Abstract
The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.
Highlights
Several nitrogen fixation genes have been shown to be involved in FeMo-co biosynthesis and the formation of an active nitrogenase enzyme
In vitro FeMo-co synthesis assays show that the molybdenum accumulated within the purified NifEN protein is suitable for FeMo-co synthesis, suggesting that NifEN serves as the entry point for molybdenum into the FeMo-co synthesis pathway
Purification of NifEN—Poly(His)-tagged NifEN was purified to ϳ98% homogeneity from A. vinelandii strain DJ1041 using Co2ϩ affinity chromatography immediately followed by desalting on a HiPrep gel filtration column (Fig. 1)
Summary
Buffers—Buffers for cell breakage, protein purification, and protein assays were made anaerobic by sparging with N2 for 30 min, followed by alternate cycles of vacuum and flushing with argon, and the addition of sodium dithionite (DTH) to a final concentration of 1 mM, unless otherwise specified. Eluted NifEN was pooled, concentrated by ultrafiltration through a YM100 membrane in an Amicon cell under a N2 atmosphere, and subjected to gel-filtration chromatography on a PD-10 column (Amersham Biosciences) to remove the residual imidazole and to exchange NifEN into buffer B (50 mM Tris buffer, pH 8, 300 mM NaCl, 10% glycerol, 5 mM -mercaptoethanol, and 2 mM DTH). The complete reaction mixtures contained: 100 l of 25 mM Tris-HCl buffer, pH 7.4, 10 l of 1 mM Na2MoO4, 20 l of 5 mM homocitrate, 200 l of ATPregenerating mixture (containing 3.6 mM ATP, 6.3 mM MgCl2, 51 mM creatine phosphate, 20 units/ml creatine phosphokinase, and 6.3 mM DTH), 10 l of a solution containing NifB-co (equivalent to 4 nmol of Fe), 200 l of A. vinelandii cell-free extract (ϳ3 mg of protein from UW45 or DJ35 W-grown cells), and 500 g of purified NifEN in a total volume of 575 l.
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