleven new co-culture combinations between methanogens and electrogens. Previously, only the most electrogenic Geobacter paired by DIET with Methanosarcinales methanogens, namely G. metallireducens and G. hydrogenophilus. Here we provide additional support, and show that five additional Methanosarcinales paired with G. metallireducens, while a strict hydrogenotroph could not. We also show that G. hydrogenophilus, which is incapable to grow with a strict hydrogenotrophic methanogen, could pair with a strict non-hydrogenotrophic Methanosarcinales. Likewise, an electrogen outside the Geobacter cluster (Rhodoferrax ferrireducens) paired with Methanosarcinales but not with strict hydrogenotrophic methanogens. The ability to interact with electrogens appears to be conserved among Methanosarcinales, the only methanogens with c-type cytochromes, including multihemes (MHC). Nonetheless, MHC, which are often linked to extracellular electron transfer, were neither unique nor universal to Methanosarcinales and only two of seven Methanosarcinales tested had MHC. Of these two, one strain had an MHC-deletion knockout available, which we hereby show is still capable to retrieve extracellular electrons from G. metallireducens or an electrode suggesting an MHC-independent strategy for extracellular electron uptake. [ABSTRACT FROM AUTHOR]

Geobacter sulfurreducens pili enable extracellular electron transfer and play a role in secretion of c-type cytochromes such as OmcZ. PilA-deficient mutants of G. sulfurreducens have previously been shown to accumulate cytochromes within their membranes. This cytochrome retaining phenotype allowed for enhanced growth of PilA-deficient mutants in electron donor and carbon-limited conditions where formate and fumarate are provided as the sole electron donor and acceptor with no supplementary carbon source. Conversely, wild-type G. sulfurreducens, which has normal secretion of cytochromes, has comparative limited growth in these conditions. This growth is further impeded for OmcZ-deficient and OmcS-deficient mutants. A PilB-deficient mutant which prevents pilin production but allows for secretion of OmcZ had moderate growth in these conditions, indicating a role for cytochrome localization to enabling survival in the electron donor and carbon-limited conditions. To determine which pathways enhanced growth using formate, Sequential Window Acquisition of all Theoretical Mass Spectra mass spectrometry (SWATH-MS) proteomics of formate adapted PilA-deficient mutants and acetate grown wild type was performed. PilA-deficient mutants had an overall decrease in tricarboxylic acid (TCA) cycle enzymes and significant upregulation of electron transport chain associated proteins including many c-type cytochromes and [NiFe]-hydrogenases. Whole genome sequencing of the mutants shows strong convergent evolution and emergence of genetic subpopulations during adaptation to growth on formate. The results described here suggest a role for membrane constrained c-type cytochromes to the enhancement of survival and growth in electron donor and carbon-limited conditions. [ABSTRACT FROM AUTHOR]

Biocompatible materials and biocarriers have attracted great attention in biological wastewater treatment owing to their excellent performance in improving pollutant removal. Graphene-based material, a biocarrier candidate, with excellent adsorbability and conductivity was increasingly applied in anaerobic digestion due to its exceptional potential in the adsorption and electron transfer process. Nevertheless, the green approach for the formation of bio-graphene complexes and their mechanism in dye removal is limited. The aim of this study is to investigate and assess the performance of biological graphene hydrogel (BGH) formed by Shewanella putrefaciens CN32 on the removal of methyl orange (MO) and methylene blue (MB). The results showed that the formation of BGH is determined by the physicochemical characteristics of graphene oxide, including sheet size, oxidation degree, and interlayer distance. BGHs significantly increased the removal efficiency of dyes in comparison to non-graphene samples, with a 24-h removal rate of MO and MB reaching 92.9% and 91%, respectively. The synergetic mechanism of BGH on the enhanced removal rate of organic dye can be ascribed to GO's ability in accelerating extracellular electron transfer and stimulating biodegradation pathways relating to c -type cytochromes, including MtrA and MtrC. These findings provided an understanding of the relationship between graphene-based nanomaterials and Shewanella , which facilitated their future application in environmental biotechnology. [Display omitted] • The reduction of GO by S. putrefaciens CN32 prompt BGH generation. • Sheet area and degree of defects of GO determine the BGH formation. • Bacteria-immobilized rGO hydrogel displayed a synergistic effect in bioremediation. • BGH increased dye removal efficiency by accelerating extracellular electron transfer. [ABSTRACT FROM AUTHOR]

