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.
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Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN.
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Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid e Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria/Consejo Superior de Investigaciones Científicas, Madrid 28223, Spain.
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Sci Adv
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Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697- 3900, USA.
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Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Pozuelo de Alarcón, Spain.
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mBio
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Department of Biochemistry, Virginia Techgrid.438526.e, Blacksburg, Virginia, USA.
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Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, CA, 92697-3900, USA.
NifEN plays a crucial role in the biosynthesis of nitrogenase, catalyzing the final step of cofactor maturation prior to delivering the cofactor to NifDK, the catalytic component of nitrogenase. The difficulty in expressing NifEN, a complex, heteromultimeric metalloprotein sharing structural/functional homology with NifDK, is a major challenge in the heterologous expression of nitrogenase. Herein, we report the expression and engineering of Azotobacter vinelandii NifEN in Escherichia coli.
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