X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 angstrom resolution

Copper-containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer-monomer interface, resembles the catalytic sites of a number of Zn enzymes. Nitrite reductase from Alcalige nes xylosoxidans has optimum activity at pH 5.2 which decreases to a neglig ible level at pH 8. The structure of this nitrite reductase has previously been determined at pH 4.6. It has now been crystallized under new condition s at pH 8.5. Its crystallographic structure provides a structural explanati on for the greatly reduced activity of the enzyme at high pH. Characterizat ion of overexpressed protein in solution by EXAFS suggested that the protei n lacked Cu in the catalytic type 2 Cu site and that the site was most prob ably occupied by Zn. Using the anomalous signals from Cu and Zn, the crysta l structure revealed that the expressed protein was devoid of Cu in the cat alytic site and that only a trace amount (<10%) of Zn was present at this s ite in the crystal. Despite the close structural similarity of the catalyti c site to a number of Zn enzymes, these data suggest that Zn, if it binds a t the catalytic copper site, binds weakly in nitrite reductase.