Size-dependent activity of carbon dots for photocatalytic H2 generation in combination with a molecular Ni cocatalyst.

Carbon dots (CDs) are low-cost light-absorbers in photocatalytic multicomponent systems, but their wide size distribution has hampered rational design and the identification of the factors that lead to their best performance. To address this challenge, we report herein the use of gel filtration size exclusion chromatography to separate amorphous, graphitic, and graphitic N-doped CDs depending on their lateral size to study the effect of their size on photocatalytic H2 evolution with a DuBois-type Ni cocatalyst. Transmission electron microscopy and dynamic light scattering confirm the size-dependent separation of the CDs, whereas UV-vis and fluorescence spectroscopy of the more monodisperse fractions show a distinct response which computational modelling attributes to a complex interplay between CD size and optical properties. A size-dependent effect on the photocatalytic H2 evolution performance of the CDs in combination with a molecular Ni cocatalyst is demonstrated with a maximum activity at approximately 2-3 nm CD diameter. Overall, size separation leads to a two-fold increase in the specific photocatalytic activity for H2 evolution using the monodisperse CDs compared to the as synthesized polydisperse samples, highlighting the size-dependent effect on photocatalytic performance.

A Cysteine Pair Controls Flavin Reduction by Extracellular Cytochromes during Anoxic/Oxic Environmental Transitions.

Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment. IMPORTANCE Bacteria that live at the oxic/anoxic interface have to rapidly adapt to changes in oxygen levels within their environment. The facultative anaerobe Shewanella oneidensis MR-1 can use EET to respire in the absence of oxygen, but on exposure to oxygen, EET could directly reduce extracellular oxygen and generate harmful reactive oxygen species that damage the bacterium. By modifying an extracellular cytochrome called MtrC, we show how preventing a redox-active disulfide from forming causes the production of cytotoxic concentrations of peroxide. The disulfide affects the affinity of MtrC for the redox-active flavin mononucleotide, which is part of the EET pathway. Our results demonstrate how a cysteine pair exposed on the surface controls the path of electron transfer, allowing facultative anaerobic bacteria to rapidly adapt to changes in oxygen concentration at the oxic/anoxic interface.

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.

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.

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

Nitrous oxide (N2O) is a potent greenhouse and ozone-reactive gas for which emissions are growing rapidly due to increasingly intensive agriculture. Synthetic catalysts for N2O 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 N2O to the climate neutral product N2. Photoexcitation of graphitic N-doped carbon dots delivers electrons to encapsulated N2O Reductase enzymes via a lipid-soluble biomolecular wire provided by the MtrCAB protein complex. Within the microreactor, electron transfer from MtrCAB to N2O Reductase is facilitated by the general redox mediator methyl viologen. The liposomal microreactors use only earth-abundant elements to catalyze N2O removal in ambient, aqueous conditions.

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.

Bespoke Biomolecular Wires for Transmembrane Electron Transfer: Spontaneous Assembly of a Functionalized Multiheme Electron Conduit.

Shewanella oneidensis exchanges electrons between cellular metabolism and external redox partners in a process that attracts much attention for production of green electricity (microbial fuel cells) and chemicals (microbial electrosynthesis). A critical component of this pathway is the outer membrane spanning MTR complex, a biomolecular wire formed of the MtrA, MtrB, and MtrC proteins. MtrA and MtrC are decaheme cytochromes that form a chain of close-packed hemes to define an electron transfer pathway of 185 Å. MtrA is wrapped inside MtrB for solubility across the outer membrane lipid bilayer; MtrC sits outside the cell for electron exchange with external redox partners. Here, we demonstrate tight and spontaneous in vitro association of MtrAB with separately purified MtrC. The resulting complex is comparable with the MTR complex naturally assembled by Shewanella in terms of both its structure and rates of electron transfer across a lipid bilayer. Our findings reveal the potential for building bespoke electron conduits where MtrAB combines with chemically modified MtrC, in this case, labeled with a Ru-dye that enables light-triggered electron injection into the MtrC heme chain.

