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. 2006 Jun 27;103(26):9779-84.
doi: 10.1073/pnas.0602278103. Epub 2006 Jun 15.

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

Jeremy A Dodsworth  1 , John A Leigh
Affiliations

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

Jeremy A Dodsworth et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Fig. 1.
Fig. 1.
Copurification of proteins with His-tagged NifI1 and NifI2 and interaction of NifI1,2 with dinitrogenase. Extracts were bound to Ni-NTA agarose, and elution fractions were analyzed by gel electrophoresis. (A) NifI2 interactions: V, strain Mm1012 [ΔnifI2 (null) background containing pLW40neo (vector control)]; I2, strain Mm711 (ΔnifI2 background containing pLW40neo nifI2 expressing His-tagged NifI2); I2ΔT, strain Mm1017 (ΔnifI2 background containing pLW40neo nifI2ΔT expressing His-tagged NifI2 with a deletion in the T-loop). Extracts were bound to Ni-NTA agarose in the absence (−) or presence (+) of 10 mM 2OG and eluted with 100 mM imidazole. Proteins corresponding to the indicated bands were identified by 2D gel electrophoresis and MALDI-TOF MS. Masses of the molecular weight markers (MW) are shown on the left. (B) NifI1 interactions: V, strain Mm1050 [ΔnifI1 (null) background containing pLCW40neo (vector control)]; I1, strain Mm1051 (ΔnifI1 background containing pLCW40 nifI1 expressing his-tagged NifI1); I1ΔT, strain Mm1067 (ΔnifI1 background containing pLCW40neo nifI1ΔT expressing His-tagged NifI1 with a deletion in the T-loop). Binding and elution are as in A. The band corresponding to NifI2 is partially obscured by His-tagged NifI1ΔT but was separated and identified on 2D gels. (C) Effect of ATP on NifI2 interactions using extract of strain Mm711. Binding, washing, and elution were done in the presence (+) or absence (−) of 5 mM ATP. Elution was done with either 100 mM imidazole (Imid) or 10 mM 2OG. NifI1 is present at the dye front of the gel.
Fig. 2.
Fig. 2.
Interaction of purified NifI1,2 and dinitrogenase. (A) Gel filtration of dinitrogenase alone, NifI1,2 alone, or a mix of the two was performed anaerobically with ATP and MgCl2, with or without 10 mM 2OG. Protein concentration in fractions, measured as A600 nm, is shown versus the elution volume. The secondary x axis shows the molecular mass corresponding to the fraction volume calibrated by known protein standards. Protein in the fractions indicated by numbers 1–6 were concentrated and run on SDS/PAGE, and the resulting Coomassie-stained gel is shown (Inset). Amounts of protein loaded on the column were dinitrogenase, 1 mg and 2 mg with and without 2OG, respectively; NifI1,2, 0.75 mg and 0.5 mg with and without 2OG, respectively; and dinitrogenase:NifI1,2 mix, 2:1 mg and 3:1.5 mg with and without 2OG, respectively. (B) Purified NifI1,2 (I1,2), dinitrogenase (DK), or a 1:2 mix (NifI1,2/NifDK, by mass) run on an 8% native PAGE gel and stained with Coomassie.
Fig. 3.
Fig. 3.
Effects of NifI1,2 and 2OG on nitrogenase activity. (A) Acetylene (C2H2) reduced per milligram of nitrogenase (75 μg each of NifH and NifDK) as a function of time, with or without 10 mM 2OG or 60 μg of NifI1,2. Data are from a representative experiment. (Inset) A Coomassie-stained SDS/PAGE gel of the purified NifH, NifDK, and NifI1,2. (B) The effect on nitrogenase activity of increasing amount of NifI1,2 as a fraction of the nitrogenase activity in the absence of NifI1,2. The concentration of NifI1,2 is expressed as a ratio to the total amount of dinitrogenase (NifDK, 75 μg) in the reaction. Results from three independent experiments (circles, diamonds, and triangles) and the average (solid line) are shown. (C) Nitrogenase activity without (filled squares) or with (open diamonds) 60–75 μg of NifI1,2 as a function of 2OG concentration, expressed as the fraction of activity without NifI1,2 or 2OG. Error bars show the SEM of at least three independent experiments.
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