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

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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...

Section: Discussionmentioning

confidence: 90%

“…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)(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).…”

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confidence: 99%

P-cluster maturation on nitrogenase MoFe protein

Fay

Lee

et al. 2007

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“…Cell growth, protein purification, preparation of apo Fe protein, FeMoco maturation assay, metal analysis, visible region absorption, EPR and x-ray spectroscopies were performed as described (13,17,18,24,33,34 AMPPNP . These were control assays with the same conditions as i except that ATP was replaced by 2.4 mM ADP, adenosine 5Ј-O-(3-thiotriphosphate) (ATP␥S) or 5Ј-adenylylimidodiphosphate (AMPPNP).…”

Section: Methodsmentioning

confidence: 99%

“…Using a biochemical͞spectroscopic approach, we identified the presence of two pairs of [4Fe-4S]-like clusters that likely represent P-cluster precursors in a FeMocodeficient MoFe protein purified from a nifH-deletion strain (13,14). This protein becomes catalytically active upon incubation with the deleted gene product, indicating that it might represent a physiologically relevant intermediate during P-cluster assembly (15).…”

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confidence: 99%

FeMo cofactor maturation on NifEN

Corbett

Fay

et al. 2006

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162

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-

“…circular dichroism (MCD) analyses (7)(8)(9) show that the [Fe 4 S 4 ]-like cluster pair (designated P*-cluster) is in fact a precursor to the P-cluster, which can be reductively coupled into a [Fe 8 S 7 ] structure upon incubation with dithionite, MgATP, and either Fe protein alone (in the case of ΔnifH MoFe protein) (12) or Fe protein plus NifZ (in the case of ΔnifBΔnifZ MoFe protein) (13).…”

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confidence: 99%

ATP-independent substrate reduction by nitrogenase P-cluster variant

Lee

Ribbe

2012

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The P-cluster of nitrogenase is largely known for its function to mediate electron transfer to the active cofactor site during catalysis. Here, we show that a P-cluster variant (designated P*-cluster), which consists of paired [Fe 4 S 4 ]-like clusters, can catalyze ATP-independent substrate reduction in the presence of a strong reductant, europium (II) diethylenetriaminepentaacetate [Eu(II)-DTPA]. The observation of a decrease of activity in the rank ΔnifH, ΔnifBΔnifZ, and ΔnifB MoFe protein, which corresponds to a decrease of the amount of P*-clusters in these cofactor-deficient proteins, firmly establishes P*-cluster as a catalytically active metal center in Eu(II)-DTPA-driven reactions. More excitingly, the fact that P*-cluster is not only capable of catalyzing the two-electron reduction of proton, acetylene, ethylene, and hydrazine, but also capable of reducing cyanide, carbon monoxide, and carbon dioxide to alkanes and alkenes, points to a possibility of developing biomimetic catalysts for hydrocarbon production under ambient conditions.N itrogenases are a family of metalloenzymes that catalyze the nucleotide-dependent reduction of dinitrogen to ammonia under ambient conditions. The best-characterized member of this enzyme family is the molybdenum (Mo)-nitrogenase of Azotobacter vinelandii, which consists of two redox-active component proteins (1). One of the proteins, termed iron (Fe) protein (encoded by nifH), is a γ 2 -dimer that contains a [Fe 4 S 4 ] cluster between the two subunits and a MgATP binding site within each subunit; the other, termed molybdenum-iron (MoFe) protein (encoded by nifDK), is an α 2 β 2 -heterotetramer that houses two complex metal clusters per αβ-dimer: the P-cluster ([Fe 8 S 7 ]), which is located at each α/β-subunit interface; and the iron-molybdenum (FeMo) cofactor, or FeMoco ([MoFe 7 S 9 C-homocitrate]), which is buried within each α-subunit (2-4). Catalysis by nitrogenase presumably involves repeated association and dissociation between the two component proteins and ATP-dependent electron transfer from the [Fe 4 S 4 ] cluster of the Fe protein, through the P-cluster, to the FeMoco of MoFe protein, where substrate reduction occurs (5).The P-cluster has long been regarded as a "capacitor" that mediates the electron transfer between the [Fe 4 S 4 ] cluster of Fe protein and the cofactor site of MoFe protein during catalysis (1). Although the question of whether the P-cluster can directly reduce substrates was raised previously, it has remained a topic of debate because of the difficulty of distinguishing the contribution of the P-cluster from that of the cofactor to catalysis. As such, MoFe protein variants carrying the P-cluster species alone need to be generated to address the catalytic capacity of P-cluster. Recent studies of the biosynthesis of nitrogenase have led to the identification of three cofactor-deficient forms of MoFe protein (Fig. 1). One form, designated ΔnifB MoFe protein, contains two intact, [Fe 8 S 7 ] P-clusters (6); another, designated ΔnifH MoFe pro...