X-RAY STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF A NITRITE REDUCTASE FROM ALCALIGENES-FAECALIS S-6 - ROLES OF 2 COPPER ATOMS IN NITRITE REDUCTION

Nitrite reductase (NIR) from the denitrifying bacterium Alcaligenes faecalis S-6 is a copper-containing enzyme which requires pseudoazurin, a low molecular weight protein containing a single type I copper atom, as a direct electron donor in vivo. Crystallographic analysis shows that NIR is a trimer composed of three identical subunits, each of which contains one atom of type I copper and one atom of type II copper, and that the ligands to the type I and type II copper atoms are the same as those of the Achromobacter cycloclastes NIR. An efficient NIR expression-secretion system in Escherichia coli was constructed and used for site-directed mutagenesis. An NIR mutant with a replacement of the type II copper ligand, His135, by Lys still retained a type II copper site as well as a type I copper atom, but it completely lost nitrite-reducing activity as measured with methyl viologen as an electron donor. On the other hand, another mutant with a replacement of the type I copper ligand, Met150, by Glu contained only a type II copper atom, but it still retained significant nitrite-reducing activity with methylviologen. When pseudoazurin was used as an electron donor for the reaction, however, Met150Glu failed to catalyze the reduction of nitrite. Kinetic analysis of the electron transfer between NIR and pseudoazurin revealed that the electron-transfer rate between Metl50Glu and pseudoazurin was reduced 1000-fold relative to that of wild-type NIR. These results clearly indicate that the type I copper site in NIR plays a crucial role for electron transfer from pseudoazurin to the type II copper site of NIR, which comprises the catalytic center of NIR for the reduction of nitrite.