PDBsum entry 2ppf

Oxidoreductase

PDB id: 2ppf

Contents

Protein chains

336 a.a. *

Ligands

ACT ×8

_NO ×4

TRS

Metals

CU1 ×5

_CU ×3

Waters ×998

* Residue conservation analysis

PDB id:

2ppf

Name:

Oxidoreductase

Title:

Reduced mutant d98n of afnir exposed to nitric oxide

Structure:

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

Source:

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

Resolution:

1.65Å R-factor: 0.151 R-free: 0.170

Authors:

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

Key ref:

E.I.Tocheva et al. (2007). Stable copper-nitrosyl formation by nitrite reductase in either oxidation state. Biochemistry, 46, 12366-12374. PubMed id: 17924665

Date:

28-Apr-07 Release date: 15-Jan-08

Protein chains

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

Seq: 376 a.a.

Struc: 338 a.a.*

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

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 = NO) corresponds exactly) = 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 46:12366-12374 (2007)

PubMed id: 17924665

Stable copper-nitrosyl formation by nitrite reductase in either oxidation state.

E.I.Tocheva, F.I.Rosell, A.G.Mauk, M.E.Murphy.

ABSTRACT

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas.