title: Single mutations that redirect internal proton transfer in the ba3 oxidase from Thermus thermophilus.
abstract: The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa3-type oxidases proton uptake to the catalytic site and "pump site" occurs simultaneously. However, with ba3 oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa3 oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba3 CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though ba3 CytcO uses only a single proton pathway for transfer of the substrate and "pumped" protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University , SE-106 91 Stockholm, Sweden.
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.forename: Hsin-Yang
authors.initials: HY
authors.lastname: Chang
authors.forename: Christoph
authors.initials: C
authors.lastname: von Ballmoos
authors.forename: Pia
authors.initials: P
authors.lastname: Ädelroth
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Peter
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D019943:Amino Acid Substitution; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
chemical_list: D017494:Proton Pumps; D011522:Protons; D003576:Electron Transport Complex IV