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We report a new technique for fabricating electrodes for electrochemical applications with lateral dimensions in the range 15-200 nm and a reproducible, well-defined geometry. This technique allows determining the electrode size by electron microscopy prior to electrochemical measurements and without contamination of the metal electrode. We measured the diffusion-limited current with stepped-current voltammetry and showed that its dependence on electrode size can be quantitatively understood if the known geometry of the electrodes is explicitly taken into account.

The purification of three membrane-bound c cytochromes from Bacillus azotoformans is described. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the Em for all is close to 150 mV.

Results show that Chromatium vinosum hydrogenase, adsorbed at a pyrolytic graphite edgeplane electrode, exhibits high level of hydrogen oxidation activity which is limited by the rate of substrate diffusion as measured by protein film voltammetry.

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 (v > 10 [k.sub.0][E.sub.step]), if the current is sampled at haft 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.

The properties and functions of redox-active centers of electron-transport enzymes in Escherichia coli were studied using voltammetry of proteins adsorbed on an electrode in an electroactive state. Protein-film voltammetry was also used to investigate catalytic electron transport in complex multicentered enzymes, particularly, succinate dehydrogenase and fumarate reductase. Findings showed that the electron exchange between electrode and one or more relay stations in the enzyme is reversible, while the catalytic activity was retained.

The interactions between methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans, are studied. The studies have shown that electron transfer from MADH to cytochrome c-551i has not involved a ternary complex but has occurred via a ping-pong mechanism in which amicyanin has used the same interface for the reactions with MADH and cytochrome c-551i.

Bacillus azotofonnans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique men aquinol-dependent nitric oxide reductase (qCuANOR) with a CuA center in its small subunit. The qCuANOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the Em for all is close to 150 mV. Only one of these c cytochromes, which exhibits an a-band maximum at 551 nm, acts as a direct electron donor to qCuANOR. Further investigation demonstrated that this cytochrome c551 possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c551 is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCuANOR. The ability of qCUANOR to accept electrons from both menaquinol and cytochrome c551 might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus den itnflcans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c551, is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b6f complex serving the bioenergetic needs of the organism. [ABSTRACT FROM AUTHOR]

This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues. Cys69 has been replaced by both an alanine and a serine residue. Redox properties of she C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred,1 = 5.39 ± 0.08. pKox = 7.29 ± 0.14, and pKred,2 = 7.84 ± 0.14 with Em,7 = -459 ± 4 mV, and a constant redox potential at high pH of -485 ± mV. The dependence of the semiquinone/hydroquionone redox potential on temperature is -0.5 ± 0.1 mV · K-1, yielding ΔH° = 28.6 ± 1.5 kJ · mol-1 and ΔS° = -50.0 ± 6.2 J · mol -1 · K-1. No significant differences in redox properties of wild-type. C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin 11. [ABSTRACT FROM AUTHOR]