Shah V K, Allen J R, Spangler N J, Ludden P W
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
J Biol Chem. 1994 Jan 14;269(2):1154-8.
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.
通过添加NIFB的低分子量产物（称为NifB辅因子，NifB-co），可以满足NIFB活性对铁钼辅因子（FeMo-co）生物合成的需求。NifB-co已通过独特的一步法纯化至同质。将NifB-co添加到FeMo-co合成系统中，产生的固氮酶活性为每分钟每纳摩尔铁形成27 - 32纳摩尔乙烯。铁是在NifB-co中检测到的唯一金属。依赖NifB-co的体外FeMo-co合成绝对依赖于反应混合物中钼酸盐、高柠檬酸和活性NIFNE蛋白的存在。该辅因子似乎是由NIFB合成的一个小的铁硫簇，作为FeMo-co的前体。在0 - 4000高斯范围内，4 K时NifB-co未显示任何电子顺磁共振信号。NifB-co溶液呈绿褐色，与FeMo-co相似。NifB-co在400至700 nm之间表现出宽泛的吸光度，无明显峰或肩峰。NifB-co对反复冻融循环稳定，在用于分离FeMo-co的溶剂N - 甲基甲酰胺中也稳定。NifB-co在60℃下热处理5分钟仍稳定。该辅因子对氧气极为敏感，在空气中半衰期小于15秒。
