Spectroscopic and electrochemical studies of the pH-Induced transition in the CuA centre from Thermus thermophilus

Abstract

A detailed study of the pH induced conformational changes at the Cu2S2 coordination site of the CuA centre of cytochrome oxidase from Thermus thermophilus has been carried out to unravel the role of the pH-induced changes in the coordination geometry on the redox potential and electron transfer kinetics of the metal centre. The pH dependence of the UV–visible absorption bands as well as of the visible circular dichroism spectra corresponding to the metal centre of the protein could be analyzed by the model involving two protonation/deprotonation steps associated with the pKa values of 3.5 and 9.6. The redox potential of the protein determined by direct electrochemistry also showed analogous pH dependence. The results suggested that the metal centre undergoes equilibrium conformational transition involving three forms, viz., ‘low pH’ form, ‘neutral pH’ form and ‘high-pH’ form characterized by different redox potentials of the metal centre. The rates of heterogeneous electron transfer were found to decrease drastically at low pH as well as at high pH. The electron transfer from cytochrome c to the CuA site of the cytochrome oxidase is proposed to be gated by conformation changes at the CuA site through a protonation-induced conformation change, which may act as a sensor for optimum proton transfer across the enzyme. The results also have been discussed in the light of understanding fast electron transfer from the CuA to the heme a in the intact enzyme.