Free radicals in biological milieu generally provoke destructive reactions owing to their high reactivity and non-specific chemistry. Recently, however, a class of enzymes has been identified in which highly oxidizing amino acid side chain radicals are generated during the normal course of catalysis. These species appear to be integral to the proper function of these enzymes. Understanding the means by which these radicals are generated, stabilized, and directed to effective catalysis is a major, long-term objective of the project. The manner in which the unpaired electron- spin density is distributed over the radical center is one means by which radical reactivity appears to be controlled. The local protein environnent around the redox active side chain is a second important factor in channeling the reactivity of these species, and the extent to which they are shielded from sovent by the overall protein structure is a third. These controlling features will be studied in the upcoming grant period in a variety of radical generating and utilizing enzymes. The specific systems upon which we intend to focus include the following: Photosystem I and II in oxygen-evolving organisms, ribonucleotide reductase, prostaglandin-H synthase, galactose oxidase, and photolyase. Work on some of these systems will be done exclusively at Michigan State; others will be studied collaboratively. The radicals generated by these enzymes include tyrosine, tryptophan, and possibly histidine. The methods we will apply include techniques from microbiology, molecular biology, biochemistry, and magnetic resonance spectroscopy. Selective isotopic labelling of the radical side chains potentially in the local environment of the radical will be accomplished, either by existing techniques or by methods that we propose to develop. EPR and ENDOR spectroscopies provide the primary analytical tools and are intended to assess the unpaired electron-spin density distribution and the local environment around the radical site. Model compounds and computational methods will be used to complement the in vivo studies. Tyrosine radicals are the best characterized thus far and will continue to be the primary focus of the proposed work. The effects of ortho substitution of the phenol, as appears to occur in galactose oxidase, will be studied. Thus far, radical-containing enzymes have been uncoverd somewhat accidentally. By characterizing these species in detail in those enzymes in which they are known to occur, we anticipate that general principles of their catalytic activity will emerge and lead to their identification in other enzymatic reactions.