X-ray structural, functional and computational studies of the O2-sensitive E. coli hydrogenase-1 C19G variant reveal an unusual [4Fe-4S] cluster.

The crystal structure of the Escherichia coli O2-sensitive C19G [NiFe]-hydrogenase-1 variant shows that the mutation results in a novel FeS cluster, proximal to the Ni-Fe active site. While the proximal cluster of the native O2-tolerant enzyme can transfer two electrons to that site, EPR spectroscopy shows that the modified cluster can transfer only one electron, this shortfall coinciding with O2 sensitivity. Computational studies on electron transfer help to explain how the structural and redox properties of the novel FeS cluster modulate the observed phenotype.

Generating single metalloprotein crystals in well-defined redox states: electrochemical control combined with infrared imaging of a NiFe hydrogenase crystal.

We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchrotron infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.

Concentrations of selected essential and non-essential elements in arctic fox (Alopex lagopus) and wolverines (Gulo gulo) from the Canadian Arctic.

Arctic fox (Alopex lagopus) and wolverine (Gulo gulo) tissues were collected in the Canadian Arctic from 1998 to 2001 and analyzed for various essential and non-essential elements. Several elements (Ag, Al, As, B, Ba, Be, Co, Cr, Mo, Ni, Sb, Sn, Sr, Tl, U and V) were near or below the detection limits in >95% arctic fox and wolverine samples. Concentrations of Cd, Cu, Fe, total Hg (THg), Mn, Pb, Se and Zn were quantifiable in >50% of the samples analyzed and reported herein. Hepatic elemental concentrations were not significantly different among arctic foxes collected at Ulukhaqtuuq (Holman), NT (n=13) and Arviat, NU (n=50), but were significantly greater than concentrations found in wolverine liver from Kugluktuk (Coppermine), NU (n=12). The mean (+/-1 S.E.) concentrations of Cd in kidney were also significantly greater in arctic fox (1.08+/-0.19 microg g(-1) wet wt.) than wolverine (0.67+/-0.18 microg g(-1) wet wt.). However, mean hepatic Cu concentrations (Ulukhaqtuuq: 5.5+/-0.64; Arviat: 7.1+/-0.49 microg g(-1) wet wt.) in arctic foxes were significantly lower than in wolverines (32+/-3.3 microg g(-1) wet wt.). Hepatic total Hg (THg) concentrations in arctic fox from this study were not significantly different from specimens collected in 1973, suggesting that THg concentrations have not changed dramatically over the past 30 years. The mono-methylmercury (MeHg) concentrations in selected (n=10) arctic fox liver samples from Arviat (0.14+/-0.07 microg g(-1) wet wt.) comprised 14% of THg. While the molar concentrations of THg were correlated with Se in arctic foxes and wolverines, the hepatic Hg/Se molar ratios were consistently lower than unity; suggesting that Se-mediated detoxification pathways of Hg are not overwhelmed at current exposure.

Influence of electrochemical properties in determining the sensitivity of [4Fe-4S] clusters in proteins to oxidative damage.

Interconversion between [4Fe-4S] cubane and [3Fe-4S] cuboidal states represents one of the simplest structural changes an iron-sulphur cluster can undertake. This reaction is implicated in oxidative damage and in modulation of the activity and regulation of certain enzymes, and it is therefore important to understand the factors governing cluster stability and the processes that activate cluster conversion. In the present study, protein film voltammetry has been used to induce and monitor the oxidative conversion of [4Fe-4S] into [3Fe-4S] clusters in different variants of Azotobacter vinelandii ferredoxin I (AvFdI; the 8Fe form of the native protein), and DeltaThr(14)/DeltaAsp(15), Thr(14)-->Cys (T14C) and C42D mutants. The electrochemical results have been correlated with the differing oxygen sensitivities of [4Fe-4S] clusters, and comparisons have been drawn with other ferredoxins (Desulfovibrio africanus FdIII, Clostridium pasteurianum Fd, Thauera aromatica Fd and Pyrococcus furiosus Fd). In contrast with high-potential iron-sulphur proteins (HiPIPs) for which the oxidized species [4Fe-4S](3+) is inert to degradation and can be isolated, the hypervalent state in these ferredoxins (most obviously the 3+ level) is very labile, and the reduction potential at which this is formed is a key factor in determining the cluster's resistance to oxidative damage.

