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 (Cu_B) 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 P_M wherein the O–O bond is fully cleaved forming an Fe^IV=O species. Two of the electrons required for the O–O cleavage are supplied by the active site heme (Fe^II→Fe^IV) while one electron each is believed to be donated by the Cu_B (Cu^I→Cu^II) and the crosslinked Tyr (Tyr→Tyr^●) to form P_M. However, direct evidence for the Cu^II and the Tyr^● remains elusive. Further e^–/H⁺ transfer steps to the active site form subsequent intermediates (P_M→ F → O_H→ E_H→ R), each coupled to pumping a proton across the membrane. Spectroscopic definition of the key intermediate P_M 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/Cu_A), obscuring features from the Cu_B and the Tyr.
In this study, we characterized P_M in UbO bo₃ from E. coli (lacks the Cu_A center) by low temperature magnetic circular dichroism spectroscopy (MCD) and observe a unique feature associated with its Fe^IV=O center. Variable-temperature, variable-field (VTVH) MCD on this excited state feature enabled us to study the ground state of P_M demonstrating that it is a three-spin exchange-coupled system: Fe^IV=O (S=1), Cu^II (S=½) and Tyr^● (S=½). These data provide direct conclusive experimental evidence that the source of the two additional e^–s are the Cu_B and the Tyr. These results further define the O–O bond cleavage mechanism in HCOs and elucidate a superexchange pathway for e^- transfer to P_M 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.

Wesley Transue

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