Purification and characterization of the inactive MoFe protein ( NifB - Kpj ) of the nitrogenase from nifB mutants of Klebsiellapneumoniae Timothy

The inactive MoFe protein of nitrogenase, NifB-Kp 1, from two distinct niB mutants of Klebsiella pneumoniae, Kp5058 (a nifB point mutant) and UNF1718 (a nijB, nifJ double mutant) has been purified and characterized. NifB-Kpl can be activated by reaction with the iron-molybdenum cofactor, FeMoco, extracted from active MoFe protein. NifB-Kpl purified from either source had similar properties and was contaminated with an approximately equimolar amount of protein of mol.wt. 21000. Like active wild-type Kp 1, it was an a2/J2 tetramer, but it was far less stable than Kpl, deteriorating rapidly at temperatures above 8°C or on mild oxidation. NifB-Kpl preparations contained 0.4-0.9 Mo and 9.0 + 0.9 Fe atoms mol-h and, when activated by FeMoco, had a specific activity of approx. 500 units mg-'. The Mo in our preparations was not associated with the e.p.r. signal normally observed from FeMoco. All preparations exhibited a weak gav = 1.95 e.p.r. signal which was probably not associated with activatable protein.