Biochemical characterization of cnx nitrate reductase-deficient mutants of Nicotiana plumbaginifolia

In cnx mutants of Nicotiana plumbaginifolia, the over-expression of nitrate reductase (NR)-associated NADH cytochrome c reductase (Ccr) activity appears to be correlated with increased levels of NR mRNA. However, in ELISA tests, cnxB, C, D, E and F extracts present low levels of cross-reacting material, whereas cnxA extracts exhibit very high levels, indicating an altered NR structure in all cnx mutants except cnxA, due to the absence of a functional molybdenum cofactor (MoCo). Detection of dimeric NR in all cnx mutants, either by gel filtration or native gel electrophoresis, shows that the cofactor is not required for enzyme dimerization, but probably for the stability of the dimer. Similarities between results obtained with cnxB through F mutants, and those nia mutants impaired in the molybdopterin domain, suggest a related NR structure.