Ligand binding reveals protonation events at the active site of cytochrome c oxidase; is the K-pathway used for the transfer of H+ or OH-?

摘要

We have investigated the CO-recombination kinetics after flash photolysis of CO from the 'half-reduced' cytochrome c oxidase as a function of pH. In addition, the reaction was investigated in mutant enzymes in which Lys(1-362) and Ser(1-299), located approximately in the middle of the K-pathway and near the enzyme surface, respectively, were modified. Laser-flash induced dissociation of CO is followed by rapid internal electron transfer from heme a, to a. At pH>7 this electron transfer is associated with proton release to the bulk solution (tau congruent to -1 ms at pH 8). Thus. the CO-recombination kinetics reflects protonation events at the catalytic site. In the wild-type enzyme, below pHsimilar to7, the main component in the CO-recombination displayed a rate of similar to20 s(-1). Above pH-7, a slow CO-recombination component developed with a rate that decreased from similar to8 s(-1) at pH 8 to similar to1 s(-1) at pH 10. This slow component was not observed with KM(I-362), while with the SD(I-299)/SG(I-299) mutant enzymes at each pH it was slower than with the wild-type enzyme. The results are interpreted in terms of proton release from H2O in the catalytic site after CO dissociation, followed by OH- binding to the oxidized heme a(3). The CO-recombination kinetics is proposed to be determined by the protonation rate of OH- and not dissociation of OH-, i.e. the K-pathway transfers protons and not OH-. With the KM(I-362) mutant enzyme the proton is not released, i.e. OH- is not formed. With the SD(I-299)/SG(I-299) mutant enzymes the proton is released, but both the release and uptake are slowed by the mutations. During reaction of the reduced enzyme with O-2 the H2O at the binuclear center is most likely involved as a proton donor in the O-O cleavage reaction.