title: Redox-linked protonation of cytochrome c oxidase: the effect of chloride bound to CuB.
abstract: The effect of bound Cl- on the redox-linked protonation of soluble beef heart cytochrome c oxidase (CcOX) has been investigated at pH 7.3-7.5 by multiwavelength stopped-flow spectroscopy, using phenol red as the pH indicator in an unbuffered medium. Reduction by Ru-II hexamine of the Cl-bound enzyme is associated with an overall apparent uptake of 1.40 +/- 0.21 H+/aa3, whereas 2.28 +/- 0.36 H+/aa3 is taken upon reduction of the Cl-free enzyme. Bound Cl- has no effect on the extent of H+ uptake coupled to heme a reduction (0.59 +/- 0.06 H+/aa3), but significantly decreases (by approximately 0.9 H+/aa3) the apparent stoichiometry of H+ uptake coupled to heme a3-Cu(B) reduction, by eliminating the net H+ uptake linked to Cu(B) reduction. To account for these results, we propose that, after the transfer of the first electron to the active site, reduction of Cu(B) is associated with Cl- dissociation, addition of a H+, and diffusion into the bulk (with subsequent dissociation) of HCl. In the physiologically competent Cl--free enzyme, an OH- likely bound to oxidized Cu(B) is protonated upon arrival of the first electron, and dissociates as H2O. The relevance of this finding to the understanding of the enzyme mechanism is discussed.
authors.affiliation: Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome La Sapienza, I-00185 Rome, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Forte
authors.forename: Maria Cecilia
authors.initials: MC
authors.lastname: Barone
authors.forename: Maurizio
authors.initials: M
authors.lastname: Brunori
authors.forename: Paolo
authors.initials: P
authors.lastname: Sarti
authors.forename: Alessandro
authors.initials: A
authors.lastname: Giuffrè
mesh_terms: D000818:Animals; D001665:Binding Sites; D002138:Calibration; D002417:Cattle; D002712:Chlorides / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D006851:Hydrochloric Acid / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D011522:Protons; D013053:Spectrophotometry
chemical_list: D002712:Chlorides; D011522:Protons; C027728:heme a; D006418:Heme; D003300:Copper; D003576:Electron Transport Complex IV; D006851:Hydrochloric Acid