Protein Film Voltammetry Research Articles

Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK a≈7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V max and the nitrite dependence of the catalytic current-potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.

Protein film voltammetry and co-factor electron transfer dynamics in spinach photosystem II core complex

Direct protein film voltammetry (PFV) was used to investigate the redox properties of the photosystem II (PSII) core complex from spinach. The complex was isolated using an improved protocol not used previously for PFV. The PSII core complex had high oxygen-evolving capacity and was incorporated into thin lipid and polyion films. Three well-defined reversible pairs of reduction and oxidation voltammetry peaks were observed at 4 °C in the dark. Results were similar in both types of films, indicating that the environment of the PSII-bound cofactors was not influenced by film type. Based on comparison with various control samples including Mn-depleted PSII, peaks were assigned to chlorophyll a (Chl a) (Em = -0.47 V, all vs. NHE, at pH 6), quinones (-0.12 V), and the manganese (Mn) cluster (Em = 0.18 V). PFV of purified iron heme protein cytochrome b-559 (Cyt b-559), a component of PSII, gave a partly reversible peak pair at 0.004 V that did not have a potential similar to any peaks observed from the intact PSII core complex. The closest peak in PSII to 0.004 V is the 0.18 V peak that was found to be associated with a two-electron process, and thus is inconsistent with iron heme protein voltammetry. The -0.47 V peak had a peak potential and peak potential-pH dependence similar to that found for purified Chl a incorporated into DMPC films. The midpoint potentials reported here may differ to various extents from previously reported redox titration data due to the influence of electrode double-layer effects. Heterogeneous electron transfer (hET) rate constants were estimated by theoretical fitting and digital simulations for the -0.47 and 0.18 V peaks. Data for the Chl a peaks were best fit to a one-electron model, while the peak assigned to the Mn cluster was best fit by a two-electron/one-proton model.

Freely diffusing versus adsorbed protein: Which better mimics the cellular state of a redox protein?

Dynamic electrochemistry of adsorbed proteins, often termed protein film electrochemistry (PFE), is widely used for the characterisation of redox proteins. The method provides a powerful alternative to spectroscopic studies that interrogate protein solutions. The reduction potential and electron stoichiometry of redox couples can be defined. The rates of catalytic redox transformations can also be quantified. Often it is considered that the behaviour of the adsorbed protein should match that displayed in solution studies if it is to be relevant to understanding the biological role of the protein. However, the environment of the protein in PFE is fundamentally different from that when it is freely diffusing in solution. As a consequence different behaviours may be expected. This raises the question, which approach is more relevant when aiming to provide insight into the cellular role of the protein? We consider this question here taking as an example the properties of a penta-heme cytochrome NrfB from Escherichia coli. The redox properties of NrfB containing solutions were presented previously (Clarke et al., Journal of Biological Chemistry (2004)). Here we present PFE of NrfB adsorbed on graphite and optically transparent mesoporous, nanocrystalline SnO2 electrodes.

Proton‐Dependence of the MitoNEET [2Fe‐2S] Cluster : An Electrochemical and Structural Investigation

MitoNEET is a novel 3‐Cys, 1‐His ligated redox active [2Fe‐2S] cluster binding protein, whose biological function remains unclear. MitoNEET is of interest due to its interaction with the anti‐diabetes drug pioglitazone, a member of the TZD drug family, and its unique cluster ligation. Here, a characterization of the clusters unique ligand environment is presented. Using protein film voltammetry, the potential of the mitoNEET cluster is found to be 0 mV at pH 7, where upon binding of pioglitazone reduces the potential to −100 mV. This is one of the first reports of PFV being used to monitor drug binding. Additionally, the mitoNEET cluster demonstrates unusual proton coupled‐electron transfer as well as marked proton‐dependent cluster instability. Through a series of site‐directed mutations of conserved residues around the [2Fe‐2S] cluster, both of these phenomena have been examined, demonstrating the presence of multiple sites of protonation that affect both cluster midpoint potential and cluster stabilization. The high conservation of these residues, which reduce cluster stability, suggest a physiological role for cluster loss and lead to a putative model, in which mitoNEET may sense cellular oxidation state, proton concentration, or iron levels. Changes in MitoNEET cluster binding could potentially mediate either cluster transfer or additional signaling events within the cell.NIH‐NIGMS R01‐GM072663

