Sites of hydroxyl radical reaction with amino acids identified by H-2 NMR detection of induced H-1/H-2 exchange

Hydroxyl radical reacts with the aliphatic C-H bonds of amino acids by H at om abstraction. Under anaerobic conditions inclusion of a H-2 atom donor re sults in H-1/H-2 exchange into these C-H bonds [Goshe et al. Biochemistry 2 000, 39, 1761-1770]. The site of H-1/H-2 exchange can be detected and quant ified by H-2 NMR. Integration of the H-2 NMR resonances within a single spe ctrum permits the relative rate of Ii atom abstraction from each position t o be determined. Analysis of the aliphatic amino acid spectra indicates tha t the methine and methylene positions were more reactive than the methyl po sitions. The H-2 NMR spectra of isoleucine and leucine show that H-atom abs traction distal to the alpha -carbon occurs preferentially. Significant H-1 /H-2 exchange was observed into the delta positions of proline and arginine and into the E-methylene of lysine, indicating that a positive charge on a geminal N does not inhibit the H-1/H-2 exchange. Comparisons of H-2 NMR in tegrations between amino acid spectra indicated that H-1/H-2 exchange occur red in the following descending order: L > I > V > R > K > Y > P > H > F > M > T > Az [C, S, D, N, E,Q, G, W]. The extent of H-1/H-2 exchange into met hionine, N-glycyl-methionine, and methionine sulfoxide suggests that a prom inent solvent exchange pathway involving hydroxyl radical mediated oxidatio n of methionine exists to account for the large H-2 incorporation into the gamma -methylene of methionine sulfoxide that is absent for N-glycyl-methio nine. Analysis of the H-1 NMR spectra of the reactions with phenylalanine a nd tyrosine indicated that hydroxyl radical addition to the phenyl ring und er the anaerobic reductive reaction conditions did not result in either exc hange or hydroxylation.