The pH-dependent redox inactivation of amicyanin from Paracoccus versutus as studied by rapid protein-film voltammetry

Abstract.

The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry. At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pK_a ^red=6.3 and pK_a ^ox≤3.2 at 22 °C. Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s^–1 at 25 °C. In order to decrease the rate of the coupled reaction, the experiments were performed at 0 °C, at which the rate constant for this conversion was determined to be 35±20 s^–1. Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.