NifI inhibits nitrogenase by competing with Fe protein for binding to the MoFe protein

Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI1,2 binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI1,2 binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI1,2, decreasing its inhibitory effect. NifI1,2 prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI1,2 was unable to bind to an AlF4--stabilized Fe protein:MoFe protein complex. NifI1,2 inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI1,2 competes with the Fe protein for binding to MoFe protein and prevents electron transfer.