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CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme from the xanthine oxidase family of molybdenum-containing enzymes, bearing a unique binuclear Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 namely MoVI/V, MoV/IV, FeSI2+/+, FeSII2+/+, FAD/FADH and FADH/FADH-. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

We demonstrate electrochemically driven catalytic voltammetry of the Mo-dependent sulfite dehydrogenase (SorT) from the α-Proteobacterium Sinorhizobium meliloti with its physiological electron acceptor, the c-type cytochrome (SorU), with both proteins co-adsorbed on a chemically modified Au working electrode. Both SorT and SorU were constrained under a perm-selective dialysis membrane with the biopolymer chitosan as a co-adsorbate, while the electrode was modified with a 3-mercaptopropionate self-assembled monolayer cast on the Au electrode. Cyclic voltammetry of the SorU protein reveals a well-defined quasireversible FeIII/II redox couple at +130 mV versus NHE in 100 mM phosphate buffer solution (pH 7.0). Introduction of wild-type sulfite dehydrogenase (SorTWT) and sulfite transforms this transient SorU voltammetric response into a sigmoidal catalytic wave, which increases with sulfite concentration before eventually saturating. In addition to the wild-type enzyme, the variants SorTR78K, SorTR78M, and SorTR78Q were also examined electrochemically in an effort to better understand the role of amino acid residue Arg78, which is in the vicinity of the Mo active site of SorT. [ABSTRACT FROM AUTHOR]

Herein, we report the electrocatalytic voltammetry of holo and heme-free human sulfite oxidase (HSO) mediated by the synthetic iron complexes 1,2-bis(1,4,7-triaza-1-cyclononyl)ethane iron(III) bromide, ([Fe(dtne)]Br3.3H2O), potassium ferricyanide (K3[Fe(CN)6]), and ferrocene methanol (FM) at a 5-(4′-pyridinyl)-1,3,4-oxadiazole-2-thiol (Hpyt) modified gold working electrode. Holo HSO contains two electroactive redox cofactors, comprising a mostly negatively charged cyt b5 (heme) domain and a Mo cofactor (Moco) domain (the site of sulfite oxidation), where the surface near the active site is positively charged. We anticipated different catalytic voltammetry based on either repulsive or attractive electrostatic interactions between the holo or heme-free enzymes and the positively or negatively charged redox mediators. Both holo and heme-free HSO experimental catalytic voltammetry has been modeled by using electrochemical simulation across a range of sweep rates and concentrations of substrate and both positive and negatively charged electron acceptors ([Fe(dtne)]3+, [Fe(CN)6]3− and FM+), which provides new insights into the kinetics of the HSO catalytic mechanism. These mediator complexes have almost the same redox potential (all lying in the range +415 to +430 mV vs. NHE) and, thus, deliver the same driving force for electron transfer with the Mo cofactor. However, differences in the electrostatic affinities between HSO and the mediator have a significant influence on the electrocatalytic response. [ABSTRACT FROM AUTHOR]

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron–sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c 2 mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c 2 accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c 2), Michaelis constants, K M,DMS and K M,cyt of 53 and 21 μM, respectively, were determined at pH 8. Further kinetic analysis revealed a “ping-pong” enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c 2 immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible FeIII/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The FeIII/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c 2 is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c 2 effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant $$ K_{{{\text{M,DMS}}}}^{\prime } $$ of 13(±1) μM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c 2 as the electron acceptor was found to be approximately 8.3. [ABSTRACT FROM AUTHOR]

Abstract: The complex molybdoenzyme xanthine oxidase (XO) catalyses the oxidation of xanthine to uric acid. Here we report the first direct (unmediated) catalytic electrochemistry of the enzyme in the presence of xanthine. The only non-turnover response (without substrate present) is a sharp two-electron wave from the FAD cofactor at −242mV vs. NHE (pH 8.0). Upon addition of xanthine to the electrochemical cell a pronounced electrocatalytic anodic current appears at ca. +300mV vs. NHE, but the FAD peak remains. This is unusual as the onset of catalysis should occur at the potential of the FAD cofactor (the site at which oxygen or NAD+ binds to the enzyme in solution). The observed electrochemical catalysis is prevented by the addition of known XO inhibitors allopurinol or cyanide. [Copyright &y& Elsevier]

Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (FeIIIFeII→ FeIIIFeIII) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Ufo) have been determined. The effect of pH on the redox potentials has been investigated in the range 3

Sulfite dehydrogenase from Starkeya novella is an αβ heterodimer comprising a 40.6 kDa subunit (containing the Mo cofactor) and a smaller 8.8 kDa heme c subunit. The enzyme catalyses the oxidation of sulfite to sulfate with the natural electron acceptor being cytochrome c[sub 550]. Its catalytic mechanism is thought to resemble that found in eukaryotic sulfite oxidases. Using protein film voltammetry and redox potentiometry, we have identified both Mo- and heme-centered redox responses from the enzyme immobilized on a pyrolytic graphite working electrode: E[sub m,8] (Fe[sup III/II]) +177 mV; E[sub m,8] (Mo[sup VI/V]) +211 mV and E[sub m,8] (Mo[sup V/IV]) -118 mV vs NHE; Upon addition of sulfite to the electrochemical cell a steady-state voltammogram is observed and an apparent Michaelis constant (K[sub m]) of 26(1) µM was determined for the enzyme immobilized on the working electrode surface, which is comparable with the value obtained from solution assays. [ABSTRACT FROM AUTHOR]

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron–sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c 2 mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c 2 accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c 2), Michaelis constants, K M,DMS and K M,cyt of 53 and 21 μM, respectively, were determined at pH 8. Further kinetic analysis revealed a “ping-pong” enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c 2 immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible FeIII/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The FeIII/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c 2 is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c 2 effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant $$ K_{{{\text{M,DMS}}}}^{\prime } $$ of 13(±1) μM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c 2 as the electron acceptor was found to be approximately 8.3. [ABSTRACT FROM AUTHOR]

Abstract: The diflavo-protein NADPH cytochrome P450 reductase (CPR) is the key electron transfer partner for all drug metabolizing cytochrome P450 enzymes in humans. The protein delivers, consecutively, two electrons to the heme active site of the P450 in a carefully orchestrated process which ultimately leads to the generation of a high valent oxo-heme moiety. Despite its central role in P450 function, no direct electrochemical investigation of the purified protein has been reported. Here we report the first voltammetric study of purified human CPR where responses from both the FMN and FAD cofactors have been identified using both cyclic and square wave voltammetry. For human CPR redox responses at −2 and −278mV (with a ratio of 1e−:3e−) vs NHE were seen at pH 7.9 while the potentials for rat CPR at pH 8.0 were −20 and −254mV. All redox responses exhibit a pH dependence of approximately −59mV/pH unit consistent with proton coupled electron transfer reactions of equal stoichiometry. [Copyright &y& Elsevier]

• A fungal nitrate reductase (NR) catalyses the reduction of nitrate to nitrite. • NR may be immobilised on a glassy carbon electrode with the biopolymer chitosan. • Electrocatalytic nitrate reduction can be achieved with NR mediated by anthraquinone sulfonate. • High selectivity and exceptional sensitivity for nitrate is found. • The NR enzyme electrode is stable for at least 3 months. We report an electrochemical nitrate biosensor incorporating the enzyme nitrate reductase (NR) from the fungus Neurospora crassa entrapped within a polymeric chitosan matrix on a glassy carbon (GC) electrode. The artificial electron mediator anthraquinone sulfonate (AQ) is utilized as an electron transfer partner (co-substrate) for NR. The NR enzyme was effectively immobilized within the chitosan matrix without the need for a dialysis membrane, and exhibited high sensitivity and selectivity for nitrate using either rotating disk cyclic voltammetry or constant potential amperometry methods. A particular feature was the longevity of the biosensor which retained more than 70% activity over a period of 3 months. The practical application of the biosensor was demonstrated by the determination of nitrate concentrations in lake and river water samples without any pre-treatment, and the results were validated with a reliable spectroscopic assay. [Display omitted] [ABSTRACT FROM AUTHOR]

