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. 1997 Oct 14;94(21):11250-5.
doi: 10.1073/pnas.94.21.11250.

ApoNifH functions in iron-molybdenum cofactor synthesis and apodinitrogenase maturation

P Rangaraj  1 , V K Shah, P W Ludden
Affiliations

ApoNifH functions in iron-molybdenum cofactor synthesis and apodinitrogenase maturation

P Rangaraj et al. Proc Natl Acad Sci U S A. .

Abstract

NifH (dinitrogenase reductase) has three important roles in the nitrogenase enzyme system. In addition to its role as the obligate electron donor to dinitrogenase, NifH is required for the iron-molybdenum cofactor (FeMo-co) synthesis and apodinitrogenase maturation. We have investigated the requirement of the Fe-S cluster of NifH for these processes by preparing apoNifH. The 4Fe-4S cluster of NifH was removed by chelation of the cluster with alpha, alpha'-bipyridyl. The resulting apoNifH was tested in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions and was found to function in both these processes. Thus, the presence of a redox active 4Fe-4S cluster in NifH is not required for its function in FeMo-co synthesis and in apodinitrogenase maturation. This, in turn, implies that the role of NifH in these processes is not one of electron transfer or of iron or sulfur donation.

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Figures

Figure 1
Figure 1
Immunoblot of an anoxic native gel developed with antibody to γ, illustrating the apoNifH- and MgATP-dependent association of γ2 with the α2β2 apodinitrogenase. FeMo-co insertion reactions containing a fraction enriched in the α2β2 apodinitrogenase and γ2 from DJ54 were subjected to anoxic native PAGE as described. Lanes: 1, reaction including NifH and MgATP; 2, reaction including NifH and excluding MgATP; 3, reaction including apoNifH and MgATP; 4, reaction including apoNifH and excluding MgATP; 5, reaction excluding NifH and MgATP.
Figure 2
Figure 2
Immunoblot of an anoxic native gel developed with antibody to γ, illustrating γ monomerization upon binding FeMo-co. FeMo-co synthesis reactions were performed as described and subjected to anoxic native PAGE. Lanes: 1, reaction excluding NifH; 2, reaction including NifH; 3, reaction including apoNifH; 4, reaction including NifH and excluding MgATP; 5, reaction including apoNifH and excluding MgATP; 6, purified γ2; 7, purified γ2 plus excess purified FeMo-co.
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