Crystallization and preliminary X-ray crystallographic studies of dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151

Dissimilatory nitrite reductase isolated from Hyphomicrobium denitrificans A3151 (HdNIR) is a novel copper‐containing nitrite reductase (CuNIR) composed of six identical subunits. One plastocyanin‐like domain and one green CuNIR‐like domain are connected to each other, suggesting that the HdNIR subunit structure resembles a complex of green CuNIR and pseudoazurin (or azurin). Recombinant HdNIR protein was crystallized using the hanging‐drop vapour‐diffusion method with PEG 4000 as the precipitant at pH 8.9. X‐ray diffraction data were collected to 2.35 Å resolution. The HdNIR crystal belonged to the tetragonal space group P41 (or P43), with unit‐cell parameters a = b = 221.9, c = 165.2 Å, giving 12 molecules (two hexamers) per asymmetric unit and a solvent content of 64%. A mutant form of HdNIR, C260A, which lacks the type I copper ion in the CuNIR‐like domain, was prepared and crystallized under wild‐type HdNIR conditions. The C260A mutant crystal belonged to the cubic space group P4332 (or P4132), with unit‐cell parameters a = b = c = 153.7 Å, giving one molecule per asymmetric unit and a solvent content of 59%. X‐ray diffraction data were collected to 3.5 Å resolution. To solve the crystal structure of HdNIR, the multiwavelength anomalous dispersion (MAD) method and the molecular‐replacement method are currently being used.