Evidence for electron transfer from the nitrogenase iron protein to the molybdenum-iron protein without MgATP hydrolysis: characterization of a tight protein-protein complex.

MgA TP hydrolysis has been proposed to be absolutely required for electron transfer from the nitrogenase iron (Fe) protein to the molybdenum-iron (MoFe) protein. This work presents evidence for primary electron transfer from the Azotobacter vinelandii nitrogenase Fe protein to the MoFe protein in the absence of MgATP hydrolysis. Deletion of an amino acid (Leu 127) in a signal transduction pathway in the Fe protein resulted in an Fe protein conformation resembling the MgATP-bound state. This altered Fe protein (L127delta) was found to bind to the MoFe protein in the absence of MgATP, forming a tight protein complex. Both steady state and stopped-flow transient kinetic measurements suggest that two L127delta Fe proteins bind to one MoFe protein with an extremely high affinity. From pre-steady state kinetic determinations of the rate of complex dissociation, the affinity was found to be at least 350 times tighter than that of the wild-type A. vinelandii nitrogenase complex and at least 20 times tighter than that of the heterologous Clostridium pasteurianum Fe protein-A. vinelandii MoFe protein complex. The L127delta Fe protein-MoFe protein complex was isolated by gel filtration liquid chromatography. Scanning densitometry of an SDS gel of the complex isolated from the gel filtration column revealed a stoichiometry of 1.7 L 127 delta Fe proteins bound per MoFe protein. The L 127 delta Fe protein was found to transfer a single electron from its [4Fe-4S] cluster to the MoFe protein at a rate of 0.2s-1. This compares with the MgATP dependent electron transfer rate of 140 s-1 observed for transfer of an electron from the wild-type Fe protein to the MoFe protein. No substrate reduction (H+ or C2H2) was detected when wild-type MoFe protein was complemented with L 127 delta Fe protein. The MgATP-independent electron transfer from the L 127 delta Fe protein to the MoFe protein required active MoFe protein and was not inhibited by MgADP. EPR spectroscopy of the complex was employed to confirm the electron transfer reaction. These results show that Fe protein in a conformation resembling the MgATP-bound state can transfer at least one electron to the MoFe protein without the need for MgATP hydrolysis.