Biomedical subjects

Fraser A Armstrong

Publications and source records attributed to Fraser A Armstrong.

At least 19 recordsLinked to original sources

PubMed2006

Electrochemical investigations of the interconversions between catalytic and inhibited states of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans.

Studies of the catalytic properties of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans by protein film voltammetry, under a H2 atmosphere, reveal and establish a variety of interesting properties not observed or measured quantitatively with other techniques. The catalytic bias (inherent ability to oxidize hydrogen vs reduce protons) is quantified over a wide pH range: the enzyme is proficient at both H2 oxidation (from pH > 6) and H2 production (pH < 6). Hydrogen production is inhibited by H2, but the effect is much smaller than observed for [NiFe]-hydrogenases from Allochromatium vinosum or Desulfovibrio fructosovorans. Under anaerobic conditions and positive potentials, the [FeFe]-hydrogenase is oxidized to an inactive form, inert toward reaction with CO and O2, that rapidly reactivates upon one-electron reduction under 1 bar of H2. The potential dependence of this interconversion shows that the oxidized inactive form exists in two pH-interconvertible states with pK(ox) = 5.9. Studies of the CO-inhibited enzyme under H2 reveals a strong enhancement of the rate of activation by white light at -109 mV (monitoring H2 oxidation) that is absent at low potential (-540 mV, monitoring H+ reduction), thus demonstrating photolability that is dependent upon the oxidation state.

Catalysis↗

PubMed2006

Electricity from low-level H2 in still air--an ultimate test for an oxygen tolerant hydrogenase.

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.

Air↗

PubMed2006

Rapid and reversible reactions of [NiFe]-hydrogenases with sulfide.

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.

Catalysis↗

PubMed2006

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.

Amino Acids↗

PubMed2006

Application of power spectra patterns in Fourier transform square wave voltammetry to evaluate electrode kinetics of surface-confined proteins.

This paper describes an application of Fourier transform (FT) voltammetry that provides a quantitative evaluation of the electron-transfer kinetics of protein molecules attached to electrode surfaces. The potential waveform applied in these experiments consists of a large-amplitude square wave of frequency f superimposed onto the traditional triangular voltage used in dc cyclic voltammetry. The resultant current-time response, when Fourier transformed into the frequency domain, provides patterns of data at the even harmonic frequencies that arise from nonlinearity in the Faradaic response. These even harmonic contributions are ideally suited for kinetic evaluation of electron-transfer processes because they are highly selective to quasi-reversible behavior (insensitive to reversible or irreversible processes) and almost devoid of background charging current. Inverse FT methods can then be used to provide the wave shapes of the dc as well as the ac voltammetric components and other characteristics employed to detect the level of nonideality present relative to theoretical models based upon noninteracting surface-confined molecules. The new form of data evaluation has been applied to the electron-transfer properties of a typical biological electron carrier, the blue copper protein azurin, immobilized on polycrystalline gold electrodes modified with self-assembled monolayers of different length alkanethiols. Details of the electrode kinetics (rates of electron transfer, dispersion, and charge-transfer coefficients) as a function of alkanethiol, apparent surface coverage, and capacitance are all deduced from the square wave (FT-inverse FT) protocol, and the implications of these findings are considered.

Azurin↗

PubMed2006

A random-sequential mechanism for nitrite binding and active site reduction in copper-containing nitrite reductase.

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO(-)(2)] reduction of the type-2 site precedes nitrite binding, at high [NO(-)(2)] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an approximately 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Alcaligenes faecalis↗

PubMed2006

Fumarate reductase and succinate oxidase activity of Escherichia coli complex II homologs are perturbed differently by mutation of the flavin binding domain.

The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily.

Alanine↗

PubMed2005

Electrochemical definitions of O2 sensitivity and oxidative inactivation in hydrogenases.

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.

Anaerobiosis↗

PubMed2005

Electrocatalytic hydrogen oxidation by an enzyme at high carbon monoxide or oxygen levels.

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 beta-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.

Biosensing Techniques↗

PubMed2005

Hydrogen cycling by enzymes: electrocatalysis and implications for future energy technology.

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 sample 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.

Carbon Monoxide↗

PubMed2005

Electron transfer and catalytic control by the iron-sulfur clusters in a respiratory enzyme, E. coli fumarate reductase.

Factors governing the efficacy of long-range electron relays in enzymes have been examined using protein film voltammetry in conjunction with site-directed mutagenesis. Investigations of the fumarate reductase from Escherichia coli, in which three Fe-S clusters relay electrons over more than 30 A, lead to the conclusion that varying the medial [4Fe-4S] cluster potential over a 100 mV range does not have a significant effect on the inherent kinetics of electron transfer to and from the active-site flavin. The results support a proposal that the reduction potential of an individual electron relay site in a multicentered enzyme is not a strong determinant of activity; instead, as deduced from the potential dependence of catalytic electron transfer, electron flow through the intramolecular relay is rapid and reversible, and even uphill steps do not limit the catalytic rate.

Amino Acid Sequence↗

PubMed2005

The mechanism of activation of a [NiFe]-hydrogenase by electrons, hydrogen, and carbon monoxide.

