title: Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase.
abstract: Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifI(1) and nifI(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 NifI(1) and NifI(2), and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the NifI proteins in switch-off, we found proteins that interact with NifI(1) and NifI(2) and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI(2) resulted in copurification of NifI(1) and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI(2) prevented copurification of dinitrogenase but did not affect copurification of NifI(1). Similar results were obtained with His-tagged NifI(1). Gel-filtration chromatography demonstrated an interaction between purified NifI(1,2) and dinitrogenase that was inhibited by 2OG. The NifI proteins themselves formed a complex of approximately 85 kDa, which appeared to further oligomerize in the presence of 2OG. NifI(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 NifI(1,2) complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifI(1) and nifI(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.
authors.affiliation: Department of Microbiology, University of Washington, Box 357242, 1959 N.E. Pacific Street, Seattle, WA 98195, USA.
authors.forename: Jeremy A
authors.initials: JA
authors.lastname: Dodsworth
authors.forename: John A
authors.initials: JA
authors.lastname: Leigh
mesh_terms: D019843:Archaeal Proteins / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000494:pharmacology*; D017017:Methanococcus / Q000201:enzymology*; D009584:Nitrogen / Q000378:metabolism; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D007656:Ketoglutaric Acids; D021122:Protein Subunits; D009591:Nitrogenase; D009584:Nitrogen