Protein film voltammetry is a relatively new approach to studying redox enzymes, the concept being that a sample of a redox protein is configured as a film on an electrode and probed by a variety of electrochemical techniques. The enzyme molecules are bound at the electrode surface in such a way that there is fast electron transfer and complete retention of the chemistry of the active site that is observed in more conventional experiments. Modulations of the electrode potential or catalytic turnover result in the movement of electrons to, from, and within the enzyme; this is detected as a current that varies in characteristic ways with time and potential. Henceforth, the potential dimension is introduced into enzyme kinetics. The presence of additional intrinsic redox centers for providing fast intramolecular electron transfer between a buried active site and the protein surface is an important factor. Centers which carry out cooperative two-electron transfer, most obviously flavins, produce a particularly sharp signal that allows them to be observed, even as transient states, when spectroscopic methods are not useful. High catalytic activity produces a large amplification of the current, and useful information can be obtained even if the coverage on the electrode is low. Certain enzymes display optimum activity at a particular potential, and this can be both mechanistically informative and physiologically relevant. This paper outlines the principles of protein film voltammetry by discussing some recent results from this laboratory.

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The rapid electron-transfer reaction of the blue copper protein azurin adsorbed on different electrodes (pyrolytic graphite "edge" (PGE) and gold modified with self-assembled monolayers of various 1-alkanethiols) has been studied by cyclic and square ware voltammetry. By using large values (0.075-0.3 V) for the square wave amplitude, the electron-transfer (ET) rate is measured over a continuously variable driving force in either direction. Values for k 0 (the standard ET "exchange" rate constant at zero driving force) depend on the nature of the electrode; by contrast, k max (the maximum rate constant at high driving force) is essentially invariant with a rate of (6 ( 3) × 10 3 s -1 at 0 °C for both oxidation and reduction, irrespective of whether the electrode is PGE or Au modified with either dodecanethiol or decanethiol. Using Marcus theory, the potential dependences yield an extremely low value for the reorganization energy (λ < 0.25); however, a good fit is obtained with an alternative model in which ET is gated by a preceding process that is common to all cases. The nature of this limitation has been probed by varying pH, H 2 O vs D 2 O, solvent viscosity, and temperature. There is little dependence on medium, thereby ruling out proton transfer or major (translational) motion in the ET-limiting step. The temperature dependences for both PGE (-40 to 0 °C) and gold (0-50 °C) electrodes give good fits to the Arrhenius equation, with activation energies E A ) 19.5 ( 1.1 and 2.6 ( 1.7 kJ mol -1 , respectively. Consequently, the gating process has an unfavorable activation entropy, suggesting that electron transfer is dependent upon prior formation of a highly ordered protein configuration on the electrode surface.

An electrode surface is presented that enables the characterization of redox-active membrane enzymes in a native-like environment. An ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), has been co-immobilized into tethered bilayer lipid membranes (tBLMs). The tBLM is formed on gold surfaces functionalized with cholesterol tethers which insert into the lower leaflet of the membrane. The planar membrane architecture is formed by self-assembly of proteoliposomes, and its structure is characterized by surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and tapping-mode atomic force microscopy (TM-AFM). The functionality of cbo(3) is investigated by cyclic voltammetry (CV) and is confirmed by the catalytic reduction of oxygen. Interfacial electron transfer to cbo(3) is mediated by the membrane-localized ubiquinol-8, the physiological electron donor of cbo(3). Enzyme coverages observed with TM-AFM and CV coincide (2-8.5 fmol.cm(-)(2)), indicating that most-if not all-cbo(3) on the surface is catalytically active and thus retains its integrity during immobilization.