Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase: Comparisons with NADPH-cytochrome P450 oxidoreductase

Jian Zhang, Pavel Martàsek, Rosemary Paschke, Thomas Shea, Bettie Sue Siler Masters, Jung Ja P Kim

Research output: Contribution to journalArticle

123 Citations (Scopus)

Abstract

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP+, has been determined at 1.9 Å resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP+ binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.

Original languageEnglish (US)
Pages (from-to)37506-37513
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number40
DOIs
StatePublished - Oct 5 2001

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NADPH-Ferrihemoprotein Reductase
Nitric Oxide Synthase Type I
Flavin-Adenine Dinucleotide
NADP
Rats
Crystal structure
Oxidoreductases
Flavin Mononucleotide
Cytochrome P-450 Enzyme System
Nitric Oxide Synthase
Flavins
Electrons
Heme
Hydrides
Proteins
Binding Sites
4,6-dinitro-o-cresol
Conservation
Peptides
Water

ASJC Scopus subject areas

  • Biochemistry

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Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase : Comparisons with NADPH-cytochrome P450 oxidoreductase. / Zhang, Jian; Martàsek, Pavel; Paschke, Rosemary; Shea, Thomas; Masters, Bettie Sue Siler; Kim, Jung Ja P.

In: Journal of Biological Chemistry, Vol. 276, No. 40, 05.10.2001, p. 37506-37513.

Research output: Contribution to journalArticle

Zhang, Jian ; Martàsek, Pavel ; Paschke, Rosemary ; Shea, Thomas ; Masters, Bettie Sue Siler ; Kim, Jung Ja P. / Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase : Comparisons with NADPH-cytochrome P450 oxidoreductase. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 40. pp. 37506-37513.
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T1 - Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase

T2 - Comparisons with NADPH-cytochrome P450 oxidoreductase

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AU - Martàsek, Pavel

AU - Paschke, Rosemary

AU - Shea, Thomas

AU - Masters, Bettie Sue Siler

AU - Kim, Jung Ja P

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AB - Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP+, has been determined at 1.9 Å resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP+ binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.

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