Rates and Equilibrium of Cu A to Heme a Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

Abstract

Intramolecular electron transfer between Cu A and heme a in solubilized bacterial (Paracoccus denitrificans) cytochrome c oxidase was investigated by pulse radiolysis. Cu A, the initial electron acceptor, was reduced by 1-methylnicotinamide radicals in a diffusion-controlled reaction, as monitored by absorption changes at 825 nm, followed by partial restoration of the absorption and paralleled by an increase in the heme a absorption at 605 nm. The latter observations indicate partial reoxidation of the Cu A center and the concomitant reduction of heme a. The rate constants for heme a reduction and Cu A reoxidation were identical within experimental error and independent of the enzyme concentration and its degree of reduction, demonstrating that a fast intramolecular electron equilibration is taking place between Cu A and heme a. The rate constants for Cu A → heme a ET and the reverse heme a → Cu A process were found to be 20,400 s −1 and 10,030 s −1, respectively, at 25°C and pH 7.5, which corresponds to an equilibrium constant of 2.0. Thermodynamic and activation parameters of these intramolecular ET reactions were determined. The significance of the results, particularly the low activation barriers, is discussed within the framework of the enzyme’s known three-dimensional structure, potential ET pathways, and the calculated reorganization energies.