Understanding differential aspects of microdiffusion (channeling) in the Coenzyme Q and Cytochrome c regions of the mitochondrial respiratory system.

Over the past decades, models of the organization of mitochondrial respiratory system have been controversial. The goal of this perspective is to assess this "conflict of models" by focusing on specific kinetic evidence in the two distinct segments of Coenzyme Q- and Cytochrome c-mediated electron transfer. Respiratory supercomplexes provide kinetic advantage by allowing a restricted diffusion of Coenzyme Q and Cytochrome c, and short-range interaction with their partner enzymes. In particular, electron transfer from NADH is compartmentalized by channeling of Coenzyme Q within supercomplexes, whereas succinate oxidation proceeds separately using the free Coenzyme Q pool. Previous evidence favoring Coenzyme Q random diffusion in the NADH-dependent electron transfer is due to downstream flux interference and misinterpretation of results. Indeed, electron transfer by complexes III and IV via Cytochrome c is less strictly dependent on substrate channeling in mammalian mitochondria. We briefly describe these differences and their physiological implications.

The Outer Membrane Cytochrome OmcA Is Essential for Infection of Shewanella oneidensis by a Zebrafish-Associated Bacteriophage.

The microbiota-the mixture of microorganisms in the intestinal tract of animals-plays an important role in host biology. Bacteriophages are a prominent, though often overlooked, component of the microbiota. The mechanisms that phage use to infect susceptible cells associated with animal hosts, and the broader role they could play in determining the substituents of the microbiota, are poorly understood. In this study, we isolated a zebrafish-associated bacteriophage, which we named Shewanella phage FishSpeaker. This phage infects Shewanella oneidensis strain MR-1, which cannot colonize zebrafish, but it is unable to infect Shewanella xiamenensis strain FH-1, a strain isolated from the zebrafish gut. Our data suggest that FishSpeaker uses the outer membrane decaheme cytochrome OmcA, which is an accessory component of the extracellular electron transfer (EET) pathway in S. oneidensis, as well as the flagellum to recognize and infect susceptible cells. In a zebrafish colony that lacks detectable FishSpeaker, we found that most Shewanella spp. are sensitive to infection and that some strains are resistant to infection. Our results suggest that phage could act as a selectivity filter for zebrafish-associated Shewanella and show that the EET machinery can be targeted by phage in the environment. IMPORTANCE Phage exert selective pressure on bacteria that influences and shapes the composition of microbial populations. However, there is a lack of native, experimentally tractable systems for studying how phage influence microbial population dynamics in complex communities. Here, we show that a zebrafish-associated phage requires both the outer membrane-associated extracellular electron transfer protein OmcA and the flagellum to infect Shewanella oneidensis strain MR-1. Our results suggest that the newly discovered phage-FishSpeaker-could exert selective pressure that restricts which Shewanella spp. colonize zebrafish. Moreover, the requirement of OmcA for infection by FishSpeaker suggests that the phage preferentially infects cells that are oxygen limited, a condition required for OmcA expression and an ecological feature of the zebrafish gut.

Heme A Synthase Deficiency Affects the Ability of Bacillus cereus to Adapt to a Nutrient-Limited Environment.

The branched aerobic respiratory chain in Bacillus cereus comprises three terminal oxidases: cytochromes aa3, caa3, and bd. Cytochrome caa3 requires heme A for activity, which is produced from heme O by heme A synthase (CtaA). In this study, we deleted the ctaA gene in B. cereus AH187 strain, this deletion resulted in loss of cytochrome caa3 activity. Proteomics data indicated that B. cereus grown in glucose-containing medium compensates for the loss of cytochrome caa3 activity by remodeling its respiratory metabolism. This remodeling involves up-regulation of cytochrome aa3 and several proteins involved in redox stress response-to circumvent sub-optimal respiratory metabolism. CtaA deletion changed the surface-composition of B. cereus, affecting its motility, autoaggregation phenotype, and the kinetics of biofilm formation. Strikingly, proteome remodeling made the ctaA mutant more resistant to cold and exogenous oxidative stresses compared to its parent strain. Consequently, we hypothesized that ctaA inactivation could improve B. cereus fitness in a nutrient-limited environment.

Enantioselective Synthesis of α-Trifluoromethyl Amines via Biocatalytic N-H Bond Insertion with Acceptor-Acceptor Carbene Donors.

