Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase

• Published in: Journal of Bacteriology 1999-05, Vol.181 (9), p.2797-2801
• Main Authors: Shah, V.K, Rangaraj, P, Chatterjee, R, Allen, R.M, Roll, J.T, Roberts, G.P, Ludden, P.W
• Format: Article
• Language: English
• Subjects: Azotobacter vinelandii; Azotobacter vinelandii - enzymology; bacterial proteins; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Bacteriology; biosynthesis; Cell-Free System; coenzymes; cofactors; Genes, Bacterial; Molybdoferredoxin - biosynthesis; Mutation; Nitrogen Fixation - genetics; nitrogenase; Nitrogenase - biosynthesis; Physiology and Metabolism; Proteins
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/JB.181.9.2797-2801.1999

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42.48 (deltanifENX deltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.