Analytical Chemistry and Spectroscopy
Wiley InterScience Backfile Collection 1832-2000
The oxidized radicals of aromatic amino acids, 4-methylimidazole and phenol were generated by UV irradiation at low temperature. The radicals were monitored by EPR spectroscopy. For the first time, infrared (IR) modes characteristic of the tyrosine, tryptophan, phenylalanine, and histidine radicals were obtained by FTIR difference spectroscopy between the ground state and the radical state. The effects of D- (on Tyr and phenol) or 15N- (on His) labeling on the IR modes of the radicals were studied, as were the influence of hydrogen bonding and of pH. These parameters were studied to model the possible radical structures and environments in proteins. The radicals obtained with tyrosine, phenol, and 4-ethylphenol present six main IR modes: a combination mode at ∼ 2110-2106 cm-1; the v8b(CC) mode at 1550-1556 cm-1, the v7a(CO) and v19b(CC) modes both at 1515-1500 cm-1, which are distinctly affected by D-labeling of the phenol ring; the 14(vCC + δCH) mode at 1290-1288 cm-1, which is strongly modified when the radical is hydrogen bonded; and the 9a(CC) mode at 1163-1159 cm-1. These IR modes partly confirm the assignments made by resonance Raman (RR) spectroscopy and should help to obtain precise structure and force field calculations for the radicals. The deprotonated 4-methylimidazole radical (4-MeIm·) is obtained at pH 12. It has characteristic IR modes at 1593 v(CC), 1425 δCH3, 1376 δCH3, 1315 cm-1, 1213 cm-1 and 1098 cm-1 δ(CH). The methyl modes seem strongly downshifted upon radical formation, while the ring modes appear less affected. In particular, the C4C5 double-bond character is conserved. The protonated 4-methylimidazole radical formed at pH ≤ 6 is characterized by signals at 1433 cm-1, 1380 cm-1, 1310 cm-1, 1227 cm-1, and 1172 cm-1. The histidine and tyrosine radicals present similar IR modes as the corresponding model of their sidechain. For all the amino acids, the vas(COO-) and vs(COO-) modes of the terminal carboxylate were respectively up- and downshifted by ∼ 20 cm-1 upon the radical formation. This effect suggests that, in a protein, the amide bond of the amino acid could also be influenced by the radical formation. © 1995 John Wiley & Sons, Inc.
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