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 O-2 reduction conditions remains elusive. Using a functional oxidase model in myoglobin called F33Y-Cu(B)Mb that contains an engineered tyrosine, we report herein direct observation of a tyrosyl radical during both reactions of H2O2 with oxidized protein and O-2 with reduced protein by electron paramagnetic resonance spectroscopy, providing a firm support for the tyrosyl radical in the HCO enzymatic mechanism.