Quantitative analysis of the proton and charge stoichiometry of cytochrome c oxidase from beef heart reconstituted into phospholipid vesicles.

The proton and charge stoichiometry of cytochrome c oxidase reconstituted into phospholipid vesicles has been analysed using a fast responding oxygen electrode and a system for the simultaneous measurement of H+, K+ or lipophilic cations, and O2. From initial rate measurements after addition of reductant (ascorbate /N,N,N',N'-tetramethyl-p-phenylenediamine) the following stoichiometries could be extrapolated: K+/e-, between 1.85 and 2.05, H+/e-, between 0.58 and 0.75. Lipophilic cations can replace valinomycin/potassium as the charge . compensating system. Net H+ extrusion was observed for up to 11 turnovers of the enzyme (11 O2/cytochrome aa3). The variation of the internal pH buffering capacity of the vesicles has an influence on the number of turnovers during which net proton translocation can be observed, but has no influence on the determined stoichiometries.