Theoretical and experimental investigation of surface-confined two-center metalloproteins by large-amplitude Fourier transformed ac voltammetry
Lee CY., Stevenson GP., Parkin A., Roessler MM., Baker RE., Gillow K., Gavaghan DJ., Armstrong FA., Bond AM.
The fourth and higher harmonic components available in large-amplitude Fourier transformed ac voltammetry have been employed in a study involving surface-confined redox proteins having multiple electron-transfer centers. Ferredoxin I from Azotobacter vinelandii (AvFdI) and two variants exhibit significant differences in the reversible reduction potentials (E o′) of their two non-interacting FeS clusters which are located at a fixed distance 1.2 nm apart. At pH 9.0 in aqueous buffered medium, FdI adsorbed on a pyrolytic graphite edge electrode displays two one-electron-transfer processes that are not complicated by coupled proton transfer. Ac voltammetric faradaic currents devoid of significant background, are detected and analyzed for the fourth to seventh harmonics of the [4Fe-4S]2+/+ and [3Fe-4S]+/0 processes. The absence of a non-faradaic background in these, but not in the dc or initial three ac harmonics, facilitates the experimental analysis. In potential regions where overlay of the [4Fe-4S]2+/+ and [3Fe-4S]+/0 processes occur, the alternating currents are not always additive because each process may exhibit different phases. The level of resolution of the two processes is governed by the separation of the Eo′ values, with the higher harmonic components giving enhanced resolution in the regimes of the reversible potentials. Simulation-experimental data comparisons have been used to determine the rate constants for each electron-transfer process. Theory-experiment comparisons are generally in satisfactory agreement, but some imperfections are consistent with the kinetic or/and thermodynamic dispersion associated with films of surface-confined proteins adhered to a heterogeneous edge plane graphite electrode. © 2011 Elsevier B.V. All rights reserved.