Effect of protein microenvironment on tyrosyl radicals. A high-field (285 GHz) EPR, resonance raman, and hybrid density functional study

The protein environment appears to regulate the biological function of tyro syl radicals (Tommos, C.; Babcock, G. T. Ace. Chem. Res. 1998, 31, 18-25). Vibrational spectroscopy and electron paramagnetic resonance (EPR) techniqu es have been used to characterize tyrosyl radicals. In this work, we have i nvestigated the relationship between the g values and the vibrational spect ra of tyrosyl radicals (Tyr) in different protein microenvironments by comb ining experimentally determined values and molecular orbital calculations. Highfield (285 GHz) electron paramagnetic resonance (HF-EPR) and resonance Raman spectroscopies were applied to obtain the g values and the vibrationa l frequencies, respectively, of the tyrosyl radical (Tyr*(CAT)) previously reported as a heme catalase intermediate [(Fe(IV)=O) Tyr*] (Ivancich, A.; J ouve, H. M.; Gaillard, J. J. Am. Chem. Sec. 1996, 118, 12852-12853. Ivancic h, A., Jouve, H. M.; Sartor, B.; Gaillard, J. Biochemistry 1997, 36, 9356-9 364). The effect of the protein microenvironment on the catalase tyrosyl ra dical was examined by varying the pH between 6.7 and 4.5. The broadness of the g(x) edge in the Tyr*(CAT) HF-EPR spectrum was interpreted as arising f rom a distribution in hydrogen bond strengths. The observed g(x) values of 2.0073(8) at pH 6.7 and 2.0076(2) at pH 3.5 indicated the presence of one o r two hydrogen bonds to the Tyr*(CAT). The asymmetric shape of the g(x) edg e of the Tyr*(CAT) spectrum was attributed to the presence of a minor featu re centered at 2.0065(5) for pH 6.7 and at 2.0082(4) for pH 4.5. These g, v alues are comparable to those reported for the hydrogen-bonded gamma-genera ted tyrosyl radical in Tyr-HCl crystals (2.00670: Fasanella, E. L.; Gordy, W. Proc. Nad. Acad Sci. U.S.A. 1969, 62, 299-303) and the non-hydrogen-bond ed Tyr* in Escherichia coli ribonucleotide reductase (RNR) (2.00866: Un, S. ; Atta, M.; Fontecave, M.; Rutherford, A. W. J. Am. Chem. Sec. 1995, 117, 1 0713-10719). One- and two-water complexes of p-methylphenoxy and phenoxy ra dicals were used to model the protein tyrosyl radical. Semiempirical MNDO m olecular orbital calculations were used to analyze the effect of hydrogen b onds on the g values of the p-methylphenoxy radical. Ab initio density func tional calculations were carried out to investigate the effect of hydrogen bond strengths on the vibrational frequencies of the radical, in particular the nu(7a)(C-O) stretching mode. The calculated g values and vibrational f requencies were in very good agreement with the experimentally observed val ues for the tyrosyl radicals in catalase, B coli RNR, and photosystem II. I n contrast to the g(x) values (g-tensor component in the C-O direction of t he radical), the density functional calculations predict a nonmonotonic beh avior of the vibrational frequency of the nu(7a)(C-O) stretching mode as a function of hydrogen bond distance. Specifically, for hydrogen bond distanc es shorter than 1.7 Angstrom, a sharp decrease of the nu(7a), vibrational f requencies was observed. In contrast, for hydrogen bond distances longer th an 1.7 Angstrom, an increase of the vibrational frequencies was observed, a s compared to the non-hydrogen-bonded situation.