Presence of the molybdenum cofactor in nitrate reductase deficient mutant cell lines of nicotiana tabacum
Mendel, R.R.; Alikulov, Z.A.; Lvov, N.P.; Mueller, A.J.
Molecular and General Genetics 181(3): 395-399
N. tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the Mo-cofactor by using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The Mo-cofactor of the tobacco wild-type line complemented efficiently the N. crassa nit-1 mutant in vitro. The Mo-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5-10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with 2 tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The 4 cnx mutants studied were shown to contain exclusively an inactive form of the Mo-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1-10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial activity. Thus the defect of the cnx mutants resides in a lack of Mo as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was independent of the Mo content of the cofactor.