My research focuses on applying crystallographic techniques to aid in understanding flavoenzyme chemistry. Flavoenzymes are proteins containing flavin nucleotide co-enzymes. They catalyze a variety of biologically important reactions using various chemical mechanisms. Factors affecting the catalytic reaction and the reactivity of the flavin are strongly dependent on the interactions between the flavin and the protein environment surrounding it. It is clear that the protein structure in the region of the active site plays a key role in the reactions carried out by these enzymes. In this proposal I aim to use a crystallography to address the question of how the redox properties of different flavoenzymes are related to their catalytic function and to structure of the protein in the active site. From structural interpretations, these relationships will be further probed and modified by genetic engineering and the effect of specific changes at the active changes at the active site both on the thermodynamics and the rates of electron transfer will be assessed. By combining redox data with structural data a structure-function redox potential correlation will be determined which will give insight into the workings of this fascinating group of enzymes. Three different proteins have been chosen for structural studies, 2 oxidases: cholesterol oxidase and L-amino acid oxidase, and one monooxygenase: tryptophan 2-monooxygenase. In all cases the first step in the reaction involves C-H bond cleavage of the substrate and transfer of a hydrogen and 2 electrons to the flavin cofactor. However, subsequent steps in the reaction vary amongst these enzymes. Thus, although these 3 enzymes all undergo a C-H bond cleavage, the chemical nature of the substrate dictates a different mechanism for this cleavage reaction. Crystallographic studies of these enzymes will address the question of how the protein structure facilitates these different mechanisms. The structure will enable us to study the different features which contribute to the stability of the flavin radical formed upon C-H bond cleavage.
