Direct EPR Observation of a Tyrosyl Radical in a Functional Oxidase Model in Myoglobin during both H₂O₂ and O₂ Reactions

Tyrosine is a conserved redox-active amino acid that plays important roles in heme–copper oxidases (HCO). Despite the widely proposed mechanism that involves a tyrosyl radical, its direct observation under O₂ reduction conditions remains elusive. Using a functional oxidase model in myoglobin called F33Y-Cu_BMb that contains an engineered tyrosine, we report herein direct observation of a tyrosyl radical during both reactions of H₂O₂ with oxidized protein and O₂ with reduced protein by electron paramagnetic resonance spectroscopy, providing a firm support for the tyrosyl radical in the HCO enzymatic mechanism