Cholesterol, a significant constituent of the endoplasmic reticulum membrane, exerts a substantial effect on the membrane's biophysical and mechanical properties. Cholesterol, however, is often neglected in model systems used to study membrane-bound proteins. For example, the influence of cholesterol on the enzymatic functions of type 2 cytochromes P450, which require a phospholipid bilayer and the redox partner P450-oxidoreductase (POR) for activity, are rarely investigated. Human aromatase (P450 19A1) catalyzes three sequential oxygenations of 19‑carbon steroids to estrogens and is widely expressed across various tissues, which are characterized by varying cholesterol compositions. Our study examined the impact of cholesterol on the functionality of the P450 19A1 complex with POR. Nanodiscs containing P450 19A1 with 20% cholesterol/80% phospholipid had similar rates and affinity of androstenedione binding as phospholipid-only P450 19A1 nanodiscs, and rates of product formation were indistinguishable among these conditions. In contrast, the rate of the first electron transfer from POR to P450 19A1 was 3-fold faster in cholesterol-containing nanodiscs than in phospholipid-only nanodiscs. These results suggest that cholesterol influences some aspects of POR interaction with P450 19A1 and might serve as an additional regulatory mechanism in this catalytic system. [Display omitted] • Cholesterol in phospholipid nanodiscs increases cytochrome P450 19A1 reduction rate 3-fold. • Cholesterol does not change the rate or affinity of substrate binding to P450 19A1. • Docking calculations suggest that cholesterol binds the proximal surface of P450 19A1. [ABSTRACT FROM AUTHOR]

• Many recent AD studies mistakenly link performance improvements exclusively to DIET. • Unequivocally proving the occurrence of DIET requires a complex set of methods. • This paper groups DIET characterization methods in four categories and discusses their strengths and weaknesses. • This paper presents promising DIET detection approaches that merit further exploration. Recent anaerobic digestion studies commonly attribute performance improvements (e.g. increased methane production, enhanced process stability, reduced startup times) to direct interspecies electron transfer (DIET), even though only indirect evidence of DIET is available and DIET alone does not explain enhanced performance in many cases. This review evaluates methods believed to confirm the occurrence of DIET in anaerobic systems. 16S rRNA gene sequencing and meta-omics approaches are necessary to further DIET knowledge but are limited in their ability to confirm the occurrence of DIET. In situ use of cyclic voltammetry should be explored further, as well as microscopy and image analysis procedures to quantify stained cytochromes. Furthermore, linking interspecies distance, interspecies mixing, and cellular activity to a DIET-based electron transfer model is promising but needs further validation for anaerobic digestion systems. In short, a combination of methods is necessary to confirm the occurrence and expand our knowledge of DIET. [ABSTRACT FROM AUTHOR]

Abstract Three-dimensional biofilm electrode reactors (3D-BERs) represent a novel technology for wastewater denitrification. Formation of mature electroactive biofilm on particle electrodes is crucial to realize successful denitrification in 3D-BERs. However, long start-up time and low electroactivity of the biofilm formed on particle electrodes limit the further application of 3D-BERs in wastewater treatment. In this work, self-assembled hybrid biofilms (SAHB) was cultivated on granular activate carbon particle electrodes of the 3D-BER by assembling nano ɑ-Fe 2 O 3 into the biofilm. ɑ-Fe 2 O 3 was selected due to its high affinity to bacterial outer-membrane cytochromes, an important mediator for microbial electron transfer. SAHB formed on particle electrodes were characterized and the denitrification performance of 3D-BERs was also investigated. Results indicate that nano ɑ-Fe 2 O 3 plays positive roles in the start-up of 3D-BER, which captures more microbes into SAHB and constructs thick biofilm on particle electrodes. Special microorganisms with denitrification function related with genera of Hydrogenophaga and Opitutus are distinctively enriched in SAHB. Nano ɑ-Fe 2 O 3 induced SAHB exhibit superior denitrification performance compared to natural biofilm. The average denitrification rate increases from 0.62 mg total nitrogen/L/h for natural biofilm to 1.73 mg total nitrogen/L/h for SAHB, mainly ascribed to accelerated nitrites reduction. Our work provides new technical solution to enhance nitrates removal in 3D-BERs and brings deep insights into application of bio-electrochemical system in wastewater treatment. Graphical abstract Unlabelled Image Highlights • SAHB was cultivated on particle electrodes of 3D-BERs by assembling nano ɑ-Fe 2 O 3. • SAHB was characterized by imaging coupled with microbial analysis. • Nano ɑ-Fe 2 O 3 facilitated to capture more functional microbes into SAHB. • SAHB accelerated denitrification mainly by enhancing nitrites reduction. [ABSTRACT FROM AUTHOR]

