Zinc-substituted pseudoazurin solved by S/Zn-SAD phasing.

Abstract

The copper(II) centre of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by zinc(II) via denaturing the protein, chelation and removal of copper and refolding the apoprotein, followed by the addition of an aqueous solution of ZnCl2. Vapour-diffusion experiments produced colourless hexagonal crystals (space group P65), which when cryocooled had unit-cell parameters a=b=49.01, c=98.08 Å. Diffraction data collected at 100 K using a copper sealed tube were phased by the weak anomalous signal of five S atoms and one Zn atom. The structure was fitted manually and refined to 1.6 Å resolution. The zinc-substituted protein exhibits similar overall geometry to the native structure with copper. Zn2+ binds more strongly to its four ligand atoms (His40 Nδ1, Cys78 Sγ, His81 Nδ1 and Met86 Sδ) and retains the tetrahedral arrangement, although the structure is less distorted than the native copper protein.