The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

Ribbe, Markus W.; Burgess, Barbara K.

doi:10.1073/pnas.101119498

Cited by 44 publications

(46 citation statements)

References 57 publications

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“…The fact that purified Av1 ⌬nifZ⌬nifB can be activated to 86% of the maximal level in the in vitro P-cluster maturation assay (Table 2, line 3) indicates that there may be other factors that further facilitate P-cluster assembly. These factors could be other nif gene-encoded products, such as NifW and NifM, which have been implicated in P-cluster assembly (25); or they could be other known chaperones, such as GroEL, which has been shown to participate in nitrogenase assembly (26). Our Av1 ⌬nifZ⌬nifB -based assay could serve as a useful tool to identify other factors required for the maturation of the second P-cluster.…”

Section: Discussionmentioning

confidence: 99%

P-cluster maturation on nitrogenase MoFe protein

Fay

Lee

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 99%

P-cluster maturation on nitrogenase MoFe protein

Fay

Lee

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…The reaction mixture was incubated and stopped as described for the FeMoco maturation assay (17). The enzymatic activities were subsequently determined as described (35). …”

Section: Methodsmentioning

confidence: 99%

FeMo cofactor maturation on NifEN

Corbett

Fay

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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-

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“…FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and the features of the P-cluster (10, 19 -21). Two types of 100% FeMoco-deficient MoFe proteins, presumably different catalytically and structurally, have been isolated and characterized.…”

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The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

Ribbe¹,

Hu²,

Guo³

et al. 2002

Journal of Biological Chemistry

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146

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The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (⌬nifH MoFe protein) was purified in large quantity. The which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.The metalloenzyme nitrogenase complex catalyzes the biological reduction of dinitrogen to ammonia (for recent reviews, see Refs. 1-6). The enzyme is composed of two separately purifiable proteins, the iron (Fe) protein and the molybdenumiron (MoFe) 1 protein. The Fe Protein is a 60-kDa dimer of two identical subunits encoded by the nifH gene. The two subunits are bridged by a [4Fe-4S] cluster, and each subunit has a binding site for MgATP. The more complicated MoFe protein is a 230-kDa ␣ 2 ␤ 2 tetramer with the ␣ and ␤ subunits encoded by the nifD and nifK genes, respectively. The MoFe protein contains two different types of metal clusters, the [8Fe-7S] cluster (P-cluster) bridged between each ␣␤ subunit pair and the [Mo7Fe-9S-homocitrate] cluster (FeMoco) located within each ␣ subunits. Substrate reduction by the enzyme requires both component proteins, with the Fe protein serving as a specific reductant of the MoFe protein, which in turn provides the site of substrate reduction. To carry out the catalytic function of nitrogenase, the reduced Fe protein first binds two molecules of MgATP and undergoes a conformational change before forming a complex with the MoFe protein. Then, coupled with MgATP hydrolysis, electrons are transferred from the Fe protein to the P-clusters of the MoFe protein within the complex. This process is followed by the dissociation and re-reduction of the oxidized Fe protein and the dissociation of MgADP from the MoFe protein. Finally, the electrons are believed to be transferred from the P-cluster to the FeMoco, where substrate reduction occurs.FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7-18) and the features of the P-cluster (

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The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

Cited by 44 publications

References 57 publications

P-cluster maturation on nitrogenase MoFe protein

P-cluster maturation on nitrogenase MoFe protein

FeMo cofactor maturation on NifEN

The FeMoco-deficient MoFe Protein Produced by a nifHDeletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

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