The fully oxidized state of cytochrome c peroxidase (CcP), called ES, contains two oxidizing equivalents, one as an oxyferryl heme and the other as an organic radical on an amino acid residue. The unusual electron paramagnetic resonance spectrum of ES has been shown to be due to a weak, distributed exchange coupling between the two paramagnetic redox centers (Houseman, A. L. P.; Doan, P. E.; Goodin, D. B.; Hoffman, B. M. Biochemistry 1993, 32, 4430—4443). Various residues have been proposed as the radical site over the years. In this paper, continuous wave and pulsed Q-band electron nuclear double resonance (ENDOR) spectroscopy on samples isotopically enriched with [13C]-, [15N]-, and [2H]tryptophan confirm that the radical is located on Trp-191, as previously proposed. The data show that an exchangeable proton with A(1H) ≈ 16 MHz is associated with N1H of the Trp π-cation radical and not with C2H that has been labilized in the radical state. In addition, through a combination of 13C and 2H labeling we have determined the hyperfine couplings at g = 2.01 for 13Cβ, and the CβH2, N1H, and C2H protons. An analysis of these couplings based on the magnetic properties of the spin-coupled heme-radical redox cluster of ES has yielded spin densities for the radical in agreement with those predicted for the cation radical, confirming the assignment and suggesting that the radical is not greatly perturbed by N1H•••O hydrogen bonding to the carboxylate of Asp-235. This paper, thus, completes the characterization of the active site of compound ES as being comprised of an oxyferryl heme coupled to the Trp-191 π-cation radical by a weak spin exchange.
ASJC Scopus subject areas
- Colloid and Surface Chemistry