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−?

Abstract

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(I-362) and Ser(I-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 a3 to a. At pH>7 this electron transfer is associated with proton release to the bulk solution (τ≅1 ms at pH 8). Thus, the CO-recombination kinetics reflects protonation events at the catalytic site. In the wild-type enzyme, below pH∼7, the main component in the CO-recombination displayed a rate of ∼20 s−1. Above pH∼7, a slow CO-recombination component developed with a rate that decreased from ∼8 s−1 at pH 8 to ∼1 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 a3. 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 O2, the H2O at the binuclear center is most likely involved as a proton donor in the O–O cleavage reaction.