title: Proton-collecting properties of bovine heart cytochrome C oxidase: kinetic and electrostatic analysis.
abstract: Proton-transfer reactions on the surface of bovine heart cytochrome c oxidase were investigated by combining a laser-induced proton-pulse technique with molecular modeling. The experimental approach simultaneously monitors the state of pyranine protonation in the bulk phase and that of a fluorescein indicator specifically attached to the native Cys(III-115) residue of subunit III of cytochrome oxidase. The reversible dynamics of the acid-base equilibration between the surface and the bulk phase were measured with submicrosecond time resolution and analyzed by numerical integration of coupled nonlinear differential rate equations. Kinetic analysis shows that carboxylates on the surface of the protein act as a proton-collecting antenna, which is able to rapidly transfer protons to nearby histidines that function as a local proton reservoir. These properties enable cytochrome oxidase to carry out its redox-linked proton translocation. Molecular modeling of the fluorescein-binding site indicates that, in addition to the covalent bond, the dye is anchored through a hydrogen bond to the hydroxyl moiety of Tyr(VII-50). The protonation of the dye is mediated through three residues that shuttle protons between the bulk and the dye. A correlation between the measured kinetic properties of the bound fluorescein and the different configurations of the dye allows us to predict the identity of the proton-binding sites in the fluorescein-binding domain.
authors.affiliation: Laser Laboratory for Fast Reactions in Biology, Department of Biology, The George S. Wise Facility of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
authors.forename: Y
authors.initials: Y
authors.lastname: Marantz
authors.forename: O
authors.initials: O
authors.lastname: Einarsdóttir O
authors.forename: E
authors.initials: E
authors.lastname: Nachliel
authors.forename: M
authors.initials: M
authors.lastname: Gutman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D009206:Myocardium / Q000201:enzymology*; D011487:Protein Conformation; D011522:Protons; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D011522:Protons; C037041:fluorescein 5-maleimide; D003576:Electron Transport Complex IV