Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase.
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.
Shah
V K
VK
Departments of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Rangaraj
P
P
Chatterjee
R
R
Allen
R M
RM
Roll
J T
JT
Roberts
G P
GP
Ludden
P W
PW
D016948:Azotobacter vinelandii / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002474:Cell-Free System; D005798:Genes, Bacterial; D008983:Molybdoferredoxin / Q000096:biosynthesis*; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000096:biosynthesis*
D001426:Bacterial Proteins; D008983:Molybdoferredoxin; C118795:NifX protein, Bacteria; D009591:Nitrogenase