Proton linkage of cytochrome a oxidoreduction in carbon monoxide-treated cytochrome c oxidase
Oxidoreduction of the low spin haem a of cytochrome c oxidase was recently reported to be coupled to release/uptake of nearly one proton from/to the enzyme at pH 7.5 in the presence of CO to block oxidoreduction of the binuclear haem a3/CuB centre (N. Capitanio et al., Biochim. Biophys. Acta, 1318 (1997) 255-265). This is difficult to reconcile with earlier findings from several laboratories that the pH-dependence of the Em of haem a is ca. 10 mV/pH unit over a wide pH range in such conditions, which implies redox coupling of only ca. 0.17 H+/e-. In order to resolve this discrepancy, we have performed careful measurements of proton release coupled to oxidation of haem a and CuA in CO-inhibited cytochrome aa3 from bovine heart mitochondria. We find that oxidation of these centres by ferricyanide leads to release of a total of 0.20 protons per enzyme molecule at pH 7.7, increasing to 0.43 protons at pH 6.6, far short of a full 1 H+/e-. Using vesicles reconstituted with cytochrome c oxidase, we also found that all this proton release occurs towards the outside of the vesicles. The observed dependence can be explained by a model in which oxidoreduction of haem a is coupled to uptake and release of ca. 0.17 H+/e-, while oxidoreduction of CuA is linked to a protonatable group which has a pKa of 6.2 when CuA is in the reduced state. In agreement with existing data, this model predicts that the Em of CuA will only be slightly pH dependent in the pH range of these measurements.
Helsinki Bioenergetics Group, Department of Medical Chemistry, Institute of Biomedical Sciences and Biocentrum Helsinki, P.O. Box 8, FIN-00014 University of Helsinki, Helsinki, Finland.
MI
Verkhovsky
N
Belevich
JE
Morgan
M
Wikstrom