Dinitrogenase is a heterotetrameric (␣ 2 ␤ 2 ) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition ␣ 2 ␤ 2 ␥ 2 , which can be activated in vitro by the addition of FeMo-co. The ␥ protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the ␥ subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now ␥. In vitro FeMo-co insertion experiments presented in this work demonstrate that ␥ stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.Nitrogenase catalyzes the reduction of nitrogen gas to ammonium, in an ATP-and reductant-dependent reaction. It is one of the best characterized metalloenzymes and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2). Dinitrogenase (also termed component I or molybdenum-iron protein) is a 240-kDa ␣ 2 ␤ 2 tetramer of the nifD and nifK gene products (3). Each ␣␤ nitrogenase dimer contains an iron-molybdenum cofactor (FeMo-co) 1 and a P cluster (3, 4). Dinitrogenase reductase (also termed component II or iron protein) is a 60-kDa ␣ 2 dimer of the nifH gene product which contains a single 4Fe-4S center coordinated between the two subunits (5). NifH has at least three roles in the nitrogenase enzyme system (6): first, it serves as electron donor to nitrogenase; second, it participates in the biosynthesis of FeMo-co; and third, it is required for maturation of apodinitrogenase to a FeMo-co-activable form.

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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 ␣, ␣-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.

Iron

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NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127⌬, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.

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 ATPregenerating 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 (⌬nifENX ⌬vnfE) 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.

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase was investigated using 99 Mo to follow the incorporation of Mo into precursors. 99 Mo 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, 99 Mo 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.99 Mo 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. 99 Mo 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 99 Mo on this protein required nucleotide.The iron-molybdenum cofactor (FeMo-co) 1 of dinitrogenase ( Fig. 1) constitutes the active site of the nif-encoded, molybdenum-containing dinitrogenase protein in Azotobacter vinelandii and other nitrogen-fixing organisms (1-3). FeMo-co can be isolated by extraction from the purified dinitrogenase protein (2), and the isolated cofactor can be used to activate FeMo-codeficient forms of dinitrogenase (referred to hereafter as "apodinitrogenase") that accumulate in strains unable to synthesize the cofactor (2, 4, 5). FeMo-co consists of Mo, Fe, and S atoms in a 1:7:9 ratio; in addition, the organic acid homocitrate is an integral component of the compound (6), serving as a nonprotein ligand to the molybdenum atom. The structure of FeMo-co in the protein was determined by Kim and Rees (7) and Chan et al. (8).Genetic studies have revealed that functional copies of the nifB, nifN, nifE, nifH, and nifV genes are required for synthesis of FeMo-co in vivo (9 -11); the nifQ gene is also required under conditions of molybdenum limitation (12). The nifKD genes, which encode the subunits of dinitrogenase (NifKD), are not required for FeMo-co synthesis, and thus FeMo-co is not synthesized "in place" but rather is preformed and then transferred to its site in dinitrogenase (13). In the absence of NifKD, completed FeMo-co accumulates on a protein called gamma, which serves as a chaperone/insertase for the maturation of NifKD and for insertion of .An in vitro FeMo-co synthesis system was devised to address the biochemical roles of these genes and to identify other factors required for FeMo-co synthesis (15). In this system, at least homocitrate, molybdenum (supplied as molybdate), MgATP, NifB-co, NifH (dinitrogenase reductase), reductant, ...