Neuronal Nitric-oxide Synthase Interaction with Calmodulin-Troponin C Chimeras

Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 273; no. 10; pp. 5451 - 5454
Main Authors:	Gachhui, Ratan, Abu-Soud, Husam M., Ghoshà, Dipak K., Presta, Anthony, Blazing, Michael A., Mayer, Bernd, George, Samuel E., Stuehr, Dennis J.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 06-03-1998 American Society for Biochemistry and Molecular Biology
Subjects:	Amino Acid Sequence Animals Brain - physiology Calmodulin - chemistry Calmodulin - pharmacology Cytochrome c Group - metabolism Electron Transport - physiology Enzyme Activation - physiology Flavoproteins - metabolism Heme - metabolism Kinetics Molecular Sequence Data NADP - metabolism Neurons - enzymology Nitric Oxide - metabolism Nitric Oxide Synthase - metabolism Rats Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - pharmacology Sequence Alignment Troponin C - chemistry Troponin C - pharmacology
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Summary:	Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS. The magnitude by which each chimera activated NO synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to ∼80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron reduction. We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0021-9258 1083-351X
DOI:	10.1074/jbc.273.10.5451