Proton-controlled electron transfer in cytochrome c oxidase: functional role of the pathways through Glu 286 and Lys...

We have used a combination of site-directed mutagenesis and spectroscopic techniques to investigate electron-transfer reactions between hemes a and a3 in cytochrome c oxidase. A state of the enzyme was prepared in which heme a/CuA are oxidized and heme a3/CuB are reduced with CO bound to heme a3, which stabilizes the reduced state of the binuclear center. In addition, in this state the pKs of protonatable groups in the vicinity of the binuclear center, interacting electrostatically with heme a3, are larger than with oxidized heme a3. Upon flash photolysis of CO from the two-electron reduced enzyme electrons at heme a3 equilibrate rapidly with heme a. In the R. sphaeroides enzyme the electron-transfer rates from heme a to a3 and from heme a3 to a were, deconvoluted and were found to be approximately 1.5.10(5) s-1 and approximately 1.4.10(5) s-1, respectively. After this rapid electron equilibration between hemes a and a3, protons are released from groups interacting electrostatically with heme a3, which is associated with additional electron transfer from heme a3 to heme a. The proton-coupled electron transfer displays a pH dependent extent and rate. In addition, it displays a deuterium-isotope effect of a factor of about three. The reaction sequence is compatible with the three-dimensional cytochrome c oxidase structure, which shows that more protonatable groups are found around heme a3 than around heme a and supports the involvement of the binuclear center in proton pumping. Proton uptake/release upon reduction/oxidation of heme a3 takes place through a proton pathway including residues Thr(I-359) and Lys(I-362) (K-pathway), but not through the pathway including residues Asp(I-132) and Glu(I-286) (D-pathway). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway.