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. Acc. 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. High-field (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. Soc. 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 4.5 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 x values are comparable to those reported for the hydrogen-bonded γ-generated tyrosyl radical in Tyr-HCl crystals (2.00670: Fasanella, E. L.; Gordy, W. Proc. Natl. Acad. Sci. U.S.A. 1969, 62, 299−304) 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. Soc. 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 ν 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, E. 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 ν 7a (C−O) stretching mode as a function of hydrogen bond distance. Specifically, for hydrogen bond distances shorter than 1.7 Å, a sharp decrease of the ν 7a vibrational frequencies was observed. In contrast, for hydrogen bond distances longer than 1.7 Å, an increase of the vibrational frequencies was observed, as compared to the non-hydrogen-bonded situation.