IN-VITRO SYNTHESIS OF THE IRON-MOLYBDENUM COFACTOR OF NITROGENASE - PURIFICATION AND CHARACTERIZATION OF NIFB COFACTOR, THE PRODUCT OF NIFBPROTEIN

The requirement of NIFB activity for the biosynthesis of iron-molybede num cofactor (FeMo-co) can be satisfied by the addition of the low mol ecular weight product of NIFB, termed NifB cofactor (NifB-co). NifB-co has been purified to homogeneity by a unique one-step method. Additio n of NifB-co into the FeMo-co synthesis system generated nitrogenase a ctivity 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-c o synthesis is absolutely dependent on the presence of molybdate, homo citrate and active NIFNE protein in the reaction mixture. The cofactor appears to be a small Fe-S cluster synthesized by NIFB, as a precurso r 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 n m with no distinctive peaks or shoulders. NifB-co is stable to repeate d freeze-thaw cycles and is also stable in N-methylformamide, the solv ent used for the isolation of FeMo-co. The NifB-co is stable to a 5-mi n heat treatment at 60-degrees-C. The cofactor is extremely O2-labile, with half-life of less then 15 s in air.