<i>In vitro</i>
            synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

Curatti, Leonardo; Hernández, José Ángel; Igarashi, Robert Y.; Soboh, Basem; Zhao, Dehua; Rubio, Luis M.

doi:10.1073/pnas.0703050104

Cited by 93 publications

(152 citation statements)

References 33 publications

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“…The inability to accomplish full activation of HydA DEFG may result from metal content heterogeneity and/or improper folding in purified HydA DEFG , either of which might result from heterologous expression in the absence of the full complement of maturation proteins. In addition, a decreased percentage of HydA DEFG activation is observed at higher HydF EG to HydA-D EFG ratios which is similar to effects observed for other systems in which scaffold proteins operate including the Isc and Nif systems [16,17].…”

Section: Investigating the Stoichiometry Of Hyda Defg Activation Bysupporting

confidence: 76%

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

et al. 2008

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In an effort to determine the specific protein component(s) responsible for in vitro activation of the [FeFe] hydrogenase (HydA), the individual maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum were purified from heterologous expressions in Escherichia coli. Our results demonstrate that HydF isolated from a strain expressing all three maturation proteins is sufficient to confer hydrogenase activity to purified inactive heterologously expressed HydA (expressed in the absence of HydE, HydF, and HydG). These results represent the first in vitro maturation of [FeFe] hydrogenase with purified proteins, and suggest that HydF functions as a scaffold upon which an H-cluster intermediate is synthesized.

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Section: Investigating the Stoichiometry Of Hyda Defg Activation Bysupporting

confidence: 76%

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

et al. 2008

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“…A minimal core of three catalytic (nifHDK) and three biosynthetic (nifENB) genes is conserved in most diazotrophs (23,26), reflecting the core protein components required for FeMo-co biosynthesis in vitro (27). Other genes required for nitrogenase biosynthesis and activity are less well conserved or are substituted by other genes in the host.…”

Section: Resultsmentioning

confidence: 99%

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

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

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All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

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“…The resulting activation of apo-NifDK was analyzed by the acetylene reduction assay after addition of 0.2 mg of NifH and 0.8 ml of ATP-regenerating mixture (41). Ammonia production by activated apo-NifDK was determined as described (14).…”

Section: Methodsmentioning

confidence: 99%

“…In this model, the [Fe-S]-containing FeMo-co precursor, NifB-co**, would be initially assembled by NifB and then transferred to NifEN for its conversion into the [Fe-S] VK-cluster (13,14). Homocitrate would be provided by the homocitrate-synthase activity of NifV (15,16).…”

mentioning

confidence: 99%

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor

Hernández

Curatti

Aznar

et al. 2008

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

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The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron-molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum-iron-sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex.nif ͉ iron-sulfur cluster ͉ Azotobacter vinelandii B iological nitrogen fixation performed by microorganisms that have nitrogenase(s) accounts for roughly two-thirds of the nitrogen fixed globally. Most nitrogen fixation is carried out by the activity of molybdenum nitrogenase (1), which is widely distributed in nature (2). The molybdenum-nitrogenase enzyme is composed of two proteins (3): a heterotetrameric NifDKprotein component (a 2 ␤ 2 dinitrogenase) and a homodimeric NifH-protein component (dinitrogenase reductase). NifDK contains one iron-molybdenum cofactor (FeMo-co) within the active site of each ␣-subunit (NifD), and one [8Fe-7S] P-cluster at the interface of the ␣-and ␤-subunits in each ␣␤ pair (4). NifH contains a [4Fe-4S] cluster bridging the two subunits and one site for Mg-ATP binding and hydrolysis in each subunit (5).FeMo-co is a unique cofactor ʈ composed of a [Mo-3Fe-3S] partial cubane bridged to a [4Fe-3S] partial cubane by three sulfur ligands and one unidentified light atom in the center of the cofactor (6). The molybdenum atom is also coordinated to the organic acid homocitrate. The biosynthesis of FeMo-co is a complex process that involves the activities of several nitrogen fixation (nif ) gene products that function as molecular scaffolds, enzymes, or escort proteins that carry FeMo-co precursors between assembly sites in the pathway (7,8). Following assembly, FeMo-co is inserted into apo-NifDK, a P-cluster-containing but FeMo-co-deficient form of NifDK that is matured into functional NifDK simply by FeMo-co insertion, to generate the mature dinitrogenase enzyme that is competent for nitrogen fixation.The NifEN scaffold protein is believed to function in the pathway as a central node to which an [Fe-S]-containing FeMo-co precursor, molybdenum, and homocitrate might converge to complete the assembly of FeMo-co (9-12). In this model, the [Fe-S]-containing FeMo-co precursor, NifB-co**, would be initially assembled by NifB and then transferred to NifEN for its conversion into the [Fe-S] VK-cluster (13,14). Homocitrate would be provided by the homocitrate-synthase ac...

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In vitro synthesis of the iron–molybdenum cofactor of nitrogenase from iron, sulfur, molybdenum, and homocitrate using purified proteins

Cited by 93 publications

References 33 publications

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

HydF as a scaffold protein in [FeFe] hydrogenase H‐cluster biosynthesis

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor

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