Identifying the Nature of Iron‐Sulfur Clusters in the CDGSH‐Family Protein AlVin0680

The CDGSH domain is a small [2Fe‐2S] cluster binding domain typified by the presence of three ligating cysteines and one ligating histidine residue. The human protein mitoNEET, an example of a CDGSH domain protein, has attracted interest due to its interaction with the type II diabetes drug pioglitazone. A greater understanding of other CDGSH domain containing proteins may provide insight into the functional role of the unique [2Fe‐2S] cluster. The Allochromatium vinosum protein, AlVin0680, has been identified as a domain fusion protein consisting of two CDGSH domains and a putative [3Fe‐4S] cluster binding DUF1271 domain. Basic characterization of AlVin0680 via UV/Vis spectroscopy, EPR, protein film voltammetry, and colorimetric assays of the iron content indicate that both the mitoNEET‐like CDGSH clusters and the DUF1271 clusters are bound by the protein. Further work will involve the exploration of the potential interactions between the two [2Fe‐2S] clusters and the [3Fe‐4S] cluster, specifically determining whether electron or cluster exchange occurs between the three clusters. A greater understanding of the iron‐sulfur clusters in AlVin0680 may provide insight into the general function of CDGSH domain proteins and their iron‐sulfur clusters.

Direct Electrochemistry of Shewanella oneidensis Cytochrome c Nitrite Reductase

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA) is a dimeric enzyme that plays a key role in bacterial nitrate respiration. The periplasmic reductase catalyzes the six‐electron reduction of nitrite to ammonia but can also catalyze the two‐electron reduction of hydroxylamine, a proposed intermediate in the nitrite reduction pathway. Here we use Protein Film Voltammetry to investigate the electrochemical and catalytic properties of soNrfA and compare its properties to the related Escherichia coli nitrite reductase (ecNrfA). Our findings highlight the similarities and differences between the two enzymes: most notably, all detected electron transfer steps are one‐electron in nature, contrary to what has been observed in ecNrfA. This suggests that for soNrfA the six‐electron reduction of nitrite is carried out in a series of one‐electron steps. Additionally we observe substrate inhibition during nitrite turnover and evidence of negative cooperativity during hydroxylamine turnover which suggests individual monomers within a ccNiR homodimer interact with one another during catalysis.

Electrostatically Driven Second-Sphere Ligand Switch between High and Low Reorganization Energy Forms of Native Cytochrome c

We have employed a combination of protein film voltammetry, time-resolved vibrational spectroelectrochemistry and molecular dynamics simulations to evaluate the electron-transfer reorganization free energy (λ) of cytochrome c (Cyt) in electrostatic complexes that mimic some basic features of protein-protein and protein-lipid interactions. The results reveal the existence of two native-like conformations of Cyt that present significantly different λ values. Conversion from the high to the low λ forms is triggered by electrostatic interactions, and involves the rupture of a weak H-bond between first- (M80) and second-sphere (Y67) ligands of the heme iron, as a distinctive feature of the conformational switch. The two flexible Ω loops operate as transducers of the electrostatic signal. This fine-tuning effect is abolished in the Y67F Cyt mutant, which presents a λ value similar to the WT protein in electrostatic complexes. We propose that interactions of Cyt with the natural redox partner proteins activate a similar mechanism to minimize the reorganization energy of interprotein electron transfer.

Principles of Sustained Enzymatic Hydrogen Oxidation in the Presence of Oxygen – The Crucial Influence of High Potential Fe–S Clusters in the Electron Relay of [NiFe]-Hydrogenases

"Hyd-1", produced by Escherichia coli , exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H(2) oxidation activity in the presence of O(2)-an intruder that normally incapacitates the sulfur- and electron-rich active site. The mechanism of "O(2) tolerance" involves a critical role for the Fe-S clusters of the electron relay, which is to ensure the availability-for immediate transfer back to the active site-of all of the electrons required to reduce an attacking O(2) molecule completely to harmless H(2)O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 Å from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H(2) oxidation activity in 1% O(2). The [3Fe-4S] cluster is essential only for the first stage of complete O(2) reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state "Ni-B" or "Ready" (Ni(III)-OH). Potentiometric titrations show that Ni-B is easily reduced (E(m) ≈ +0.1 V at pH 6.0); this final stage of the O(2)-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme's response to O(2), viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H(2) oxidation activity; thus, O(2) behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe-S clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase.

