Localized control of proton transfer through the D-pathway in cytochrome c oxidase: application of the proton-inventory technique.

In the reaction cycle of cytochrome c oxidase from Rhodobacter sphaeroides, one of the steps that are coupled to proton pumping, the oxo-ferryl-to-oxidized transition (F → O), displays a large kinetic deuterium isotope effect of about 7. In this study we have investigated in detail the dependence of the kinetics of this reaction step [kFO(χ)] on the fraction (χ) D2O in the enzyme solution (proton-inventory technique). According to a simplified version of the Gross−Butler equation, from the shape of the graph describing kFO(χ)/kFO(0), conclusions can be drawn concerning the number of protonatable sites involved in the rate-limiting proton-transfer reaction step. Even though the proton-transfer reaction during the F → O transition takes place over a distance of at least 30 A and involves a large number of protonatable sites, the proton-inventory analysis displayed a linear dependence, which indicates that the entire deuterium isotope effect of 7 is associated with a single protonatable site. On the basis of...