Time-Resolved ATR-FTIR Spectroscopy of the Oxygen Reaction in the D124N Mutant of Cytochrome c Oxidase from Paracoccus denitrificans

Real-time measurements of the cytochrome c oxidase reaction with oxygen were performed by ATR-FTIR spectroscopy, using a mutant with a blocked D-pathway of proton transfer (D124N, Paracoccus denitrificans numbering). The complex spectrum of the ferryl-->oxidized transition together with other bands showed protonation of Glu 278 with a peak position at 1743 cm-1. Since our time resolution was not sufficient to follow the earlier reaction steps, the FTIR spectrum of the CO-inhibited fully reduced-->ferryl transition was obtained as a difference between the spectrum before the laser flash and the first spectrum after it. A trough at 1735 cm-1 due to deprotonation of Glu 278 was detected in this spectrum. These observations confirm the proposal [Smirnova I.A., et al. (1999) Biochemistry 38, 6826-6833] that the proton required for chemistry at the binuclear site is taken from Glu 278 in the perroxy-->ferryl step, and that the rate of the next step (ferryl-->oxidized) is limited by reprotonation of Glu 278 from the N-side of the membrane in the D124N mutant enzyme. The blockage of the D-pathway in this mutant for the first time allowed direct detection of deprotonation of Glu 278 and its reprotonation during oxidation of cytochrome oxidase by O2.