Induced peroxidase activity of haem containing nitrate reductases revealed by protein film electrochemistry

Direct voltammetry of adsorbed redox enzymes at pyrolytic graphite electrodes has shown to be very useful to probe the catalytic activity of several nitrate reductases in the presence of nitrate. In this work we demonstrated that in some cases an electrode-induced haem alteration leads to a loss of nitrate reductase activity.Nitrate reductases are key enzymes in the biological nitrogen cycle. In particular, NapAB from Cupriavidus necator has an important role in the scavenging of nitrate and NarGHI from Marinobacter hydrocarbonoclasticus 617 is essential for the anaerobic respiration. These enzymes present haem groups among their redox centres, which are essential for the electron transfer from the reducing to the oxidising substrate. When adsorbed at graphite electrodes, both enzymes displayed a non-turnover signal corresponding to a one-electron redox process, with formal reduction potentials at pH 7.6 of −159mV and −139mV vs. SHE for Nap and Nar, respectively. Both enzymes displayed peroxidase activity at a potential close to that of the non-turnover response. Experiments with the whole enzymes and the haem free NapA from Desulfovibrio desulfuricans and NarGH from M. hydrocarbonoclasticus 617 were a valuable tool to get information about the cofactor undergoing electron transfer. We confirmed that this behaviour is related with the haems present in subunit B and subunit I of NapAB and NarGHI, respectively.

A unified electrocatalytic description of the action of inhibitors of nickel carbon monoxide dehydrogenase.

Several small molecules and ions, notably carbon monoxide, cyanide, cyanate, and hydrogen sulfide, are potent inhibitors of Ni-containing carbon monoxide dehydrogenases (Ni-CODH) that catalyze very rapid, efficient redox interconversions of CO(2) and CO. Protein film electrochemistry, which probes the dependence of steady-state catalytic rate over a wide potential range, reveals how these inhibitors target particular oxidation levels of Ni-CODH relating to intermediates (C(ox), C(red1), and C(red2)) that have been established for the active site. The following properties are thus established: (1) CO suppresses CO(2) reduction (CO is a product inhibitor), but its binding affinity decreases as the potential becomes more negative. (2) Cyanide totally inhibits CO oxidation, but its effect on CO(2) reduction is limited to a narrow potential region (between -0.5 and -0.6 V), below which CO(2) reduction activity is restored. (3) Cyanate is a strong inhibitor of CO(2) reduction but inhibits CO oxidation only within a narrow potential range just above the CO(2)/CO thermodynamic potential--EPR spectra confirm that cyanate binds selectively to C(red2). (4) Hydrogen sulfide (H(2)S/HS(-)) inhibits CO oxidation but not CO(2) reduction--the complex on/off characteristics are consistent with it binding at the same oxidation level as C(ox) and forming a modified version of this inactive state rather than reacting directly with C(red1). The results provide a new perspective on the properties of different catalytic intermediates of Ni-CODH--uniting and clarifying many previous investigations.

Electrochemical Investigation of the Radical SAM Enzyme, BtrN from Bacillus Circulans

Radical SAM enzymes catalyze a variety of reactions involved in biological pathways including the biosynthesis of antibiotics, cofactors, and biosynthesis and repair of DNA. Measurement of the electrochemical characteristics of radical SAM enzymes has been limited due to the typically buried location of the radical SAM cluster within the enzyme. The midpoint potential of a radical SAM cluster has only been determined for lysine aminomutase using spectroelectrochemical titrations with the use of mediators. This study presents the first direct electrochemical measurement of a radical SAM enzyme using the technique of protein film voltammetry (PFV). BtrN from Bacillus circulans is an emerging class of radical SAM dehydrogenases, which catalyze the third step in the biosynthetic pathway of the antibiotic butirosin. BtrN has been shown to contain a second [4Fe-4S] in addition to the canonical radical SAM [4Fe-4S] cluster. Nonturnover PFV has characterized the electrochemical properties of these clusters and provided insight into the mechanism of electron transfer. Additionally, PFV has been used to characterize the effect of substrate binding on the clusters. These results provide insight into the catalytic mechanism of BtrN and the electrochemical characteristics of radical SAM enzymes in general.

