title: The cytochrome ba3 oxidase from Thermus thermophilus does not generate a tryptophan radical during turnover: Implications for the mechanism of proton pumping.
abstract: Oxygen reduction by cytochrome ba3 oxidase from Thermus thermophilus was studied by stopped-flow and microsecond freeze-hyperquenching analyzed with UV-Vis and EPR spectroscopy. In the initial phase, the low-spin heme b560 is rapidly and almost completely oxidized (kobs>33,000s(-1)) whereas CuA remains nearly fully reduced. The internal equilibrium between CuA and heme b560 with forward and reverse rate constants of 4621s(-1) and 3466s(-1), respectively, indicates a ~7.5mV lower midpoint potential for CuA compared to heme b560. The formation of the oxidized enzyme is relatively slow (693s(-1)). In contrast to the Paracoccus denitrificans cytochrome aa3 oxidase, where in the last phase of the oxidative half cycle a radical from the strictly conserved Trp272 is formed, no radical is formed in the cytochrome ba3 oxidase. Mutation of the Trp229, the cytochrome ba3 oxidase homologue to the Trp272, did not abolish the activity, again in contrast to the Paracoccus cytochrome aa3 oxidase. Differences in the proton pumping mechanisms of Type A and Type B oxidases are discussed in view of the proposed role of the strictly conserved tryptophan residue in the mechanism of redox-linked proton pumping in Type A oxidases. In spite of the differences between the Type A and Type B oxidases, we conclude that protonation of the proton-loading site constitutes the major rate-limiting step in both catalytic cycles.
authors.affiliation: Laboratory of Biotechnology, Section Biocatalysis, Delft University of Technology, Julianalaan 67, 2628BC Delft, The Netherlands.
authors.forename: Angela
authors.initials: A
authors.lastname: Paulus
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Werner
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Ludwig
authors.affiliation: Laboratory of Biotechnology, Section Biocatalysis, Delft University of Technology, Julianalaan 67, 2628BC Delft, The Netherlands. Electronic address: s.devries@tudelft.nl.
authors.forename: Simon
authors.initials: S
authors.lastname: de Vries
keywords: Catalytic mechanism; Cytochrome c oxidase; Electron transfer; Kinetics; Proton pumping