Enzyme electrokinetics: energetics of succinate oxidation by fumarate reductase and succinate dehydrogenase.

Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD. Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II.

A distal histidine mutant (H52Q) of yeast cytochrome c peroxidase catalyzes the oxidation of H(2)O(2) instead of its reduction.

A H52Q variant of yeast cytochrome c peroxidase (CcP), in which the distal histidine is replaced by glutamine, catalyzes oxidation of H(2)O(2) instead of reduction. This redirection of catalytic action is detected by protein film voltammetry. In the presence of H(2)O(2), wild-type CcP, adsorbed on a graphite electrode, shows a strong catalytic reduction wave commencing at about 0.8V (pH 5.4); by contrast, H52Q does not exhibit this activity but instead shows a catalytic oxidation current at potentials in the region of 0.9 V. The oxidation current is partly suppressed in the presence of tetranitromethane (a superoxide scavenger) and is not observed for other mutants studied, including H52A. The only significant structural change in the H52Q variant is that the Q-52 side chain occupies the space vacated by the H-52 imidazole; specifically, the N-epsilon atom that is believed to transfer a proton and induce O--O cleavage is replaced, to within 0.75 A, by the carbamide-O. Thus, while the weakly basic amide functionality is unable to serve in the reorganization of bound H(2)O(2), it is able to facilitate its oxidation, most obviously by serving as a H-bond acceptor to assist formation of a labile superoxide intermediate.

Determination of an optimal potential window for catalysis by E. coli dimethyl sulfoxide reductase and hypothesis on the role of Mo(V) in the reaction pathway.

Protein film voltammetry (PFV) of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC) adsorbed at a graphite electrode reveals that the catalytic activity of this complex Mo-pterin/Fe-S enzyme is optimized within a narrow window of electrode potential. The upper and lower limits of this window are determined from the potential dependences of catalytic activity in reducing and oxidizing directions; i.e., for reduction of DMSO (or trimethylamine-N-oxide) and oxidation of trimethylphosphine (PMe(3)). At either limit, the catalytic activity drops despite the increase in driving force: as the potential is lowered below -200 mV (pH 7.0-8.9), the rate of reduction of DMSO decreases abruptly, while for PMe(3), an oxidative current is observed that vanishes as the potential is raised above +20 mV (pH 9.0). Analysis of the waveshapes reveals that both activity thresholds result from one-electron redox reactions that arise, most likely, from groups within the enzyme; if so, they represent "switches" that reflect the catalytic mechanism and may be of physiological relevance. The potential window of activity coincides approximately with the appearance of the Mo(V) EPR signal observed in potentiometric titrations, suggesting that crucial stages of catalysis are facilitated while the active site is in the intermediate Mo(V) oxidation state.

Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster.

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.

Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry.

Rapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two [4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from [3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal [3Fe-4S] formation (in excess over [4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The [3Fe-4S]:[4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that [3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of [4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with [3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of [3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to [4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the [3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts.

Atomically defined mechanism for proton transfer to a buried redox centre in a protein.

The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as 'proton pumps. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins. Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is 'gated' by proton release to the solvent. Studies of this 'proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica.

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.

Ferredoxin III of Desulfovibrio africanus: sequencing of the native gene and characterization of a histidine-tagged form.

