Purification and characterization of the inactive MoFe protein (NifB-Kp1) of the nitrogenase from nifB mutants of Klebsiella pneumoniae

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Abstract
The inactive MoFe protein of nitrogenase, NifB-Kp1, from 2 distinct nifB mutants of K. pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nifB, nifJ double mutant) was purified and characterized. NifB-Kp1 can be activated by reaction with the Fe-Mo cofactor, FeMoco, extracted from active MoFe protein. NifB-Kp1 purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of Mw 21,000. Like active wild-type Kp1, it was an .alpha.2.beta.2 tetramer, but it was far less stable than Kp1, deteriorating rapidly at temperatures above 8.degree. C or on mild oxidaiton. NifB-Kp1 preparations contained 0.4-0.9 Mo and 9.0 .+-. 0.9 Fe atoms.cntdot.mol-1 and, when activated by FeMoco, had a specific activity of .apprx. 500 U.cntdot.mg-1. The Mo in the preparations was not associated with the EPR signal normally observed from FeMoco. All preparations exhibited a weak gav. = 1.95 EPR signal which was probably not associated with activatable protein.