title: Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase. Studies with the carbon monoxide inhibited enzyme.
abstract: A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.
authors.affiliation: Department of Medical Biochemistry and Biology, University of Bari, Piazza G. Cesare, 70124 Bari, Italy.
authors.forename: N
authors.initials: N
authors.lastname: Capitanio
authors.forename: G
authors.initials: G
authors.lastname: Capitanio
authors.forename: M
authors.initials: M
authors.lastname: Minuto
authors.forename: E
authors.initials: E
authors.lastname: De Nitto
authors.forename: L L
authors.initials: LL
authors.lastname: Palese
authors.forename: P
authors.initials: P
authors.lastname: Nicholls
authors.forename: S
authors.initials: S
authors.lastname: Papa
mesh_terms: D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry* / Q000494:pharmacology*; D002417:Cattle; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D008956:Models, Chemical; D010084:Oxidation-Reduction; D011522:Protons
chemical_list: D011522:Protons; C027728:heme a; D006418:Heme; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV