Ferrocyanide-peroxidase activity of cytochrome c oxidase

Abstract

Redox interaction of mitochondrial cytochrome c oxidase (COX) with ferrocyanide/ferricyanide couple is greatly accelerated by polycations, such as poly- l-lysine [Musatov et al. (1991) Biological Membranes 8, 229–234]. This has allowed us to study ferrocyanide oxidation by COX at very high redox potentials of the ferrocyanide/ferricyanide couple either following spectrophotometrically ferricyanide accumulation or measuring proton uptake associated with water formation in the reaction. At low [ferrocyanide]/[ferricyanide] ratios ( E h values around 500 mV) and ambient oxygen concentration, the ferrocyanide-oxidase activity of COX becomes negligibly small as compared to the reaction rate observed with pure ferrocyanide. Oxidation of ferrocyanide under these conditions, is greatly stimulated by H 2O 2 or ethylhydroperoxide indicating peroxidatic reaction involved. The ferrocyanide-peroxidase activity of COX is strictly polylysine-dependent and is inhibited by heme a 3 ligands such as KCN and NaN 3. Apparently the reaction involves normal electron pathway, i.e. electron donation through Cu A and oxidation via heme a 3. The peroxidase reaction shows a pH-dependence similar to that of the cytochrome c oxidase activity of COX. When COX is preequilibrated with excess H 2O 2, addition of ferrocyanide shifts the initial steady-state concentrations of the Ferryl–Oxo and Peroxy compounds towards approximately 2:1 ratio of the two intermediates. It is suggested that in the peroxidase cycle ferrocyanide donates electrons to both P and F intermediates with a comparable efficiency. Isolation of a partial redox activity of COX opens a possibility to study separately proton translocation coupled to the peroxidase half-reaction of the COX reaction cycle.