As recently as 2006 the mitochondrial amidoxime reducing component (mARC) was identified as the fourth and last Mo enzyme present in humans. Its physiological role remains unknown. mARC is capable of reducing a variety of N -hydroxylated compounds such as amidoximes to their corresponding amidine and there is considerable interest in this enzyme from a pharmaceutical perspective. mARC is a target for N -hydroxylated pro-drugs that may be reductively activated intracellularly to release potent drugs such as cationic amidinium ions, which exhibit a broad spectrum of activity as antithrombotics and against various bacteria and parasites. In the quest for a rapid screen of new mARC substrates and inhibitors we present an electrochemical method which utilizes the natural electron partner of mARC, cytochrome b 5 , coupled to an electrochemical electrode. Mediated electron transfer from the electrode via cytochrome b 5 to mARC results in a catalytic current in the presence of substrate. [ABSTRACT FROM AUTHOR]

We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa , is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant ( K M ) of 39 μM (pH 7). The catalytic cathodic current increases with temperature from 5 to 35 °C and an activation enthalpy of 26 kJ mol − 1 was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. [ABSTRACT FROM AUTHOR]

Mediated electrocatalytic voltammetry of human sulfite oxidase (HSO) is demonstrated with synthetic one electron transfer iron complexes bis(1,4,7-triazacyclononane)iron(III) ([Fe(tacn) 2 ] 3+ ) and 1,2-bis(1,4,7-triaza-1-cyclononyl)ethane iron(III) ([Fe(dtne)] 3+ ) at a glassy carbon working electrode. The two synthetic electron acceptors for HSO, differing in redox potential by 270 mV, deliver different driving forces for electrocatalysis. Digital simulation of the catalytic voltammetry was achieved with single set of enzyme-dependent kinetic parameters that reproduced the experimental data across a range of sweep rates, and sulfite and mediator concentrations. Amperometry carried out in a stirred solution with the lower potential mediator [Fe(tacn) 2 ] 3+ was optimised and exhibited a linear increase in steady state current in the sulfite concentration range 5.0 × 10 −6 to 8.0 × 10 −4 M with a detection limit of 0.2 pM (S/N = 3). The HSO coupled electrode was successfully used for the determination of sulfite concentration in white wine and beer samples and the results validated with a standard spectrophotometric method. [ABSTRACT FROM AUTHOR]

Abstract: We describe the catalytic voltammograms of the periplasmic arsenite oxidase (Aio) from the chemolithoautotrophic bacterium Rhizobium sp. str. NT-26 that oxidizes arsenite to arsenate. Electrochemistry of the enzyme was accomplished using its native electron transfer partner, cytochrome c 552 (cyt c 552), as a mediator. The protein cyt c 552 adsorbed on a mercaptoundecanoic acid (MUA) modified Au electrode exhibited a stable, reversible one-electron voltammetric response at +275mV vs NHE (pH6). In the presence of arsenite and Aio the voltammetry of cyt c 552 is transformed from a transient response to an amplified sigmoidal (steady state) wave consistent with an electro-catalytic system. Digital simulation was performed using a single set of parameters for all catalytic voltammetries obtained at different sweep rates and various substrate concentrations. The obtained kinetic constants from digital simulation provide new insight into the kinetics of the NT-26 Aio catalytic mechanism. [Copyright &y& Elsevier]

