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

Ole Farver ^*, Ernst Grell , Bernd Ludwig , Hartmut Michel  and Israel Pecht 

^* Institute of Analytical Chemistry, The Danish University of Pharmaceutical Sciences, 2100 Copenhagen, Denmark; Max-Planck Institute of Biophysics, D-60439 Frankfurt, Germany; Biocenter, Johann Wolfgang Goethe-Universität, D-60439 Frankfurt, Germany; and Department of Immunology, The Weizmann Institute of Science, 76100 Rehovot, Israel

Correspondence: Address reprint requests to Ole Farver, E-mail: of{at}dfuni.dk.

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.