In the present study, we found that our isolate Shewanella decolorationis NTOU1 is able to degrade acetate under anaerobic condition with concomitant implementation of extracellular electron transfer (EET). With +0.63 V (vs. SHE) poised on the anode, in a 72-h experiment digesting acetate, only 2 mM acetate was consumed, which provides 6% of the electron equivalents derived from the initial substrate mass to support biomass (5%) and current generation (1%). To clarify the effects on EET of the addition of electron-shuttles, riboflavin, anthraquinone-2,6-disulfonate (AQDS), hexaammineruthenium, and hexacyanoferrate were selected to be spiked into the electrochemical cell in four individual experiments. It was found that the mediators with proton-associated characteristics (i.e., riboflavin and AQDS) would not enhance current generation, but the metal-complex mediators (i.e., hexaammineruthenium, and hexacyanoferrate) significantly enhanced current generation as the concentration increased. According to the results of electrochemical analyses, the i - V graphs represent that the catalytic current induced by the primitive electron shuttles started at the onset potential of −0.27 V and continued increasing until +0.73 V. In the riboflavin-addition experiment, the catalytic current initiated at the same potential but rapid saturated beyond −0.07 V; this indicated that the addition of riboflavin affects mediator secretion by S. decolorationis NTOU1. It was also found that the current was eliminated after adding 48 mM N -acetyl-L-methionine (i.e., the cytochrome inhibitor) when using acetate as a substrate, indicating the importance of outer-membrane cytochrome. [ABSTRACT FROM AUTHOR]

The direct electron transfer (DET) of enzymes has been utilized to develop biosensors and enzymatic biofuel cells on micro‐ and nanostructured electrodes. Whereas some enzymes exhibit direct electron transfer between their active‐site cofactor and an electrode, other oxidoreductases depend on acquired cytochrome domains or cytochrome subunits as built‐in redox mediators. The physiological function of these cytochromes is to transfer electrons between the active‐site cofactor and a redox partner protein. The exchange of the natural electron acceptor/donor by an electrode has been demonstrated for several cytochrome carrying oxidoreductases. These multi‐cofactor enzymes have been applied in third generation biosensors to detect glucose, lactate, and other analytes. This review investigates and classifies oxidoreductases with a cytochrome domain, enzyme complexes with a cytochrome subunit, and covers designed cytochrome fusion enzymes. The structurally and electrochemically best characterized proponents from each enzyme class carrying a cytochrome, that is, flavoenzymes, quinoenzymes, molybdenum‐cofactor enzymes, iron‐sulfur cluster enzymes, and multi‐haem enzymes, are featured, and their biochemical, kinetic, and electrochemical properties are compared. The cytochromes molecular and functional properties as well as their contribution to the interdomain electron transfer (IET, between active‐site and cytochrome) and DET (between cytochrome and electrode) with regard to the achieved current density is discussed. Protein design strategies for cytochrome‐fused enzymes are reviewed and the limiting factors as well as strategies to overcome them are outlined. It′s classified: Multi‐cofactor enzymes with a cytochrome domain use it to transfer electrons to physiological redox partner proteins, but the cytochrome can also establish direct electron transfer (DET) to electrodes. The enzymes are classified, compared, and analyzed in terms of their redox and DET properties to obtain guidelines for selecting and designing suitable enzymes for third‐generation biosensors. [ABSTRACT FROM AUTHOR]

