title: Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R. sphaeroides.
abstract: In this study we have combined the use of site-directed mutants with time-resolved optical absorption spectroscopy to investigate the role of the protonatable subunit-I residues lysine-362 (K(I-362)) and threonine-359 (T(I-359)) in cytochrome c oxidase from Rhodobacter sphaeroides in electron and proton transfer. These residues have been proposed to be part of a proton-transfer pathway in cytochrome oxidases from Paracoccus denitrificans and bovine heart. Mutation of K(I-362) and T(I-359) to methionine and alanine, respectively, results in reduction of the overall turnover activities to <2% and approximately 35%, respectively, of those in the wild-type enzyme. The results show that in the absence of dioxygen, electron transfer between hemes a3 and a with a time constant of approximately 3 micros, not coupled to protonation reactions, is not affected in the mutant enzymes. However, the slower electron transfer between hemes a3 and a, coupled to proton release with a time constant of approximately 3 ms (at pH 9.0) is impaired in the KM(I-362) and TA(I-359) mutant enzymes. This is consistent with the slow reduction rate of heme a3 in the oxidized KM(I-362) enzyme because in the wild-type enzyme reduction of heme a3 is coupled to proton uptake. On the other hand, when reacting with O2, both the wild-type and mutant fully reduced enzymes become oxidized in approximately 5 ms, and proton uptake on this time scale is not affected. Hence, the results indicate that the KM(I-362) mutant enzyme is inactive because the proton-transfer pathway through K(I-362) and T(I-359) is involved in proton uptake during reduction of the oxidized binuclear center. Proton uptake during oxidation of the fully reduced enzyme takes place through a different pathway [through E(I-286) (Adelroth, P., et al. (1997) Biochemistry 36, 13824-13829)].
authors.affiliation: Department of Biochemistry and Biophysics, Goteborg University and Chalmers University of Technology, Medicinaregatan 9C, S-413 90 Goteborg, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Adelroth
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
authors.forename: P
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology; D017354:Point Mutation; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
chemical_list: D011522:Protons; D006418:Heme; D003576:Electron Transport Complex IV