Ronda M. Allen

Incorporation of Iron and Sulfur from NifB Cofactor into the Iron-Molybdenum Cofactor of Dinitrogenase

NifB-co is an iron- and sulfur-containing precursor to the iron-molybdenum cofactor (FeMo-co) of dinitrogenase. The synthesis of NifB-co requires at least the product of the nifB gene. Incorporation of 55Fe and 35S from NifB-co into FeMo-co was observed only when all components of the in vitro FeMo-co synthesis system were present. Incorporation of iron and sulfur from NifB-co into dinitrogenase was not observed in control experiments in which the apodinitrogenase (lacking FeMo-co) was initially activated with purified, unlabeled FeMo-co or in assays where NifB-co was oxygen-inactivated prior to addition to the synthesis system. These data clearly demonstrate that iron and sulfur from active NifB-co are specifically incorporated into FeMo-co of dinitrogenase and provide direct biochemical identification of an iron-sulfur precursor of FeMo-co. Under different in vitro FeMo-co synthesis conditions, iron and sulfur from NifB-co were associated with at least two other proteins (NIFNE and gamma) that are involved in the formation of active dinitrogenase. The results presented here suggest that multiple FeMo-co processing steps might occur on NIFNE and that FeMo-co synthesis is most likely completed prior to the association of FeMo-co with gamma.

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.

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.

In Vitro Synthesis of the Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase EFFECT OF SUBSTITUTION OF VNFH FOR NIFH

NIFH (the nifH gene product) has several functions in the nitrogenase enzyme system. In addition to reducing dinitrogenase during nitrogenase turnover, NIFH functions in the biosynthesis of the iron-molybdenum cofactor (FeMo-co), and in the processing of α2β2 apodinitrogenase 1 (a catalytically inactive form of dinitrogenase 1 that lacks the FeMo-co) to the FeMo-co-activatable α2β2γ2 form. The molybdenum-independent nitrogenase 2 (vnf-encoded) has a distinct dinitrogenase reductase protein, VNFH. We investigated the ability of VNFH to function in the in vitro biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. VNFH can replace NIFH in both the biosynthesis of FeMo-co and in the maturation of apodinitrogenase 1. These results suggest that the dinitrogenase reductase proteins do not specify the heterometal incorporated into the cofactors of the respective nitrogenase enzymes. The specificity for the incorporation of molybdenum into FeMo-co was also examined using the in vitro FeMo-co synthesis assay system.

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).

Incorporation of the Iron and Sulfur from NifB-co into the Iron-Molibdenum Cofactor of Dinitrogenase

NifB-co, the product of the nifB gene, is involved in the biosynthesis of the ironmolibdenum cofactor (FeMo-co) of dinitrogenase. Incorporation of 55Fe and 35S from NifB-co into FeMo-co was investigated using the in vitro FeMo-co synthesis system. Fe and S from NifB-co were incorporated into dinitrogenase only under conditions where all components required for FeMo-co synthesis were present. Incorporation of Fe and S from NifB-co into dinitrogenase was not observed in control experiments in which apodinitrogenase was initially activated with purified, unlabelled FeMo-co or in assays where NifB-co was oxygen-inactivated prior to addition to the synthesis system. These data clearly demonstrate that Fe and S from active NifB-co are specifically incorporated into FeMo-co of dinitrogenase and provide the first biochemical identification of an FeS donor for FeMo-co synthesis.