nifU gene product from Azotobacter vinelandii is a homodimer that contains two identical [2Fe-2S] clusters.
The nifU gene product is required for the full activation of the metalloenzyme nitrogenase, the catalytic component of biological nitrogen fixation. In the present work, a hybrid plasmid that contains the Azotobacter vinelandii nifU gene was constructed and used to hyperexpress the NIFU protein in Escherichia coli. Recombinant NIFU was purified to homogeneity and was found to be a homodimer of 33-kDa subunits with approximately two Fe atoms per subunit. The combination of UV/visible absorption, variable-temperature magnetic circular dichroism, EPR, and resonance Raman spectroscopies shows the presence of a [2Fe-2S]2+,+ center (Em = -254 mV) with complete cysteinyl coordination in each subunit. The electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center do not conform to those established for any of the spectroscopically distinct types of 2Fe ferredoxins. These distinctive properties appear to be a consequence of a novel arrangement of coordinating cysteinyl residues in NIFU, and the residues likely to be involved in cluster coordination are discussed in light of primary sequence comparisons to other putative [2Fe-2S] proteins. The observed physicochemical properties of NIFU and its constituent [2Fe-2S] cluster also provide insight into the role of this protein in nitrogenase metallocluster biosynthesis.
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Department of Chemistry, University of Georgia, Athens 30602.
Jack
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RF
Morgan
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TV
Dean
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Johnson
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MK
D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics*; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D017931:DNA Primers; C088637:NifU protein, Bacteria; D011994:Recombinant Proteins