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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036490-15
Application #
7151925
Study Section
Special Emphasis Panel (ZRG1-BMT (02))
Program Officer
Ikeda, Richard A
Project Start
1988-12-01
Project End
2008-12-31
Budget Start
2007-06-01
Budget End
2008-12-31
Support Year
15
Fiscal Year
2007
Total Cost
$212,629
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Kasim, Mumtaz; Chen, Huai-Chun; Swenson, Richard P (2009) Functional characterization of the re-face loop spanning residues 536-541 and its interactions with the cofactor in the flavin mononucleotide-binding domain of flavocytochrome P450 from Bacillus megaterium. Biochemistry 48:5131-41
Murray, Tracey Arnold; Swenson, Richard P (2003) Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 1. Kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5'-phosphate moiety of the cofactor. Biochemistry 42:2307-16
Murray, Tracey Arnold; Foster, Mark P; Swenson, Richard P (2003) Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 2. Evidence for cooperative conformational changes involving tryptophan 60 in the interaction between the phosphate- and ring-binding subsites. Biochemistry 42:2317-27
Bradley, L H; Swenson, R P (2001) Role of hydrogen bonding interactions to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Biochemistry 40:8686-95
Kasim, M; Swenson, R P (2001) Alanine-scanning of the 50's loop in the Clostridium beijerinckii flavodoxin: evaluation of additivity and the importance of interactions provided by the main chain in the modulation of the oxidation-reduction potentials. Biochemistry 40:13548-55
Kasim, M; Swenson, R P (2000) Conformational energetics of a reverse turn in the Clostridium beijerinckii flavodoxin is directly coupled to the modulation of its oxidation-reduction potentials. Biochemistry 39:15322-32
Bradley, L H; Swenson, R P (1999) Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin. Glutamate-59 is not responsible for the pH dependency but contributes to the stabiliz Biochemistry 38:12377-86
Chang, F C; Swenson, R P (1999) The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as me Biochemistry 38:7168-76
Druhan, L J; Swenson, R P (1998) Role of methionine 56 in the control of the oxidation-reduction potentials of the Clostridium beijerinckii flavodoxin: effects of substitutions by aliphatic amino acids and evidence for a role of sulfur-flavin interactions. Biochemistry 37:9668-78
Feng, Y; Swenson, R P (1997) Evaluation of the role of specific acidic amino acid residues in electron transfer between the flavodoxin and cytochrome c3 from Desulfovibrio vulgaris. Biochemistry 36:13617-28

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