Graphical abstract Perspective views of diheme cytochrome c (without protein chain) and its synthetic diheme analog are shown here. The structure-function relationship has been explored using a series of such synthetic dihemes with a variety of imidazoles and pyridines as axial ligands which thereby provide a large structural and functional diversity. Highlights • Structure-function relationship of the diheme cytochrome c using a series of synthetic diheme analogs. • The solid-state structure determinations of both the oxidized and reduced forms show only a small structural change. • Axial ligands are perpendicularly oriented in both the forms. • FeIII/FeII couple is shifted more positive on going from mono-heme to dihemes. • Effects of heme-heme interactions are scrutinized. Abstract Multiheme cytochromes c are among the most fascinating molecular machineries evolved by Nature with essential functions in electron transfer and enzymatic catalysis. The multiheme architecture ensures fast intramolecular electron transfer over long distances. We explore here the structure-function relationship of the diheme cytochrome c (DHC2), the simplest member of such family, using a series of synthetic diheme analogs with a variety of imidazoles and pyridines as axial ligands which thereby enable structural and functional diversity. The molecules, both in the oxidized and reduced forms, have been synthesized and their structure and properties are scrutinized at the molecular level without any interference from large protein superstructure unlike in the native enzyme. The solid-state structure of such model diheme shows similar spatial orientation and heme-to-heme separation as also observed in the native DHC2 isolated from Geobacter sulfurreducens and Haemophilus influenza. Also, the UV–visible spectra of the synthetic dihemes are similar to the DHC2 both in the oxidized and reduced forms. The solid-state X-ray structures of four such diheme analogs have been determined which show only a small structural change between the oxidized and reduced forms and hence smaller reorganization energy is required during electronic communications in dihemes. Spectroscopic characterizations through solid-state Mössbauer, EPR, and 1H NMR studies confirm the presence of low-spin diiron centers in all the complexes. The Fe(III)/Fe(II) redox couple shows a linear relationship to the p K a of coordinated axial ligands and it is found to be dependent on the nature of the bridging group between two heme centers. These observations are further supported by DFT calculations. [ABSTRACT FROM AUTHOR]

Abstract Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for self-monitoring of blood glucose sensors; however, the achievement of direct electron transfer (DET) with FADGDHs is difficult. In this study, a designer FADGDH was constructed by fusing Aspergillus flavus derived FADGDH (AfGDH) and a Phanerochaete chrisosporium CDH (PcCDH)-derived heme b -binding cytochrome domain to develop a novel FADGDH that is capable of direct electron transfer with an electrode. A structural prediction suggested that the heme in the CDH may exist in proximity to the FAD of AfGDH if the heme b -binding cytochrome domain is fused to the AfGDH N-terminal region. Spectroscopic observations of recombinantly produced designer FADGDH confirmed the intramolecular electron transfer between FAD and the heme. A decrease in pH and the presence of a divalent cation improved the intramolecular electron transfer. An enzyme electrode with the immobilized designer FADGDH showed an increase in current immediately after the addition of glucose in a glucose concentration-dependent manner, whereas those with wild-type AfGDH did not show an increase in current. Therefore, the designer FADGDH was confirmed to be a novel GDH that possesses electrode DET ability. The difference in the surface electrostatic potentials of AfGDH and the catalytic domain of PcCDH might be why their intramolecular electron transfer ability is inferior to that of CDH. These relevant and consistent findings provide us with a novel strategic approach for the improvement of the DET properties of designer FADGDH. (241 words) Highlights • The structural prediction suggested that the GDH N-terminal fusion of cytochrome domain of CDH may provide DET enzyme. • A designer FADGDH was constructed by fusing a fungal FADGDH and CDH derived a heme b -binding cytochrome domain. • The spectroscopic observation confirmed that the intra-molecular electron transfer between FAD and heme occurred. • The decrease in pH and  the addition of divalent cation improved the intra-molecular electron transfer of designer FADGDH. • The designer FADGDH is confirmed as a novel GDH possessing DET ability to electrode. [ABSTRACT FROM AUTHOR]

