PDBsum entry: 1et7

Oxidoreductase

PDB id: 1et7

Contents

Protein chain

339 a.a. *

Metals

_CD ×3

_CU ×2

Waters ×243

* Residue conservation analysis

PDB id:

1et7

Name:

Oxidoreductase

Title:

Crystal structure of nitrite reductase his255asp mutant from alcaligenes faecalis s-6

Structure:

Nitrite reductase. Chain: a. Fragment: 40 - 376. Synonym: cu-nir. Engineered: yes. Mutation: yes

Source:

Alcaligenes faecalis. Organism_taxid: 511. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PDB file)

Resolution:

1.70Å R-factor: 0.188 R-free: 0.210

Authors:

M.J.Boulanger,M.Kukimoto,M.Nishiyama,S.Horinouchi,M.E.P.Murp

Key ref:

M.J.Boulanger et al. (2000). Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase. J Biol Chem, 275, 23957-23964. PubMed id: 10811642 DOI: 10.1074/jbc.M001859200

Date:

12-Apr-00 Release date: 24-Aug-00

Protein chain

P38501  (NIR_ALCFA) -  Copper-containing nitrite reductase

Seq: 376 a.a.

Struc: 339 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 + H₂O + ferricytochrome c = nitrite + ferrocytochrome c + 2 H⁺
• Cofactor: Copper or iron; FAD

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

 Gene Ontology (GO) functional annotation 

• Cellular component: periplasmic space (1 term)
• Biological process: nitrogen compound metabolic process (3 terms)
• Biochemical function: oxidoreductase activity (4 terms)

reference

DOI no: 10.1074/jbc.M001859200

J Biol Chem 275:23957-23964 (2000)

PubMed id: 10811642

Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase.

M.J.Boulanger, M.Kukimoto, M.Nishiyama, S.Horinouchi, M.E.Murphy.

ABSTRACT

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1. 9-A resolution. In the native structure, His-255 Nepsilon2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.

Selected figure(s)

Figure 2.
Fig. 2. Structures of native and mutant forms of AfNIR. Upper left, the active site of native AfNIR is depicted with the type II copper atom as a brown sphere and the water molecules as aquamarine spheres. The side chains of the three histidine copper ligands and His-255 are blue. Asp-98 is colored red. The backbone secondary structure elements are light blue (N-terminal domain) and yellow (C-terminal domain of the neighboring subunit). Potential hydrogen bonds are in dotted lines. Upper right, an analogous representation of D98N AfNIR in which Asn-98 is colored light red. Lower left, the structure of H255N AfNIR is depicted with Asn-255 in light red. Lower right, Asp-255 is drawn in red in the H255D AfNIR structure. Images were created using Raster3D (34) and Molscript (35).

Figure 3.
Fig. 3. The proposed active site hydrogen bond networks in native AfNIR and the D98N, H255N, and H255D mutants.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 23957-23964) copyright 2000.

Figures were selected by the author.