Glassy carbon tubular electrodes for the reduction of oxygen to hydrogen peroxide.

The oxygen reduction reaction (ORR) to produce hydrogen peroxide (H2O2) is of great industrial interest. Herein, a hydrodynamic electrochemical method is explored for use as a continuous method to produce H2O2 at the point-of-use. The ORR was studied in a tubular glassy carbon flow cell under a laminar flow regime. A generalised theoretical model was developed to explore the conditions, such as volume flow rates and tubular lengths etc., for which a near-full electrolysis may be achieved. The parameters probed, transfer coefficient, half-wave potentials, volume flow rates, etc., provide physical insights into the irreversible oxygen reduction process. Thereafter, the surface modification of the tubular electrode with an electrocatalyst, 2-anthraquinonyl group (AQ-), is investigated for the mediated reduction of oxygen. This is shown to usefully decrease the required overpotential for the reduction process.

Exploring the biochemistry at the extracellular redox frontier of bacterial mineral Fe(III) respiration.

Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.

Electrolyte tuning of electrode potentials: the one electron vs. two electron reduction of anthraquinone-2-sulfonate in aqueous media.

The electrode potentials of quinone redox centres in aqueous solutions can be tuned by varying the electrolyte cation identity. The phenomenon is due to the ion pairing effect of the tetra-n-butylammonium cation with the semiquinone intermediate species.

Semiquinone intermediates in the two-electron reduction of quinones in aqueous media and their exceptionally high reactivity towards oxygen reduction.

We report the catalytic anthraquinone-mediated reduction of oxygen at a boron-doped diamond electrode. Scheme of squares modelling confirms the existence of and reveals the role of the semiquinone intermediates, which are shown to have an exceptional reactivity towards oxygen (as compared to the di-reduced anthraquinone).

Quinol-cytochrome c oxidoreductase and cytochrome c4 mediate electron transfer during selenate respiration in Thauera selenatis.

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E(m) + 234 +/- 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified ( approximately 24 and approximately 6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c(4) family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 +/- 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c(4) is a novel route for a member of the DMSO reductase family of molybdoenzymes.

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 beta-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.

The role of multihaem cytochromes in the respiration of nitrite in Escherichia coli and Fe(III) in Shewanella oneidensis.

The periplasmic nitrite reductase system from Escherichia coli and the extracellular Fe(III) reductase system from Shewanella oneidensis contain multihaem c-type cytochromes as electron carriers and terminal reductases. The position and orientation of the haem cofactors in multihaem cytochromes from different bacteria often show significant conservation despite different arrangements of the polypeptide chain. We propose that the decahaem cytochromes of the iron reductase system MtrA, MtrC and OmcA comprise pentahaem 'modules' similar to the electron donor protein, NrfB, from E. coli. To demonstrate this, we have isolated and characterized the N-terminal pentahaem module of MtrA by preparing a truncated form containing five covalently attached haems. UV-visible spectroscopy indicated that all five haems were low-spin, consistent with the presence of bis-His ligand co-ordination as found in full-length MtrA.

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors.

MtrC is a decaheme c-type cytochrome associated with the outer cell membrane of Fe(III)-respiring species of the Shewanella genus. It is proposed to play a role in anaerobic respiration by mediating electron transfer to extracellular mineral oxides that can serve as terminal electron acceptors. The present work presents the first spectropotentiometric and voltammetric characterization of MtrC, using protein purified from Shewanella oneidensis MR-1. Potentiometric titrations, monitored by UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopy, reveal that the hemes within MtrC titrate over a broad potential range spanning between approximately +100 and approximately -500 mV (vs. the standard hydrogen electrode). Across this potential window the UV-vis absorption spectra are characteristic of low-spin c-type hemes and the EPR spectra reveal broad, complex features that suggest the presence of magnetically spin-coupled low-spin c-hemes. Non-catalytic protein film voltammetry of MtrC demonstrates reversible electrochemistry over a potential window similar to that disclosed spectroscopically. The voltammetry also allows definition of kinetic properties of MtrC in direct electron exchange with a solid electrode surface and during reduction of a model Fe(III) substrate. Taken together, the data provide quantitative information on the potential domain in which MtrC can operate.

Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1.

The interaction of proteins implicated in dissimilatory metal reduction by Shewanella oneidensis MR-1 (outer membrane [OM] proteins OmcA, MtrB, and MtrC; OM-associated protein MtrA; periplasmic protein CctA; and cytoplasmic membrane protein CymA) were characterized by protein purification, analytical ultracentrifugation, and cross-linking methods. Five of these proteins are heme proteins, OmcA (83 kDa), MtrC (75 kDa), MtrA (32 kDa), CctA (19 kDa), and CymA (21 kDa), and can be visualized after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by heme staining. We show for the first time that MtrC, MtrA, and MtrB form a 198-kDa complex with a 1:1:1 stoichiometry. These proteins copurify through anion-exchange chromatography, and the purified complex has the ability to reduce multiple forms of Fe(III) and Mn(IV). Additionally, MtrA fractionates with the OM through sucrose density gradient ultracentrifugation, and MtrA comigrates with MtrB in native gels. Protein cross-linking of whole cells with 1% formaldehyde show new heme bands of 160, 151, 136, and 59 kDa. Using antibodies to detect each protein separately, heme proteins OmcA and MtrC were shown to cross-link, yielding the 160-kDa band. Consistent with copurification results, MtrB cross-links with MtrA, forming high-molecular-mass bands of approximately 151 and 136 kDa.

A dedicated haem lyase is required for the maturation of a novel bacterial cytochrome c with unconventional covalent haem binding.

In bacterial c-type cytochromes, the haem cofactor is covalently attached via two cysteine residues organized in a haem c-binding motif. Here, a novel octa-haem c protein, MccA, is described that contains only seven conventional haem c-binding motifs (CXXCH), in addition to several single cysteine residues and a conserved CH signature. Mass spectrometric analysis of purified MccA from Wolinella succinogenes suggests that two of the single cysteine residues are actually part of an unprecedented CX15CH sequence involved in haem c binding. Spectroscopic characterization of MccA identified an unusual high-potential haem c with a red-shifted absorption maximum, not unlike that of certain eukaryotic cytochromes c that exceptionally bind haem via only one thioether bridge. A haem lyase gene was found to be specifically required for the maturation of MccA in W. succinogenes. Equivalent haem lyase-encoding genes belonging to either the bacterial cytochrome c biogenesis system I or II are present in the vicinity of every known mccA gene suggesting a dedicated cytochrome c maturation pathway. The results necessitate reconsideration of computer-based prediction of putative haem c-binding motifs in bacterial proteomes.