I review recent experimental measurements probing electron transfer (ET) and electron transport (ETp) through multi-heme cytochromes (MHCs) as well as their theoretical interpretation. Examples include pump-probe spectroscopy of Ru-labeled MHCs aimed at determining heme–heme ET rates in MHCs and the measurement of the I – V characteristics of MHCs in bioelectronic junctions. While the ET mechanism appears to be well established for MHCs in aqueous solution, the ETp mechanism in bioelectronic junctions such as STM remains elusive partly due to the complexities of the electrode–protein interface. [ABSTRACT FROM AUTHOR]

Abstract Microorganisms can acquire energy from the environment by extending their electron transport chains to external solid electron donors or acceptors. This process, known as extracellular electron transfer (EET), is now being heavily pursued for wiring microbes to electrodes in bioelectrochemical renewable energy technologies. Recent studies highlight the crucial role of multi-heme cytochromes in facilitating biotic–abiotic EET both for cellular electron export and uptake. Here we explore progress in understanding the range and function of these biological electron conduits in the context of fuel-to-electricity and electricity-to-bioproduct conversion. We also highlight emerging topics, including the role of multi-heme cytochromes in inter-species electron transfer and in inspiring the design and synthesis of a new generation of protein-based bioelectronic components. [ABSTRACT FROM AUTHOR]

Classical electron transfer in polar glasses is described by a theory based on a model microscopic Hamiltonian which includes the discreteness and randomness of the glassy polar modes with distinct orientation. When configurational dynamics is fast, the reaction is described by exponential kinetics with a rate constant of non-Arrhenius type. The temperature dependent rate constant resembles the tunneling rate, despite the classical transfer of the electron. This effect is called ‘‘false tunneling.’’ In this limit the possibility of a self-acceleration of the reaction is pointed out. When configurational dynamics is very slow the reaction kinetics are nonexponential with multirelaxation time behavior. The reaction is shown to be almost insensitive to temperature change pointing out on a possible explanation of a broad temperature-independent range in the ‘‘rate constant’’ in an electron transfer in cytochrome c oxidation in chromatium. At short times, the reaction accelerates compared to the exponential behavior, while at long times it becomes slower. For strongly exothermic reactions the kinetics are always slower than an exponential decay. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The electron transfer process from cytochrome c to the special pair is revisited. In the present model the transfer rate in photosynthetic reaction centers of Chromatium vinosum is modulated by fluctuations of the dielectric constant of the protein environment between the donor and acceptor site. The fluctuations enter the rate expression as the inverse of a Lamb–Mössbauer factor. The strong temperature dependence of the transfer rate can thus be related to protein specific dynamics. Experimental evidence about such dynamics is provided by recently recorded Mössbauer spectra of iron containing proteins [Parak et al., J. Mol. Biol. 161, 177 (1982)]. [ABSTRACT FROM AUTHOR]

• 58 screened cytochromes of five protein folds could wire enzymes to electrodes. • Electron uptake of eight cytochromes from a glucose dehydrogenase was studied. • c-Type cytochromes showed faster electron uptake than b-type cytochromes. • Complementary surface charges were not a predictor for fast electron shuttling. • Enzyme/cytochrome redox potential difference correlates to fast electron uptake. Cytochromes are a large and well-known group of electron transfer proteins, but only very few are used in bioelectrochemistry, e.g. to serve as electron shuttles between biocatalysts and electrodes. There, cytochromes generate an artificial electron transfer pathway if their structural and electrochemical properties are fitting. Cytochromes considered suitable are small and feature an exposed heme cofactor, a suitable redox potential, an electrochemically reversible redox process, sufficient protein production, and a fast electron transfer between other enzymes and hemoproteins. Out of 850 screened hemoproteins, 58 cytochromes met the requirements to function as an electron shuttle. They can be classified into five distinguished protein folds. Four representative b -type and three c -type cytochromes were selected for further analyses. The proteins were obtained in a yield of 0.075 to 375 mg L-1 in Escherichia coli or Komagataella phaffii. The electrochemical reversibility on 1-thioglycerol functionalized gold electrodes of all cytochromes was confirmed. The pH-dependent redox potentials range from -50 mV to +298 mV vs. SHE at pH 7.0. The electronic wiring ability was tested using the biocatalyst FAD-dependent glucose dehydrogenase (GDH). Cytochrome reduction by the GDH was monitored by spectrophotometry and amperometry. c -Type cytochromes showed a faster electron uptake from GDH than b -type cytochromes. Complementary surface charges at the cytochrome/GDH interface were not a predictor for a fast electron transfer but a higher difference in the redox potential of the cofactors was. This study supports the design of bioelectrocatalysts with high electron transfer rates, which are of great interest in 3rd generation biosensors and for direct bioelectrocatalysis. [Display omitted] [ABSTRACT FROM AUTHOR]

