Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase

Essam A. Sheta, Kirk McMillan, Bettie Sue Siler Masters

Research output: Contribution to journalArticle

182 Citations (Scopus)

Abstract

Nitric oxide synthase (NOS) catalyzes the NADPH-dependent, Ca2+/calmodulin-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The localization of the heme-binding consensus sequence in the NH2-terminal half of NOS and of the binding sequences for nucleotides (FMN and FAD) in the COOH-terminal half suggests a bidomain structure. In addition, the presence of a putative calmodulin-binding sequence between the heme- and flavin-binding domains of the enzyme suggests a role for calmodulin in modulating a spatial orientation of these domains that is required for catalytic activity. First, to determine the effects of calmodulin and the functionality of the separated domains, Ca2+/calmodulin binding-induced conformational changes in NOS were measured by fluorescence quenching, from which a binding constant of ~1 nM for calmodulin was calculated. Second, electron transport to various artificial acceptors was measured. The addition of Ca2+/calmodulin increased cytochrome c reduction from 10-15-fold while stimulating the rate of 2,6-dichlorophenolindophenol and ferricyanide reduction only slightly, if at all. Calmodulin stimulation of NOS results in NADPH-mediated cytochrome c reduction, which is sensitive to superoxide dismutase, and the reduction of acetylated cytochrome c, which is only weakly reducible by unstimulated NOS. Thus, this stimulated activity is presumably superoxide anion-mediated. Third, limited proteolysis of NOS in the absence of calmodulin resulted in a time-dependent increase in cytochrome c reductase activity, which was not inhibitable by superoxide dismutase, and a decrease in catalysis of NO formation. SDS-polyacrylamide gel electrophoresis analysis of the tryptic digest demonstrated the formation of ~89- and ~79-kDa fragments. Sequence analysis of the peptides confirmed that trypsin cleaves the enzyme in the putative calmodulin-binding region beginning with Ala728. This region was protected from proteolysis by the addition of Ca2+/calmodulin. The separated NH2-terminal domain exhibited the characteristic spectrum of bound heme, while the COOH-terminal domain showed the characteristic spectrum of bound flavins. Other cleavage patterns were obtained in the presence of calmodulin. The data demonstrate that the heme- and flavin-binding domains of NOS can be isolated in functionally intact forms.

Original languageEnglish (US)
Pages (from-to)15147-15153
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number21
StatePublished - May 27 1994

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Calmodulin
Nitric Oxide Synthase
Cytochromes c
Heme
Proteolysis
NADP
Superoxide Dismutase
Flavins
2,6-Dichloroindophenol
Cytochrome Reductases
Flavin Mononucleotide
Citrulline
Flavin-Adenine Dinucleotide
Molecular oxygen
Protein Sequence Analysis
Consensus Sequence
Enzymes
Electron Transport
Electrophoresis
Catalysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sheta, E. A., McMillan, K., & Masters, B. S. S. (1994). Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. Journal of Biological Chemistry, 269(21), 15147-15153.

Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. / Sheta, Essam A.; McMillan, Kirk; Masters, Bettie Sue Siler.

In: Journal of Biological Chemistry, Vol. 269, No. 21, 27.05.1994, p. 15147-15153.

Research output: Contribution to journalArticle

Sheta, EA, McMillan, K & Masters, BSS 1994, 'Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase', Journal of Biological Chemistry, vol. 269, no. 21, pp. 15147-15153.
Sheta, Essam A. ; McMillan, Kirk ; Masters, Bettie Sue Siler. / Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 21. pp. 15147-15153.
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