Evidence for a molybdenum cofactor common to all molybdenum enzymes based on the in vitro assembly of assimilatory NADPH-nitrate reductase using the Neurospora mutant nit-1

Abstract

The in vitro assembly of an active Neurospora-like assimilatory NADPH—nitrate reductase (E.C. 1.6.6.2) has been effected by incubation of cell-free extracts of the Neurospora crassa nitrate reductase mutant nit-1 with (a) cell-free preparation of certain non-allelic mutants or uninduced wild type or (b) acid-treated molybdenum enzyme from diverse phylogenetic sources (20 different non-molybdenum enzymes were found to be inactive under the same experimental conditions). There is also a specific stimulatory effect of added molybdenum compounds on the in vitro assembly of NADPH-nitrate reductase. The incorporation of 99Mo, or its in vivo competitive inhibitor 185W, occurred in the in vitro assembly of nitrate reductase from a labelled, cell-free preparation of uninduced wild type (obtained by growth on a 99MoO 4 2− or 185WO 4 2−-containing medium) when incubated with an extract of unlabelled induced nit-1. Reciprocal experiments using labelled nit-1 and unlabelled, uninduced wild type showed no incorporation of radioactivity in the in vitro assembly of the enzyme. The results provide direct evidence for the view that molybdenum, presumably as a component of a larger, and as yet, unidentified cofactor, is furnished exclusively by uninduced wild type or acid-treated molybdenum enzymes in the in-vitro assembly system. It is postulated that the molybdenum cofactor of molecular weight approximately 1000 serves as both a link that binds the enzyme sub-units of nit-1 to yield the active enzyme and as an electron carrier.