Cytochrome c and Cytochrome c Oxidase: Monolayer Assemblies and Catalysis

Abstract

A novel electrochemical system has been designed and assembled to study the kinetic activity of cytochrome c oxidase. Gold electrodes coated with 3-mercapto-1-propanol formed the surface for the physisorption of monolayers of cytochrome c and cytochrome c oxidase or a preformed cytochrome c−cytochrome c oxidase complex. The films were investigated by cyclic voltammetry at scanning at rates slow enough to permit near redox equilibrium between electrode and redox protein and hence obtain redox midpoint potentials. Cytochrome c monolayers alone displayed a reversible midpoint potential at pH 8 (Em8 vs NHE) at +240 mV, close to the native cytochrome c value observed in solution. In contrast, oxidase monolayers alone failed to support any detectable redox contact between electrode and protein, implying that the distances between the oxidase redox cofactors in the adsorbed oxidase are too far away from the electrode to promote significant electron transfer rates. However, adsorption of a preformed cytochrome c−cytochrome c oxidase complex promoted effective redox contact, demonstrating electron transfer with an apparent onset halfpoint potential at +225 mV. This effect is consistent with the mandatory requirement for cytochrome c to mediate electrons from the electrode to cytochrome c oxidase and presumably in a way reflecting the physiological pathway. Cyclic voltammetric measurements arranged to determine the rates of electron transfer between electrode and the complex showed that at scan rates up to 50 mV/s, extraordinary kinetic turnover is displayed attributable to the catalysis of oxygen reduction. Thus it is established that the protein complex can be assembled and enable the natural mediation of electron transfer from the electrode by cytochrome c to the enzyme at a rate fast enough for catalysis to be observed and controlled.