Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself

Ratan Gachhui, Anthony Presta, Dennis F. Bentley, Husam M. Abu-Soud, Ryan McArthur, Gary W Brudvig, Dipak K. Ghosh, Dennis J. Stuehr

Research output: Contribution to journalArticle

128 Citations (Scopus)

Abstract

Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6- oxidized reductase domain with NADPH indicated the flavin semiquinone re- formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full- length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

Original languageEnglish
Pages (from-to)20594-20602
Number of pages9
JournalJournal of Biological Chemistry
Volume271
Issue number34
DOIs
Publication statusPublished - 1996

Fingerprint

Nitric Oxide Synthase Type I
Pichia
Calmodulin
Yeast
Rats
Oxidoreductases
Yeasts
Nitric Oxide Synthase
Flavins
Electrons
NADP
Cytochromes c
Heme
Proteins
Chemical activation
2,6-Dichloroindophenol
Affinity chromatography
Flavin Mononucleotide
Heme Oxygenase (Decyclizing)
Oxygenases

ASJC Scopus subject areas

  • Biochemistry

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Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself. / Gachhui, Ratan; Presta, Anthony; Bentley, Dennis F.; Abu-Soud, Husam M.; McArthur, Ryan; Brudvig, Gary W; Ghosh, Dipak K.; Stuehr, Dennis J.

In: Journal of Biological Chemistry, Vol. 271, No. 34, 1996, p. 20594-20602.

Research output: Contribution to journalArticle

Gachhui, Ratan ; Presta, Anthony ; Bentley, Dennis F. ; Abu-Soud, Husam M. ; McArthur, Ryan ; Brudvig, Gary W ; Ghosh, Dipak K. ; Stuehr, Dennis J. / Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 34. pp. 20594-20602.
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