Desulfovibrio africanus ferredoxin III (Da FdIII) contains one [4Fe-4S](2+/1+) cluster and one [3Fe-4S](1+/0) cluster, bound by seven Cys residues, in which the [3Fe-4S] cluster is co-ordinated by the unusual sequence, Cys(11)-Xaa-Xaa-Asp(14)-Xaa-Xaa-Cys(17)-Xaa(n)-Cys(51)-Glu. The [3Fe-4S] core of this ferredoxin is so far unique in showing rapid bi-directional [3Fe-4S]<-->[4Fe-4S] cluster interconversion with a wide range of metal ions. In order to obtain protein for mutagenesis studies Da FdIII has been cloned, sequenced, and expressed as a hexa-histidine tagged (ht) polypeptide in Escherichia coli strain BL21(DE3) pLysS. Expression of ht Da FdIII, whether translated from a synthetic gene (pJB10) or from the native nucleotide sequence (pJB11), occurred at similar levels (approx. 6 mg.l(-1)), but without incorporation of metal clusters. The nucleotide sequence confirms the protein sequence reported previously [Bovier-Lapierre, Bruschi, Bonicel and Hatchikian (1987) Biochim. Biophys. Acta 913, 20-26]. Cluster incorporation was achieved using FeCl(3) together with cysteine sulphur transferase, NifS, plus cysteine to generate low levels of sulphide ions. Absorption and EPR spectroscopy show that both [3Fe-4S] and [4Fe-4S] clusters are correctly inserted. Thin-film electrochemistry provides evidence that the [3Fe-4S] cluster undergoes reversible cluster transformation in the presence of Fe(II) and Zn(II) ions with properties identical to the native protein. Nevertheless the protein has lower stability than native Da FdIII during chromatography. The one-dimensional 600 MHz NMR spectrum of the apoprotein indicates an unstructured protein with random coil chemical shifts whereas spectra of the reconstituted ht protein show secondary structural elements and 18 peaks shifted downfield of 9.6 p.p.m. The spectra are unique but have similarities with the shift patterns seen with 7Fe Desulfurolobus ambivalens Fd. The ht does not affect iron-sulphur cluster incorporation, but NMR evidence suggests that excess Fe binds to the tag. This may account for the lower stability of the ht compared with the native protein.

Fast voltammetric studies of the kinetics and energetics of coupled electron-transfer reactions in proteins.

A wealth of information on the reactions of redox-active sites in proteins can be obtained by voltammetric studies in which the protein sample is arranged as a layer on an electrode surface. By carrying out cyclic voltammetry over a wide range of scan rates and exploiting the ability to poise or pulse the electrode potential between cycles, data are obtained that are conveniently (albeit simplistically) analysed in terms of plots of peak potentials against scan rate. A simple reversible electron-transfer process gives rise to a 'trumpet'-shaped plot because the oxidation and reduction peaks separate increasingly at high scan rate; the electrochemical kinetics are then determined by fitting to Butler-Volmer or Marcus models. Much more interesting though are the ways in which this 'trumpet plot' is altered, often dramatically, when electron transfer is coupled to biologically important processes such as proton transfer, ligand exchange, or a change in conformation. It is then possible to derive particularly detailed information on the kinetics, energetics and mechanism of reactions that may not revealed clearly or even at all by other methods. In order to interpret the voltammetry of coupled systems, it is important to be able to define 'ideal behaviour' for systems that are expected to show simple and uncoupled electron transfer. Accordingly, this paper describes results we have obtained for several proteins that are expected to show such behaviour, and compares these results with theoretical predictions.

Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I.

The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0').

Voltammetric studies of bidirectional catalytic electron transport in Escherichia coli succinate dehydrogenase: comparison with the enzyme from beef heart mitochondria.

The succinate dehydrogenases (SDH: soluble, membrane-extrinsic subunits of succinate:quinone oxidoreductases) from Escherichia coli and beef heart mitochondria each adsorb at a pyrolytic graphite 'edge' electrode and catalyse the interconversion of succinate and fumarate according to the electrochemical potential that is applied. E. coli and beef heart mitochondrial SDH share only ca. 50% homology, yet the steady-state catalytic activities, when measured over a continuous potential range, display very similar catalytic operating potentials and energetic biases (the relative ability to catalyse succinate oxidation vs. fumarate reduction). Importantly, E. coli SDH also exhibits the interesting 'tunnel-diode' behaviour previously reported for the mitochondrial enzyme. Thus as the potential is lowered below ca. -60 mV (pH 7, 38 degrees C) the rate of catalytic fumarate reduction decreases abruptly despite an increase in driving force. Since the homology relates primarily to residues associated with active site regions, the marked similarity in the voltammetry reaffirms our previous conclusions that the tunnel-diode behaviour is a characteristic property of the enzyme active site. Thus, succinate dehydrogenase is an excellent fumarate reductase, but its activity in this direction is limited to a very specific range of potential.

Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H2 value.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.

Thermodynamic influences on the fidelity of iron-sulphur cluster formation in proteins.

In an organism, two thermodynamic factors are important in ensuring that homometallic [4Fe-4S] cubane clusters are formed in preference to clusters containing heterometals such as Zn or Cu. These are the electronic resonance stabilisation, which boosts the binding of Fe(II) within an Fe-S cluster relative to its normally low position in the Irving-Williams order, and attenuation of the cytoplasmic concentrations of competing metals such as Zn or Cu by specific ligands.

Redox properties of flavocytochrome c3 from Shewanella frigidimarina NCIMB400.

The thermodynamic and catalytic properties of flavocytochrome c3 from Shewanella frigidimarina have been studied using a combination of protein film voltammetry and solution methods. As measured by solution kinetics, maximum catalytic efficiencies for fumarate reduction (kcat/Km = 2.1 x 10(7) M-1 s-1 at pH 7.2) and succinate oxidation (kcat/Km = 933 M-1 s-1 at pH 8.5) confirm that flavocytochrome c3 is a unidirectional fumarate reductase. Very similar catalytic properties are observed for the enzyme adsorbed to monolayer coverage at a pyrolytic graphite "edge" electrode, thus confirming the validity of the electrochemical method for providing complementary information. In the absence of fumarate, the adsorbed enzyme displays a complex envelope of reversible redox signals which can be deconvoluted to yield the contributions from each active site. Importantly, the envelope is dominated by the two-electron signal due to FAD [E degrees ' = -152 mV vs the standard hydrogen electrode (SHE) at pH 7.0 and 24 degrees C] which enables quantitative examination of this center, the visible spectrum of which is otherwise masked by the intense absorption bands due to the hemes. The FAD behaves as a cooperative two-electron center with a pH-dependent reduction potential that is modulated (pKox at 6.5) by ionization of a nearby residue. In conjunction with the kinetic pKa values determined for the forward and reverse reactions (7.4 and 8.6, respectively), a mechanism for fumarate reduction, incorporating His365 and an anionic form of reduced FAD, is proposed. The reduction potentials of the four heme groups, estimated by analysis of the underlying envelope, are -102, -146, -196, and -238 mV versus the SHE at pH 7.0 and 24 degrees C and are comparable to those determined by redox potentiometry.

Using the pulsed nature of staircase cyclic voltammetry to determine interfacial electron-transfer rates of adsorbed species.

Staircase cyclic voltammetry (SCV) is the digital counterpart of analog cyclic voltammetry (CV). However, when the redox-active species is adsorbed at the electrode surface, the voltammetric peak shapes (width, height, area, and to a lesser extent the reduction potentials) obtained with SCV can be very different from those of CV, even when small potential steps are used. Like analog CV, SCV provides a straightforward method to estimate and subtract the background and charging currents from the desired Faradaic current, while the pulsed nature of SCV provides the time-dependent decay of the Faradaic current, similar to chronoamperometry. Thus, electron-transfer rate constants can be directly measured as a function of applied potential, and no a priori model is required. An SCV equivalent of the square wave "quasi-reversible maximum" of observed peak height versus sampling moment and step size is predicted. The SCV response can only become independent of potential step size and similar to CV at high scan rates (ν > 10 k(0)E(step)), if the current is sampled at half the step interval. The applicability of SCV to studies of redox centers in proteins is illustrated for the two-electron oxidation/reduction of yeast cytochrome c peroxidase, adsorbed at a pyrolytic graphite edge-plane electrode.