Electron and proton transfer in the ba 3 oxidase from Thermus thermophilus

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Abstract
The ba 3-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa 3–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O2 to water is catalysed at a haem a 3-CuB catalytic site. The three-dimensional structure of the ba 3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa 3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba 3 -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k≅6.8 × 104 s−1) and formation of the peroxy (PR) intermediate. In the next step a proton was taken up from solution with a rate constant of ∼1.7 × 104 s−1, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ∼1,100 s−1, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the PR and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba 3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa 3-type oxidases.

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