Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic reso nance (EPR) spectroscopy has been used to characterize radical intermediate s in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high -field EPR spectra of the exchange-coupled oxoferryl-trytophanyl radical pa ir that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp(.+)] we re analyzed using a spin Hamiltonian that incorporated a general anisotropi c spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discuss ed. Using numerical solutions of the completely anisotropic Hamiltonian, it s was possible to simulate accurately the experimental data from 9 to 285 G Hz using a single set of spin parameters. The results are also consistent w ith previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a tr ansient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degreesC and disappeared aft er 1 h. High-field EPR was also used to characterize the radical intermedia te that was generated by hydrogen peroxide addition to the W191G CcP mutant . The g-values of this radical (g(x) = 2.00660, g(y) = 2.00425, and g(z) = 2.00208), as well as the wild-type transient tyrosyl radical, are essential ly identical to those obtained from the high-field EPR spectra of the tyros yl radical generated by y-irradiation of crystals of tyrosine hydrochloride (g(x) = 2.00658, g(y) = 2.00404, and g(z) 2.00208). The low g(x)-value ind icated that ail three of the tyrosyl radicals were in electropositive envir onments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discus sed.