Reconstitution of NADH-nitrate reductase in vitro from nitrate reductase-deficient Nicotiana tabacum mutants

Abstract

Two types of nitrate reductase-deficient mutant cell lines (nia and cnx) of Nicotiana tabacum have been used for in vitro reconstitution of NADH-nitrate reductase. The cnx mutants simultaneously lack NADH-,FADH2-, red benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are interpreted to be defective in the molybdenum-containing cofactor necessary for nitrate reductase activity. In the nia lines xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities of nitrate reductase, including NADH-cytochrome c reductase. When cnx cells (induced by nitrate) were homogenized together with nia cells (induced by nitrate or uninduced), NADH-nitrate reductase activity was detectable in the cell extract. No nitrate reductase was observed when the cnx mutants were homogenized together, or after cohomogenization of the nia mutants. Thus, the inactive nitrate reductase molecule formed in the cnx mutants has been complemented in vitro with the molybdenum-containing cofactor supplied by nia extracts, thus giving rise to NADH-nitrate reductase activity. This result gives additional support to the interpretation that the active nitrate reductase of Nicotiana tabacum is composed of at least the NADH-cytochrome c reductase moiety and a molybdenum-containing cofactor which is formed by the action of the cnx gene product(s).