Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase

David Li, Mariam Kabir, Dennis J. Stuehr, Denis L. Rousseau, Syun-Ru Yeh

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Nitric oxide synthase (NOS) catalyzes the formation of NO via a consecutive two-step reaction. In the first step, L-arginine (Arg) is converted to N-hydroxy-L-arginine (NOHA). In the second step, NOHA is further converted to citrulline and nitric oxide (NO). To assess the mechanistic differences between the two steps of the reaction, we have used resonance Raman spectroscopy combined with a homemade continuous-flow rapid solution mixer to study the structural properties of the metastable dioxygen-bound complexes of the oxygenase domain of inducible NOS (iNOSoxy). We identified the O-O stretching frequency of the substrate-free enzyme at 1133 cm-1. This frequency is insensitive to the presence of tetrahydrobiopterin, but it shifts to 1126 cm-1 upon binding of Arg, which we attribute to H-bonding interactions to the terminal oxygen atom of the heme iron-bound dioxygen. In contrast, the addition of NOHA to the enzyme did not bring about a shift in the frequency of the O-O stretching mode, because, unlike Arg, there is no H-bond associated with the terminal oxygen atom of the dioxygen. The substrate-specific H-bonding interactions play a critical role in determining the fate of the key peroxy intermediate. In the first step of the reaction, the H-bonds facilitate the rupture of the O-O bond, leading to the formation of the active ferryl species, which is essential for the oxidation of the Arg. On the other hand, in the second step of the reaction, the absence of the H-bonds prevents the premature O-O bond cleavage, such that the peroxy intermediate can perform a nucleophilic addition reaction to the substrate, NOHA.

Original languageEnglish (US)
Pages (from-to)6943-6951
Number of pages9
JournalJournal of the American Chemical Society
Volume129
Issue number21
DOIs
StatePublished - May 30 2007

Fingerprint

Nitric oxide
Nitric Oxide Synthase
Protein Isoforms
Oxygen
Arginine
Substrates
Stretching
Enzymes
Nitric Oxide
Atoms
Addition reactions
Citrulline
Oxygenases
Raman Spectrum Analysis
Raman spectroscopy
Structural properties
Nitric Oxide Synthase Type II
Heme
Iron
Rupture

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase. / Li, David; Kabir, Mariam; Stuehr, Dennis J.; Rousseau, Denis L.; Yeh, Syun-Ru.

In: Journal of the American Chemical Society, Vol. 129, No. 21, 30.05.2007, p. 6943-6951.

Research output: Contribution to journalArticle

@article{d4f6d34b7af74c92ab05a323448a18f8,
title = "Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase",
abstract = "Nitric oxide synthase (NOS) catalyzes the formation of NO via a consecutive two-step reaction. In the first step, L-arginine (Arg) is converted to N-hydroxy-L-arginine (NOHA). In the second step, NOHA is further converted to citrulline and nitric oxide (NO). To assess the mechanistic differences between the two steps of the reaction, we have used resonance Raman spectroscopy combined with a homemade continuous-flow rapid solution mixer to study the structural properties of the metastable dioxygen-bound complexes of the oxygenase domain of inducible NOS (iNOSoxy). We identified the O-O stretching frequency of the substrate-free enzyme at 1133 cm-1. This frequency is insensitive to the presence of tetrahydrobiopterin, but it shifts to 1126 cm-1 upon binding of Arg, which we attribute to H-bonding interactions to the terminal oxygen atom of the heme iron-bound dioxygen. In contrast, the addition of NOHA to the enzyme did not bring about a shift in the frequency of the O-O stretching mode, because, unlike Arg, there is no H-bond associated with the terminal oxygen atom of the dioxygen. The substrate-specific H-bonding interactions play a critical role in determining the fate of the key peroxy intermediate. In the first step of the reaction, the H-bonds facilitate the rupture of the O-O bond, leading to the formation of the active ferryl species, which is essential for the oxidation of the Arg. On the other hand, in the second step of the reaction, the absence of the H-bonds prevents the premature O-O bond cleavage, such that the peroxy intermediate can perform a nucleophilic addition reaction to the substrate, NOHA.",
author = "David Li and Mariam Kabir and Stuehr, {Dennis J.} and Rousseau, {Denis L.} and Syun-Ru Yeh",
year = "2007",
month = "5",
day = "30",
doi = "10.1021/ja070683j",
language = "English (US)",
volume = "129",
pages = "6943--6951",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "21",

}

TY - JOUR

T1 - Substrate- and isoform-specific dioxygen complexes of nitric oxide synthase

AU - Li, David

AU - Kabir, Mariam

AU - Stuehr, Dennis J.