Redox Enzymology of Shewanella Oneidensis Cytochrome C Nitrite Reductase

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has previously been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) adsorbed to a graphite electrode, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA that is characterized by ‘boosts’ and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed, e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which have previously been observed in any cytochrome c nitrite reductase. Collectively these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

Redox Sensor-Like Behavior in the Human [2Fe-2S] Binding Protein Mitoneet

MitoNEET, a novel 3-Cys, 1-His ligated redox active [2Fe-2S] cluster binding protein, is found at the cytosolic face of the outer mitochondrial membrane in humans. While its biological function remains unclear, mitoNEET is of interest due to its interaction with the anti-diabetes drug pioglitazone, a member of the TZD drug family. Here, a characterization of both drug binding and the unique ligand environment are presented. Midpoint potentials of the mitoNEET [2Fe-2S] cluster are reported based on protein film voltammetry measurements. The potential of the mitoNEET cluster is 0 mV at pH 7, where upon binding of pioglitazone the potential is markedly reduced to −100 mV. This is one of the first reports of PFV being used to monitor drug binding. Due to its unique His-ligand and conserved hydrogen bonding networks the mitoNEET cluster demonstrates unusual proton coupled-electron transfer as well as marked cluster instability below neutral pH. Through a series of site-directed mutations both of these phenomena have been examined, demonstrating a) the presence of multiple sites of protonation that affect cluster midpoint potential, b) the cluster destabilizing affect of oxidation, protonation, and the presence of conserved residues His-87 and Lys-55, and c) the reversible loss of structure concomitant with cluster loss. The conservation of residues that reduce cluster stability, suggest a physiological role for cluster loss and lead to a putative model, in which mitoNEET may sense cellular oxidation state, proton concentration, and iron levels. Changes in MitoNEET cluster binding may mediate either cluster transfer or additional signaling events within the cell.

Electron transfer with azurin at Au–SAM junctions in contact with a protic ionic melt: impact of glassy dynamics

Gold electrodes were coated with alkanethiol SAM-azurin (Az, blue cupredoxin) assemblies and placed in contact with a water-doped and buffered protic ionic melt as the electrolyte, choline dihydrogen phosphate ([ch][dhp]). Fast-scan protein-film voltammetry was applied to explore interfacial biological electron transfer (ET) under conditions approaching the glass-transition border. The ET rate was studied as a function of the water amount, temperature (273-353 K), and pressure (0.1-150 MPa). Exposure of the Az films to the semi-solid electrolyte greatly affected the protein's conformational dynamics, hence the ET rate, via the mechanism occurring in the extra complicated dynamically-controlled regime, is compared to the earlier studies on the reference system with a conventional electrolyte (D. E. Khoshtariya et al., Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 2757-2762), allowing for the disclosure of even more uncommon mechanistic motifs. For samples with low water content (ca. 3 or less waters per [ch][dhp]), at moderately low temperatures (below ca. 298 K) and/or high pressure (150 MPa), the voltammetric profiles systematically deviated from the standard Marcus current-overvoltage pattern, deemed as attributable to a breakdown of the linear response approximation through the essential steepening of the Gibbs energy wells near the glass-forming threshold. Electrolytes with a higher water content (6 to 15 waters per [ch][dhp]) display anomalous temperature and pressure performances, suggesting that the system crosses a broad nonergodic zone which arises from the interplay of ET-coupled large-scale conformational (highly cooperative) modes of the Az protein, inherently linked to the electrolyte's (water-doped [ch][dhp]) slowest collective relaxation(s).

