title: Regulation of interdomain interactions by calmodulin in inducible nitric-oxide synthase.
abstract: Nitric-oxide synthases (NOSs) catalyze the conversion of l-arginine to nitric oxide and citrulline. There are three NOS isozymes, each with a different physiological role: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). NOSs consist of an N-terminal oxygenase domain and a C-terminal reductase domain, linked by a calmodulin (CaM)-binding region. CaM is required for NO production, but unlike other NOS isozymes, iNOS binds CaM independently of the exogenous Ca(2+) concentration. We have co-expressed CaM and the FMN domain of human iNOS, which includes the CaM-binding region. The Ca(2+)-bound protein complex (CaCaMxFMN) forms an air-stable semiquinone when reduced with NADPH and reduces cytochrome c when reconstituted with the iNOS FAD/NADPH domain. We have solved the crystal structure of the CaCaMxFMN complex in four different conformations, each with a different relative orientation, between the FMN domain and the bound CaM. The CaM-binding region together with bound CaM forms a hinge, pivots on the conserved Arg(536), and regulates electron transfer from FAD to FMN and from FMN to heme by adjusting the relative orientation and distance among the three cofactors. In addition, the relative orientations of the N- and C-terminal lobes of CaM are also different among the four conformations, suggesting that the flexibility between the two halves of CaM also contributes to the fine tuning of the orientation/distance between the redox centers. The data demonstrate a possible mode for precise control of electron transfer by altering the distance and orientation of redox centers in a protein displaying domain movement.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Chuanwu
authors.initials: C
authors.lastname: Xia
authors.forename: Ila
authors.initials: I
authors.lastname: Misra
authors.forename: Takashi
authors.initials: T
authors.lastname: Iyanagi
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004579:Electron Transport; D005486:Flavin Mononucleotide; D006801:Humans; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D002147:Calmodulin; D005486:Flavin Mononucleotide; D052247:Nitric Oxide Synthase Type II