MgATP-independent hydrogen evolution catalysed by nitrogenase: an explanation for the missing electron(s) in the MgADP-AlF4 transition-state complex

When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumonia e nitrogenase are incubated with MgADP and AlF4- in the presence of dithion ite as a reducing agent, a stable putative transition-state complex is prod uced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, th e EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S = 3/2 EPR signal arising from the dithionite reduced state of the Mo Fe cofactor centre of the protein. This is consistent with the [Fe4S4] cent re of the Fe protein in the complex being oxidized, and similar observation s have been made with the complex of Azotobacter vinelandii [Spee, Arendsen , Wassink, Marritt, Hagen and Haaker (1998) FEES Lett. 432, 55-58]. No sati sfactory explanation for the fate of the electrons lost by Kp2 has been for thcoming. However, we report here that during the preparation of the MgADP- AlF4 K. pneumoniae complex under argon, H-2 was evolved in amounts correspo nding to one half of the FeMoco content of the Kp1 (FeMoco is the likely ca talytic site of nitrogenase with a composition Mo:Fe-7:S-9: homocitrate). T his is surprising, since activity is observed during incubation in the abse nce of MgATP, normally regarded as being essential for nitrogenase function , and in the presence of MgADP, a strong competitive inhibitor of nitrogena se. The formation of H-2 by nitrogenase in the absence of AlF4- was also ob served in reaction mixtures containing MgADP but not MgATP. The reaction sh owed saturation kinetics when Kp1 was titrated with increasing amounts of K p2 and, at saturation, the amount of H-2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H-2 on [ MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase s ince MgADP supports a very low rate of hydrogen evolution.