title: Kinetic and structural characterization of the interaction between the FMN binding domain of cytochrome P450 reductase and cytochrome c.
abstract: Cytochrome P450 reductase (CPR) is a diflavin enzyme that transfers electrons to many protein partners. Electron transfer from CPR to cyt c has been extensively used as a model reaction to assess the redox activity of CPR. CPR is composed of multiple domains, among which the FMN binding domain (FBD) is the direct electron donor to cyt c. Here, electron transfer and complex formation between FBD and cyt c are investigated. Electron transfer from FBD to cyt c occurs at distinct rates that are dependent on the redox states of FBD. When compared with full-length CPR, FBD reduces cyt c at a higher rate in both the semiquinone and hydroquinone states. The NMR titration experiments reveal the formation of dynamic complexes between FBD and cyt c on a fast exchange time scale. Chemical shift mapping identified residues of FBD involved in the binding interface with cyt c, most of which are located in proximity to the solvent-exposed edge of the FMN cofactor along with other residues distributed around the surface of FBD. The structural model of the FBD-cyt c complex indicates two possible orientations of complex formation. The major complex structure shows a salt bridge formation between Glu-213/Glu-214 of FBD and Lys-87 of cyt c, which may be essential for the formation of the complex, and a predicted electron transfer pathway mediated by Lys-13 of cyt c. The findings provide insights into the function of CPR and CPR-cyt c interaction on a structural basis.
authors.affiliation: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and.
authors.forename: Rui
authors.initials: R
authors.lastname: Huang
authors.affiliation: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Anesthesiology, University of Michigan and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105.
authors.forename: Freeborn
authors.initials: F
authors.lastname: Rwere
authors.affiliation: Department of Anesthesiology, University of Michigan and Veterans Affairs Medical Center, Ann Arbor, Michigan 48105.
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
authors.affiliation: Department of Chemistry and Biophysics, University of Michigan, Ann Arbor, Michigan 48109-1055 and. Electronic address: ramamoor@umich.edu.
authors.forename: Ayyalusamy
authors.initials: A
authors.lastname: Ramamoorthy
mesh_terms: D000818:Animals; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D005486:Flavin Mononucleotide; D045304:Cytochromes c; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Cytochrome P450; Cytochrome c; Electron Transfer Complex; Enzyme Kinetics; Nuclear Magnetic Resonance (NMR); Protein Structure; Protein-Protein Interaction