Structure of a Cofactor-Deficient Nitrogenase MoFe Protein

et al. 2006

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101

107

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein is a highly complex metallocluster that provides the catalytically essential site for biological nitrogen fixation. FeMoco is assembled outside the MoFe protein in a stepwise process requiring several components, including NifB-co, an iron-and sulfurcontaining FeMoco precursor, and NifEN, an intermediary assembly protein on which NifB-co is presumably converted to FeMoco. Through the comparison of Azotobacter vinelandii strains expressing the NifEN protein in the presence or absence of the nifB gene, the structure of a NifEN-bound FeMoco precursor has been analyzed by x-ray absorption spectroscopy. The results provide physical evidence to support a mechanism for FeMoco biosynthesis. The NifEN-bound precursor is found to be a molybdenum-free analog of FeMoco and not one of the more commonly suggested cluster types based on a standard [4Fe-4S] architecture. A facile scheme by which FeMoco and alternative, non-molybdenum-containing nitrogenase cofactors are constructed from this common precursor is presented that has important implications for the biosynthesis and biomimetic chemical synthesis of FeMoco.biosynthesis ͉ extended x-ray absorption fine structure ͉ nitrogenase ͉ x-ray absorption spectroscopy

Section: Resultsmentioning

confidence: 99%

Structural insights into a protein-bound iron-molybdenum cofactor precursor

Corbett

et al. 2006

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101

107

“…The impact of homocitrate on Mo binding to NifEN (Tables 1 and 2) indicates that homocitrate and Mo are possibly preassociated and concurrently incorporated into the precursor. It has been speculated that homocitrate, the organic moiety of FeMoco responsible for the overall negative charge of the cluster, may assist FeMoco insertion by ''steering'' it down a positively charged ''FeMoco insertion funnel'' into its final location in the MoFe protein (32). It is interesting that some of the NifD residues (␣ subunit of MoFe protein) providing the ''FeMoco insertion funnel'' are also positively charged in the corresponding NifE residues (␣ subunit of NifEN), indicating the possible presence of an analogous, positively charged ''Mo͞homocitrate insertion funnel'' in the NifEN complex (11).…”

Section: Discussionmentioning

confidence: 99%

“…To test whether the NifENЈ proteins (above) contain mature FeMoco, the following assay was designed to reconstitute the ⌬nifB MoFe protein. Such an assay contained, in a 0.8 ml total volume, 25 mM Tris⅐HCl (pH 8.0), 20 mM Na 2 S 2 O 4 , 0.5 mg of purified ⌬nifB MoFe protein from A. vinelandii strain DJ1143 (32). FeMoco insertion was initiated with the addition of 2 mg of isolated NifENЈ to the mixture above.…”

Section: Methodsmentioning

confidence: 99%

FeMo cofactor maturation on NifEN

Corbett

et al. 2006

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163

FeMo cofactor (FeMoco) biosynthesis is one of the most complicated processes in metalloprotein biochemistry. Here we show that Mo and homocitrate are incorporated into the Fe͞S core of the FeMoco precursor while it is bound to NifEN and that the resulting fully complemented, FeMoco-like cluster is transformed into a mature FeMoco upon transfer from NifEN to MoFe protein through direct protein-protein interaction. Our findings not only clarify the process of FeMoco maturation, but also provide useful insights into the other facets of nitrogenase chemistry. (9), which is located at the ␣␤-interface and ligated to six protein residues; and the [Mo-7Fe-9S-X-homocitrate] (the identity of X is unknown but is considered to be C, O, or N; ref. 10) FeMo cofactor (FeMoco), which is situated within the ␣-subunit and bound to only two protein residues and an exogenous homocitrate ligand. Both P-cluster and FeMoco are composed of smaller substructures: the P-cluster comprises two subclusters that share a 6 -sulfide (9) and FeMoco consists of [Mo-3Fe-3S] and [4Fe-3S] subcubanes that are bridged by three 2 -sulfides and share a central 6 -light atom (10). These metal clusters are essential for nitrogenase reaction, a process that involves ATP-dependent electron transfer from the [4Fe-

“…1B, I), was obtained by deleting nifH, the gene encoding the subunit of Fe protein (13)(14)(15)(16), whereas another of them, designated Av1 ⌬nifB (Fig. 1B, III), was obtained by deleting nifB, the gene specifically involved in FeMoco biosynthesis (17). Compared with Av1 ⌬nifB , which has two intact P-clusters (17), Av1 ⌬nifH contains two pairs of [4Fe-4S]-like clusters in their places (14).…”

mentioning

confidence: 99%

P-cluster maturation on nitrogenase MoFe protein

Lee

et al. 2007

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Biosynthesis of nitrogenase P-cluster has attracted considerable attention because it is biologically important and chemically unprecedented. Previous studies suggest that P-cluster is formed from a precursor consisting of paired [4Fe-4S]-like clusters and that P-cluster is assembled stepwise on MoFe protein, i.e., one cluster is assembled before the other. Here, we specifically tackle the assembly of the second P-cluster by combined biochemical and spectroscopic approaches. By using a P-cluster maturation assay that is based on purified components, we show that the maturation of the second P-cluster requires the concerted action of NifZ, Fe protein, and MgATP and that the action of NifZ is required before that of Fe protein/MgATP, suggesting that NifZ may act as a chaperone that facilitates the subsequent action of Fe protein/ MgATP. Furthermore, we provide spectroscopic evidence that the [4Fe-4S] cluster-like fragments can be converted to P-clusters, thereby firmly establishing the physiological relevance of the previously identified P-cluster precursor. (Fig. 1A) contains two unique clusters per ␣␤-subunit pair: the [8Fe-7S] P-cluster (6), which is located at the ␣␤-subunit interface, and the [Mo-7Fe-9S-X-homocitrate] † iron-molybdenum cofactor (FeMoco) (7), which is positioned within the ␣-subunit. Nitrogenase catalysis involves a series of complex formation and dissociation between the Fe protein and the MoFe protein, during which process the electrons are sequentially transferred from the [4Fe-4S] cluster of the Fe protein, through the P-cluster, to the FeMoco within the MoFe protein, where substrate reduction eventually occurs. The complexity of nitrogenase reaction mechanism is undoubtedly associated with the presence of complex metalloclusters in the enzyme, namely, the P-cluster and FeMoco, the structure and function of which have served as one of the central topics in nitrogenase research for decades.The P-cluster holds a unique place in nitrogenase chemistry. Catalytically, it serves as a ''hub'' that mediates the shuffling of electrons between the metal centers of the Fe protein and the MoFe protein. Structurally, it represents a high-nuclearity, Fe/ S-only cluster that can be viewed as two [4Fe-4S] subclusters sharing a 6 -sulfide. Such a ''modular'' composition suggests that the P-cluster is formed through the fusion of its substructural units, a reaction mechanism that is well established in synthetic inorganic chemistry (8) and further supported by recent advances toward successful synthesis of P-cluster topologs (9-12). Biological evidence of such a fusion mechanism came from studies of three FeMoco-deficient forms of A. vinelandii MoFe protein (Fig. 1B) that allowed analysis of P-cluster species without the interference of FeMoco. One of these MoFe proteins, designated Av1 ⌬nifH (Fig. 1B, I), was obtained by deleting nifH, the gene encoding the subunit of Fe protein (13-16), whereas another of them, designated Av1 ⌬nifB (Fig. 1B, III), was obtained by deleting nifB, the gene specifically invo...