Proton and electron transfer during the reduction of molecular oxygen by fully reduced cytochrome c oxidase: A flow-flash investigation using opticalmultichannel detection

Proton and electron transfer events during the reaction of solubilized full y reduced bovine heart cytochrome c oxidase with molecular oxygen were inve stigated using the flow-flash technique. Time-resolved spectral changes res ulting from ligand binding and electron transfer events were detected simul taneously with pH changes in the bulk. The kinetics and spectral changes in the visible region (450-750 nm) were probed by optical multichannel detect ion, allowing high spectral resolution on time scales from 50 ns to 50 ms. Experiments were carried out in the presence and absence of pH-sensitive dy es (carboxyfluorescein at pH 6.5, phenol red at pH 7.5, and m-cresol purple at pH 8.5) which permitted separation of spectral changes due to proton tr ansfer from those caused by ligand binding and electron transfer. The trans ient spectra recorded in the absence of dye were analyzed by singular value decomposition and multiexponential fitting. Five apparent lifetimes (0.93 mu s, 10 mu s, 36 mu s, 90 mu s, and 1.3 ms at pH 7.5) could consistently b e distinguished and provided a basis for a reaction mechanism consistent wi th our most recent kinetic model [Sucheta, A., Szundi, I., and Einarsdottir , O. (1999) Biochemistry 37, 17905-17914]. The dye response indicated that proton uptake occurred concurrently with the two slowest electron transfer steps, in agreement with previous results based on single-wavelength detect ion [Hallen, S., and Nilsson, T. (1992) Biochemistry 31, 11853-11859]. The stoichiometry of the proton uptake reactions was approximately 1.3 +/- 0.3, 1.4 +/- 0.3, and 1.6 +/- 0.5 protons per enzyme at pH 6.5, 7.5, and 8.5, r espectively. The electron transfer between heme a and Cu-A was limited by p roton uptake on a 90 mu s time scale. We have established the lower limit o f the true rate constant for the electron transfer between CuA and heme a t o be similar to 2 x 10(5) s(-1).