Activation of the oxidized inactive state (termed Unready or Ni(u)) of the [NiFe]-hydrogenase from Allochromatium vinosum requires removal of an unidentified oxidizing entity [O], produced by partial reduction of O(2). Dynamic electrochemical kinetic studies, subjecting enzyme molecules on an electrode to sequences of potential steps and gas injections, establish the order of events in an otherwise complex sequence of reactions that involves more than one intermediate retaining [O] or its redox equivalent; fast and reversible electron transfer precedes the rate-determining step which is followed by a reaction with H(2), or the inhibitor CO, that renders the reductive activation process irreversible.

Carbon Monoxide↗

PubMed2005

[NiFe]-hydrogenases: spectroscopic and electrochemical definition of reactions and intermediates.

Production and usage of di-hydrogen, H2, in micro-organisms is catalysed by highly active, 'ancient' metalloenzymes known as hydrogenases. Based on the number and identity of metal atoms in their active sites, hydrogenases fall into three main classes, [NiFe]-, [FeFe]- and [Fe]-. All contain the unusual ligand CO (and in most cases CN- as well) making them intriguing examples of 'organometallic' cofactors. These ligands render the active sites superbly 'visible' using infrared spectroscopy, which complements the use of electron paramagnetic resonance spectroscopy in studying mechanisms and identifying intermediates. Hydrogenases are becoming a focus of attention for research into future energy technologies, not only H2 production but also H2 oxidation in fuel cells. Hydrogenases immobilized on electrodes exhibit high electrocatalytic activity, providing not only an important new technique for their investigation, but also a basis for novel fuel cells either using the enzyme itself, or inspired synthetic catalysts. Favourable comparisons have been made with platinum electrocatalysts, an advantage of enzymes being their specificity for H2 and tolerance of CO. A challenge for exploiting hydrogenases is their sensitivity to O2, but some organisms are known to produce enzymes that overcome this problem by subtle alterations of the active site and gas access channels.

Catalysis↗

PubMed2005

Investigating metalloenzyme reactions using electrochemical sweeps and steps: fine control and measurements with reactants ranging from ions to gases.

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 examples 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 examples 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.

Binding Sites↗

PubMed2005

Recent developments in dynamic electrochemical studies of adsorbed enzymes and their active sites.

This review outlines recent developments in electrochemical investigations of proteins adsorbed on electrodes. The important point about 'protein film voltammetry' is that it addresses the rates of reactions that occur in enzymes - catalysis, inhibition, electron flow - as a function of potential; in other words, it introduces the 'potential dimension' into enzyme kinetics. Some surprisingly subtle, yet significant observations are made, including demonstration of a special role for Mo(V) in the catalytic cycle of Mo enzymes, quantitation of the catalytic bias in multi-centred enzymes such as mitochondrial Complex I, insight into mechanisms of proton transfer in enzymes, and properties of proteins that are covalently attached directly to a gold surface.

Adsorption↗

PubMed2004

Electrochemical potential-step investigations of the aerobic interconversions of [NiFe]-hydrogenase from Allochromatium vinosum: insights into the puzzling difference between unready and ready oxidized inactive states.

Dynamic electrochemical studies, incorporating catalytic voltammetry and detailed potential-step manipulations, provide compelling evidence that the oxidized inactive state of [NiFe]-hydrogenases termed Unready (or Ni-A) contains a product of partial reduction of O(2) that is trapped in the active site.

Aerobiosis↗

PubMed2004

Electrochemical studies of arsenite oxidase: an unusual example of a highly cooperative two-electron molybdenum center.

Arsenite oxidase from Alcaligenes faecalis, an unusual molybdoenzyme that does not exhibit a Mo(V) EPR signal during oxidative-reductive titrations, has been investigated by protein film voltammetry. A film of the enzyme on a pyrolytic graphite edge electrode produces a sharp two-electron signal associated with reversible reduction of the oxidized Mo(VI) molybdenum center to Mo(IV). That reduction or oxidation of the active site occurs without accumulation of Mo(V) is consistent with the failure to observe a Mo(V) EPR signal for the enzyme under a variety of conditions and is indicative of an obligate two-electron center. The reduction potential for the molybdenum center, 292 mV (vs SHE) at pH 5.9 and 0 degrees C, exhibits a linear pH dependence for pH 5-10, consistent with a two-electron reduction strongly coupled to the uptake of two protons without a pK in this range. This suggests that the oxidized enzyme is best characterized as having an L(2)MoO(2) rather than L(2)MoO(OH) center in the oxidized state and that arsenite oxidase uses a "spectator oxo" effect to facilitate the oxo transfer reaction. The onset of the catalytic wave observed in the presence of substrate correlates well with the Mo(VI/IV) potential, consistent with catalytic electron transport that is limited only by turnover at the active site. The one-electron peaks for the iron-sulfur centers are difficult to observe by protein film voltammetry, but spectrophotometric titrations have been carried out to measure their reduction potentials: at pH 6.0 and 20 degrees C, that of the [3Fe-4S] center is approximately 260 mV and that of the Rieske center is approximately 130 mV.

Alcaligenes faecalis↗

PubMed2004

Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site.

The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli.

Azides↗