Julea N. Butt

Characterization of an electron conduit between bacteria and the extracellular environment

A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning β-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.

Structure of a bacterial cell surface decaheme electron conduit

Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular intercytochrome electron exchange along “nanowire” appendages. We present a 3.2-Å crystal structure of one of these decaheme cytochromes, MtrF, that allows the spatial organization of the 10 hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65-Å octaheme chain transects the length of the protein and is bisected by a planar 45-Å tetraheme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g., minerals), soluble substrates (e.g., flavins), and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

The ‘porin–cytochrome’ model for microbe-to-mineral electron transfer

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via ‘porin–cytochrome’ electron transport modules. The molecular structure of an outer‐membrane extracellular‐facing deca‐haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer‐membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as ‘nanowires’, or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram‐negative bacteria.

Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals

The mineral-respiring bacterium Shewanella oneidensis uses a protein complex, MtrCAB, composed of two decaheme cytochromes, MtrC and MtrA, brought together inside a transmembrane porin, MtrB, to transport electrons across the outer membrane to a variety of mineral-based electron acceptors. A proteoliposome system containing a pool of internalized electron carriers was used to investigate how the topology of the MtrCAB complex relates to its ability to transport electrons across a lipid bilayer to externally located Fe(III) oxides. With MtrA facing the interior and MtrC exposed on the outer surface of the phospholipid bilayer, the established in vivo orientation, electron transfer from the interior electron carrier pool through MtrCAB to solid-phase Fe(III) oxides was demonstrated. The rates were 103 times higher than those reported for reduction of goethite, hematite, and lepidocrocite by S. oneidensis, and the order of the reaction rates was consistent with those observed in S. oneidensis cultures. In contrast, established rates for single turnover reactions between purified MtrC and Fe(III) oxides were 103 times lower. By providing a continuous flow of electrons, the proteoliposome experiments demonstrate that conduction through MtrCAB directly to Fe(III) oxides is sufficient to support in vivo, anaerobic, solid-phase iron respiration.

Identification and Characterization of MtoA: A Decaheme c-Type Cytochrome of the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1

The Gram-negative bacterium Sideroxydans lithotrophicus ES-1 (ES-1) grows on FeCO3 or FeS at oxic–anoxic interfaces at circumneutral pH, and the ES-1-mediated Fe(II) oxidation occurs extracellularly. However, the molecular mechanisms underlying ES-1’s ability to oxidize Fe(II) remain unknown. Survey of the ES-1 genome for candidate genes for microbial extracellular Fe(II) oxidation revealed that it contained a three-gene cluster encoding homologs of Shewanella oneidensis MR-1 (MR-1) MtrA, MtrB, and CymA that are involved in extracellular Fe(III) reduction. Homologs of MtrA and MtrB were also previously shown to be involved in extracellular Fe(II) oxidation by Rhodopseudomonas palustris TIE-1. To distinguish them from those found in MR-1, the identified homologs were named MtoAB and CymAES-1. Cloned mtoA partially complemented an MR-1 mutant without MtrA with regards to ferrihydrite reduction. Characterization of purified MtoA showed that it was a decaheme c-type cytochrome and oxidized soluble Fe(II). Oxidation of Fe(II) by MtoA was pH- and Fe(II)-complexing ligand-dependent. Under conditions tested, MtoA oxidized Fe(II) from pH 7 to pH 9 with the optimal rate at pH 9. MtoA oxidized Fe(II) complexed with different ligands at different rates. The reaction rates followed the order Fe(II)Cl2 >  Fe(II)–citrate > Fe(II)–NTA > Fe(II)–EDTA with the second-order rate constants ranging from 6.3 × 10−3 μM−1 s−1 for oxidation of Fe(II)Cl2 to 1.0 × 10−3 μM−1 s−1 for oxidation of Fe(II)–EDTA. Thermodynamic modeling showed that redox reaction rates for the different Fe(II)-complexes correlated with their respective estimated reaction-free energies. Collectively, these results demonstrate that MtoA is a functional Fe(II)-oxidizing protein that, by working in concert with MtoB and CymAES-1, may oxidize Fe(II) at the bacterial surface and transfer released electrons across the bacterial cell envelope to the quinone pool in the inner membrane during extracellular Fe(II) oxidation by ES-1.

The Nitric Oxide Reductase Activity of Cytochrome c Nitrite Reductase from Escherichia coli

Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO· than the parent strain led to the proposal that NrfA might also participate in NO· detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO· reductase activity. NO· reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO· reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO· reduction by NrfA is described by a Km of the order of 300 μm. The predicted turnover number of ∼840 NO· s–1 is much higher than that of the dedicated respiratory NO· reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO· detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO· encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO· levels such that any NO· reaching the cytoplasm is efficiently removed by flavorubredoxin (Km ∼ 0.4 μm).

