Structural Basis for Isozyme-specific Regulation of Electron Transfer in Nitric-oxide Synthase[boxs]

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and bioch...

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Published inThe Journal of biological chemistry Vol. 279; no. 36; pp. 37918 - 37927
Main Authors Garcin, Elsa D., Bruns, Christopher M., Lloyd, Sarah J., Hosfield, David J., Tiso, Mauro, Gachhui, Ratan, Stuehr, Dennis J., Tainer, John A., Getzoff, Elizabeth D.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 03.09.2004
American Society for Biochemistry and Molecular Biology
Subjects
Amino Acid Sequence
Animals
Binding Sites
Catalysis
Electron Transport
Flavins - metabolism
Humans
Isoenzymes - chemistry
Isoenzymes - metabolism
Models, Molecular
Molecular Sequence Data
Mutagenesis
Nitric Oxide Synthase - chemistry
Nitric Oxide Synthase - metabolism
Nitric Oxide Synthase Type I
Protein Conformation
Rats
Sequence Homology, Amino Acid
X-Ray Diffraction
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Summary:Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca2+/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg1400, and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M406204200