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.

Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature

“…This, however, is not the case for ba 3 C c O. It was recently proposed that the transient formation of the tryptophan radical is involved in proton pumping, and consequently the lack of such a radical in ba 3 C c O could be related to the lower proton pumping stoichiometry displayed by this enzyme 39 . In light of this debate, it is interesting to note that the SFX structure and all previously published ba 3 C c O structures differ from the aa 3 -type C c O structures in the vicinity of this conserved tryptophan residue (Fig.…”

Specific inhibition of proton pumping by the T315V mutation in the K channel of cytochrome ba from Thermus thermophilus

“…Similar to aa 3 oxidases, the third transition of the ba 3 oxidase includes re-reduction of the lowspin heme, while during the fourth transition the electron is transferred from heme b to the BNC and the ferric/cupric state of BNC is formed. However the third transition is not accompanied by changes of the BNC spectrum, so the spectrum of the intermediate, corresponding in time to the F state, is identical to that of the P R state [9,[19][20]. It was proposed that the proton taken up in F does not reside on the Cu B ligand but is transferred either to the cross-linked tyrosine Y237 near Cu B [9], or to the proton-loading site (PLS) above the BNC [21].…”

Photosystem II and terminal respiratory oxidases molecular machines operating in opposite directions

“…These changes are believed to influence on the proton transfer from E286 to the PLS (129). A fundamentally different model of the proton pump in the bacterial oxidases were recently proposed based on experimental observations of tryptophan W272* radical (W280 in COX from R. sphaeroides) during the F→O transition (311)(312). This model also assumes that the pumped proton is transferred after the substrate proton.…”