Isolation of an iron-molybdenum cofactor from nitrogenase

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.

Section: Methodsmentioning

confidence: 99%

Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

Rubio¹,

Rangaraj²,

Homer³

et al. 2002

“…Azotobacter vinelandii mutants lacking any one of the nifH, nifN, nifE, or nifB gene products produce a FeMo cofactorless dinitrogenase (2,5), referred to as apodinitrogenase (this form of apodinitrogenase contains the P-clusters). At least seven nif gene products are known to be involved in FeMo-co biosynthesis: nifQ, nifV, nifX, nifN, nifE, nifB, and nifH (2,16,17).…”

mentioning

confidence: 99%

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Rangaraj

Ryle²,

Lanzilotta³

et al. 1999

Self Cite

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.

“…1, lanes 3 and 6); these data also show that the antibodies to VNFG did not cross-react with VNFDK (present in extract of strain CA119; Ref. 32). …”

Section: Accumulation Of Vnfg (␦) In Various Mutantmentioning

confidence: 53%

“…Fractions were monitored for FeV-co using the FeV-co insertion assay. In a second control experiment, 200 l of a solution of FeMo-co in NMF were chromatographed on the Superose 12 column both in the presence and absence of VNFG as described above; fractions were monitored for FeMo-co using the FeMo-co insertion assay (32) and for VNFG by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of VNFG, the δ Subunit of the vnf-Encoded Apodinitrogenase from Azotobacter vinelandii

Chatterjee

Ludden

Shah³

1997

Self Cite

The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an ␣ 2 ␤ 2 ␦ 2 hexamer. The ␦ subunit (the VNFG protein) has been characterized in order to further delineate its function in the nitrogenase 2 enzyme system. Two species of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG associated with the VNFDK polypeptides, and the other is a homodimer of the VNFG protein.Both species of VNFG are observed in extracts of A. vinelandii strains that accumulate dinitrogenase 2, whereas extracts of strains impaired in the biosynthetic pathway of the iron-vanadium cofactor (FeV-co) that accumulate apodinitrogenase 2 (a catalytically inactive form of dinitrogenase 2 that lacks FeV-co) exhibit only the VNFG dimer on native gels. FeV-co and nucleotide are required for the stable association of VNFG with the VNFDK polypeptides; this stable association can be correlated with the formation of active dinitrogenase 2. The iron-molybdenum cofactor was unable to replace FeV-co in promoting the stable association of VNFG with VN-FDK. FeV-co specifically associates with the VNFG dimer in vitro to form a complex of unknown stoichiometry; combination of this VNFG-FeV-co species with apodinitrogenase 2 results in its reconstitution to dinitrogenase 2. The results presented here suggest that VNFG is required for processing apodinitrogenase 2 to functional dinitrogenase 2.