Identification by ENDOR of Trp¹⁹¹ as the free-radical site in cytochrome c peroxidase compound ES

The chemical identity of the amino acid free-radical site that represents one of the two oxidizing equivalents stored in the H₂O ₂-oxidized intermediate (compound ES) of the mitochondrial heme enzyme, cytochrome c peroxidase (CcP) has been sought for almost a quarter of a century. Site-directed mutagenesis alone cannot yield this answer. Low-temperature 35-gigahertz (Q-band) electron nuclear double resonance (ENDOR) spectroscopy was used to examine compound ES prepared from proteins containing specifically deuterated methionine or tryptophan, as well as the amino acid replacement Trp⁵¹ → Phe. The results definitely identify the site of the radical in compound ES as tryptophan, most likely Trp¹⁹¹.