The SIR provided L-[B_2 H2]Tryptophan; 100mg and L-[4' _17 O]Tyrosine Tyrosine radicals occur in a variety of enzyme systems including ribonucleotide, reductase, prostaglandin synthase, Photosystern 111, and galactose oxidase. Spin density distributions can be altered by environment factors, some of which may be critical for the function of enzyme-bound radicals. We have been involved in characterizing these radicals by EPR spectroscopy in order to determine spin-density. Recent work suggests substantial variation in r at the phenol oxygen position in tyrosines in ribonucleotide reductase, in Photosystem H, and in a chemical model of tyrosine. This study aims at elucidating particular protein-radical interactions that contribute to enzyme function. We propose to access the spin density at the phenolic oxygen by labeling with [4'- 170]tyrosine. By collaborating with the Stockholm group of Prof Britt-Marie Sj6berg, we have obtained the EPR spectrum of the '70_ labeled tyrosine radical in ribonucleotide reductase, and we seek to obtain complementary model and Photosystem H data. Model 17 O-labeled tyrosine radicals will be generated by the UV techniques we have developed [Barry et al., 1990 JBC 265, 20139-20141). 17 O-labeled tyrosine will be incorporated into the cyanobacterium Synechocystis by using the auxotrophic condition that we have already described (Barry and Babcock, 1987 Proc. Natl. Acad. Sci. USA, 84, 7099-7103). EPR spectroscopy will be done as in these earlier studies. The active site of bacterial methylamine dehydrogenases contains a novel redox prosthetic group tryptophan tryptophanyl quinone that is made up of two tryptophan redidues. P-Deuterated tryptophan will be used to probe the spin density distribution in the serniquinone fon-n of the cofactor.
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