AU - Rousseau, Denis L.

AU - Yeh, Syun-Ru

PY - 2007/5/30

Y1 - 2007/5/30

N2 - Nitric oxide synthase (NOS) catalyzes the formation of NO via a consecutive two-step reaction. In the first step, L-arginine (Arg) is converted to N-hydroxy-L-arginine (NOHA). In the second step, NOHA is further converted to citrulline and nitric oxide (NO). To assess the mechanistic differences between the two steps of the reaction, we have used resonance Raman spectroscopy combined with a homemade continuous-flow rapid solution mixer to study the structural properties of the metastable dioxygen-bound complexes of the oxygenase domain of inducible NOS (iNOSoxy). We identified the O-O stretching frequency of the substrate-free enzyme at 1133 cm-1. This frequency is insensitive to the presence of tetrahydrobiopterin, but it shifts to 1126 cm-1 upon binding of Arg, which we attribute to H-bonding interactions to the terminal oxygen atom of the heme iron-bound dioxygen. In contrast, the addition of NOHA to the enzyme did not bring about a shift in the frequency of the O-O stretching mode, because, unlike Arg, there is no H-bond associated with the terminal oxygen atom of the dioxygen. The substrate-specific H-bonding interactions play a critical role in determining the fate of the key peroxy intermediate. In the first step of the reaction, the H-bonds facilitate the rupture of the O-O bond, leading to the formation of the active ferryl species, which is essential for the oxidation of the Arg. On the other hand, in the second step of the reaction, the absence of the H-bonds prevents the premature O-O bond cleavage, such that the peroxy intermediate can perform a nucleophilic addition reaction to the substrate, NOHA.

AB - Nitric oxide synthase (NOS) catalyzes the formation of NO via a consecutive two-step reaction. In the first step, L-arginine (Arg) is converted to N-hydroxy-L-arginine (NOHA). In the second step, NOHA is further converted to citrulline and nitric oxide (NO). To assess the mechanistic differences between the two steps of the reaction, we have used resonance Raman spectroscopy combined with a homemade continuous-flow rapid solution mixer to study the structural properties of the metastable dioxygen-bound complexes of the oxygenase domain of inducible NOS (iNOSoxy). We identified the O-O stretching frequency of the substrate-free enzyme at 1133 cm-1. This frequency is insensitive to the presence of tetrahydrobiopterin, but it shifts to 1126 cm-1 upon binding of Arg, which we attribute to H-bonding interactions to the terminal oxygen atom of the heme iron-bound dioxygen. In contrast, the addition of NOHA to the enzyme did not bring about a shift in the frequency of the O-O stretching mode, because, unlike Arg, there is no H-bond associated with the terminal oxygen atom of the dioxygen. The substrate-specific H-bonding interactions play a critical role in determining the fate of the key peroxy intermediate. In the first step of the reaction, the H-bonds facilitate the rupture of the O-O bond, leading to the formation of the active ferryl species, which is essential for the oxidation of the Arg. On the other hand, in the second step of the reaction, the absence of the H-bonds prevents the premature O-O bond cleavage, such that the peroxy intermediate can perform a nucleophilic addition reaction to the substrate, NOHA.

UR - http://www.scopus.com/inward/record.url?scp=34249776667&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34249776667&partnerID=8YFLogxK

U2 - 10.1021/ja070683j

DO - 10.1021/ja070683j

M3 - Article

C2 - 17488012

AN - SCOPUS:34249776667

VL - 129

SP - 6943

EP - 6951

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 21

ER -