ORCID Profile
0000-0001-6444-9382
Current Organisation
University of Oxford
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Microbial genetics | Enzymes | Biochemistry and cell biology
Publisher: Wiley
Date: 30-12-2003
Abstract: The electron-transfer chemistry of the isolated iron-molybdenum cofactor of nitrogenase (FeMoco) has been studied by electrochemical and spectroelectrochemical methods. Two interconverting forms of the cofactor arise from a redox-linked ligand isomerism at the terminal iron atom this is attributed to rotamerism of an anionic N-methyl formamide ligand bound at this site. FeMoco in its EPR-silent oxidised state is shown to undergo three successive one-electron transfer steps. We argue that the first and second redox processes are associated with electron-transfer delocalised over the iron-sulfur core of the cofactor, whilst the third irreversible process is localised on molybdenum. This is strongly reinforced by spectroelectrochemical studies under (12)CO and (13)CO which reveal two independent carbon monoxide binding sites that are specifically associated with the second (iron core) and third (molybdenum) electron-transfer processes and which give rise to terminal nu((12)CO) bands at 1885 and 1920 cm(-1) respectively. Moreover, in parallel with earlier studies on the enzyme system, it is shown that at low CO concentration, carbon monoxide binds to the cofactor in bridging modes, with nu(CO) bands at 1835 and 1808 cm(-1) that are interconverted by single-electron transfer. Importantly we show that the contentious overall 2e difference in the assignment of the metal oxidation levels in the resting state of the enzyme-bound cofactor, arising from analysis of (57)Fe ENDOR and Mössbauer data, can be resolved in the light of the electron-transfer chemistry of the isolated cofactor described herein.
Publisher: Wiley
Date: 11-05-2021
Abstract: A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H 2 as a simple, completely atom‐economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25–70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH 2 or FADH 2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme “ene‐reductases” to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min −1 and total turnover numbers up to 20 200 were recorded during flavin recycling.
Publisher: Royal Society of Chemistry (RSC)
Date: 2022
DOI: 10.1039/D2CC02411J
Abstract: Soluble hydrogenase enables atom efficient, H 2 -driven, recycling of synthetic nicotinamide cofactors.
Publisher: American Chemical Society (ACS)
Date: 08-2007
DOI: 10.1021/JA073643O
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1DT02219A
Abstract: The distribution of redox species in single crystals of [FeFe]-hydrogenase can be tuned electrochemically. All catalytic intermediates are observed by simultaneous infrared microspectroscopy, providing a roadmap for enriching specific redox states.
Publisher: American Chemical Society (ACS)
Date: 18-06-2003
DOI: 10.1021/JA035296Y
Abstract: The cycling between active and inactive states of the catalytic center of [NiFe]-hydrogenase from Allochromatium vinosum has been investigated by dynamic electrochemical techniques. Adsorbed on a rotating disk pyrolytic graphite "edge" electrode, the enzyme is highly electroactive: this allows precise manipulations of the complex redox chemistry and facilitates quantitative measurements of the interconversions between active catalytic states and the inactive oxidized form Ni(r) (also called Ni-B or "ready") as functions of pH, H(2) partial pressure, temperature, and electrode potential. Cyclic voltammograms for catalytic H(2) oxidation (current is directly related to turnover rate) are highly asymmetric (except at pH > 8 and high temperature) due to inactivation being much slower than activation. Controlled potential-step experiments show that the rate of oxidative inactivation increases at high pH but is independent of potential, whereas the rate of reductive activation increases as the potential becomes more negative. Indeed, at 45 degrees C, activation takes just a few seconds at -288 mV. The cyclic asymmetry arises because interconversion is a two-stage reaction, as expected if the reduced inactive Ni(r)-S state is an intermediate. The rate of inactivation depends on a chemical process (rearrangement and uptake of a ligand) that is independent of potential, but sensitive to pH, while activation is driven by an electron-transfer process, Ni(III) to Ni(II), that responds directly to the driving force. The potentials at which fast activation occurs under different conditions have been analyzed to yield the potential-pH dependence and the corresponding entropies and enthalpies. The reduced (active) enzyme shows a pK of 7.6 thus, when a one-electron process is assumed, reductive activation at pH 8, there is no net exchange of protons with solvent. Activation is favored by a large positive entropy, consistent with the release of a ligand and/or relaxation of the structure around the active site.
Publisher: IOP Publishing
Date: 22-06-2022
Abstract: Renewable fuel generation is essential for a low carbon footprint economy. Thus, over the last five decades, a significant effort has been dedicated towards increasing the performance of solar fuels generating devices. Specifically, the solar to hydrogen efficiency of photoelectrochemical cells has progressed steadily towards its fundamental limit, and the faradaic efficiency towards valuable products in CO 2 reduction systems has increased dramatically. However, there are still numerous scientific and engineering challenges that must be overcame in order to turn solar fuels into a viable technology. At the electrode and device level, the conversion efficiency, stability and products selectivity must be increased significantly. Meanwhile, these performance metrics must be maintained when scaling up devices and systems while maintaining an acceptable cost and carbon footprint. This roadmap surveys different aspects of this endeavor: system benchmarking, device scaling, various approaches for photoelectrodes design, materials discovery, and catalysis. Each of the sections in the roadmap focuses on a single topic, discussing the state of the art, the key challenges and advancements required to meet them. The roadmap can be used as a guide for researchers and funding agencies highlighting the most pressing needs of the field.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C6SC02860H
Abstract: Electrochemical control over nitrogenase allows us to examine electrocatalytic proton reduction and potential-triggered CO inhibition using infrared spectroscopy.
Publisher: Elsevier BV
Date: 12-2011
Publisher: Royal Society of Chemistry (RSC)
Date: 2013
DOI: 10.1039/C3CP00119A
Abstract: We report a versatile infrared spectroscopic method for studying redox chemistry of metalloproteins, and demonstrate for the first time electrochemically-induced changes to the active site of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha. A carbon particle network working electrode allows control over a wide potential window without the need for solution mediators.
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1SC00295C
Abstract: Heterogeneous chemo-bio catalytic hydrogenation is an attractive strategy for clean, enantioselective CX reduction.
Publisher: Springer Science and Business Media LLC
Date: 08-03-2023
DOI: 10.1038/S41586-023-05781-7
Abstract: Diverse aerobic bacteria use atmospheric H 2 as an energy source for growth and survival 1 . This globally significant process regulates the composition of the atmosphere, enhances soil bio ersity and drives primary production in extreme environments 2,3 . Atmospheric H 2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily 4,5 . However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H 2 amid ambient levels of the catalytic poison O 2 and how the derived electrons are transferred to the respiratory chain 1 . Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H 2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H 2 at the expense of O 2 , and 3 [3Fe–4S] clusters modulate the properties of the enzyme so that atmospheric H 2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H 2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H 2 in ambient air.
Publisher: American Chemical Society (ACS)
Date: 12-2005
DOI: 10.1021/JA055160V
Abstract: A new strategy is described for comparing, quantitatively, the ability of hydrogenases to tolerate exposure to O2 and anoxic oxidizing conditions. Using protein film voltammetry, the inherent sensitivities to these challenges (thermodynamic potentials and rates of reactions) have been measured for enzymes from a range of mesophilic microorganisms. In the absence of O2, all the hydrogenases undergo reversible inactivation at various potentials above that of the H+/H2 redox couple, and H2 oxidation activities are thus limited to characteristic "potential windows". Reactions with O2 vary greatly the [FeFe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757, an anaerobe, is irreversibly damaged by O2, surviving only if exposed to O2 in the anaerobically oxidized state (which therefore affords protection). In contrast, the membrane-bound [NiFe]-hydrogenase from the aerobe, Ralstonia eutropha, reacts reversibly with O2 even during turnover and continues to catalyze H2 oxidation in the presence of O2.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7CC04465H
Abstract: We describe the implementation of a system of immobilised enzymes for H 2 -driven NADH recycling coupled to a selective biotransformation to enable H 2 -driven biocatalysis in flow.
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C1CC14826E
Abstract: Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase.
Publisher: Wiley
Date: 12-06-2020
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9DT00924H
Abstract: Iron–sulfur clusters are exceptionally tuneable protein cofactors, and as one of their many roles they are involved in biological responses to nitrosative stress.
Publisher: Royal Society of Chemistry (RSC)
Date: 2011
DOI: 10.1039/C004274A
Abstract: Direct electrochemical methods have been productive in revealing mechanistic details of catalysis by a range of metalloenzymes including hydrogenases and carbon and nitrogen cycling enzymes. In this approach, termed protein film electrochemistry, the protein is attached or adsorbed on the electrode surface and exchanges electrons directly, providing precise control over redox states or catalysis and avoiding diffusion-limited electron transfer. The 'edge' surface of pyrolytic graphite has proved to be a particularly good surface for adsorption of proteins in electroactive conformations. We now describe development of an approach that combines the precise control achieved in direct electrochemical measurements at a graphite electrode with surface infrared (IR) spectroscopic analysis of chemistry occurring at metallocentres in proteins. Hydrogenases are of particular interest: their unusual organo-metallic active sites--iron or nickel-iron centres coordinated by CO and CN(-)--give rise to IR v(CO) and v(CN) bands that are detected readily because these ligands are strong vibrational oscillators and are sensitive to changes in electron density and coordination at the metals. Small diatomic species also bind as exogenous ligands (as substrate, product, activator or inhibitor) to a range of other important metalloproteins, and understanding their reactivity and binding selectivity is critical in building up a multidimensional picture of enzyme chemistry and evolutionary history. The surface IR spectroelectrochemical approach we describe is based around Attenuated Total Reflectance (ATR) mode s ling of a film of pyrolytic graphite particles modified with a protein of interest. The particle network extends the electrode into three-dimensional space, providing sufficient adsorbed protein for spectroscopic analysis under precise electrochemical control. This strategy should open up new opportunities for detection of redox-dependent chemistry at metal centres in proteins, including short-lived catalytic intermediates and time-resolved details of catalysis and inhibition.
