Nitrogenase: the reaction between iron protein and bathophenanthrolinedisulfonate as a probe for interactions with MgATP

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Abstract
The reaction between the Fe(II) chelating agent, bathophenanthrolinedisulfonate and the Fe-S cluster in the Fe protein of nitrogenase from Clostridium pasteurianum was studied. This reaction is greatly accelerated by the presence of MgATP. Analysis of the relationship between reaction rate and concentration of MgATP supports a model in which both of 2 binding sites for MgATP on the Fe protein must be occupied before the protein undergoes a conformational change, allowing the Fe-S site to react rapidly with chelator. This model is also consistent with presently available data on equilibrium binding of MgATP to the Fe protein. MgADP inhibits the effect of MgATP on the chelator reaction in a manner which suggests that MgADP binds strongly to 1 of the MgATP sites and more weakly to the other. Loss of enzymic activity due to exposure to O2 or 0.degree. C is accompanied by a decrease in the ATP-specific chelator reaction. Hence, this reaction was used to estimate the concentration of active Fe-S centers for the purpose of computing the extinction coefficient of the Fe protein, giving the value .DELTA..epsilon.430nm(ox-red) = 6600 M-1 cm-1.