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 tyrosyl radicals (Tommos, C.; Babcock, G. T. Ace. Chem. Res. 1998, 31, 18-25). Vibrational spectroscopy and electron paramagnetic resonance (EPR) techniques have been used to characterize tyrosyl radicals. In this work, we have investigated the relationship between the g values and the vibrational spectra of tyrosyl radicals (Tyr) in different protein microenvironments by combining 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 vibrational frequencies, respectively, of the tyrosyl radical (Tyr*(CAT)) previously reported as a heme catalase intermediate [(Fe(IV)=O) Tyr*] (Ivancich, A.; Jouve, H. M.; Gaillard, J. J. Am. Chem. Sec. 1996, 118, 12852-12853. Ivancich, A., Jouve, H. M.; Sartor, B.; Gaillard, J. Biochemistry 1997, 36, 9356-9364). The effect of the protein microenvironment on the catalase tyrosyl radical 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 from 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 or two hydrogen bonds to the Tyr*(CAT). The asymmetric shape of the g(x) edge of the Tyr*(CAT) spectrum was attributed to the presence of a minor feature centered at 2.0065(5) for pH 6.7 and at 2.0082(4) for pH 4.5. These g, values are comparable to those reported for the hydrogen-bonded gamma-generated 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-bonded Tyr* in Escherichia coli ribonucleotide reductase (RNR) (2.00866: Un, S.; Atta, M.; Fontecave, M.; Rutherford, A. W. J. Am. Chem. Sec. 1995, 117, 10713-10719). One- and two-water complexes of p-methylphenoxy and phenoxy radicals were used to model the protein tyrosyl radical. Semiempirical MNDO molecular orbital calculations were used to analyze the effect of hydrogen bonds on the g values of the p-methylphenoxy radical. Ab initio density functional 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 frequencies were in very good agreement with the experimentally observed values for the tyrosyl radicals in catalase, B coli RNR, and photosystem II. In contrast to the g(x) values (g-tensor component in the C-O direction of the radical), the density functional calculations predict a nonmonotonic behavior of the vibrational frequency of the nu(7a)(C-O) stretching mode as a function of hydrogen bond distance. Specifically, for hydrogen bond distances shorter than 1.7 Angstrom, a sharp decrease of the nu(7a), vibrational frequencies was observed. In contrast, for hydrogen bond distances longer than 1.7 Angstrom, an increase of the vibrational frequencies was observed, as compared to the non-hydrogen-bonded situation.