The Crystal Structure of a Biological Insulated Transmembrane Molecular Wire.

A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.

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.

Reaction of Thiosulfate Dehydrogenase with a Substrate Mimic Induces Dissociation of the Cysteine Heme Ligand Giving Insights into the Mechanism of Oxidative Catalysis.

Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys-/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (Em, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (Cα-CH2-S-SO3-) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (Cα-CH2-S-SSO3-). The active site heme in the stable sulfonated protein displays an Em approximately 200 mV more positive than the Cys-/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.

The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni.

Campylobacter jejuni is a microaerophilic zoonotic pathogen with an atypical respiratory Complex I that oxidizes a flavodoxin (FldA) instead of NADH. FldA is essential for viability and is reduced via pyruvate and 2-oxoglutarate oxidoreductases (POR/OOR). Here, we show that FldA can also be reduced by FqrB (Cj0559), an NADPH:FldA reductase. An fqrB deletion mutant was viable but displayed a significant growth defect. FqrB is related to flavoprotein reductases from Gram-positive bacteria that can reduce NrdI, a specialized flavodoxin that is needed for tyrosyl radical formation in NrdF, the beta subunit of class 1b-type (Mn) ribonucleotide reductase (RNR). However, C. jejuni possesses a single class Ia-type (Fe) RNR (NrdAB) that would be expected to be ferredoxin dependent. We show that CjFldA is an unusually high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild-type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in an NADPH, FqrB, and FldA dependent manner, as evidenced by both optical and electron paramagnetic resonance (EPR) spectroscopy. Thus, FldA activates RNR in C. jejuni, partly explaining its essentiality.

Photocatalytic Removal of the Greenhouse Gas Nitrous Oxide by Liposomal Microreactors.

Nitrous oxide (N2 O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2 O decomposition typically contain precious metals and/or operate at elevated temperatures driving a desire for more sustainable alternatives. Here we demonstrate self-assembly of liposomal microreactors enabling catalytic reduction of N2 O to the climate neutral product N2 . Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2 O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2 O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2 O removal in ambient, aqueous conditions.

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes.

Thiosulfate dehydrogenases (TsdAs) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic CD (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a microaerobic human pathogen, and from the purple sulfur bacterium Allochromatium vinosum In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase, respectively. The active site Heme 1 in both enzymes has His/Cys ligation in the ferric and ferrous states and the midpoint potentials (Em ) of the corresponding redox transformations are similar, -185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni, TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em , -129 mV) to His/Met (Em , +266 mV), but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s).

Quantum dot interactions with and toxicity to Shewanella oneidensis MR-1.

Combining abiotic photosensitisers such as quantum dots (QDs) with non-photosynthetic bacteria presents an intriguing concept into the design of artificial photosynthetic organisms and solar-driven fuel production. Shewanella oneidensis MR-1 (MR-1) is a versatile bacterium concerning respiration, metabolism and biocatalysis, and is a promising organism for artificial photosynthesis as the bacterium's synthetic and catalytic ability provides a potential system for bacterial biohydrogen production. MR-1's hydrogenases are present in the periplasmatic space. It follows that for photoenergised electrons to reach these enzymes, QDs will need to be able to enter the periplasm, or electrons need to enter the periplasm via the Mtr pathway that is responsible for MR-1's extracellular electron transfer ability. As a step towards this goal, various QDs were tested for their photo-reducing potential, nanotoxicology and further for their interaction with MR-1. CdTe/CdS/TGA, CdTe/CdS/Cysteamine, a commercial, negatively charged CdTe and CuInS2/ZnS/PMAL QDs were examined. The photoreduction potential of the QDs was confirmed by measuring their ability to photoreduce methyl viologen with different sacrificial electron donors. The commercial CdTe and CuInS2/ZnS/PMAL QDs showed no toxicity towards MR-1 as evaluated by a colony-forming units method and a fluorescence viability assay. Only the commercial negatively charged CdTe QDs showed good interaction with MR-1. With transmission electron microscopy, QDs were observed both in the cytoplasm and periplasm. These results inform on the possibilities and bottlenecks when developing bionanotechnological systems for the photosynthetic production of biohydrogen by MR-1.

