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 e nzyme at pH 7.5 in the presence of CO to block oxidoreduction of the binucl ear haem a(3)/Cu-B centre (N. Capitanio et al., Biochim. Biophys. Acta, 131 8 (1997) 255-265). This is difficult to reconcile with earlier findings fro m several laboratories that the pH-dependence of the E-m of haem a is ca. 1 0 mV/pH unit over a wide pH range in such conditions, which implies redox c oupling 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 Cu-A in CO-inhibited cytochrome aa(3) from bovine heart mitoc hondria. We find that oxidation of these centres by ferricyanide leads to r elease 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 r econstituted 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 t o uptake and release of ca. 0.17 H+/e(-) , while oxidoreduction of CuA is l inked to a protonatable group which has a pK(a) of 6.2 when Cu-A is in the reduced state. In agreement with existing data, this model predicts that th e E-m of Cu-A will only be slightly pH dependent in the pH range of these m easurements. (C) 1999 Elsevier Science B.V. All rights reserved.