Publisher: Wiley
Date: 28-10-2015
Publisher: Portland Press Ltd.
Date: 06-01-2017
DOI: 10.1042/BCJ20160513
Abstract: The present study considers the ways in which redox enzyme modules are coupled in living cells for linking reductive and oxidative half-reactions, and then reviews ex les in which this concept can be exploited technologically in applications of coupled enzyme pairs. We discuss many ex les in which enzymes are interfaced with electronically conductive particles to build up heterogeneous catalytic systems in an approach which could be termed synthetic biochemistry. We focus on reactions involving the H+/H2 redox couple catalysed by NiFe hydrogenase moieties in conjunction with other biocatalysed reactions to assemble systems directed towards synthesis of specialised chemicals, chemical building blocks or bio-derived fuel molecules. We review our work in which this approach is applied in designing enzyme-modified particles for H2-driven recycling of the nicotinamide cofactor NADH to provide a clean cofactor source for applications of NADH-dependent enzymes in chemical synthesis, presenting a combination of published and new work on these systems. We also consider related photobiocatalytic approaches for light-driven production of chemicals or H2 as a fuel. We emphasise the techniques available for understanding detailed catalytic properties of the enzymes responsible for in idual redox half-reactions, and the importance of a fundamental understanding of the enzyme characteristics in enabling effective applications of redox biocatalysis.
Publisher: Wiley
Date: 10-08-2020
Publisher: Royal Society of Chemistry (RSC)
Date: 2021
DOI: 10.1039/D1SC01734A
Abstract: Electrochemically-coupled IR microspectroscopy of single crystals provides insight into proton-coupled electron transfer in [NiFe] hydrogenase.
Publisher: Wiley
Date: 28-08-2018
Abstract: The sluggish kinetics of oxygen reduction to water remains a significant limitation in the viability of proton-exchange-membrane fuel cells, yet details of the four-electron oxygen reduction reaction remain elusive. Herein, we apply in situ infrared spectroscopy to probe the surface chemistry of a commercial carbon-supported Pt nanoparticle catalyst during oxygen reduction. The IR spectra show potential-dependent appearance of adsorbed superoxide and hydroperoxide intermediates on Pt. This strongly supports an associative pathway for oxygen reduction. Analysis of the adsorbates alongside the catalytic current suggests that another pathway must also be in operation, consistent with a parallel dissociative pathway.
Publisher: Royal Society of Chemistry (RSC)
Date: 2014
DOI: 10.1039/C4FD00058G
Abstract: We present a study of electrocatalysis by an enzyme adsorbed on a range of carbon materials, with different size, surface area, morphology and graphitic structure, which are either commercially available or prepared via simple, established protocols. We choose as our model enzyme the hydrogenase I from E. coli (Hyd-1), which is an active catalyst for H 2 oxidation, is relatively robust and has been demonstrated in H 2 fuel cells and H 2 -driven chemical synthesis. The carbon materials were characterised according to their surface area, surface morphology and graphitic character, and we use the electrocatalytic H 2 oxidation current for Hyd-1 adsorbed on these materials to evaluate their effectiveness as enzyme electrodes. Here, we show that a variety of carbon materials are suitable for adsorbing hydrogenases in an electroactive configuration. This unified study provides insight into selection and design of carbon materials for study of redox enzymes and different applications of enzyme electrocatalysis.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3SC01718D
Publisher: Proceedings of the National Academy of Sciences
Date: 31-10-2005
Abstract: Use of hydrogen in fuel cells requires catalysts that are tolerant to oxygen and are able to function in the presence of poisons such as carbon monoxide. Hydrogen-cycling catalysts are widespread in the bacterial world in the form of hydrogenases, enzymes with unusual active sites composed of iron, or nickel and iron, that are buried within the protein. We have established that the membrane-bound hydrogenase from the β-proteobacterium Ralstonia eutropha H16, when adsorbed at a graphite electrode, exhibits rapid electrocatalytic oxidation of hydrogen that is completely unaffected by carbon monoxide [at 0.9 bar (1 bar = 100 kPa), a 9-fold excess] and is inhibited only partially by oxygen. The practical significance of this discovery is illustrated with a simple fuel cell device, thus demonstrating the feasibility of future hydrogen-cycle technologies based on biological or biologically inspired electrocatalysts having high selectivity for hydrogen.