His/Met heme ligation in the PioA outer membrane cytochrome enabling light-driven extracellular electron transfer by Rhodopseudomonas palustris TIE-1.

A growing number of bacterial species are known to move electrons across their cell envelopes. Naturally this occurs in support of energy conservation and carbon-fixation. For biotechnology it allows electron exchange between bacteria and electrodes in microbial fuel cells and during microbial electrosynthesis. In this context Rhodopseudomonas palustris TIE-1 is of much interest. These bacteria respond to light by taking electrons from their external environment, including electrodes, to drive CO2-fixation. The PioA cytochrome, that spans the bacterial outer membrane, is essential for this electron transfer and yet little is known about its structure and electron transfer properties. Here we reveal the ten c-type hemes of PioA are redox active across the window +250 to -400 mV versus Standard Hydrogen Electrode and that the hemes with most positive reduction potentials have His/Met and His/H2O ligation. These chemical and redox properties distinguish PioA from the more widely studied family of MtrA outer membrane decaheme cytochromes with ten His/His ligated hemes. We predict a structure for PioA in which the hemes form a chain spanning the longest dimension of the protein, from Heme 1 to Heme 10. Hemes 2, 3 and 7 are identified as those most likely to have His/Met and/or His/H2O ligation. Sequence analysis suggests His/Met ligation of Heme 2 and/or 7 is a defining feature of decaheme PioA homologs from over 30 different bacterial genera. His/Met ligation of Heme 3 appears to be less common and primarily associated with PioA homologs from purple non-sulphur bacteria belonging to the alphaproteobacteria class.

Structural modeling of an outer membrane electron conduit from a metal-reducing bacterium suggests electron transfer via periplasmic redox partners.

Many subsurface microorganisms couple their metabolism to the reduction or oxidation of extracellular substrates. For example, anaerobic mineral-respiring bacteria can use external metal oxides as terminal electron acceptors during respiration. Porin-cytochrome complexes facilitate the movement of electrons generated through intracellular catabolic processes across the bacterial outer membrane to these terminal electron acceptors. In the mineral-reducing model bacterium Shewanella oneidensis MR-1, this complex is composed of two decaheme cytochromes (MtrA and MtrC) and an outer-membrane ß-barrel (MtrB). However, the structures and mechanisms by which porin-cytochrome complexes transfer electrons are unknown. Here, we used small-angle neutron scattering (SANS) to study the molecular structure of the transmembrane complexes MtrAB and MtrCAB. Ab initio modeling of the scattering data yielded a molecular envelope with dimensions of ∼105 × 60 × 35 Å for MtrAB and ∼170 × 60 × 45 Å for MtrCAB. The shapes of these molecular envelopes suggested that MtrC interacts with the surface of MtrAB, extending ∼70 Å from the membrane surface and allowing the terminal hemes to interact with both MtrAB and an extracellular acceptor. The data also reveal that MtrA fully extends through the length of MtrB, with ∼30 Å being exposed into the periplasm. Proteoliposome models containing membrane-associated MtrCAB and internalized small tetraheme cytochrome (STC) indicate that MtrCAB could reduce Fe(III) citrate with STC as an electron donor, disclosing a direct interaction between MtrCAB and STC. Taken together, both structural and proteoliposome experiments support porin-cytochrome-mediated electron transfer via periplasmic cytochromes such as STC.

Ultrafast Light-Driven Electron Transfer in a Ru(II)tris(bipyridine)-Labeled Multiheme Cytochrome.

Multiheme cytochromes attract much attention for their electron transport properties. These proteins conduct electrons across bacterial cell walls and along extracellular filaments and when purified can serve as bionanoelectronic junctions. Thus, it is important and necessary to identify and understand the factors governing electron transfer in this family of proteins. To this end we have used ultrafast transient absorbance spectroscopy, to define heme-heme electron transfer dynamics in the representative multiheme cytochrome STC from Shewanella oneidensis in aqueous solution. STC was photosensitized by site-selective labeling with a Ru(II)(bipyridine)3 dye and the dynamics of light-driven electron transfer described by a kinetic model corroborated by molecular dynamics simulation and density functional theory calculations. With the dye attached adjacent to STC Heme IV, a rate constant of 87 × 106 s-1 was resolved for Heme IV → Heme III electron transfer. With the dye attached adjacent to STC Heme I, at the opposite terminus of the tetraheme chain, a rate constant of 125 × 106 s-1 was defined for Heme I → Heme II electron transfer. These rates are an order of magnitude faster than previously computed values for unlabeled STC. The Heme III/IV and I/II pairs exemplify the T-shaped heme packing arrangement, prevalent in multiheme cytochromes, whereby the adjacent porphyrin rings lie at 90° with edge-edge (Fe-Fe) distances of ∼6 (11) Å. The results are significant in demonstrating the opportunities for pump-probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes.

Towards compartmentalized photocatalysis: multihaem proteins as transmembrane molecular electron conduits.

The high quantum efficiency of natural photosynthesis has inspired chemists for solar fuel synthesis. In photosynthesis, charge recombination in photosystems is minimized by efficient charge separation across the thylakoid membrane. Building on our previous bioelectrochemical studies of electron transfer between a light-harvesting nanoparticle (LHNP) and the decahaem subunit MtrC, we demonstrate photo-induced electron transfer through the full transmembrane MtrCAB complex in liposome membranes. Successful photoelectron transfer is demonstrated by the decomposition of a redox dye, Reactive Red 120 (RR120), encapsulated in MtrCAB proteoliposomes. The photoreduction rates are found to be dependent on the identity of the external LHNPs, specifically, dye-sensitized TiO2, amorphous carbon dots (a-CD) and graphitic carbon dots with core nitrogen doping (g-N-CDs). Agglomeration or aggregation of TiO2 NPs likely reduces the kinetics of RR120 reductive decomposition. In contrast, with the dispersed a-CD and g-N-CDs, the kinetics of the RR120 reductive decomposition are observed to be faster with the MtrCAB proteoliposomes and we propose that this is due to enhancement in the charge-separated state. Thus, we show a proof-of-concept for using MtrCAB as a lipid membrane-spanning building block for compartmentalised photocatalysis that mimics photosynthesis. Future work is focused on incorporation of fuel generating redox catalysts in the MtrCAB proteoliposome lumen.

Photosensitised Multiheme Cytochromes as Light-Driven Molecular Wires and Resistors.

Multiheme cytochromes possess closely packed redox-active hemes arranged as chains spanning the tertiary structure. Here we describe five variants of a representative multiheme cytochrome engineered as biohybrid phototransducers for converting light into electricity. Each variant possesses a single Cys sulfhydryl group near a terminus of the heme chain, and this was efficiently labelled with a RuII (2,2'-bipyridine)3 photosensitiser. When irradiated in the presence of a sacrificial electron donor (SED) the proteins exhibited different types of behaviour. Certain proteins were rapidly and fully reduced. Other proteins were rapidly semi-reduced but resisted complete photoreduction. These findings reveal that photosensitised multiheme cytochromes can be engineered to act as resistors, with intrinsic regulation of light-driven electron accumulation, and also as molecular wires with essentially unhindered photoreduction. It is proposed that the observed behaviour arises from interplay between the site of electron injection and the distribution of heme reduction potentials along the heme chain.

Characterization of Anammox Hydrazine Dehydrogenase, a Key N2-producing Enzyme in the Global Nitrogen Cycle.

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P460) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms.

Electron Accepting Units of the Diheme Cytochrome c TsdA, a Bifunctional Thiosulfate Dehydrogenase/Tetrathionate Reductase.

