Direct evidence for a tyrosine radical in the reaction of cytochrome c oxidase with hydrogen peroxide

Cytochrome c oxidase (COX) catalyzes the reduction of oxygen to water, a pr ocess which is accompanied by the pumping of four protons across the membra ne. Elucidation of the structures of intermediates in these processes is cr ucial for understanding the mechanism of oxygen reduction. In the work pres ented here, the reaction of H2O2 With the fully oxidized protein at pH 6.0 has been investigated with electron paramagnetic resonance (EPR) spectrosco py. The results reveal an EPR signal with partially resolved hyperfine stru cture typical of an organic radical. The yield of this radical based on com parison with other paramagnetic centers in COX was similar to 20%. Recent c rystallographic data have shown that one of the CUB ligands, His 276 (in th e bacterial case), is cross-linked to Tyr 280 and that this cross-linked ty rosine is ideally positioned to participate in dioxygen activation. Here se lectively deuterated tyrosine has been incorporated into the protein, and a drastic change in the line shape of the EPR signal observed above has been detected. This would suggest that the observed EPR signal does indeed aris e from a tyrosine radical species. It would seem also quite possible that t his radical is an intermediate in the mechanism of oxygen reduction.