Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation

Joseph Sabat, Tsuyoshi Egawa, Changyuan Lu, Dennis J. Stuehr, Gary J. Gerfen, Denis L. Rousseau, Syun-Ru Yeh

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-Larginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retardNOsynthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498-33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the L-Arg reaction but also during the N-hydroxy-L-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS.

Original languageEnglish (US)
Pages (from-to)6095-6106
Number of pages12
JournalJournal of Biological Chemistry
Volume288
Issue number9
DOIs
StatePublished - Mar 1 2013

Fingerprint

Nitric Oxide Synthase Type II
Heme
Nitric Oxide Synthase
Mutation
Arginine
Nitric Oxide
Citrulline
Hydroxylation
Histidine
Structural analysis
Tryptophan
Cations
Catalytic Domain
Oxygen
Ligands
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation. / Sabat, Joseph; Egawa, Tsuyoshi; Lu, Changyuan; Stuehr, Dennis J.; Gerfen, Gary J.; Rousseau, Denis L.; Yeh, Syun-Ru.

In: Journal of Biological Chemistry, Vol. 288, No. 9, 01.03.2013, p. 6095-6106.

Research output: Contribution to journalArticle

@article{2cb2334ac7dd4afc8983a5ab275e50d3,
title = "Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation",
abstract = "Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-Larginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retardNOsynthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498-33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the L-Arg reaction but also during the N-hydroxy-L-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS.",
author = "Joseph Sabat and Tsuyoshi Egawa and Changyuan Lu and Stuehr, {Dennis J.} and Gerfen, {Gary J.} and Rousseau, {Denis L.} and Syun-Ru Yeh",
year = "2013",
month = "3",
day = "1",
doi = "10.1074/jbc.M112.403238",
language = "English (US)",
volume = "288",
pages = "6095--6106",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "9",

}

TY - JOUR

T1 - Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation

AU - Sabat, Joseph

AU - Egawa, Tsuyoshi

AU - Lu, Changyuan

AU - Stuehr, Dennis J.

AU - Gerfen, Gary J.

AU - Rousseau, Denis L.

AU - Yeh, Syun-Ru

PY - 2013/3/1

Y1 - 2013/3/1

N2 - Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-Larginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retardNOsynthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498-33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the L-Arg reaction but also during the N-hydroxy-L-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS.

AB - Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-Larginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retardNOsynthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498-33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the L-Arg reaction but also during the N-hydroxy-L-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS.

UR - http://www.scopus.com/inward/record.url?scp=84874777557&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84874777557&partnerID=8YFLogxK

U2 - 10.1074/jbc.M112.403238

DO - 10.1074/jbc.M112.403238

M3 - Article

C2 - 23269673

AN - SCOPUS:84874777557

VL - 288

SP - 6095

EP - 6106

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 9

ER -