The biocatalytic toolbox has recently been expanded to include enzyme-catalyzed carbene transfer reactions not occurring in Nature. Herein, we report the development of a biocatalytic strategy for the synthesis of enantioenriched α-trifluoromethyl amines through an asymmetric N-H carbene insertion reaction catalyzed by engineered variants of cytochrome c552 from Hydrogenobacter thermophilus. Using a combination of protein and substrate engineering, this metalloprotein scaffold was redesigned to enable the synthesis of chiral α-trifluoromethyl amino esters with up to >99% yield and 95:5 er using benzyl 2-diazotrifluoropropanoate as the carbene donor. When the diazo reagent was varied, the enantioselectivity of the enzyme could be inverted to produce the opposite enantiomers of these products with up to 99.5:0.5 er. This methodology is applicable to a broad range of aryl amine substrates, and it can be leveraged to obtain chemoenzymatic access to enantioenriched ß-trifluoromethyl-ß-amino alcohols and halides. Computational analyses provide insights into the interplay of protein- and reagent-mediated control on the enantioselectivity of this reaction. This work introduces the first example of a biocatalytic N-H carbenoid insertion with an acceptor-acceptor carbene donor, and it offers a biocatalytic solution for the enantioselective synthesis of α-trifluoromethylated amines as valuable synthons for medicinal chemistry and the synthesis of bioactive molecules.

Expression of Cytochrome c3 from Desulfovibrio vulgaris in Plant Leaves Enhances Uranium Uptake and Tolerance of Tobacco.

Cytochrome c3 (uranyl reductase) from Desulfovibrio vulgaris can reduce uranium in bacterial cells and in cell-free systems. This gene was introduced in tobacco under control of the RbcS promoter, and the resulting transgenic plants accumulated uranium when grown on a uranyl ion containing medium. The uptaken uranium was detected by EM in chloroplasts. In the presence of uranyl ions in sublethal concentration, the transgenic plants grew phenotypically normal while the control plants' development was impaired. The data on uranium oxidation state in the transgenic plants and the possible uses of uranium hyperaccumulation by plants for environmental cleanup are discussed.

Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme.

Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 µm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme-heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis We observed rates of heme-to-heme electron transfer on the order of 109 s-1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser-Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-µs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.

The bacterial protective armor against stress: The cis-trans isomerase of unsaturated fatty acids, a cytochrome-c type enzyme.

Bacteria have evolved several outstanding strategies to resist to compounds or factors that compromise their survival. The first line of defense of the cell against environmental stresses is the membrane with fatty acids as fundamental building blocks of phospholipids. In this review, we focus on a periplasmic heme enzyme that catalyzes the cis-trans isomerization of unsaturated fatty acids to trigger a decrease in the fluidity of the membrane in order to rapidly counteract the danger. We particularly detailed the occurrence of such cis-trans isomerase in Nature, the different stresses that are at the origin of the double bond isomerization, the first steps in the elucidation of the mechanism of this peculiar metalloenzyme and some aspects of its regulation.

C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

Identification and Validation of Four Novel Promoters for Gene Engineering with Broad Suitability across Species.

The transcriptional capacities of target genes are strongly influenced by promoters, whereas few studies have focused on the development of robust, high-performance and cross-species promoters for wide application in different bacteria. In this work, four novel promoters (Pk.rtufB, Pk.r1, Pk.r2, and Pk.r3) were predicted from Ketogulonicigenium robustum and their inconsistency in the -10 and -35 region nucleotide sequences indicated they were different promoters. Their activities were evaluated by using green fluorescent protein (gfp) as a reporter in different species of bacteria, including K. vulgare SPU B805, Pseudomonas putida KT2440, Paracoccus denitrificans PD1222, Bacillus licheniformis and Raoultella ornithinolytica, due to their importance in metabolic engineering. Our results showed that the four promoters had different activities, with Pk.r1 showing the strongest activity in almost all of the experimental bacteria. By comparison with the commonly used promoters of E. coli (tufB, lac, lacUV5), K. vulgare (Psdh, Psndh) and P. putida KT2440 (JE111411), the four promoters showed significant differences due to only 12.62% nucleotide similarities, and relatively higher ability in regulating target gene expression. Further validation experiments confirmed their ability in initiating the target minCD cassette because of the shape changes under the promoter regulation. The overexpression of sorbose dehydrogenase and cytochrome c551 by Pk.r1 and Pk.r2 resulted in a 22.75% enhancement of 2-KGA yield, indicating their potential for practical application in metabolic engineering. This study demonstrates an example of applying bioinformatics to find new biological components for gene operation and provides four novel promoters with broad suitability, which enriches the usable range of promoters to realize accurate regulation in different genetic backgrounds.