Publisher: Wiley
Date: 26-05-2015
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D2SC05641K
Abstract: Changing the conserved active-site arginine of [NiFe]-hydrogenases into a lysine greatly lowers the rates of catalytic H 2 activation in each direction and results in the extremely tight binding of a diatomic ligand.
Publisher: Springer International Publishing
Date: 2016
DOI: 10.1007/10_2016_3
Abstract: A more complete understanding of bioelectrochemical interfaces is of increasing importance in both fundamental studies and biotechnological applications of proteins. Bioelectrochemical methods provide detailed information about the activity or rate of a process, but in situ spectroscopic methods are needed to gain direct structural insight into functionally relevant states. A number of methods have been reported that allow electrochemical and spectroscopic data to be collected from the same electrode, providing direct spectroscopic 'snapshots' of protein function, and here we focus on the application of infrared and Raman spectroscopies to the study of electrode-immobilised species. The ability to probe coordination at metal centres, protonation changes in amino acid side chains, reaction-induced changes in organic cofactors or substrates, protein orientation and subtle changes in protein secondary structure simultaneously, rapidly and at room temperature means that vibrational spectroscopic approaches are almost uniquely applicable to answering a wide range of questions in bioelectrochemistry.
Publisher: Wiley
Date: 17-04-2013
DOI: 10.1111/FEBS.12245
Abstract: A large number of industrially relevant enzymes depend upon nicotinamide cofactors, which are too expensive to be added in stoichiometric amounts. Existing NAD(P)H-recycling systems suffer from low activity, or the generation of side products. H₂-driven cofactor regeneration has the advantage of 100% atom efficiency and the use of H₂ as a cheap reducing agent, in a world where sustainable energy carriers are increasingly attractive. The state of development of H₂-driven cofactor-recycling systems and ex les of their integration with enzyme reactions are summarized in this article. The O₂-tolerant NAD⁺-reducing hydrogenase from Ralstonia eutropha is a particularly attractive candidate for this approach, and we therefore discuss its catalytic properties that are relevant for technical applications.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6CC05955D
Abstract: Adsorbed formate is observed on a supported Pt nanoparticle for the first time during formic acid electro-oxidation.
Publisher: American Chemical Society (ACS)
Date: 11-09-2007
DOI: 10.1021/CR050191U
Publisher: American Chemical Society (ACS)
Date: 12-04-2019
DOI: 10.26434/CHEMRXIV.7982864.V1
Abstract: Chemicals labelled with the heavy hydrogen isotope deuterium ( 2 H) have long been used in chemical and biochemical mechanistic studies, spectroscopy, and as analytical tracers. More recently, demonstration of selectively deuterated drug candidates that exhibit advantageous pharmacological traits has spurred innovations in metal-catalysed 2 H insertion at targeted sites, but asymmetric deuteration remains a key challenge. Here we demonstrate an easy-to-implement biocatalytic deuteration strategy, achieving high chemo-, enantio- and isotopic selectivity, requiring only 2 H 2 O (D 2 O) and unlabelled dihydrogen under ambient conditions. The vast library of enzymes established for NADH-dependent C=O, C=C, and C=N bond reductions have yet to appear in the toolbox of commonly employed 2 H-labelling techniques due to requirements for suitable deuterated reducing equivalents. By facilitating transfer of deuterium atoms from 2 H 2 O solvent to NAD + , with H 2 gas as a clean reductant, we open up biocatalysis for asymmetric reductive deuteration as part of a synthetic pathway or in late stage functionalisation. We demonstrate enantioselective deuteration via ketone and alkene reductions and reductive amination, as well as exquisite chemo-control for deuteration of compounds with multiple unsaturated sites.
Publisher: Springer Science and Business Media LLC
Date: 11-11-2007
Abstract: A new concept for enzyme-catalyzed redox transformations features pairs of electron donor and acceptor enzymes attached to conducting particles. Electrons furnished by oxidation at one enzyme are used at the other. Graphite microparticles modified with hydrogenase and nitrate reductase or fumarate reductase catalyze reductions of nitrate or fumarate by H2.
Publisher: American Chemical Society (ACS)
Date: 07-2008
DOI: 10.1021/CR0680639
Publisher: Wiley
Date: 22-09-2004
Abstract: The first electrochemical and infra-red data on the binding of cyanide to the isolated iron-molybdenum cofactor of nitrogenase, FeMoco, is described. It is shown that cyanide stabilises a hitherto unrecognised, low-spin, EPR-active (S= 1/2), superoxidised form of FeMoco, and we provide the first evidence that carbon monoxide and cyanide bind synergically to the oxidised and semireduced states of the isolated cofactor, states which are unreactive to carbon monoxide alone.
