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

doi:10.1073/pnas.0602278103

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

Jeremy A. Dodsworth and
John A. Leigh*

Department of Microbiology, University of Washington, Box 357242, 1959 N.E. Pacific Street, Seattle, WA 98195

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved May 17, 2006 (received for review March 20, 2006)

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 NifI₁ and NifI₂, 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 NifI₁ and NifI₂ and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI₂ resulted in copurification of NifI₁ and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI₂ prevented copurification of dinitrogenase but did not affect copurification of NifI₁. Similar results were obtained with His-tagged NifI₁. 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 ≈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.

Footnotes

*To whom correspondence should be addressed. E-mail: leighj{at}u.washington.edu
Author contributions: J.A.D. and J.A.L. designed research; J.A.D. performed research; J.A.D. analyzed data; and J.A.D. and J.A.L. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

2OG,

2-oxoglutarate.