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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.

SUBMITTER: Xia C

PROVIDER: S-EPMC2781624 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Regulation of interdomain interactions by calmodulin in inducible nitric-oxide synthase.

Xia Chuanwu C Misra Ila I Iyanagi Takashi T Kim Jung-Ja P JJ

The Journal of biological chemistry 20090908 44

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 ...[more]

PMID: 19737939

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Project description:Nitric oxide synthase (NOS), a flavo-hemoprotein, tightly regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a key signaling molecule for vasodilatation and neurotransmission at low concentrations, and also as a defensive cytotoxin at higher concentrations. Three NOS isoforms, iNOS, eNOS and nNOS (inducible, endothelial, and neuronal NOS), achieve their key biological functions by tight regulation of interdomain electron transfer (IET) process via interdomain interactions. In particular, the FMN-heme IET is essential in coupling electron transfer in the reductase domain with NO synthesis in the heme domain by delivery of electrons required for O(2) activation at the catalytic heme site. Compelling evidence indicates that calmodulin (CaM) activates NO synthesis in eNOS and nNOS through a conformational change of the FMN domain from its shielded electron-accepting (input) state to a new electron-donating (output) state, and that CaM is also required for proper alignment of the domains. Another exciting recent development in NOS enzymology is the discovery of importance of the the FMN domain motions in modulating reactivity and structure of the catalytic heme active site (in addition to the primary role of controlling the IET processes). In the absence of a structure of full-length NOS, an integrated approach of spectroscopic (e.g. pulsed EPR, MCD, resonance Raman), rapid kinetics (laser flash photolysis and stopped flow) and mutagenesis methods is critical to unravel the molecular details of the interdomain FMN/heme interactions. This is to investigate the roles of dynamic conformational changes of the FMN domain and the docking between the primary functional FMN and heme domains in regulating NOS activity. The recent developments in understanding of mechanisms of the NOS regulation that are driven by the combined approach are the focuses of this review. An improved understanding of the role of interdomain FMN/heme interaction and CaM binding may serve as the basis for the design of new selective inhibitors of NOS isoforms.

Dataset Information

Regulation of interdomain interactions by calmodulin in inducible nitric-oxide synthase.

Publications

Regulation of interdomain interactions by calmodulin in inducible nitric-oxide synthase.

Similar Datasets

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