Publisher: American Chemical Society (ACS)
Date: 10-07-2015
Abstract: The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.
Publisher: Royal Society of Chemistry (RSC)
Date: 2009
DOI: 10.1039/B801144N
Abstract: This tutorial review describes studies of hydrogen production and oxidation by biological catalysts--metalloenzymes known as hydrogenases--attached to electrodes. It explains how the electrocatalytic properties of hydrogenases are studied using specialised electrochemical techniques and how the data are interpreted to allow assessments of catalytic rates and performance under different conditions, including the presence of O2, CO and H2S. It concludes by drawing some comparisons between the enzyme active sites and platinum catalysts and describing some novel proof-of-concept applications that demonstrate the high activities and selectivities of these 'alternative' catalysts for promoting H2 as a fuel.
Publisher: American Chemical Society (ACS)
Date: 25-01-2005
DOI: 10.1021/IC048519+
Abstract: Protein film voltammetry is a powerful method for probing the chemistry of redox-active sites in metalloproteins. The technique affords precise potential control over a tiny quantity of material that is manipulated on an electrode surface, providing information on ligand- or metal-exchange reactions coupled to electron transfer. This is illustrated by ex les of transformations of the iron-sulfur clusters in ferredoxins. Protein film voltammetry is particularly advantageous in studies of metalloenzymes for which the current response is proportional to catalytic activity: kinetic data of extremely high signal/noise ratio are obtained for highly active enzymes. We present a series of interesting ex les in which catalytic activity varies in unusual ways with applied potential, surveying information that can be obtained from cyclic voltammetry and then looking beyond this method to controlled potential-step experiments that yield kinetic and mechanistic details. Recent results on the voltammetry of the highly active [NiFe]-hydrogenase from Allochromatium vinosum illustrate how it is possible to use the precise kinetic information from potential-step experiments to diagnose subtle details of transformations between catalytically active and inactive states of an enzyme. Protein film voltammetry thus complements spectroscopic techniques and other physical methods, revealing the chemistry of systems that might appear intractable or convoluted by other means.
Publisher: Royal Society of Chemistry (RSC)
Date: 2006
DOI: 10.1039/B614272A
Abstract: We demonstrate an extreme test of O(2) tolerance for a biological hydrogen-cycling catalyst: the generation of electricity from just 3% H(2) released into still, ambient air using an open fuel cell comprising an anode modified with the unusual hydrogenase from Ralstonia metallidurans CH34, that oxidizes trace H(2) in atmospheric O(2), connected via a film of electrolyte to a cathode modified with the fungal O(2) reductase, laccase.
Publisher: American Chemical Society (ACS)
Date: 31-01-2020
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D2FD00170E
Abstract: We associate potentials with the binding of substrates and inhibitors, H + , C 2 H 2 , CO and MeNC, to nitrogenase MoFe protein by coupling electrochemical control with gas chromatography analysis and in situ infrared spectroscopy.
Publisher: Elsevier BV
Date: 2009
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7CC02591B
Abstract: We manipulate and verify the redox state of single metalloprotein crystals by combining electrochemical control with synchrotron infrared microspectroscopy.
Publisher: American Chemical Society (ACS)
Date: 08-07-2014
DOI: 10.1021/JA504942H
Abstract: Cyanide reacts rapidly with [NiFe]-hydrogenases (hydrogenase-1 and hydrogenase-2 from Escherichia coli) under mild oxidizing conditions, inhibiting the electrocatalytic oxidation of hydrogen as recorded by protein film electrochemistry. Electrochemical, EPR, and FTIR measurements show that the final enzyme product, formed within a second (even under 100% H2), is the resting state known as Ni-B, which contains a hydroxido-bridged species, Ni(III)-μ(OH)-Fe(II), at the active site. "Cyanide inhibition" is easily reversed because it is simply the reductive activation of Ni-B. This paper brings back into focus an observation originally made in the 1940s that cyanide inhibits microbial H2 oxidation and addresses the interesting mechanism by which cyanide promotes the formation of Ni-B. As a much stronger nucleophile than hydroxide, cyanide binds more rapidly and promotes oxidation of Ni(II) to Ni(III) however, it is quickly replaced by hydroxide which is a far superior bridging ligand.
