Kinetic control of internal electron transfer in cytochrome c oxidase

Two alternative hypotheses have been proposed to account for the relatively slow (ms) internal eT observed in the oxidized cyt c oxidase. The thermodynamic control hypothesis states that eT between cyt a and a3 is very fast (microsecond), but the apparent reduction of cyt a3 is slow because thermodynamics favors reduced cyt a. Whereas the kinetic control hypothesis states that inter-heme eT is intrinsically slow (ms), for the oxidized binuclear center. Monitoring by stopped flow the anaerobic reduction of the oxidized enzyme by ruthenium hexamine in the absence and presence of CO or NO, used as "trapping" ligands for cyt a3(2+), we found that the rate of formation of the cyt a3(2+)-NO adduct (k' approximately 20-25 s-1) is independent of the concentration of ruthenium hexamine and NO. We conclude that in the oxidized enzyme the two hemes are not in very rapid redox equilibrium and internal eT is kinetically controlled.