We present a mechanism by which oxygen is reduced to water in living organisms.

The ba 3 cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K B pathway. It was previously shown that the ba 3 oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated variants in which two amino acids of the K B proton pathway leading to the catalytic site were exchanged; Tyr-248 (located~23 Å below the active site towards the cytoplasm) in subunit I (Y248T) and Glu-15 (~26 Å below the active site,~16 Å from Tyr-248) in subunit II (E15 II Q). Even though the overall catalytic turnover in these two variants is similar and very low (< 1 % of wildtype), the substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site. The results indicate that the Glu-15 II is the only essentially crucial residue of the K B pathway, but that the Tyr-248 also plays a distinct role in defining an internal proton donor and controlling the dynamics of proton transfer to the pump site and the catalytic site. time constant; WT, wildtype. Unless indicated by a superscript II for subunit II, all residues discussed are located in subunit I.[a] C.