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.

Coherent Electron Transport across a 3 nm Bioelectronic Junction Made of Multi-Heme Proteins.

Multi-heme cytochromes (MHCs) are fascinating proteins used by bacterial organisms to shuttle electrons within, between, and out of their cells. When placed in solid-state electronic junctions, MHCs support temperature-independent currents over several nanometers that are 3 orders of magnitude higher compared to other redox proteins of similar size. To gain molecular-level insight into their astonishingly high conductivities, we combine experimental photoemission spectroscopy with DFT+Σ current-voltage calculations on a representative Gold-MHC-Gold junction. We find that conduction across the dry, 3 nm long protein occurs via off-resonant coherent tunneling, mediated by a large number of protein valence-band orbitals that are strongly delocalized over heme and protein residues. This picture is profoundly different from the electron hopping mechanism induced electrochemically or photochemically under aqueous conditions. Our results imply that the current output in solid-state junctions can be even further increased in resonance, for example, by applying a gate voltage, thus allowing a quantum jump for next-generation bionanoelectronic devices.

Which Multi-Heme Protein Complex Transfers Electrons More Efficiently? Comparing MtrCAB from Shewanella with OmcS from Geobacter.

Microbial nanowires are fascinating biological structures that allow bacteria to transport electrons over micrometers for reduction of extracellular substrates. It was recently established that the nanowires of both Shewanella and Geobacter are made of multi-heme proteins; but, while Shewanella employs the 20-heme protein complex MtrCAB, Geobacter uses a redox polymer made of the hexa-heme protein OmcS, begging the question as to which protein architecture is more efficient in terms of long-range electron transfer. Using a multiscale computational approach we find that OmcS supports electron flows about an order of magnitude higher than MtrCAB due to larger heme-heme electronic couplings and better insulation of hemes from the solvent. We show that heme side chains are an essential structural element in both protein complexes, accelerating rate-limiting electron tunnelling steps up to 1000-fold. Our results imply that the alternating stacked/T-shaped heme arrangement present in both protein complexes may be an evolutionarily convergent design principle permitting efficient electron transfer over very long distances.

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.

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.

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.

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.

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).

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.

Membrane-spanning electron transfer proteins from electrogenic bacteria: Production and investigation.

Certain bacterial species have a natural ability to exchange electrons with extracellular redox partners. This behavior allows coupling of catalytic transformations inside bacteria to complementary redox transformations of catalysts and electrodes outside the cell. Electricity generation can be coupled to waste-water remediation. Industrially relevant oxidation reactions can proceed exclusively when electrons are released to anodes. Reduced products such as fuels can be generated when electrons are provided from (photo)cathodes. Rational development of these opportunities and inspiration for novel technologies is underpinned by resolution at the molecular level of pathways supporting electron exchange across bacterial cell envelopes. This chapter describes methods for purification, engineering, and in vitro characterization of proteins providing the primary route for electron transport across the outer membrane lipid bilayer of Shewanella oneidensis MR-1, a well-described electrogenic bacterium and chassis organism for related biotechnologies.

Direct evidence for heme-assisted solid-state electronic conduction in multi-heme c-type cytochromes.

Multi-heme cytochrome c (Cytc) proteins are key for transferring electrons out of cells, to enable intracellular oxidation to proceed in the absence of O2. In these proteins most of the hemes are arranged in a linear array suggesting a facile path for electronic conduction. To test this, we studied solvent-free electron transport across two multi-heme Cytc-type proteins: MtrF (deca-heme Cytc) and STC (tetra-heme Cytc). Transport is measured across monolayers of these proteins in a solid state configuration between Au electrodes. Both proteins showed 1000× higher conductance than single heme, or heme-free proteins, but similar conductance to monolayers of conjugated organics. Conductance is found to be temperature-independent (320-80 K), suggesting tunneling as the transport mechanism. This mechanism is consistent with I-V curves modelling, results of which could be interpreted by having protein-electrode coupling as rate limiting, rather than transport within the proteins.