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems ofα-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the γ-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 μm determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the α-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo–O bond length is 2.6 Å, which is indicative of a water ligand. The potential range over which the Mo6+ state is reduced to the Mo5+ state in either NapA (between +100 and -100 mV) or the NapAB complex (-150 to -350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo6+/5+ > +350 mV), and the form of the Mo5+ EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo5+ state could not be further reduced to Mo4+. We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo6+ species may participate.

Methylene blue as an electrochemical discriminator of single- and double-stranded oligonucleotides immobilised on gold substrates

Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient and versatile route to DNA-functionalised surfaces. Here we show that the square-wave voltammetric peak position of methylene blue complexed to thiol-terminated single-stranded oligonucleotides immobilised on gold electrodes differs from that of methylene blue complexed to thiol-terminated double-stranded oligonucleotides immobilised on gold electrodes. The peak potential of methylene blue at the single-stranded oligonucleotide array was consistently found to occur at potentials ca. 10-15 mV more positive than that at double-stranded oligonucleotide arrays, the precise difference being dependent on the direction of the voltammetry. This voltammetric behaviour mirrors that found for methylene blue bound to freely diffusing single- and double-stranded calf thymus DNA and suggests that the immobilised oligonucleotides retain the methylene blue binding properties of their freely diffusing counterparts. Thus methylene blue provides a simple electrochemical indicator for the status of oligonucleotide-functionalised gold surfaces.

Multi-haem cytochromes in Shewanella oneidensis MR-1: structures, functions and opportunities

Multi-haem cytochromes are employed by a range of microorganisms to transport electrons over distances of up to tens of nanometres. Perhaps the most spectacular utilization of these proteins is in the reduction of extracellular solid substrates, including electrodes and insoluble mineral oxides of Fe(III) and Mn(III/IV), by species of Shewanella and Geobacter. However, multi-haem cytochromes are found in numerous and phylogenetically diverse prokaryotes where they participate in electron transfer and redox catalysis that contributes to biogeochemical cycling of N, S and Fe on the global scale. These properties of multi-haem cytochromes have attracted much interest and contributed to advances in bioenergy applications and bioremediation of contaminated soils. Looking forward, there are opportunities to engage multi-haem cytochromes for biological photovoltaic cells, microbial electrosynthesis and developing bespoke molecular devices. As a consequence, it is timely to review our present understanding of these proteins and we do this here with a focus on the multitude of functionally diverse multi-haem cytochromes in Shewanella oneidensis MR-1. We draw on findings from experimental and computational approaches which ideally complement each other in the study of these systems: computational methods can interpret experimentally determined properties in terms of molecular structure to cast light on the relation between structure and function. We show how this synergy has contributed to our understanding of multi-haem cytochromes and can be expected to continue to do so for greater insight into natural processes and their informed exploitation in biotechnologies.

A trans‐outer membrane porin‐cytochrome protein complex for extracellular electron transfer by Geobacter sulfurreducens PCA

The multi-heme, outer membrane c-type cytochrome (c-Cyt) OmcB of Geobacter sulfurreducens was previously proposed to mediate electron transfer across the outer membrane. However, the underlying mechanism has remained uncharacterized. In G. sulfurreducens, the omcB gene is part of two tandem four-gene clusters, each is predicted to encode a transcriptional factor (OrfR/OrfS), a porin-like outer membrane protein (OmbB/OmbC), a periplasmic c-type cytochrome (OmaB/OmaC) and an outer membrane c-Cyt (OmcB/OmcC) respectively. Here, we showed that OmbB/OmbC, OmaB/OmaC and OmcB/OmcC of G. sulfurreducens PCA formed the porin-cytochrome (Pcc) protein complexes, which were involved in transferring electrons across the outer membrane. The isolated Pcc protein complexes reconstituted in proteoliposomes transferred electrons from reduced methyl viologen across the lipid bilayer of liposomes to Fe(III)-citrate and ferrihydrite. The pcc clusters were found in all eight sequenced Geobacter and 11 other bacterial genomes from six different phyla, demonstrating a widespread distribution of Pcc protein complexes in phylogenetically diverse bacteria. Deletion of ombB-omaB-omcB-orfS-ombC-omaC-omcC gene clusters had no impact on the growth of G. sulfurreducens PCA with fumarate but diminished the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite. Complementation with the ombB-omaB-omcB gene cluster restored the ability of G. sulfurreducens PCA to reduce Fe(III)-citrate and ferrihydrite.