Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifi(1) and nifl(2), which encode members of the PII family of nitrogen-regulatory proteins. Previous work demonstrated that nitrogenase activity in cell extracts was inhibited in the presence of Nifl(1) and Nifl(2), and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the Nifl proteins in switch-off, we found proteins that interact with Nifl, and Nifl(2) and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged Nifl(2) resulted in copurification of Nifl(1) and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of Nifl(2) prevented copurification of dinitrogenase but did not affect copurification of Nifl(1). Similar results were obtained with His-tagged Nifl(1). Gel-filtration chromatography demonstrated an interaction between purified Nifl(1,2) and dinitrogenase that was inhibited by 2OG. The Nifl proteins themselves formed a complex of approximate to 85 kDa, which appeared to further oligomerize in the presence of 2OG. Nifl(1,2) inhibited activity of purified nitrogenase when present in a 1:1 molar ratio to dinitrogenase, and 2OG fully relieved this inhibition. These results suggest a model for switch-off of nitrogenase activity, where direct interaction of a Nifl(1,2) complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifi(1) and nifl(2) in the nif operons of all nitrogen-fixing Archaea and some anaerobic Bacteria suggests that this mode of nitrogenase regulation may operate in a wide variety of diazotrophs.