Electron and proton transfer in the ba3 oxidase from Thermus thermophilus

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 O 2 to water is catalysed at a haem a 3 -Cu B 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 × 10 4 s −1 ) and formation of the peroxy (P R ) intermediate. In the next step a proton was taken up from solution with a rate constant of ∼1.7 × 10 4 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 P R 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.