Abstract
Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of alpha(2)beta(2) apo-dinitrogenase (apo-dinitrogenase maturation). In an attempt to understand the role of NifH in FeMo-co biosynthesis, a site-specific altered form of NifH in which leucine at position 127 has been deleted, L127Delta, was employed in in vitro FeMo-co synthesis assays. This altered form of NifH has been shown to inhibit substrate reduction by the wild-type nitrogenase complex, forming a tight protein complex with dinitrogenase. The L127Delta NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the gamma gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Delta NifH. The formation of a complex of L127Delta NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Delta NifH and NifNE in the presence of NifB-co. This work presents evidence for one of the possible roles for NifH in FeMo-co biosynthesis, i.e. the interaction of NifH with a NifNE.NifB-co complex.
Highlights
Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of ␣22 apo-dinitrogenase
The features of NifH required for its function in substrate reduction include the ability of NifH to interact with low potential electron donors, to bind MgATP, to undergo the nucleotide-induced conformational change, to bind dinitrogenase, to transfer electrons to dinitrogenase, and to hydrolyze MgATP
The assays were carried out by incubating wild-type and L127⌬ NifH (50 g of protein) with aliquots of extract of strain CA11.1 (2.5 mg of protein), as described under “Experimental Procedures.”. The results of these assays are shown in Fig. 1. ␥ in the extract of strain CA11.1 is a dimer (␥2) because the strain is impaired in FeMo-co synthesis (Fig. 1, lane 1)
Summary
Besides serving as the obligate electron donor to dinitrogenase during nitrogenase turnover, dinitrogenase reductase (NifH) is required for the biosynthesis of the iron-molybdenum cofactor (FeMo-co) and for the maturation of ␣22 apo-dinitrogenase (apo-dinitrogenase maturation). In Vitro FeMo-co Synthesis by Wild-type and L127⌬ NifH— The site-specific altered form of NifH, L127⌬, was tested in FeMo-co biosynthesis by the ␥ gel shift assay [16].
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