Structure of Pseudomonas aeruginosai zinc azurin mutant Asn47Asp at 2.4 Å resolution

The Pseudomonas aeruginosa azurin mutant Asn47Asp has been isolated, its spectroscopic and kinetic properties characterized, and the X-ray crystal structure of its zinc derivative determined. While the optical and electron paramagnetic resonance spectra as well as the electron-transfer activity of the mutant are very similar to the wild-type values, the Asn47Asp reduction potential is slightly increased by 20 mV. The mutant crystallized in the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 57.8, b = 81.5 and c = 112.6 Å. There are four molecules in the asymmetric unit, packed as a tetramer which consists of two independent dimers. The zinc site of this mutant structure is similar to the wild-type zinc azurin and, in particular, the metal-binding site is almost identical to the site found in the wild-type zinc-azurin structure [Nar, Huber, Messerschmidt, Filippou, Barth, Jaquinod, Kamp & Canters (1992). Eur. J. Biochem. 205, 1123–1129]. The Asp47 side chain at that mutation site takes on a very similar orientation to Asn47 in the wild-type structure preserving the two hydrogen bonds with the neighbouring Thr113 NH and O^γH. Therefore, the increased reduction potential of the mutant is probably a result of an altered charge distribution close to the metal site.