Dr. Karplus proposes to investigate the structure and structure- function relationships of two flavoenzymes: ferredoxin:NADP+ reductase (FNR), which is an FAD-dependent enzyme which produces NADPH during photosynthesis, and Old Yellow Enzyme (OYE), an FMN- dependent NADPH oxidase of unknown physiological function. The main methods to be used in these studies are single crystal x- ray diffraction for structure determination combined with protein chemistry, for purifying the individual enzymes, preparing relevant crystals and complexes, and testing hypotheses based on the structures. The time is ripe for the study of both of these enzymes as they have been cloned and expressed within the last year, so that site-directed mutagenesis studies can be used to probe functional aspects of the structure as revealed by crystallography. %%% The overall goal is to elucidate the specific structural factors involved in catalysis for both of these enzymes. The results will give significant insight into general features of flavoprotein catalysis because FNR is the only structurally known NAD(P) dependent dehydrogenase-electron transferase and OYE has a flavin environment which is unique with respect to other characterized flavoenzymes.
