Hexaheme nitrite reductase from Desulfovibrio desulfuricans (ATCC 27774)

This chapter focuses on purification and various properties of hexaheme nitrite reductase from Desulfovibrio desulfuricans. Hexaheme nitrite reductase is the second enzyme involved in dissimilatory nitrate reduction performed by a number of anaerobically grown bacteria. The assay procedure for the nitrite reductase involves two separate steps (1) conversion of nitrite to ammonia by the nitrite reductase and (2) quantitative determination of ammonia by the indophenol reaction. One unit of enzyme activity is the amount of enzyme that catalyzes the reduction of one μmol of nitrite to ammonia per minute at 37〫. All purification procedures described in the chapter were carried out at 4〫 and the buffers used were at pH 7.6. The purified nitrite reductase exhibits a typical c-type cytochrome absorption spectrum. The heme of the purified nitrite reductase could not be dissociated from the polypeptide backbone by extraction with HCl–acetone, but a hemin is released from the protein on treatment with Ag₂SO₄. The purified nitrite reductase could catalyze the reduction of nitrite or hydroxylamine to form ammonia.