PDBsum entry 2ppa

Oxidoreductase

PDB id: 2ppa

Contents

Protein chains

336 a.a. *

Ligands

ACT ×10

N2O ×2

TRS

Metals

CU1 ×3

_CU ×3

Waters ×776

* Residue conservation analysis

PDB id:

2ppa

Name:

Oxidoreductase

Title:

Anaerobically manipulated wild type oxidized afnir bound to nitrous oxide

Structure:

Copper-containing nitrite reductase. Chain: a, b, c. Synonym: cu-nir. Engineered: yes

Source:

Alcaligenes faecalis. Organism_taxid: 511. Strain: s-6. Gene: nirk, nir. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.69Å R-factor: 0.193 R-free: 0.224

Authors:

E.I.Tocheva,M.E.P.Murphy

Key ref:

E.I.Tocheva et al. (2008). Conserved active site residues limit inhibition of a copper-containing nitrite reductase by small molecules. Biochemistry, 47, 4452-4460. PubMed id: 18358002

Date:

28-Apr-07 Release date: 01-Apr-08

Protein chains

P38501  (NIR_ALCFA) -  Copper-containing nitrite reductase from Alcaligenes faecalis

Seq: 376 a.a.

Struc: 336 a.a.

Enzyme reactions

• Enzyme class: E.C.1.7.2.1 - nitrite reductase (NO-forming).
• Reaction: nitric oxide + Fe(III)-[cytochrome c] + H2O = Fe(II)-[cytochrome c] + nitrite + 2 H⁺

nitric oxide + Fe(III)-[cytochrome c] + H2O (Bound ligand (Het Group name = N2O) matches with 66.67% similarity) = Fe(II)-[cytochrome c] + nitrite + 2 × H(+)

• Cofactor: Cu cation or Fe cation; FAD

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

reference

Biochemistry 47:4452-4460 (2008)

PubMed id: 18358002

Conserved active site residues limit inhibition of a copper-containing nitrite reductase by small molecules.

E.I.Tocheva, L.D.Eltis, M.E.Murphy.

ABSTRACT

The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.