The main aim of the research project is an understanding of the chemical reactivity of flavin coenzymes, and how this reactivity is used and controlled in biological systems. The methods of approach will be the study of specific enzymes representative of the various classes of flavoproteins and studies of model systems. Considerable use will be made of steady state kinetics coupled with rapid reactior kinetics studies, and of the technique of replacing the natural coenzymes of flavoproteins with synthetic flavins, in order to test possible mechanisms, and to probe the nature of the environment immediately around the protein-bound flavin. The latter is particularly important in providing information about the active site region in proteins where the structure is not available from X-ray crystallography. It also offers a powerful way for investigating dynamic aspects of protein structure in the case of enzymes where the crystal structure is known. We also plan to clone the gene for L-lactate oxidase in order to determine the amino acid sequence of the protein, to aid in the crystallographic determination of its three-dimensional structure, and to use site- directed mutagenesis in study of the enzyme mechanism.