Molybdenum and tungsten in nitrate reductase

Abstract

Spinach plants grown in purified sand culture without added molybdenum have no nitrate reductase. Enzyme activity in healthy plants also declines rapidly on nitrate starvation. When these plants are transferred to radioactively-labelled molybdate solution or nitrate solution containing the metal isotope, respectively, enzyme activity is induced in 24 h and subsequent enzyme purification shows specific association of radioactivity with nitrate reductase. Tungsten, which inhibits production of enzyme activity, was shown to form a tungsten analogue of nitrate reductase which was inactive with respect to nitrate reduction but retained the ability to act as an NADH 2-dehydrogenase. Vanadium, however, was not incorporated and failed to form such an analogue. Nitrate reductase protein-containing molybdenum or tungsten showed no in vitro exchange with molybdenum and neither metal could be removed by cyanide. Inhibition of enzyme activity of the molybdenum protein was freely reversed by removal of the cyanide. The differential effect of two inhibitors of nitrate reductase induction, L-azetidine-2-carboxylic acid (an analogue of proline), and puromycin (a peptide chain terminator), was illustrated using the 185W-labelled nitrate reductase. Puromycin inhibited 185W incorporation, whereas L-azetidine-2-carboxylic acid did not.