Characterization of nonlinear background components in voltammetry by use of large amplitude periodic perturbations and fourier transform analysis.

Under most experimental conditions, a distinctly nonlinear background current is encountered in all forms of voltammetry which arises from the potential dependence of the capacitance. The nonlinear background current has been successfully modeled under large amplitude sinusoidal ac voltammetric conditions with a fourth order polynomial. The model was applied to a dummy cell containing a nonideal ceramic capacitor and commonly used electrodes. The nonlinearity in behavior of the background capacitance is particularly significant when considering the discrimination between the Faradaic and background contributions in the higher order harmonics resolved in ac voltammetry by Fourier transform-inverse Fourier transform approaches and in the simulation of the background current and hence double-layer capacitance as a function of potential. Typically, measurable background current under large amplitude conditions is detectable in the dc and fundamental to fourth harmonic components in large amplitude ac voltammetry. For analytical purposes, this background current can be corrected on a per harmonic basis without the need for any model. Background correction has been successfully applied to the first four harmonics for the oxidation of ferrocenemonocarboxylic acid over the concentration range of 5-500 microM in aqueous 0.5 M NaCl solution.

Systematic evaluation of electrode kinetics and impact of surface heterogeneity for surface-confined proteins using analysis of harmonic components available in sinusoidal large-amplitude Fourier transformed ac voltammetry.

A systematic approach to quantifying the electrode kinetics of surface-confined proteins and identifying the impact of surface heterogeneity is presented. The evaluation approach is based on analysis of individual harmonics derived from Fourier transformed large-amplitude ac voltammetry, and their peak current magnitude, I(p)(nomegat) versus frequency, f, relationships. Effectively, variability in the time-scale of each harmonic is expected, and advantage is taken of the fact that each individual harmonic displays a different level of sensitivity with respect to the kinetic evaluation. The data strategy protocols have been examined for the azurin Cu(II)/Cu(I) process when this metalloprotein is immobilized on gold electrodes modified alkanethiols having different chain lengths, using both pure and mixed thiol systems. I(p)(nomegat) versusf relationships also offer the advantage of the ability to detect and allow for the ohmic IR(u) drop effect and allow analyses that are independent of protein surface coverage. Estimation of an electron transfer rate is achievable from this form of analysis. However, experimentally observed waveshapes for each individual harmonic are consistently broader than that deduced theoretically on the basis of their rate constants because of kinetic and/or thermodynamic dispersion. In the mixed thiol systems, and with use of the ac method, kinetic discrimination is achieved for fast processes. This systematic study based on a model protein indicates that a more comprehensive level of evaluation of electrode kinetics can be derived from analysis of the ac harmonics available in large-amplitude ac voltammetry, by initially using I(p)(nomegat)-f data to evaluate the electrode kinetics followed by waveshape analysis to detect heterogeneity effects that give rise to kinetic or thermodynamic dispersion.

Electrochemical quartz crystal microbalance study of azurin adsorption onto an alkanethiol self-assembled monolayer on gold.

A quartz crystal microbalance coupled with electrochemistry was used to examine the adsorption of azurin on a gold electrode modified with a self-assembled monolayer of octanethiol. Azurin adsorbed irreversibly to form a densely packed monolayer. The rate of azurin adsorption was related to the bulk concentration of azurin in solution within the concentration range studied. At a high azurin concentration (2.75 muM), adsorption was rapid with a stable adsorption maximum attained in 2-3 min. At a lower azurin solution concentration (0.35 muM), the time to reach a stable adsorption maximum was approximately 30 min. Interestingly, the maximum surface concentration attained for all solution concentrations studied by the QCM method was 25 +/- 1 pmol cm-2, close to that predicted for monolayer coverage. The dissipation was monitored during adsorption, and only small changes were detected, implying a rigid adsorption model, as needed when using the Sauerbrey equation. Cyclic voltammetric data were consistent with a one-electron, surface-confined CuII/CuI azurin process with fast electron-transfer kinetics. The electroactive surface concentration calculated using voltammetry was 7 +/- 1 pmol cm-2. The differences between the QCM and voltammetrically determined surface coverage values reflect, predominantly, the different measurement methods but imply that all surface-confined azurin is not electrochemically active on the time scale of cyclic voltammetry.

Detailed analysis of the electron-transfer properties of azurin adsorbed on graphite electrodes using DC and large-amplitude Fourier transformed AC voltammetry.