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA).

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

Catalytic Properties of Flavocytochrome c Sulfide Dehydrogenase from Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus.

Flavocytochrome c sulfide dehydrogenase (FCC) is one of the central enzymes of the respiratory chain in sulfur-oxidizing bacteria. FCC catalyzes oxidation of sulfide and polysulfide ions to elemental sulfur accompanied by electron transfer to cytochrome c. The catalytically active form of the enzyme is a non-covalently linked heterodimer composed of flavin- and heme-binding subunits. The Thioalkalivibrio paradoxus ARh1 genome contains five copies of genes encoding homologous FCCs with an amino acid sequence identity from 36 to 54%. When growing on thiocyanate or thiosulfate as the main energy source, the bacterium synthesizes products of different copies of FCC genes. In this work, we isolated and characterized FCC synthesized during the growth of Tv. paradoxus on thiocyanate. FCC was shown to oxidize exclusively sulfide but not other reduced sulfur compounds, such as thiosulfate, sulfite, tetrathionate, and sulfur, and it also does not catalyze the reverse reaction of sulfur reduction to sulfide. Kinetic parameters of the sulfide oxidation reaction are characterized.

A Case of Gene Fragmentation in Plant Mitochondria Fixed by the Selection of a Compensatory Restorer of Fertility-Like PPR Gene.

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme-lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmFN2 gene encoding a heme-lyase complex subunit which derives from the split of the ccmFN gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.

Outer membrane compositions enhance the rate of extracellular electron transport via cell-surface MtrC protein in Shewanella oneidensis MR-1.

While cell membrane composition is critical for the function of membrane proteins, membrane modification has not been used to control the rate of extracellular electron transfer (EET) via the outer membrane protein complexes. Here, the rate of electron flow via the cell-surface redox protein, MtrC, was highly enhanced upon change in the outer membrane composition in Shewanella oneidensis MR-1. The MR-1 strain was pre-cultured at 4 °C and 30 °C to initiate differentiation of membrane composition. The whole-cell difference electrochemical assay of wild-type and mutant strains lacking MtrC suggested that the rate of EET via MtrC increased approximately 18 times at 4 °C than 30 °C. Circular dichroism spectroscopy showed that the molar exciton coupling coefficient for inter-heme interaction in MtrC increased in MR-1 at 4 °C than 30 °C. Results suggest that membrane modification may be a novel strategy for improving the efficiency of EET-based technologies.

Heterologous production and functional characterization of Bradyrhizobium japonicum copper-containing nitrite reductase and its physiological redox partner cytochrome c550.

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions.

Outer Membrane c-Type Cytochromes OmcA and MtrC Play Distinct Roles in Enhancing the Attachment of Shewanella oneidensis MR-1 Cells to Goethite.

The outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC in Shewanella are key terminal reductases that bind and transfer electrons directly to iron (hydr)oxides. Although the amounts of OmcA and MtrC at the cell surface and their molecular structures are largely comparable, MtrC is known to play a more important role in dissimilatory iron reduction. To explore the roles of these outer membrane c-Cyts in the interaction of Shewanella oneidensis MR-1 with iron oxides, the processes of attachment of S. oneidensis MR-1 wild type and c-type cytochrome-deficient mutants (the ΔomcA, ΔmtrC, and ΔomcA ΔmtrC mutants) to goethite are compared via quartz crystal microbalance with dissipation monitoring (QCM-D). Strains with OmcA exhibit a rapid initial attachment. The quantitative model for QCM-D responses reveals that MtrC enhances the contact area and contact elasticity of cells with goethite by more than one and two times, respectively. In situ attenuated total reflectance Fourier transform infrared two-dimensional correlation spectroscopic (ATR-FTIR 2D-CoS) analysis shows that MtrC promotes the initial interfacial reaction via an inner-sphere coordination. Atomic force microscopy (AFM) analysis demonstrates that OmcA enhances the attractive force between cells and goethite by about 60%. As a result, OmcA contributes to a higher attractive force with goethite and induces a rapid short-term attachment, while MtrC is more important in the longer-term interaction through an enhanced contact area, which promotes interfacial reactions. These results reveal that c-Cyts OmcA and MtrC adopt different mechanisms for enhancing the attachment of S. oneidensis MR-1 cells to goethite. It improves our understanding of the function of outer membrane c-Cyts and the influence of cell surface macromolecules in cell-mineral interactions.IMPORTANCEShewanella species are one group of versatile and widespread dissimilatory iron-reducing bacteria, which are capable of respiring insoluble iron minerals via six multiheme c-type cytochromes. Outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC are the terminal reductases in this pathway and have comparable protein structures. In this study, we elucidate the different roles of OmcA and MtrC in the interaction of S. oneidensis MR-1 with goethite at the whole-cell level. OmcA confers enhanced affinity toward goethite and results in rapid attachment. Meanwhile, MtrC significantly increases the contact area of bacterial cells with goethite and promotes the interfacial reaction, which may explain its central role in extracellular electron transfer. This study provides novel insights into the role of bacterial surface macromolecules in the interfacial interaction of bacteria with minerals, which is critical to the development of a comprehensive understanding of cell-mineral interactions.