Publisher: American Chemical Society (ACS)
Date: 29-07-2008
DOI: 10.1021/JA8027668
Abstract: Studies have been carried out to establish the ability of O2-tolerant membrane-bound [NiFe] hydrogenases (MBH) from Ralstonia sp. to catalyze H2 production in addition to H2 oxidation. These hydrogenases are not noted for H2-evolution activity, and this is partly due to strong product inhibition. However, when adsorbed on a rotating disk graphite electrode the enzymes produce H2 efficiently, provided the H2 product is continuously removed by rapidly rotating the electrode and flowing N2 through the gastight electrochemical cell. Electrocatalytic H2 production proceeds with minimal overpotentiala significant observation because lowering the overpotential (the electrochemically responsive activation barrier) is seen as crucial in developing small-molecule catalysts for H2 production. A mutant having a high KM for H2 oxidation did not prove to be a better H2 producer relative to the wild type, thus suggesting that weak binding of H2 does not itself confer a tendency to be a H2 producer. Inhibition by H2 is much stronger than inhibition by CO and, most significantly, even O2. Consequently, H2 can be produced sustainably in the presence of O2 as long as the H2 is removed continuously, thereby proving the feasibility for biological H2 production in air.
Publisher: Sociedade Brasileira de Quimica (SBQ)
Date: 2014
Publisher: American Chemical Society (ACS)
Date: 18-05-2006
DOI: 10.1021/JA061732F
Abstract: Rapid and reversible binding of sulfide to [NiFe]-hydrogenases (particularly the enzyme from Desulfovibrio vulgaris) under weakly acidic conditions (pH 6) has been studied by protein film voltammetry, which tracks the formation of different species as a function of potential. Sulfide (most likely entering as H2S) rapidly attacks the active site during H2 oxidation. The inactive adduct is formed (and is stable) only at potentials substantially more positive than the comparable species formed with oxygen species and is easily reactivated upon reduction. The sulfide adduct also reacts further with O2 to produce a new species that undergoes reductive activation very slowly. The results clarify complex and controversial chemistry reported in the literature and provide insight into how these enzymes would cope with sulfide production in sulfate-reducing bacteria.
Publisher: Wiley
Date: 26-01-2021
DOI: 10.1002/JLCR.3899
Publisher: Royal Society of Chemistry (RSC)
Date: 2012
DOI: 10.1039/C1CC15871F
Abstract: This article reviews recent developments in spectroscopic analysis of electrode-immobilised enzymes under direct, unmediated electrochemical control. These methods unite the suite of spectroscopic methods available for characterisation of structural, electronic and coordination changes in proteins with the exquisite control over complex redox enzymes that can be achieved in protein film electrochemistry in which immobilised protein molecules exchange electrons directly with an electrode. This combination is particularly powerful in studies of highly active enzymes where redox states can be controlled even under fast electrocatalytic turnover. We examine ex les in which UV-visible, IR, Raman and MCD spectroscopy have been combined with direct electrochemistry to probe redox-dependent chemistry, and consider future opportunities for 'direct' spectroelectrochemistry of immobilised enzymes.
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6CC07212G
Abstract: Metals on polyaniline (PANI) exhibit excellent activity in the electro-reduction of CO 2 to HCOOH or CH 3 OH due to tunable properties: N atoms on PANI capture CO 2 while Pd atoms facilitate fast proton and electron transfers along the PANI in cooperative manner.
Publisher: Royal Society of Chemistry (RSC)
Date: 2020
DOI: 10.1039/D0DT90059A
Abstract: Sam de Visser, Jonathan Rourke and Kylie Vincent introduce this Dalton Transactions themed collection on inorganic reaction mechanisms.
Publisher: American Chemical Society (ACS)
Date: 02-09-2010
DOI: 10.1021/JA1067178
Publisher: American Chemical Society (ACS)
Date: 07-2020
Publisher: Wiley
Date: 28-10-2008
Publisher: American Chemical Society (ACS)
Date: 02-08-2018
DOI: 10.1021/JACS.8B04798
Abstract: Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O
Publisher: American Chemical Society (ACS)
Date: 11-11-2010
DOI: 10.1021/BI1016167
Abstract: A molecular wire is used to connect two proteins through their physiologically relevant redox cofactors to facilitate direct electron transfer. Photosystem I (PS I) and an [FeFe]-hydrogenase (H(2)ase) serve as the test bed for this new technology. By tethering a photosensitizer with a hydrogen-evolving catalyst, attached by Fe-S coordination bonds between the F(B) iron-sulfur cluster of PS I and the distal iron-sulfur cluster of H(2)ase, we assayed electron transfer between the two components via light-induced hydrogen generation. These hydrogen-producing nanoconstructs self-assemble when the PS I variant, the H(2)ase variant, and the molecular wire are combined.
