X-ray crystallography will be the primary method for studies of reactivity, specificity, and stability in metallo- and flavoenzymes which catalyze biological oxidations. The choice of enzymes for analysis has been based on the potential ability of the structures to furnish new information on enzyme-cofactor and enzyme-substrate interactions. The structures of Fe superoxide dismutase from E. coli and Mn superoxide dismutase from T. thermophilus will be refined, and the Me (II) states and inhibitor complexes will be examined at high resolution. Site-directed mutagenesis combined with analysis of the resultant structures will assess the roles of active center residues and of dimer interactions in the Fe enzyme. Mutagenesis will also be applied to flavodoxin from A. nidulans in quantitative studies of the FMN-protein interactions that affect the oxidation-reduction potentials of this electron carrier. Structures of three other enzymes, phthalate oxygenase reductase and protocatechuate dioxygenase from P. cepacia and lactate oxidase from M. smegmatis, for which suitable crystals have been obtained, will be determined. Phthalate oxygenase reductase, a monomeric [2Fe-2S] flavoprotein, is an attractive model for studies of intramolecular electron transfer. Protocatechuate dioxygenase, an iron-containing enzyme with the subunit structure (AlphaBetaFe)4, plays a key role in the microbial utilization of aromatics; compounds related to the reaction cycle, including transition state analogs and halo-aromatics that form abortive complexes, are accessible to structural study. Determination of the structure of lactate oxidase, an octamer of 43,600d units, affords an opportunity to understand the reactivity of flavoproteins which rapidly reduce O2. The preparation of large crystals of phthalate dioxygenase, orotate reductase, and components of the microsomal P-450 hydroxylase system, involved in drug detoxification, will be explored.
