Direct measurement of the initial and early ratios of proton extrusion to oxygen uptake accompanying cytochrome c oxidation by rat liver mitoplasts

Abstract

We have recently described new methods that enable the sharp initiation of a respiratory pulse by photolysis of the CO complex of cytochrome oxidase in a stirred suspension of mitochondria, succinate, O2, and CO (Setty, O. H., R. I. Shrager, B. Bunow, B. Reynafarje, A. L. Lehninger, and R. W. Hendler. 1986. Biophys. J. 50:391-404). Data are collected directly into a microcomputer at 10-ms intervals from fast responding O2 and pH electrodes. These procedures eliminate delays and uncertainties due to mixing times, recorder response, and recovery of the O2 electrode from responding to the injection of O2. Correction procedures were also described for the inherent electrode delays. These procedures revealed an initial burst in medium acidification and a lag in O2 uptake that led to H+/O rates of 20-30 during the first 50 ms and relaxed to "normal" levels by 300 ms. Subsequent changes in [H+] and [O2] followed time courses that appeared to be, but were not strictly, first order. We describe here similar studies in which cytochrome c served as electron donor to site III of rat liver mitoplasts. A qualitatively similar but quantitatively smaller burst in medium acidification and H+/O ratio was seen in these studies. Implications of the previous (Setty et al., 1986) and current studies on defining "mechanistic" H+/O ratios are discussed.