Publisher: Elsevier BV
Date: 09-2009
Publisher: The Royal Society
Date: 13-08-2010
Abstract: Recent developments in infrared (IR) spectroscopic time resolution, sensitivity and s le manipulation make this technique a powerful addition to the suite of complementary approaches for the study of time-resolved chemistry at metal centres within proteins. Application of IR spectroscopy to proteins has often targeted the amide bands as probes for gross structural change. This article focuses on the possibilities arising from recent IR technical developments for studies that monitor localized vibrational oscillators in proteins—native or exogenous ligands such as NO, CO, SCN − or CN − , or genetically or chemically introduced probes with IR-active vibrations. These report on the electronic and coordination state of metals, the kinetics, intermediates and reaction pathways of ligand release, hydrogen-bonding interactions between the protein and IR probe, and the electrostatic character of sites in a protein. Metalloprotein reactions can be triggered by light/dark transitions, an electrochemical step, a change in solute composition or equilibration with a new gas atmosphere, and spectra can be obtained over a range of time domains as far as the sub-picosecond level. We can expect to see IR spectroscopy exploited, alongside other spectroscopies, and crystallography, to elucidate reactions of a wide range of metalloprotein chemistry with relevance to cell metabolism, health and energy catalysis.
Publisher: American Chemical Society (ACS)
Date: 07-03-2017
Publisher: American Chemical Society (ACS)
Date: 05-08-2014
DOI: 10.1021/JA505291J
Abstract: We use infrared spectroscopy to demonstrate the critical role that trace O2 plays in determining the products formed when a [2Fe2S] cluster protein reacts with nitric oxide (NO). The observed importance of O2 may have physiological relevance, as many pathogens sense NO using iron-sulfur proteins and will be exposed to NO in an aerobic environment during a mammalian immune response. We show that the [2Fe2S]-containing spinach ferredoxin I undergoes reaction with NO at pH 6.0, with the proportion of protein-bound Roussin's Red Ester compared to the dinitrosyl iron complex product favored by trace O2. Roussin's Red Ester is also favored on nitrosylation in the presence of the thiolate scavenging reagent, iodoacetamide, suggesting that the role of O2 is in oxidative sequestration of cysteine thiolates. Infrared spectroscopy has been overlooked as a tool for studying iron-sulfur protein nitrosylation despite the fact that there exists a wealth of infrared spectroscopic data on small-molecule nitrosyl clusters which serve as models for the identification of protein-bound nitrosyl clusters.
Publisher: Public Library of Science (PLoS)
Date: 10-10-2011
Publisher: Wiley
Date: 29-04-2015
Publisher: Wiley
Date: 28-08-2018
Publisher: Springer Science and Business Media LLC
Date: 10-11-2016
DOI: 10.1038/NCOMMS13303
Abstract: Optoelectronic devices based on hybrid halide perovskites have shown remarkable progress to high performance. However, despite their apparent success, there remain many open questions about their intrinsic properties. Single crystals are often seen as the ideal platform for understanding the limits of crystalline materials, and recent reports of rapid, high-temperature crystallization of single crystals should enable a variety of studies. Here we explore the mechanism of this crystallization and find that it is due to reversible changes in the solution where breaking up of colloids, and a change in the solvent strength, leads to supersaturation and subsequent crystallization. We use this knowledge to demonstrate a broader range of processing parameters and show that these can lead to improved crystal quality. Our findings are therefore of central importance to enable the continued advancement of perovskite optoelectronics and to the improved reproducibility through a better understanding of factors influencing and controlling crystallization.
Publisher: Royal Society of Chemistry (RSC)
Date: 2005
DOI: 10.1039/B508520A
Abstract: Hydrogenases provide an inspiration for future energy technologies. The active sites of these microbial enzymes contain Fe or Ni and Fe coordinated by CO and CN ligands: yet they have activities for hydrogen cycling that compare with Pt catalysts. Is there a future for enzymes in technological H2 cycling? There are obviously going to be disadvantages, perhaps overwhelming, as enzymes are notoriously fragile yet what are the positive aspects and can we learn any chemistry that might be applied to produce the electrolytic and fuel cell catalysts of the future? We have developed a suite of novel electrochemical experiments to probe the chemistry of hydrogenases. The reactions are controlled and monitored at the surface of a small electrode, and characteristic catalytic properties are discernible from tiny amounts of s le material, so this approach can be used to search the microbial world for the best catalysts. Although electrochemistry does not provide structural information directly, it does give a "road map" by which to navigate the pathways and conditions that lead to particular states of the enzymes. This has prompted many interdisciplinary collaborations with other scientists who have provided microbiological, spectroscopic and structural contexts for this work. This article describes how these electrochemical experiments are set up, the data are analysed, and the results interpreted. We have determined mechanisms of catalysis, electron transfer, activation and inactivation, and defined important properties such as O2 tolerance and CO resistance in physical terms. Using an O2-tolerant hydrogenase, we have demonstrated a "proof of concept" miniature fuel cell that will run on a mixed H2/O2 feed in aqueous solution.
