Superoxide reductase from Nanoarchaeum equitans: expression, purification, crystallization and preliminary X-ray crystallographic analysis

Abstract

Superoxide reductases (SORs) are the most recent oxygen-detoxification system to be identified in anaerobic and microaerobic bacteria and archaea. SORs are metalloproteins that are characterized by their possession of a catalytic nonhaem iron centre in the ferrous form coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is the only organism known to date to serve as a host for Nanoarchaeum equitans, a nanosized hyperthermophilic archaeon isolated from a submarine hot vent which completely depends on the presence of and contact with I. hospitalis cells for growth to occur. Similarly to I. hospitalis, N. equitans has a neelaredoxin (a 1Fe-type SOR) that keeps toxic oxygen species under control, catalysing the one-electron reduction of superoxide to hydrogen peroxide. Blue crystals of recombinant N. equitans SOR in the oxidized form (12.7 kDa, 109 residues) were obtained using polyethylene glycol (PEG 2000 MME) as precipitant. These crystals diffracted to 1.9 Å resolution at 100 K and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.88, b = 82.01, c = 91.30 Å. Cell-content analysis suggested the presence of four monomers in the asymmetric unit. The Matthews coefficient (V(M)) was determined to be 1.9 Å(3) Da(-1), corresponding to an estimated solvent content of 36%. Self-rotation function and native Patterson calculations suggested a tetramer with 222 point-group symmetry, similar to other 1Fe-SORs. The three-dimensional structure will be determined by the molecular-replacement method.

Figure 1

(a) SDS–PAGE. Lane 1, low-molecular-weight markers; lane 2, N. equitans SOR protein sample eluted from the gel-filtration column. (b) UV–Vis absorption spectrum of pure N. equitans SOR; the region from 400 to 800 nm is amplified 5× in order to highlight the characteristic band with a maximum at ∼550 nm. (c) N. equitans SOR protein elution profile from a Superdex 200 (10/300) column. The 280 nm peak corresponds to a retention volume of 15.4 ml. The y axis corresponds to absorbance at 280 nm.

Figure 2

Blue crystals of N. equitans superoxide reductase (neelaredoxin) grown in 20%(v/v) PEG 2000 MME. The largest crystal dimensions are 0.1 × 0.08 × 0.04 mm.

Figure 3

Diffraction images collected on ESRF beamline ID23-1 from an N. equitans 1Fe-SOR single crystal (inset). A composite image of the first (top half, frame 1) and the last (bottom half, frame 241) diffraction images collected is shown, demonstrating the effect of crystal mosaicity on spot shape. The green scale bars in the inset are 100 µm in length. The mosaicity range of the collected data was 0.249–0.642°.

Figure 4

MOLREP self-rotation function calculation showing the presence of twofold NCS axes perpendicular to the crystallographic c axis. The resolution range of the self-rotation search was 15–3 Å.

Figure 5

w = 1/2 section of the native Patterson map showing a large non-origin peak corresponding to a noncrystallographic twofold axis parallel to the crystallographic c axis. The map was calculated using all data and contoured at 5σ intervals starting at 3σ. The origin peak is 133σ and the NCS peak is 38σ.