Analysis of protein film voltammograms as Michaelis–Menten saturation curves yield the electron cooperativity number for deconvolution

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 aa3 (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.

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► The applied potential is equivalent to log(substrate concentration).
► A catalytic voltammogram thus is equivalent to a Michaelis–Menten saturation curve.
► Voltammetric Lineweaver–Burk, Eadie–Hofstee, and Hanes–Woolf plots can be drawn.
► These provide new information on enzyme kinetics.
► These provide the electron cooperativity number needed for deconvolution.