Priya Rangaraj

Requirement of NifX and Other nif Proteins for In Vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

The iron-molybdenum cofactor (FeMo-co) of nitrogenase contains molybdenum, iron, sulfur, and homocitrate in a ratio of 1:7:9:1. In vitro synthesis of FeMo-co has been established, and the reaction requires an ATP-regenerating system, dithionite, molybdate, homocitrate, and at least NifB-co (the metabolic product of NifB), NifNE, and dinitrogenase reductase (NifH). The typical in vitro FeMo-co synthesis reaction involves mixing extracts from two different mutant strains of Azotobacter vinelandii defective in the biosynthesis of cofactor or an extract of a mutant strain complemented with the purified missing component. Surprisingly, the in vitro synthesis of FeMo-co with only purified components failed to generate significant FeMo-co, suggesting the requirement for one or more other components. Complementation of these assays with extracts of various mutant strains demonstrated that NifX has a role in synthesis of FeMo-co. In vitro synthesis of FeMo-co with purified components is stimulated approximately threefold by purified NifX. Complementation of these assays with extracts of A. vinelandii DJ42. 48 (DeltanifENX DeltavnfE) results in a 12- to 15-fold stimulation of in vitro FeMo-co synthesis activity. These data also demonstrate that apart from the NifX some other component(s) is required for the cofactor synthesis. The in vitro synthesis of FeMo-co with purified components has allowed the detection, purification, and identification of an additional component(s) required for the synthesis of cofactor.

Accumulation of 99Mo-containing iron-molybdenum cofactor precursors of nitrogenase on NifNE, NifH, and NifX of Azotobacter vinelandii.

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase was investigated using the purified in vitro FeMo-co synthesis system and 99Mo. The purified system involves the addition of all components that are known to be required for FeMo-co synthesis in their purified forms. Here, we report the accumulation of a 99Mo-containing FeMo-co precursor on NifNE. Apart from NifNE, NifH and NifX also accumulate 99Mo label. We present evidence that suggests NifH may serve as the entry point for molybdenum incorporation into the FeMo-co biosynthetic pathway. We also present evidence suggesting a role for NifX in specifying the organic acid moiety of FeMo-co.

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase in Azotobacter vinelandii*

The vnf-encoded nitrogenase fromAzotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the iron-molybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in thevnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe7–8Sx precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe3S4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from anifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from thenifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

Incorporation of Molybdenum into the Iron-Molybdenum Cofactor of Nitrogenase

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using99Mo to follow the incorporation of Mo into precursors. 99Mo label accumulates on dinitrogenase only when all known components of the FeMo-co synthesis system, NifH, NifNE, NifB-cofactor, homocitrate, MgATP, and reductant, are present. Furthermore, 99Mo label accumulates only on the gamma protein, which has been shown to serve as a chaperone/insertase for the maturation of apodinitrogenase when all known components are present. It appears that only completed FeMo-co can accumulate on the gamma protein. Very little FeMo-co synthesis was observed when all known components are used in purified forms, indicating that additional factors are required for optimal FeMo-co synthesis. 99Mo did not accumulate on NifNE under any conditions tested, suggesting that Mo enters the pathway at some other step, although it remains possible that a Mo-containing precursor of FeMo-co that is not sufficiently stable to persist during gel electrophoresis occurs but is not observed. 99Mo accumulates on several unidentified species, which may be the additional components required for FeMo-co synthesis. The molybdenum storage protein was observed and the accumulation of 99Mo on this protein required nucleotide.

Inhibition of Iron-Molybdenum Cofactor Biosynthesis by L127Δ NifH and Evidence for a Complex Formation between L127Δ NifH and NifNE

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 α2β2 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, L127Δ, 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 L127Δ NifH was found to inhibit in vitro FeMo-co synthesis by wild-type NifH as detected by the γ gel shift assay. Increasing the concentration of NifNE and NifB-cofactor (NifB-co) relieved the inhibition of FeMo-co synthesis by L127Δ NifH. The formation of a complex of L127Δ NifH with NifNE was investigated by gel filtration chromatography. We herein report the formation of a complex between L127Δ 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.

Biosynthesis of the Iron-Molybdenum and Iron-Vanadium Cofactors

The iron-molybdenum cofactor (FeMo-co) (Shah, Brill, 1977) is the prototype of a small family of cofactors that constitute the active sites of the known nitrogenases. No other Mo, V or Fe-containing enzyme is known to employ FeMo-co or its analogs and all known nitrogenases contain one of these cofactors. FeMo-co (MoFe7S9homocitrate) is found at the active site of the nif-encoded, molybdenum nitrogenase, and its structure (Fig 1) was determined as a component of the dinitrogenase protein (NifDK) of Azotobacter vinelandii (Kim, Rees, 1992; Chan et al, 1993). The vnf-encoded nitrogenase-2 contains FeV-co (Smith et al, 1988) and the anf-encoded nitrogenase-3 contains FeFe-co (Davis et al, 1996) as dissociable cofactors that are thought to have structures that differ from FeMo-co only at the position of the heteroatom (Mo, V or Fe) as shown in Fig 1. The structures of FeV-co and FeFe-co have not been determined and the argument that their structures are similar to that of FeMo-co is based on the ability of isolated FeV-co and FeFe-co to replace FeMo-co in the nif-encoded dinitrogenase proteins When apodinitrogenase protein (NifDK, lacking FeMo-co but containing the P clusters) is reconstituted with FeV-co or FeFe-co in vitro, it functions to reduce acetylene and protons effectively, but not N2. Furthermore, FeMo-co is found associated with the anf-encoded dinitrogenase protein (AnfDGK) when cells are supplied with Mo (Gollan et al, 1993; Pau et al, 1993).

Inhibition of Iron-Molybdenum Cofactor Synthesis by L127Δ, an Altered form of Dinitrogenase Reductase

Dinitrogenase reductase (NifH) has three important roles in the nitrogenase enzyme system: (i) as the obligate physiological electron donor to dinitrogenase during catalysis; (ii) as a required participant for the synthesis of FeMo-co, which uses at least six nif genes, nifQ, V, B, H, N and E (but not nifK and D); and (iii) as a participant in the maturation of apodinitrogenase, when NifH mediates the association of a γ protein with the α2β2 form of apodinitrogenase to yield a hexameric (α2β2γ2) FeMo-co-activatable species (Allen et al, 1993). γ is a non-nif protein and has been shown to function as a chaperone-insertase during the formation of dinitrogenase (Homer et al, 1995). Mutations in nifH that render dinitrogenase reductase inactive in substrate reduction do not necessarily affect its ability to function in cofactor synthesis and apodinitrogenase maturation. This observation suggests that NifH contains distinct domains associated with each of its functions. The 4Fe-4S cluster of NifH is not required for its function in cofactor synthesis and apodinitrogenase maturation (Rangaraj et al, unpublished).