Internal electron transfer in cytochrome c oxidase from Rhodobacter sphaeroides.

Absorbance changes following CO dissociation by flash photolysis from mixed-valence aa3 cytochrome oxidase from Rhodobacter sphaeroides have been followed in the Soret and alpha regions. They reflect internal electron transfer in the partially reduced enzyme, and the kinetics of the reactions has been determined. As with the bovine enzyme, three kinetic phases are found with relaxation time constants at neutral pH of about 3 microseconds, 35 microseconds, and 1 ms. The first reaction phase represents electron transfer from cytochrome a3 to cytochrome a, and the extent of this reaction is about 3 times larger compared to the bovine enzyme. The energetics of the reaction has been analyzed on the basis of measurements of its temperature dependence. The reorganization energy is close to 120 kJ mol-1, and it is suggested that this rather high value is the result of changes in solvation at the cytochrome a3-CuB site. The subsequent electron transfer between cytochrome a and CuA, with a time constant of 35 microseconds, is almost activationless and has a very low reorganization energy. The final phase, with a time constant close to 1 ms at neutral pH, represents a further shift in the equilibrium between cytochrome a3 and cytochrome a, and it is limited by proton-transfer reactions. The pKa values of the groups involved are significantly shifted in the bacterial oxidase compared to the bovine one. The total extent of electron transfer in the three backflow reactions has also been determined by a comparison of the CO-recombination rates in the mixed-valence and fully reduced enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)