A New Type of Metalloprotein: The Mo Storage Protein from <i>Azotobacter vinelandii</i> Contains a Polynuclear Molybdenum–Oxide Cluster

Fenske, Dirk; Gnida, Manuel; Schneider, Klaus; Meyer‐Klaucke, Wolfram; Schemberg, Jörg; Henschel, Volker; Meyer, A.S.; Knöchel, A.; Müller, Achim

doi:10.1002/cbic.200400263

Cited by 51 publications

(71 citation statements)

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“…Molbindin domains are found in the ModA component of molybdatetransport systems, in the molybdate-responsive ModE transcriptional regulator, and in the A. vinelandii ModG protein involved in molybdenum homeostasis. Additionally, NifQ does not exhibit significant sequence similarity to the molybdenum-storageprotein (Mosto) MosA and MosB polypeptide components, which are able to bind large amounts of molybdenum, presumably as heptamolybdate (24).In this work, we show that NifQ carries a molybdenum-ironsulfur cluster that serves as a specific molybdenum donor for the synthesis of FeMo-co in vitro. Mobilization of molybdenum from ʈ In addition to its presence in the FeMo-cofactor of nitrogenase, biologically active molybdenum can be found constituting molybdopterin cofactors (Mo-co).…”

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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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confidence: 79%

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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 primary amino acid sequence of NifQ is unique and unrelated to other proteins involved in molybdenum trafficking, such as molbindins or molybdenum storage proteins (22,30). NifQ proteins have a conserved Cx 4 Cx 2 Cx 5 C amino acid motif that was proposed to be a binding site for an [Fe-S] cluster, a molybdenumcontaining metal cluster, or a Mo-S intermediate for FeMo-co synthesis (17,67).…”

Section: Molybdenum Processing For Femo-co Biosynthesis Possible Rolementioning

confidence: 99%

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

385

311

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The iron-molybdenum cofactor (FeMo-co), located at the active site of the molybdenum nitrogenase, is one of the most complex metal cofactors known to date. During the past several years, an intensive effort has been made to purify the proteins involved in FeMo-co synthesis and incorporation into nitrogenase. This effort is starting to provide insights into the structures of the FeMo-co biosynthetic intermediates and into the biochemical details of FeMo-co synthesis. 93

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“…R. sphaeroides, S. meliloti, and A. tumefaciens possibly produce siderophores that bind and thus detoxify excess HMO in the medium, as shown for Azotobacter vinelandii (20). It is noteworthy that none of these strains has a homolog of the A. vinelandii Mo/W storage protein MosAB, which binds up to 100 Mo or W atoms as polyoxometalates (21,22). The two Rhodobacter strains have Mop-type Mo storage proteins (23), which are absent in S. meliloti and A. tumefaciens.…”

Section: Resultsmentioning

confidence: 99%

Bacterial PerO Permeases Transport Sulfate and Related Oxyanions

et al. 2017

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Rhodobacter capsulatus synthesizes the high-affinity ABC transporters CysTWA and ModABC to specifically import the chemically related oxyanions sulfate and molybdate, respectively. In addition, R. capsulatus has the low-affinity permease PerO acting as a general oxyanion transporter, whose elimination increases tolerance to molybdate and tungstate. Although PerO-like permeases are widespread in bacteria, their function has not been examined in any other species to date. Here, we present evidence that PerO permeases from the alphaproteobacteria Agrobacterium tumefaciens, Dinoroseobacter shibae, Rhodobacter sphaeroides, and Sinorhizobium meliloti and the gammaproteobacterium Pseudomonas stutzeri functionally substitute for R. capsulatus PerO in sulfate uptake and sulfate-dependent growth, as shown by assimilation of radioactively labeled sulfate and heterologous complementation. Disruption of perO genes in A. tumefaciens, R. sphaeroides, and S. meliloti increased tolerance to tungstate and, in the case of R. sphaeroides, to molybdate, suggesting that heterometal oxyanions are common substrates of PerO permeases. This study supports the view that bacterial PerO permeases typically transport sulfate and related oxyanions and, hence, form a functionally conserved permease family.IMPORTANCE Despite the widespread distribution of PerO-like permeases in bacteria, our knowledge about PerO function until now was limited to one species, Rhodobacter capsulatus. In this study, we showed that PerO proteins from diverse bacteria are functionally similar to the R. capsulatus prototype, suggesting that PerO permeases form a conserved family whose members transport sulfate and related oxyanions.KEYWORDS transport, sulfate, molybdate, tungstate, oxyanions, permease, PerO, Rhodobacter, ABC transporters T he oxyanions sulfate (SO 4 2-) and molybdate (MoO 4 2-) are the preferred sources for sulfur and molybdenum, respectively, in bacteria. To specifically import these structurally similar oxyanions, Escherichia coli synthesizes the ATP-binding cassette (ABC) transporters CysPTWA and ModABC (1, 2). ABC uptake systems consist of a periplasmic substrate binding protein, a transmembrane channel protein, and a cytoplasmic ATP-binding protein, which provides the energy for active substrate transport across the cytoplasmic membrane against a concentration gradient (3). In addition to molybdate, ModABC imports tungstate (WO 4 2-) but not sulfate (4, 5). In addition to sulfate, CysPTWA imports molybdate, albeit less efficiently than its primary substrate sulfate (6, 7).Recently, we described the permease PerO mediating transport of sulfate, molybdate, and tungstate in the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus (8). PerO belongs to the ArsB/NhaD ion transporter superfamily, whose members encompass 10 to 13 transmembrane domains and have diverse functions such as arsenite export, citrate/succinate antiport, and Na ϩ /H ϩ antiport. PerO contains two central TrkA_C domains, making this permease considerably larg...

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A New Type of Metalloprotein: The Mo Storage Protein from Azotobacter vinelandii Contains a Polynuclear Molybdenum–Oxide Cluster

Cited by 51 publications

References 46 publications

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

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

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Bacterial PerO Permeases Transport Sulfate and Related Oxyanions

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