In vitro synthesis of the iron-molybdenum cofactor of nitrogenase. Purification and characterization of NifB cofactor, the product of NIFB protein.
The requirement of NIFB activity for the biosynthesis of iron-molybdenum cofactor (FeMo-co) can be satisfied by the addition of the low molecular weight product of NIFB, termed NifB cofactor (NifB-co). NifB-co has been purified to homogeneity by a unique one-step method. Addition of NifB-co into the FeMo-co synthesis system generated nitrogenase activity of 27-32 nmol of ethylene formed/min/nmol of iron. Iron is the only metal detected in the NifB-co. NifB-co-dependent in vitro FeMo-co synthesis is absolutely dependent on the presence of molybdate, homocitrate and active NIFNE protein in the reaction mixture. The cofactor appears to be a small Fe-S cluster synthesized by NIFB, as a precursor of FeMo-co. NifB-co did not display any EPR signal at 4 K in 0-4000 gauss range. A solution of NifB-co is greenish-brown in color, similar to FeMo-co. NifB-co exhibits a broad absorbance between 400 and 700 nm with no distinctive peaks or shoulders. NifB-co is stable to repeated freeze-thaw cycles and is also stable in N-methylformamide, the solvent used for the isolation of FeMo-co. The NifB-co is stable to a 5-min heat treatment at 60 degrees C. The cofactor is extremely O2-labile, with half-life of less then 15 s in air.
Shah
V K
VK
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Allen
J R
JR
Spangler
N J
NJ
Ludden
P W
PW
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism; D058085:Iron Compounds / Q000302:isolation & purification*; D007711:Klebsiella pneumoniae / Q000737:chemistry*; D008983:Molybdoferredoxin; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000737:chemistry*; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D058085:Iron Compounds; D008983:Molybdoferredoxin; C085381:NifB cofactor; D007501:Iron; D009591:Nitrogenase