title: Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.
abstract: Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate 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 essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. 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 discussed.
authors.affiliation: Section de Bioénergétique, URA 2096 CNRS, Département de Biologie Cellulaire et Moléculaire, CEA Saclay, 91191 Gif-sur-Yvette, France.
authors.forename: A
authors.initials: A
authors.lastname: Ivancich
authors.forename: P
authors.initials: P
authors.lastname: Dorlet
authors.forename: D B
authors.initials: DB
authors.lastname: Goodin
authors.forename: S
authors.initials: S
authors.lastname: Un
mesh_terms: D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D009154:Mutation; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D014364:Tryptophan; D003578:Cytochrome-c Peroxidase