Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12

Abstract

Nitrate reductase was purified 134-fold from E. coli K12. The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope. The purified enzyme exists in aqueous solution either as a monomer (mol. wt.[molecular weight] about 220,000) or as an associated form (probably a tetrameter; mol.wt. about 880,000). The purified enzyme has 3 subunits with apparent mol. wts. of 150,000, 67,000 and 65,000. An additional subunit of apparent mol. wt. 20,000 is present in a haeme-containing fraction that is also produced by the preparative procedure described. None of the enzyme subunits is present in cell envelope of cells grown in the absence of nitrate. Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3- activity.