Using electrophoresis to observe the interaction of nitrogenase with ions.

Abstract

The two protein components of nitrogenase from Klebsiella pneumoniae were shown to interact with metal ions and ADP, altering their electrophoretic mobility in polyacrylamide gel electrophoresis. Both Mg+2 and Mn+2 caused reduced mobility of Fe protein relative to other proteins. The effect was about 50% complete at concentrations around 0.2 mM. Other ions including Fe+2, Ni+2 and Co+2 had no observable effect at levels up to 1 _mM. Both Cd+2 and Zn+2 appeared to interact with the protein; Cd+2 at 0.5 mM dramatically destabilized the protein. The effects of more than a dozen different mutations of the Fe protein on Mg+2 interaction were examined. All mutated proteins appeared to interact with Mg+2 similarly to wild-type. Using relative mobility differences of charge-changed mutants it was estimated that two to three Mg+2 interact with each Fe protein monomer. The MoFe protein also showed interaction with metal ions but the alteration of mobility was much smaller than for the Fe protein because it is larger and less acidic, so that it runs much more slowly than the Fe protein in standard gels. The interaction of ADP with Fe protein was examined in the presence of Mg+2. Increasing ADP partially reversed the mobility decrease observed on Mg+2 binding, and produced a more diffuse protein band indicative of a reaction zone of interconverting conformers. No alteration of MoFe protein mobility was observed with ADP added during electrophoresis.