Analysis of protein film voltammograms as Michaelis-Menten saturation curves yield the electron cooperativity number...

Deconvolution of protein film voltammetric data by fitting multiple components (sigmoids, derivative peaks) often is ambiguous when features are partially overlapping, due to exchangeability between the width and the number of components. Here, a new method is presented to obtain the width of the components. This is based on the equivalence between the sigmoidal catalytic response as function of electrode potential, and the classical saturation curve obtained for the enzyme activity as function of the soluble substrate concentration, which is also sigmoidal when plotted versus log[S]. Thus, analysis of the catalytic voltammogram with Lineweaver-Burk, Eadie-Hofstee, and Hanes-Woolf plots is feasible. This provides a very sensitive measure of the cooperativity number (Hill coefficient), which for electrons equals the apparent (fractional) number of electrons that determine the width, and thereby the number of components (kinetic phases). This analysis is applied to the electrocatalytic oxygen reduction by Paracoccus denitrificans cytochrome aa(3) (cytochrome c oxidase). Four partially overlapping kinetic phases are observed that (stepwise) increase the catalytic efficiency with increasingly reductive potential. Translated to cell biology, the activity of the terminal oxidase stepwise adapts to metabolic demand for oxidative phosphorylation.