The onset of the deuterium isotope effect in cytochrome c oxidase

Abstract

We have investigated the dynamics of proton equilibration within the proton-transfer pathway of cytochrome c oxidase from bovine heart that is used for the transfer of both substrate and pumped protons during reaction of the reduced enzyme with oxygen (D-pathway). The kinetics of the slowest phase in the oxidation of the enzyme (the oxo-ferryl → oxidized transition, F→ O), which is associated with proton uptake, were studied by monitoring absorbance changes at 445 nm. The rate constant of this transition, which is 800 s-1 in H2O (at pH ~7.5), displayed a kinetic deuterium isotope effect of ~4 (i.e., the rate was ~200 s-1 in 100% D2O). To investigate the kinetics of the onset of the deuterium isotope effect, fully reduced, solubilized CO-bound cytochrome c oxidase in H2O was mixed rapidly at a ratio of 1:5 with a D2O buffer saturated with oxygen. After a well-defined time period, CO was flashed off using a short laser flash. The time between mixing and flashing off CO was varied within the range 0.04-10 s. The results show that for the bovine enzyme, the onset of the deuterium isotope effect takes place within two time windows of ≤ 100 ms and ~1 s, respectively. The slow onset of the deuterium isotope effect indicates that the rate-limiting step during the F → O transition is internal proton transfer from a site, proposed to be Glu (I-286) (R. sphaeroides amino acid residue numbering), to the binuclear center. The spontaneous equilibration of protons/deuterons with this site in the interior of the protein is slow (~1 s).