Publisher: American Chemical Society (ACS)
Date: 07-08-2017
Publisher: American Chemical Society (ACS)
Date: 15-06-2016
Publisher: Informa UK Limited
Date: 25-07-2022
DOI: 10.1080/15438627.2022.2139618
Abstract: We aimed to report the epidemiology of lower-limb and lumbosacral injuries in Police Force recruits. We performed a cohort study of Police Force recruits undergoing a six-month training program with prospective injury data collected between 2018 and 2021. Cardiorespiratory fitness was quantified by the beep-test and police-specific-functional-capacity was quantified using a specifically designed physical performance evaluation (PPE) tool. Injury frequency and prevalence were reported. Fifteen percent (n = 180) of study Police Force recruits (n = 1,181) sustained a lower-limb or lumbosacral injury. The six-month training program significantly improved cardiorespiratory fitness (p < 0.001) and functional capacity (p < 0.001). Increased cardiorespiratory fitness at baseline decreased injury risk (OR = 0.8, 95%CI: 0.66-0.97, p = 0.019). Injury rates decreased over time and females were injured significantly earlier than males (HR = 0.70, 95%CI: 0.52 to 0.95, p = 0.021). Interventions that can pre-condition Police Force recruits prior to the commencement of their basic physical training may reduce the number of lower-limb and lumbosacral injuries.
Publisher: Springer Science and Business Media LLC
Date: 19-03-2020
DOI: 10.1038/S41467-020-15310-Z
Abstract: Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium ( 2 H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2 H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4- 2 H]-NADH). Here we report on a strategy that combines a clean reductant (H 2 ) with a cheap source of 2 H-atoms ( 2 H 2 O) to generate and recycle [4- 2 H]-NADH. By coupling [4- 2 H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1 S ,3’ R )-[2’,2’,3’- 2 H 3 ]-solifenacin fumarate on a preparative scale.
Publisher: Wiley
Date: 11-05-2021
Abstract: A new activity for the [NiFe] uptake hydrogenase 1 of Escherichia coli (Hyd1) is presented. Direct reduction of biological flavin cofactors FMN and FAD is achieved using H 2 as a simple, completely atom‐economical reductant. The robust nature of Hyd1 is exploited for flavin reduction across a broad range of temperatures (25–70 °C) and extended reaction times. The utility of this system as a simple, easy to implement FMNH 2 or FADH 2 regenerating system is then demonstrated by supplying reduced flavin to Old Yellow Enzyme “ene‐reductases” to support asymmetric alkene reductions with up to 100 % conversion. Hyd1 turnover frequencies up to 20.4 min −1 and total turnover numbers up to 20 200 were recorded during flavin recycling.
Publisher: Wiley
Date: 02-02-2011
Publisher: American Chemical Society (ACS)
Date: 11-2019
DOI: 10.1021/ACS.ACCOUNTS.9B00293
Abstract: Achieving a unified understanding of the mechanism of a multicenter redox enzyme such as [NiFe] hydrogenase is complicated by difficulties in reconciling information obtained by using different techniques and on s les in different physical forms. Measurements of the activity of the enzyme, and of factors which perturb activity, are generally carried out using biochemical assays in solution or with electrode-immobilized enzymes using protein film electrochemistry (PFE). Conversely, spectroscopy aimed at reporting on features of the metalloclusters in the enzyme, such as electron paramagnetic resonance (EPR) or X-ray absorption spectroscopy (XAS), is often conducted on frozen s les and is thus difficult to relate to catalytically relevant states as information about turnover and activity has been lost. To complicate matters further, most of our knowledge of the atomic-level structure of metalloenzymes comes from X-ray diffraction studies in the solid, crystalline state, which are again difficult to link to turnover conditions. Taking [NiFe] hydrogenases as our case study, we show here how it is possible to apply infrared (IR) spectroscopic s ling approaches to unite direct spectroscopic study with catalytic turnover. Using a method we have named protein film IR electrochemistry (PFIRE), we reveal the steady-state distribution of intermediates during catalysis and identify catalytic "bottlenecks" introduced by site-directed mutagenesis. We also show that it is possible to study dynamic transitions between active site states of enzymes in single crystals, uniting solid state and solution spectroscopic information. In all of these cases, the spectroscopic data complement and enhance interpretation of purely activity-based measurements by providing direct chemical insight that is otherwise hidden. The [NiFe] hydrogenases possess a bimetallic [NiFe] active site, coordinated by CO and CN
Publisher: American Chemical Society (ACS)
Date: 19-12-2008
DOI: 10.1021/JA078299+
Publisher: American Chemical Society (ACS)
Date: 11-02-2021
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2023
End Date: 12-2025
Amount: $694,096.00
Funder: Australian Research Council
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