Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus marivaludis occurs when the regulatory protein NifI(1.2) binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI(1.2) binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI(1.2), decreasing its inhibitory effect. NifI(1.2) 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 NifI(1.2) was unable to bind to an AIF(4)(-)-stabilized Fe protein:MoFe protein complex. NifI(1.2) inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI(1.2) competes with the Fe protein for binding to MoFe protein and prevents electron transfer. (C) 2007 Elsevier Inc. All rights reserved.