Comparative characterisation of nitrate reductase from wild-type and molybdenum cofactor-defective cell cultures of Nicotiana tabacum

Abstract

Affinity-purified nitrate reductase (EC 1.6.6.1) from wild-type and the molybdenum cofactor-defective cnx-68/2 cell line of Nicotiana tabacum has been comparatively characterised with respect to enzymological properties. K m values and pH optima for the 4 nitrate reductase-associated activities, including diaphorase, are presented. Exposure to different inhibitors and moderate heat (45°C) reveal the tobacco nitrate reductase to be functionally divided into two distinct parts. Both the wild-type and cnx mutant nitrate reductase contain a heme group of cytochrome b type, possess an identical sedimentation coefficient of 7.6S and an identical gel filtration derived molecular weight of 200 000. Both enzymes were found to exhibit similar enzymatic parameters, inhibitor specificities and heat stabilities. Upon affinity chromatography, varying portions of the 7.6S holoenzyme dissociate into slower sedimenting (about 4S) subunits with cytochrome c reductase activity, which has been established for the wild-type as well as the cnx mutant enzyme. From the data obtained it can be concluded that (1) the apoprotein of the cnx mutant nitrate reductase is unaffected by the mutation while the defect of this enzyme type resides exclusively in the molybdenum-cofactor; (2) the cnx mutant nitrate reductase possesses a molybdenum-cofactor, defective in its catalytic properties, but still able to mediate the assembly of the cytochrome c reductase subunits to form the 7.6S holoenzyme.