STRUCTURE OF ALCALIGENES-FAECALIS NITRITE REDUCTASE AND A COPPER SITEMUTANT, M150E, THAT CONTAINS ZINC

The structures at 2.0 and 2.25 Angstrom resolution of native and recom binant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Ach romobacter cycloclastes. The crystallographic structure of a mutant, M 150E, which unlike the wildtype protein cannot be reduced by pseudoazu rin, shows that the glutamate replacement for methionine binds to a me tal at the type I Cu site via only one oxygen. Anomalous scattering da ta collected at wavelengths of 1.040 and 1.377 Angstrom reveal that th e metal at the type I site is a Zn. No significant differences from th e native structure other than local perturbations at the type I site a re seen, A local pseudo 2-fold axis relates the two domains of differe nt monomers which form the active site. The two residues, Asp98 and Hi s255, believed to be involved in catalysis are related by this 2-fold. An unusual + - + charge interaction between Lys269, Glu279, and His10 0 helps to orient the active site Cu ligand, His100. A number of negat ively charged surface residues create an electrostatic field whose sha pe suggests that it may serve to direct incoming negatively charged ni trite as well as to dock the electron donor partner, pseudoazurin.