Direct Electrochemistry of Shewanella oneidensis Cytochrome c Nitrite Reductase: Evidence of Interactions across the Dimeric Interface

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed; e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA [Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) J. Biol. Chem. 277, 23374-23381]. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which has previously been observed in any cytochrome c nitrite reductase. Collectively, these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

Mind the gap: diversity and reactivity relationships among multihaem cytochromes of the MtrA/DmsE family

Shewanella oneidensis MR-1 has the ability to use many external terminal electron acceptors during anaerobic respiration, such as DMSO. The pathway that facilitates this electron transfer includes the decahaem cytochrome DmsE, a paralogue of the MtrA family of decahaem cytochromes. Although both DmsE and MtrA are decahaem cytochromes implicated in the long-range electron transfer across a ~300 Å (1 Å=0.1 nm) wide periplasmic 'gap', MtrA has been shown to be only 105 Å in maximal length. In the present paper, DmsE is further characterized via protein film voltammetry, revealing that the electrochemistry of the DmsE haem cofactors display macroscopic potentials lower than those of MtrA by 100 mV. It is possible this tuning of the redox potential of DmsE is required to shuttle electrons to the outer-membrane proteins specific to DMSO reduction. Other decahaem cytochromes found in S. oneidensis, such as the outer-membrane proteins MtrC, MtrF and OmcA, have been shown to have electrochemical properties similar to those of MtrA, yet possess a different evolutionary relationship.

Immobilization of azurin with retention of its native electrochemical properties at alkylsilane self-assembled monolayer modified indium tin oxide

Indium tin oxide (ITO) is a promising material for developing spectroelectrochemical methods due to its combination of excellent transparency in the visible region and high conductivity over a broad range of potential. However, relatively few examples of immobilization of redox proteins at ITO with retention of the ability to transfer electrons with the underlying material with native characteristics have been reported. In this work, we utilize an alkylsilane functionalized ITO surface as a biocompatible interface for immobilization of the blue copper protein azurin. Adsorption of azurin at ITO as well as ITO coated with self-assembled monolayers of (3-mercaptopropyl)trimethoxysilane (MPTMS) and n-decyltrimethoxysilane (DTMS) was achieved, and immobilized protein probed using protein film electrochemistry. The native redox properties of the protein were perturbed by adsorption directly to ITO or to the MPTMS layer on an ITO surface. However, azurin adsorbed at a DTMS covered ITO surface retained native electrochemical properties (E1/2=122±5mV vs. Ag/AgCl) and could exchange electrons directly with the underlying ITO layer without need for an intervening chemical mediator. These results open new opportunities for immobilizing functional redox proteins at ITO and developing spectroelectrochemical methods for investigating them.

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

The extraordinary ability of Fe- and Ni-containing enzymes to catalyze rapid and efficient H(+)/H(2) interconversion--a property otherwise exclusive to platinum metals--has been investigated in a series of experiments combining variable-temperature protein film voltammetry with mathematical modeling. The results highlight important differences between the catalytic performance of [FeFe]-hydrogenases and [NiFe]-hydrogenases and justify a simple model for reversible catalytic electron flow in enzymes and electrocatalysts that should be widely applicable in fields as diverse as electrochemistry, catalysis, and bioenergetics. The active site of [FeFe]-hydrogenases, an intricate Fe-carbonyl complex known as the "H cluster," emerges as a supreme catalyst.

Electrochemical Single‐Molecule AFM of the Redox Metalloenzyme Copper Nitrite Reductase in Action

We studied the electrochemical behavior of the redox metalloenzyme copper nitrite reductase (CNiR, Achromobacter xylosoxidans) immobilized on a Au(111)-electrode surface modified by a self-assembled cysteamine molecular monolayer (SAM) using a combination of cyclic voltammetry and electrochemically-controlled atomic force microscopy (in situ AFM). The enzyme showed no voltammetric signals in the absence of nitrite substrate, whereas a strong reductive electrocatalytic signal appeared in the presence of nitrite. Such a pattern is common in protein film and monolayer voltammetry and points to conformational changes in the enzyme upon substrate binding. Binding thus either improves the enzyme/electrode contact, or opens intramolecular electron-transfer channels between the redox center for electron inlet (a type I copper center) and the catalytic site for nitrite reduction (a type II copper center). The in situ AFM data are at the level of the single CuNiR enzyme molecule. The voltammetric patterns were paralleled by a clear increase (swelling) of the molecular height when the electrochemical potential traversed the region from resting to the electrocatalytically active redox enzyme function in the presence of nitrite. No change in size was observed in the absence of nitrite over the same potential range. The enzyme size variation is suggested to offer clues to the broadly observed substrate triggering in metalloenzyme monolayer voltammetry.

Substrate-dependent modulation of the enzymatic catalytic activity: Reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.