The analysis of dc cyclic voltammograms of surface-confined metalloproteins is complicated by large background currents, significant ohmic iRu drop, and frequency dispersion related to protein and electrode surface inhomogeneity. The use of large-amplitude Fourier transform ac voltammetry for the quantification of the electron-transfer properties of a thin film of redox-active protein azurin adsorbed onto edge-plane, basal-plane, and highly oriented pyrolytic graphite electrode surfaces has been evaluated and compared to results obtained by dc cyclic voltammetry. In principle, it has been established that fourth and higher harmonic sine-wave data are ideally suited for analysis of electron-transfer processes as they are almost completely devoid of background capacitance current contributions. However, uncompensated resistance has a higher impact on these components, as is the case with fast scan rate dc techniques, so strategies to include this term in the simulations have been investigated. Application of recommended strategies for the evaluation of the electron-transfer properties of azurin adsorbed onto three forms of graphite, each having different background or uncompensated resistance values, is described and compared to results obtained by traditionally used forms of cyclic voltammetry. The electron-transfer rate constant, k0', of azurin at a highly oriented pyrolytic graphite electrode surface was approximately 250 s(-1), compared with > or =1000 s(-1) at edge-plane and basal-plane graphite electrodes. The significantly lower k0' value found at the highly oriented pyrolytic graphite electrode was related to the relatively low level of edge-plane defect sites present at the surface of this electrode. However, analysis of high ac harmonics suggests that frequency dispersion is substantial at all electrode surfaces. Such effects in these diffusionless situations are significantly enhanced relative to solution-phase voltammetry, where overlay of diffusion layers minimizes the impact of heterogeneity.

Recent progress in cytochrome P450 enzyme electrochemistry.

Cytochrome P450 (CYP) enzymes perform crucial functions in humans, including the metabolism of drugs and hormone synthesis. The catalytic reactions performed by these enzymes (typically monoxygenation) require the transfer of electrons. Thermodynamic and mechanistic detail of the electron transfer component of these catalytic processes has been obtained traditionally from potentiometric titrations. More recently, voltammetric approaches (that are inherently simpler and require less sample) have been used. This has been made possible by the creation of biocompatible electrode surfaces at which the P450 enzyme is confined and able to undergo physiologically relevant electron transfer processes. The continuing challenge has been to obtain an in vivo-like enzyme response, and to provide the basis for the creation of an artificial bioprocess in vitro. A powerful instrumental electrochemical method, employing Fourier-transformed large-amplitude ac voltammetry, offers the potential for greater insight and new opportunities to understand the nuances of the electron transfer process. This review highlights several recent advances in the electrochemistry of P450 enzymes rather than providing a comprehensive review of P450 electrochemistry.

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.

Separation of electron-transfer and coupled chemical reaction components of biocatalytic processes using Fourier transform ac voltammetry.

The underlying electron-transfer and coupled chemical processes associated with biologically important catalytic reactions can be resolved using a combination of Fourier transform ac voltammetry with an analysis of the separated dc and ac components. This outcome can be achieved because the response associated with generation of the catalytic current is essentially confined to the steady-state dc component, whereas the electron-transfer step is dominant in the fundamental and higher harmonics. For the mediated oxidation of glucose with glucose oxidase, it was found that the underlying reversible redox chemistry of the mediator, ferrocenemonocarboxylic acid, as detected in the third and higher harmonics, was totally unaffected by introduction of the catalytic process. In contrast, for the catalytic reduction of molecular oxygen by cytochrome P450, slight changes in the P450 redox process were detected when the catalytic reaction was present. Simulations of a simple catalytic reaction scheme support the fidelity of this novel FT ac voltammetric approach for examining mechanistic nuances of catalytic forms of electrochemical reaction schemes.

Redox properties of cytochrome p450BM3 measured by direct methods.

Cytochrome p450BM3 is a self-sufficient fatty acid monooxygenase consisting of a diflavin (FAD/FMN) reductase domain and a heme domain fused together in a single polypeptide chain. The multidomain structure makes it an ideal model system for studying the mechanism of electron transfer and for understanding p450 systems in general. Here we report the redox properties of the cytochrome p450BM3 wild-type holoenzyme, and its isolated FAD reductase and p450 heme domains, when immobilized in a didodecyldimethylammonium bromide film cast on an edge-plane graphite electrode. The holoenzyme showed cyclic voltammetric peaks originating from both the flavin reductase domain and the FeIII/FeII redox couple contained in the heme domain, with formal potentials of -0.388 and -0.250 V with respect to a saturated calomel electrode, respectively. When measured in buffer solutions containing the holoenzyme or FAD-reductase domain, the reductase response could be maintained for several hours as a result of protein reorganization and refreshing at the didodecyldimethylammonium modified surface. When measured in buffer solution alone, the cyclic voltammetric peaks from the reductase domain rapidly diminished in favour of the heme response. Electron transfer from the electrode to the heme was measured directly and at a similarly fast rate (ks' = 221 s-1) to natural biological rates. The redox potential of the FeIII/FeII couple increased when carbon monoxide was bound to the reduced heme, but when in the presence of substrate(s) no shift in potential was observed. The reduced heme rapidly catalysed the reduction of oxygen to hydrogen peroxide.