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

• Published in: Journal of bioenergetics and biomembranes 2008-08, Vol.40 (4), p.281-287
• Main Authors: Smirnova, Irina A, Zaslavsky, Dmitry, Fee, James A, Gennis, Robert B, Brzezinski, Peter
• Format: Article
• Language: English
• Subjects: Paracoccus denitrificans; Rhodobacter sphaeroides; Thermus thermophilus
• ISSN: 0145-479X; 1573-6881
• DOI: 10.1007/s10863-008-9157-7

The ba sub(3)-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa sub(3)-type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O sub(2) to water is catalysed at a haem a sub(3)-Cu sub(B) catalytic site. The three-dimensional structure of the ba sub(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa sub(3) oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba sub( 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 (k6.8 10 super(4) s super(-1)) and formation of the peroxy (P sub(R)) intermediate. In the next step a proton was taken up from solution with a rate constant of 1.7 10 super(4) s super(-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 super(-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 sub(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 sub(3) oxidases, the proton-uptake reactions occur over the same time scales as in the aa sub(3)-type oxidases.