Aerobic organisms use O2 to drive the membrane-bound respiratory electron transport chain for synthesis of ATP. The 4e– reduction of O2 to H2O underlying this biological process is catalyzed by the superfamily of metalloenzymes called heme–copper oxidases (HCO), most notably cytochrome c oxidases (CcO) and ubiquinol oxidases (UbO). The free energy gained from this reaction is used by HCOs to drive a proton pumping process for ATP synthesis. The HCO active site consists of a heme center ligated by a histidine residue (His) and a copper center (CuB) ligated by three His, one of which is covalently linked to a tyrosine residue (Tyr). Binding of O2 to the fully reduced active site generates intermediate PM wherein the O–O bond is fully cleaved forming an FeIV=O species. Two of the electrons required for the O–O cleavage are supplied by the active site heme (FeII→FeIV) while one electron each is believed to be donated by the CuB (CuI→CuII) and the crosslinked Tyr (Tyr→Tyr●) to form PM. However, direct evidence for the CuII and the Tyr● remains elusive. Further e–/H+ transfer steps to the active site form subsequent intermediates (PM → F → OH → EH → R), each coupled to pumping a proton across the membrane. Spectroscopic definition of the key intermediate PM at the O–O cleavage and proton pumping junction had been precluded by the dominant features of other redox centers in these enzymes (low-spin heme/CuA), obscuring features from the CuB and the Tyr. In this study, we characterized PM in UbO bo3 from E. coli (lacks the CuA center) by low temperature magnetic circular dichroism spectroscopy (MCD) and observe a unique feature associated with its FeIV=O center. Variable-temperature, variable-field (VTVH) MCD on this excited state feature enabled us to study the ground state of PM demonstrating that it is a three-spin exchange-coupled system: FeIV=O (S=1), CuII (S=½) and Tyr● (S=½). These data provide direct conclusive experimental evidence that the source of the two additional e–s are the CuB and the Tyr. These results further define the O–O bond cleavage mechanism in HCOs and elucidate a superexchange pathway for e- transfer to PM which initiates proton pumping. This study also sets the foundation to investigate subsequent intermediates in the catalytic cycle to understand the mechanistic coupling of molecular oxygen reduction to proton pumping by HCOs during cellular respiration.

The three-spin intermediate at the O–O cleavage and proton pumping junction in heme–Cu oxidases

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