The extracellular electron transfer (EET) process of Shewanella species is believed to be indispensable for their anaerobic respiration with an electrode. However, the function of outer membrane c -type cytochromes (OM c -Cyts, the primary components of the EET pathway) is still controversial. In this study, we investigated the effect of two OM c -Cyts (MtrC and UndA) of Shewanella putrefaciens CN32 with respect to electricity production and anodic EET efficiency. Deletion of the mtrC gene severely prolonged the microbial fuel cell (MFC) start-up time and decreased electricity production due to depressed flavin-mediated electron transfer, whereas deletion of the undA gene did not have a significant impact. Strikingly, the depression of EET by the deletion of mtrC could be partially relieved by acclimation, which might be due to an increase in the transmembrane transport of electron shuttles and/or the activation of other redox proteins. These results suggested that MtrC may be the primary reductase of flavins to ensure fast indirect EET, which plays a crucial role in MFC electricity generation. [ABSTRACT FROM AUTHOR]

A key feature of the modified Q-cycle of the cytochrome bc 1 and related complexes is a bifurcation of QH 2 oxidation involving electron transfer to two different acceptor chains, each coupled to proton release. We have studied the kinetics of proton release in chromatophore vesicles from Rhodobacter sphaeroides , using the pH-sensitive dye neutral red to follow pH changes inside on activation of the photosynthetic chain, focusing on the bifurcated reaction, in which 4H + are released on complete turnover of the Q-cycle (2H + /ubiquinol (QH 2 ) oxidized). We identified different partial processes of the Q o -site reaction, isolated through use of specific inhibitors, and correlated proton release with electron transfer processes by spectrophotometric measurement of cytochromes or electrochromic response. In the presence of myxothiazol or azoxystrobin, the proton release observed reflected oxidation of the Rieske iron‑sulfur protein. In the absence of Q o -site inhibitors, the pH change measured represented the convolution of this proton release with release of protons on turnover of the Q o -site, involving formation of the ES -complex and oxidation of the semiquinone intermediate. Turnover also regenerated the reduced iron-sulfur protein, available for further oxidation on a second turnover. Proton release was well-matched with the rate limiting step on oxidation of QH 2 on both turnovers. However, a minor lag in proton release found at pH 7 but not at pH 8 might suggest that a process linked to rapid proton release on oxidation of the intermediate semiquinone involves a group with a p K in that range. [ABSTRACT FROM AUTHOR]

