The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin

Linda J. Roman, Pavel Martásek, R. Timothy Miller, Dawn E. Harris, Melissa A. De La Garza, Thomas M. Shea, Jung Ja P Kim, Bettie Sue Siler Masters

Research output: Contribution to journalArticle

112 Citations (Scopus)

Abstract

The sequences of nitric-oxide synthase flavin domains closely resemble that of NADPH-cytochrome P450 reductase (CPR). However, all nitric-oxide synthase (NOS) isoforms are 20-40 residues longer in the C terminus, forming a "tail" that is absent in CPR. To investigate its function, we removed the 33 and 42 residue C termini from neuronal NOS (nNOS) and endothelial NOS (eNOS), respectively. Both truncated enzymes exhibited cytochrome c reductase activities without calmodulin that were 7-21-fold higher than the nontruncated forms. With calmodulin, the truncated and wild-type enzymes reduced cytochrome c at approximately equal rates. Therefore, calmodulin functioned as a nonessential activator of the wild-type enzymes and a partial noncompetitive inhibitor of the truncated mutants. Truncated nNOS and eNOS plus calmodulin catalyzed NO formation at rates that were 45 and 33%, respectively, those of their intact forms. Without calmodulin, truncated nNOS and eNOS synthesized NO at rates 14 and 20%, respectively, those with calmodulin. By using stopped-flow spectrophotometry, we demonstrated that electron transfer into and between the two flavins is faster in the absence of the C terminus. Although both CPR and intact NOS can exist in a stable, one-electron-reduced semiquinone form, neither of the truncated enzymes do so. We propose negative modulation of FAD-FMN interaction by the C termini of both constitutive NOSs.

Original languageEnglish (US)
Pages (from-to)29225-29232
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number38
DOIs
StatePublished - Sep 22 2000

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Calmodulin
Heme
Nitric Oxide Synthase
Modulation
Electrons
NADPH-Ferrihemoprotein Reductase
Enzymes
Cytochromes c
Flavins
Enzyme Activators
Cytochrome Reductases
Flavin Mononucleotide
Flavin-Adenine Dinucleotide
Nitric Oxide Synthase Type III
Spectrophotometry
4,6-dinitro-o-cresol
Oxidoreductases
Protein Isoforms

ASJC Scopus subject areas

  • Biochemistry

Cite this

Roman, L. J., Martásek, P., Miller, R. T., Harris, D. E., De La Garza, M. A., Shea, T. M., ... Masters, B. S. S. (2000). The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin. Journal of Biological Chemistry, 275(38), 29225-29232. https://doi.org/10.1074/jbc.M004766200

The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin. / Roman, Linda J.; Martásek, Pavel; Miller, R. Timothy; Harris, Dawn E.; De La Garza, Melissa A.; Shea, Thomas M.; Kim, Jung Ja P; Masters, Bettie Sue Siler.

In: Journal of Biological Chemistry, Vol. 275, No. 38, 22.09.2000, p. 29225-29232.

Research output: Contribution to journalArticle

Roman, LJ, Martásek, P, Miller, RT, Harris, DE, De La Garza, MA, Shea, TM, Kim, JJP & Masters, BSS 2000, 'The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin', Journal of Biological Chemistry, vol. 275, no. 38, pp. 29225-29232. https://doi.org/10.1074/jbc.M004766200
Roman, Linda J. ; Martásek, Pavel ; Miller, R. Timothy ; Harris, Dawn E. ; De La Garza, Melissa A. ; Shea, Thomas M. ; Kim, Jung Ja P ; Masters, Bettie Sue Siler. / The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 38. pp. 29225-29232.
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