The enzymes of the thiosulfate dehydrogenase (TsdA) family are wide-spread diheme c-type cytochromes. Here, redox carriers were studied mediating the flow of electrons arising from thiosulfate oxidation into respiratory or photosynthetic electron chains. In a number of organisms, including Thiomonas intermedia and Sideroxydans lithotrophicus, the tsdA gene is immediately preceded by tsdB encoding for another diheme cytochrome. Spectrophotometric experiments in combination with enzymatic assays in solution showed that TsdB acts as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. Although TsdA covers a range from -300 to +150 mV, TsdB is redox active between -100 and +300 mV, thus enabling electron transfer between these hemoproteins. The three-dimensional structure of the TsdB-TsdA fusion protein from the purple sulfur bacterium Marichromatium purpuratum was solved by X-ray crystallography to 2.75 Å resolution providing insights into internal electron transfer. In the oxidized state, this tetraheme cytochrome c contains three hemes with axial His/Met ligation, whereas heme 3 exhibits the His/Cys coordination typical for TsdA active sites. Interestingly, thiosulfate is covalently bound to Cys330 on heme 3. In several bacteria, including Allochromatium vinosum, TsdB is not present, precluding a general and essential role for electron flow. Both AvTsdA and the MpTsdBA fusion react efficiently in vitro with high potential iron-sulfur protein from A. vinosum (Em +350 mV). High potential iron-sulfur protein not only acts as direct electron donor to the reaction center in anoxygenic phototrophs but can also be involved in aerobic respiratory chains.

High Performance Reduction of H2O2 with an Electron Transport Decaheme Cytochrome on a Porous ITO Electrode.

The decaheme cytochrome MtrC from Shewanella oneidensis MR-1 immobilized on an ITO electrode displays unprecedented H2O2 reduction activity. Although MtrC showed lower peroxidase activity in solution compared to horseradish peroxidase, the ten heme cofactors enable excellent electronic communication and a superior activity on the electrode surface. A hierarchical ITO electrode enabled optimal immobilization of MtrC and a high current density of 1 mA cm-2 at 0.4 V vs SHE could be obtained at pH 6.5 (Eonset = 0.72 V). UV-visible and Resonance Raman spectroelectrochemical studies suggest the formation of a high valent iron-oxo species as the catalytic intermediate. Our findings demonstrate the potential of multiheme cytochromes to catalyze technologically relevant reactions and establish MtrC as a new benchmark in biotechnological H2O2 reduction with scope for applications in fuel cells and biosensors.

Carbon Dots as Versatile Photosensitizers for Solar-Driven Catalysis with Redox Enzymes.

Light-driven enzymatic catalysis is enabled by the productive coupling of a protein to a photosensitizer. Photosensitizers used in such hybrid systems are typically costly, toxic, and/or fragile, with limited chemical versatility. Carbon dots (CDs) are low-cost, nanosized light-harvesters that are attractive photosensitizers for biological systems as they are water-soluble, photostable, nontoxic, and their surface chemistry can be easily modified. We demonstrate here that CDs act as excellent light-absorbers in two semibiological photosynthetic systems utilizing either a fumarate reductase (FccA) for the solar-driven hydrogenation of fumarate to succinate or a hydrogenase (H2ase) for reduction of protons to H2. The tunable surface chemistry of the CDs was exploited to synthesize positively charged ammonium-terminated CDs (CD-NHMe2+), which were capable of transferring photoexcited electrons directly to the negatively charged enzymes with high efficiency and stability. Enzyme-based turnover numbers of 6000 mol succinate (mol FccA)-1 and 43,000 mol H2 (mol H2ase)-1 were reached after 24 h. Negatively charged carboxylate-terminated CDs (CD-CO2-) displayed little or no activity, and the electrostatic interactions at the CD-enzyme interface were determined to be essential to the high photocatalytic activity observed with CD-NHMe2+. The modular surface chemistry of CDs together with their photostability and aqueous solubility make CDs versatile photosensitizers for redox enzymes with great scope for their utilization in photobiocatalysis.

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII -dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.

Layer-by-Layer Assembly of Supported Lipid Bilayer Poly-L-Lysine Multilayers.

Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.

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 10(3) 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 10(3) 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.

Structural basis of biological NO generation by octaheme oxidoreductases.

Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.