Flavoproteins constitute one of the largest groups of related proteins known. Linked through their common utilization of riboflavin-based cofactors, these proteins catalyze essential oxidation-reduction steps in almost every metabolic pathway in the prokaryotic and eukaryotic cell, including crucial steps in biosynthesis, biodegradation, and energy transduction. Their involvement in important signal transduction mechanisms such as phototropism, chemo- and aerotaxis has been more recent discoveries. This family of proteins fully exploits the entire gamut of the diverse chemistry available to the isoalloxazine ring system of the cofactor, yet the protein must exercise exquisite control of the chemical and redox properties of the bound flavin cofactor so as to optimize the specific reaction or process catalyzed. Should something go amiss with this process, serious metabolic problems may result. How proteins are able to tightly regulate the biochemical properties of flavin cofactors is a fundamental and critical question in biochemistry. Such control is evident in the unique ability of flavoproteins to bridge electron transfer between obligatory two- and one-electron donor-acceptor molecules using all three redox states of the flavin. Perhaps no better excellent examples of this property can be found than for the cytochrome P450 reductase, cytochrome P450BM-3_monooxygenase, and nitric oxide synthase systems. These flavoproteins catalyze important reactions in xenobiotic, steroid, and prostaglandin biosynthesis; in fatty acid metabolism; and in neurotransmission, blood pressure homeostasis, and inflammatory responses. The manner by which these flavoproteins promote both the one- and two-electron reactions so efficiently is not well understood. A multifaceted approach that couples a systematic protein engineering approach with detailed biochemical and biophysical characterization that has been used so effectively in the study of the factors that regulate the redox properties in the flavodoxin is applied in this proposal to describe the unique properties of the flavodoxin-like domain within cytochrome P450BM-3 monooxygenase and microsomal cytochrome P450 reductase. These covalent, multi-redox centered enzymes will serve as excellent systems in which to more fully investigate the fundamental relationships between the regulation of flavin reduction potentials and the control the inter-flavin and inter-domain electron transfer mechanisms and enzymatic activity. These studies are also designed to expand our understanding of the fundamentally different way that the one electron reduced state (the flavin semiquinone) is stabilized and is utilized in these two different systems.
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