PDBsum entry: 3dwj

Oxidoreductase

PDB id: 3dwj

Contents

Protein chains

408 a.a. *

Ligands

HEM ×2

H4B ×2

SO4

NH4 ×2

Waters ×323

* Residue conservation analysis

PDB id:

3dwj

Name:

Oxidoreductase

Title:

Heme-proximal w188h mutant of inducible nitric oxide synthase

Structure:

Nitric oxide synthase, inducible. Chain: a, b. Fragment: oxygenase domain 66-496. Synonym: nos type ii, inducible no synthase, inducible nos, inos, macrophage nos, mac- nos. Engineered: yes. Mutation: yes

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Gene: nos2, inosl. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.75Å R-factor: 0.229 R-free: 0.294

Authors:

J.Tejero,A.Biswas,Z.-Q.Wang,M.M.Haque,C.Hemann,J.L.Zweier, R.C.Page,S.Misra,D.J.Stuehr

Key ref:

J.Tejero et al. (2008). Stabilization and Characterization of a Heme-Oxy Reaction Intermediate in Inducible Nitric-oxide Synthase. J Biol Chem, 283, 33498-33507. PubMed id: 18815130 DOI: 10.1074/jbc.M806122200

Date:

22-Jul-08 Release date: 30-Sep-08

Protein chains

P29477  (NOS2_MOUSE) -  Nitric oxide synthase, inducible

Seq: 1144 a.a.

Struc: 408 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.14.13.39 - Nitric-oxide synthase.
• Reaction: L-arginine + n NADPH + n H⁺ + m O₂ = citrulline + nitric oxide + n NADP⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: oxidation-reduction process (2 terms)
• Biochemical function: calmodulin binding (7 terms)

reference

DOI no: 10.1074/jbc.M806122200

J Biol Chem 283:33498-33507 (2008)

PubMed id: 18815130

Stabilization and Characterization of a Heme-Oxy Reaction Intermediate in Inducible Nitric-oxide Synthase.

J.Tejero, A.Biswas, Z.Q.Wang, R.C.Page, M.M.Haque, C.Hemann, J.L.Zweier, S.Misra, D.J.Stuehr.

ABSTRACT

Nitric-oxide synthases (NOS) are heme-thiolate enzymes that N-hydroxylate l-arginine (l-Arg) to make NO. NOS contain a unique Trp residue whose side chain stacks with the heme and hydrogen bonds with the heme thiolate. To understand its importance we substituted His for Trp(188) in the inducible NOS oxygenase domain (iNOSoxy) and characterized enzyme spectral, thermodynamic, structural, kinetic, and catalytic properties. The W188H mutation had relatively small effects on l-Arg binding and on enzyme heme-CO and heme-NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild-type iNOSoxy, indicating it decreased heme-thiolate electronegativity. The protein crystal structure showed that the His(188) imidazole still stacked with the heme and was positioned to hydrogen bond with the heme thiolate. Analysis of a single turnover l-Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Its build up coincided kinetically with the disappearance of the enzyme heme-dioxy species and with the formation of a tetrahydrobiopterin (H(4)B) radical in the enzyme, whereas its subsequent disappearance coincided with the rate of l-Arg hydroxylation and formation of ferric enzyme. We conclude: (i) W188H iNOSoxy stabilizes a heme-oxy species that forms upon reduction of the heme-dioxy species by H(4)B. (ii) The W188H mutation hinders either the processing or reactivity of the heme-oxy species and makes these steps become rate-limiting for l-Arg hydroxylation. Thus, the conserved Trp residue in NOS may facilitate formation and/or reactivity of the ultimate hydroxylating species by tuning heme-thiolate electronegativity.

Selected figure(s)

Figure 1.
Reaction scheme for the single turnover l-Arg hydroxylation of NOS enzymes. After formation of the ferrous dioxygen complex (I) the subsequent steps are fast and none of the three putative intermediates (II, III, and IV; dashed boxes) have been spectroscopically observed in single turnover reactions. See text for details.

Figure 2.
Spectral properties of the W188H iNOSoxy mutant in the presence of H[4]B and l-Arg. Representative spectra for the enzyme in the oxidized and reduced states, as well as the Fe^II-CO, Fe^II-NO, and Fe^III-NO complexes are shown.

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 33498-33507) copyright 2008.

Figures were selected by an automated process.