PDBsum entry 2vm3

Oxidoreductase

PDB id: 2vm3

Contents

Protein chain

333 a.a. *

Ligands

PG4

Metals

_CU ×2

_ZN

Waters ×230

* Residue conservation analysis

PDB id:

2vm3

Name:

Oxidoreductase

Title:

Structure of alcaligenes xylosoxidans in space group r3 - 1 of 2

Structure:

Dissimilatory copper-containing nitrite reductase. Chain: a. Fragment: residues 25-360. Synonym: nitrite reductase, nir. Engineered: yes

Source:

Achromobacter xylosoxidans. Organism_taxid: 85698. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.80Å R-factor: 0.186 R-free: 0.210

Authors:

M.A.Hough,S.V.Antonyuk,R.W.Strange,R.R.Eady,S.S.Hasnain

Key ref:

M.A.Hough et al. (2008). Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase. J Mol Biol, 378, 353-361. PubMed id: 18353369 DOI: 10.1016/j.jmb.2008.01.097

Date:

22-Jan-08 Release date: 05-Feb-08

Protein chain

O68601  (O68601_ALCXX) -  Copper-containing nitrite reductase from Alcaligenes xylosoxydans xylosoxydans

Seq: 360 a.a.

Struc: 333 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⁺
• Cofactor: Cu cation or Fe cation; FAD

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

reference

DOI no: 10.1016/j.jmb.2008.01.097

J Mol Biol 378:353-361 (2008)

PubMed id: 18353369

Crystallography with online optical and X-ray absorption spectroscopies demonstrates an ordered mechanism in copper nitrite reductase.

M.A.Hough, S.V.Antonyuk, R.W.Strange, R.R.Eady, S.S.Hasnain.

ABSTRACT

Nitrite reductases are key enzymes that perform the first committed step in the denitrification process and reduce nitrite to nitric oxide. In copper nitrite reductases, an electron is delivered from the type 1 copper (T1Cu) centre to the type 2 copper (T2Cu) centre where catalysis occurs. Despite significant structural and mechanistic studies, it remains controversial whether the substrates, nitrite, electron and proton are utilised in an ordered or random manner. We have used crystallography, together with online X-ray absorption spectroscopy and optical spectroscopy, to show that X-rays rapidly and selectively photoreduce the T1Cu centre, but that the T2Cu centre does not photoreduce directly over a typical crystallographic data collection time. Furthermore, internal electron transfer between the T1Cu and T2Cu centres does not occur, and the T2Cu centre remains oxidised. These data unambiguously demonstrate an 'ordered' mechanism in which electron transfer is gated by binding of nitrite to the T2Cu. Furthermore, the use of online multiple spectroscopic techniques shows their value in assessing radiation-induced redox changes at different metal sites and demonstrates the importance of ensuring the correct status of redox centres in a crystal structure determination. Here, optical spectroscopy has shown a very high sensitivity for detecting the change in T1Cu redox state, while X-ray absorption spectroscopy has reported on the redox status of the T2Cu site, as this centre has no detectable optical absorption.

Selected figure(s)

Figure 1.
Fig. 1. Combined spectroscopic and crystallographic measurements on an AxNiR crystal. (a) Optical spectra of AxNiR crystals. The crystal prior to X-ray exposure shows an absorption spectrum with a peak at 595 nm, characteristic of T1Cu(II) in oxidised AxNiR (solid line). This peak is no longer present at the end of the collection of the first 1.90 Å resolution crystallographic dataset (dotted line), indicating that the T1Cu centre has been reduced during the experiment. (b) XAS spectrum measured following the collection of the AxNiR2 crystal structure (dotted line). The solid line spectrum is from a similar crystal, which had not been exposed to X-ray radiation prior to the XAS scan. A small shift in the edge to lower energy is apparent, consistent with photoreduction of the T1Cu site, but the spectrum indicates that T2Cu is still in the Cu(II) form. Inset: XAS spectra of the crystal following the AxNiR2 data collection (red line) compared with oxidised (solid black line) and reduced (dashed black line) AxNiR solutions, and XAS spectra of the ‘oxidised’ AxNiR crystal spectrum (blue line) and a ‘fully reduced’ AxNiR crystal following exposure to a dose of 3 × 10^7 Gy of 1.37 Å X-rays (green line) in which the ‘edge-shoulder’ feature attributed to reduced T2Cu is clearly present. Note that the ‘oxidised’ crystal has undergone some X-ray-induced reduction of the T1Cu site during the XAS data collection. The X-ray dose for solution data is more than an order of magnitude less than on the crystallographic beamline.

Figure 4.
Fig. 4. Re-examination of nitrite-soaked crystal structures of NiR. (a) Electron density maps suggesting the presence of Cu-bound NO at the T2Cu site of nitrite-soaked AcNiR (PDB accession code 2bwi^2). The 2F[o] − F[c] map contoured at 1σ is shown in cyan, where the model includes 0.35 occupancy of NO[2 ]^−, 0.25 occupancy of NO and 0.25 occupancy of water. The F[o] − F[c] omit map contoured at 4σ, calculated by omitting NO from the model, is shown in red, suggesting a significant fraction of NO at this site. (b) The structure of AcNiR with endogenously bound NO[2 ]^− and NO (PDB accession code 2bw5^2). NO was removed from the model for the calculation of the F[o] − F[c] omit map (red; contoured at 4σ). (c) 2F[o] − F[c] electron density map contoured at 1σ showing Cu-coordinated NO in the structure of nitrite-soaked RsNiR (PDB accession code 2dws^31). NO is modelled with an occupancy of 0.7.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 378, 353-361) copyright 2008.

Figures were selected by the author.