The Mo-dependent enzyme YiiM enzyme from Escherichia coli is a member of the sulfite oxidase family and shares many similarities with the well-studied human mitochondrial amidoxime reducing component (mARC). We have investigated YiiM catalysis using electrochemical and spectroscopic methods. EPR monitored redox potentiometry found the active site redox potentials to be MoVI/V –0.02 V and MoV/IV –0.12 V vs NHE at pH 7.2. In the presence of methyl viologen as an electrochemically reduced electron donor, YiiM catalysis was studied with a range of potential substrates. YiiM preferentially reduces N-hydroxylated compounds such as hydroxylamines, amidoximes, N-hydroxypurines and N-hydroxyureas but shows little or no activity against amine-oxides or sulfoxides. The pH optimum for catalysis was 7.1 and a bell-shaped pH profile was found with pK a values of 6.2 and 8.1 either side of this optimum that are associated with protonation/deprotonations that modulate activity. Simulation of the experimental voltammetry elucidated kinetic parameters associated with YiiM catalysis with the substrates 6–hydroxyaminopurine and benzamidoxime. • Broad substrate activity for electrocatalytic reduction of N-hydroxylated organic compounds • Characterization of active site redox potentials • In depth electrochemical simulation of experimental data to obtain kinetic data [ABSTRACT FROM AUTHOR]

Herein we report the mediated electrocatalyticvoltammetry of theplant molybdoenzyme nitrate reductase (NR) from Arabidopsisthalianausing the established truncated molybdenum-hemefragment at a glassy carbon (GC) electrode. Methyl viologen (MV),benzyl viologen (BV), and anthraquinone-2-sulfonic acid (AQ) are employedas effective artificial electron transfer partners for NR, differingin redox potential over a range of about 220 mV and delivering differentreductive driving forces to the enzyme. Nitrate is reduced at theMo active site of NR, yielding the oxidized form of the enzyme, whichis reactivated by the electro-reduced form of the mediator. Digitalsimulation was performed using a single set of enzyme dependent parametersfor all catalytic voltammetry obtained under different sweep ratesand various substrate or mediator concentrations. The kinetic constantsfrom digital simulation provide new insight into the kinetics of theNR catalytic mechanism. [ABSTRACT FROM AUTHOR]

Abstract: Sulfite dehydrogenase (SDH) from Starkeya novella is a heterodimeric enzyme comprising a Mo active site and a heme c electron relay, which mediates electron transfer from the Mo cofactor to cytochrome c following sulfite oxidation. Studies on the wild type enzyme (SDHWT) and its variants have identified key amino acids at the active site, specifically Arg-55 and His-57. We report the MoVI/V, MoV/IV and FeIII/II (heme) redox potentials of the variants SDHR55K, SDHR55M, SDHR55Q and SDHH57A in comparison with those of SDHWT. For SDHR55M, SDHR55Q and SDHH57A the heme potentials are lowered from ca. 240mV in SDHWT to ca. 200mV, while the heme potential in SDHR55K remains unchanged and the Mo redox potentials are not affected significantly in any of these variants. Protein film voltammetry reveals a pH dependence of the electrochemical catalytic half-wave potential (E cat ) of −59mV/pH in SDHWT and SDHR55K which tracks the pH dependence of the MoVI/V redox potential. By contrast, the catalytic potentials for SDHR55M and SDHH57A are pH-independent and follow the potential of the heme cofactor. These results highlight a switch in the pathway of electron exchange as a function of applied potential that is revealed by protein film voltammetry where an actuation of rate limiting intramolecular electron transfer (IET, Mo to heme) at high potential attenuates the catalytic current relative to faster, direct electron transfer (Mo to electrode) at lower potential. The same change in electron transfer pathway is linked to an unusual peak-shaped profile of the ideally sigmoidal steady state voltammogram in SDHWT alone, which has been associated with a potential dependent change in the orientation of the enzyme on the electrode surface. All other variants show purely sigmoidal voltammetry due to their inherently slower turnover numbers which are always lower than IET rates. [Copyright &y& Elsevier]

Abstract: The human cytochromes P450 are responsible for the clearance of ∼90% of xenobiotics yet comparatively little is known about their electrochemistry. Here we report the first direct electrochemistry of P450s from the 2C subfamily; one of the major groups of enzymes from this family. Specifically, the proteins that we have examined are recombinant human P450s 2C9, 2C18 and 2C19 and reversible FeIII/II couples are seen in the absence of dioxygen. Even in the presence of trace amounts of dioxygen, a pronounced cathodic response is seen which is assigned to catalytic reduction of the bound dioxygen ligand by the ferrous P450. [Copyright &y& Elsevier]