Tuning Extracellular Electron Transfer by Shewanella oneidensis Using Transcriptional Logic Gates.

Extracellular electron transfer (EET) pathways, such as those in the bacterium Shewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux in S. oneidensis has proven challenging because a functional understanding of its EET pathway proteins and their effect on engineering parametrizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway of S. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriate S. oneidensis knockout strains, we pinpointed construct/strain pairings that expressed cymA, mtrA, and mtrC with maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures that predictably turn on and turn off EET flux, respectively, in response to isopropyl ß-D-1-thiogalactopyranoside (IPTG). Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

Disparity of Cytochrome Utilization in Anodic and Cathodic Extracellular Electron Transfer Pathways of Geobacter sulfurreducens Biofilms.

Extracellular electron transfer (EET) in microorganisms is prevalent in nature and has been utilized in functional bioelectrochemical systems. EET of Geobacter sulfurreducens has been extensively studied and has been revealed to be facilitated through c-type cytochromes, which mediate charge between the electrode and G. sulfurreducens in anodic mode. However, the EET pathway of cathodic conversion of fumarate to succinate is still under debate. Here, we apply a variety of analytical methods, including electrochemistry, UV-vis absorption and resonance Raman spectroscopy, quartz crystal microbalance with dissipation, and electron microscopy, to understand the involvement of cytochromes and other possible electron-mediating species in the switching between anodic and cathodic reaction modes. By switching the applied bias for a G. sulfurreducens biofilm coupled to investigating the quantity and function of cytochromes, as well as the emergence of Fe-containing particles on the cell membrane, we provide evidence of a diminished role of cytochromes in cathodic EET. This work sheds light on the mechanisms of G. sulfurreducens biofilm growth and suggests the possible existence of a nonheme, iron-involving EET process in cathodic mode.

Extracellular electron uptake in Methanosarcinales is independent of multiheme c-type cytochromes.

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.

Structure and redox properties of the diheme electron carrier cytochrome c4 from Pseudomonas aeruginosa.

At low oxygen concentrations, respiration of Pseudomonas aeruginosa (Pa) and other bacteria relies on activity of cytochrome cbb3 oxidases. A diheme cytochrome c4 (cyt c4) donates electrons to Pa cbb3 oxidases to enable oxygen reduction and proton pumping by these enzymes. Given the importance of this redox pathway for bacterial pathogenesis, both cyt c4 and cbb3 oxidase are potential targets for new antibacterial strategies. The structural information about these two proteins, however, is scarce, and functional insights for Pa and other bacteria have been primarily drawn from analyses of the analogous system from Pseudomonas stutzeri (Ps). Herein, we describe characterization of structural and redox properties of cyt c4 from Pa. The crystal structure of Pa cyt c4 has revealed that this protein is organized in two monoheme domains. The interdomain interface is more hydrophobic in Pa cyt c4, and the protein surface does not show the dipolar distribution of charges found in Ps cyt c4. The reduction potentials of the two hemes are similar in Pa cyt c4 but differ by about 100 mV in Ps cyt c4. Analyses of structural models of these and other cyt c4 proteins suggest that multiple factors contribute to the potential difference of the two hemes in these proteins, including solvent accessibility of the heme group, the distribution of surface charges, and the nature of the interdomain interface. The distinct properties of cyt c4 proteins from closely-related Pa and Ps bacteria emphasize the importance of examining the cbb3/cyt c4 redox pathway in multiple species.