One of the most prominent glucose dehydrogenases (GDHs) capable of direct electron transfer with electrodes is the FADGDH complex derived from Burkholderia cepacia . This FADGDH complex comprises the following three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, a hitch-hiker protein from the bacterial TAT secretion system (γ subunit), and the electron transfer subunit (β subunit). The electron transfer subunit (β subunit) of the FADGDH complex is a three-heme c containing cytochrome c like molecule (heme 1, heme 2 and heme 3 from the N-terminal). In this study, an FADGDH complex harboring a truncated electron transfer subunit composed of only heme 3 was constructed, and its enzymatic activity and electrochemical properties were investigated to elucidate the role of heme 3 and its region. A truncated electron transfer subunit, trβ subunit, was designed using the 3D structures of homologous cytochrome c proteins. The designed trβ subunit was expressed as soluble and functional heme c molecules forming complexes with γα catalytic complexes. Thus, the formed FADGDH complex has inter-molecular electron transfers from the FAD to the trβ subunit, and from the trβ subunit to the external electron acceptor, showing electron transfer subunit-mediated characteristic dye-mediated dehydrogenase activity with a Ru-complex. Therefore, heme 3 in the electron transfer subunit is responsible for accepting the electron from the γα catalytic complex. Moreover, the FADGDH complex harboring the trβ subunit showed DET activity toward the electrode. Spectroelectrochemical observations revealed that the trβ subunit possessed a lower formal potential than any of the 3 hemes in the whole electron transfer subunit. These unexpected electrochemical properties of the heme in the trβ subunit may potentially result in the construction of a DET principle-based glucose sensor, which can be operated at a much lower potential than those achieved using the FADGDH complex with a whole electron transfer subunit. [ABSTRACT FROM AUTHOR]

Abstract: Shewanella oneidensis MR‐1 gains energy by extracellular electron transfer to solid surfaces. They employ c‐type cytochromes in two Mtr transmembrane complexes, forming a multiheme wire for electron transport across the cellular outer membrane. We investigated electron‐ and hole‐transfer mechanisms in the external terminal of the two complexes, MtrC and MtrF. Comparison of computed redox potentials with previous voltammetry experiments in distinct environments (isolated and electrode‐bound conditions of PFV or in vivo) suggests that these systems function in different regimes depending on the environment. Analysis of redox potential shifts in different regimes indicates strong coupling between the hemes via an interplay between direct Coulomb and indirect interactions through local structural reorganization. The latter results in the screening of Coulomb interactions and explains poor correlation of the strength of the heme‐to‐heme interactions with the distance between the hemes. [ABSTRACT FROM AUTHOR]

Connection of photosystem I (PSI) with electrodes has been shown to create artificial photosynthetic systems that hold promise for the synthesis of solar fuels. The high quantum yields of PSI require efficient electron transfer from the electrode to the reaction center of PSI in order to restock the light-induced holes, a task which in nature is performed by small redox proteins. Here, we have investigated the potential “wiring” properties of a dihemic c -type cytochrome (cyt c 4 ), in order to efficiently connect PSI with electrodes. Cyt c 4 has shown direct electron transfer (DET) with both hemes in electrical communication with two different electrode materials (ITO and Au) and on the basis of cyt c 4 -multilayer electrodes “self-exchange” properties can also be deduced. Investigation of cyt c 4 in combination with PSI within an inverse opal ITO electrode has shown the dihemic protein to be a valuable molecular electron conduit, able to interconnect the photoenzymatic reaction with the 3D electrode. The properties have been compared with those of electrodes based on monohemic cyt c derived from horse heart. [ABSTRACT FROM AUTHOR]

Abstract: Microbial extracellular electron transfer (EET) with high performance and good controllability is always desired in various bioelectric applications. Depending on the redox state, C‐type cytochromes located across the outer membranes (OMCs) mediate EET with different behaviors. Here, by incorporating Sn‐doped In2O3 nanowire array with flat F‐doped In2O3 (FTO), a composite electrode is developed that can highly boost EET by over 60 times at a certain potential of 0.2 V, where normally only limited EET current is observed on a conductive electrode. It is proposed that the underlay FTO with bias 0.2 V promotes EET via OMCs by physical contacting with microbes, while the semiconductor nanowires provide a suitable energy level to facilitate the EET via OMC‐flavins cofactor, breaking the stereotype of the certain redox state of OMCs at a given potential. As a synergistic effect, EET via both OMCs and flavins is highly boosted simultaneously to achieve the dual‐pathway EET, which could be further amplified by the nanostructured topology of the as‐prepared electrode. Moreover, the EET current shows a positive correlation with the nanowire length, showing good controllability. This work provides an applicable method for constructing various highly efficient bioelectric devices. [ABSTRACT FROM AUTHOR]