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 (E m8 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.

Iron(III) protoporphyrin IX (Fe(III)PP) and iron(III) hematoporphyrin (Fe(III)HP) were selectively and covalently attached to dimercaptoalkane-modified gold electrodes. Reaction of their vinyl or hydroxyethyl groups with the surface-immobilized thiols produced thioether linkages, reminiscent of the heme macrocycle attachment in c-type cytochromes. Cyclic voltammetry revealed reversible electrochemistry of self-assembled monolayers (SAMs) of FePPs and FeHPs on the thiol-modified gold substrates. The surface coverage estimated from the charges transferred corresponds to 30% of a monolayer. The heterogeneous rate constant of electron transfer between the Fe(III)PPs and the gold substrate decreases exponentially with the length of the spacer, demonstrating a value of 1.0 Å -1 for the tunneling length coefficient, β. At pH 8, a linear increase of the formal redox potential (E°′) with the length of the linker was also observed. This suggests that in the film, there is a close contact between the porphyrins and the alkane SAM: the E°′ is affected by the drop of the electrostatic potential from the electrode to the surface of the alkane SAM, and also that there is a strong ion pairing of the Fe(III)PPs in the film with the anions of the solution. The E°′ of Fe(III)PPs in the SAM shows a strong and complex dependency on the pH of the solution, explained by variations in the coordination of the iron, involving hydroxyl ions, water, and eventually dioxygen molecules. Interactions of the iron with either functional groups present at the surface of the substrate or with the propionate groups attached to the porphyrin ring, do not appear to be involved in the electron-proton transfer coupling mechanisms.