Accumulation of 99Mo-containing Iron-Molybdenum Cofactor Precursors of Nitrogenase on NifNE, NifH, and NifX ofAzotobacter vinelandii

Rangaraj, Priya; Ludden, Paul W.

doi:10.1074/jbc.m204581200

Cited by 33 publications

(52 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: Discussionmentioning

confidence: 90%

“…In vitro FeMo-co synthesis reactions using Na 2 MoO 4 as source of molybdenum showed mutual NifH and NifEN dependence in the incorporation of molybdenum into FeMo-co precursors (38,39). Based on these results, NifH was suggested to be an entry point for molybdenum (38) or even asserted to be a molybdenum/ homocitrate insertase (39). On the other hand, the existence of a NifH-independent molybdenum binding site within NifEN was proposed based on the presence of substoichiometric amounts of molybdenum in NifEN purified from a ⌬nifH strain (11).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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.

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

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.

View full text Add to dashboard Cite

show abstract

“…EXAFS analysis indicates that molybdenum is part of an [Fe-S] cluster at this stage (25). Subsequently, molybdenum is mobilized into the VK-clusterderived FeMo-co precursor in a reaction that requires NifH and Mg · ATP (36,38,57). Reports on whether ATP hydrolysis is required at this step are contradictory.…”

Section: Role Of Nifen and Its Association With Nifhmentioning

confidence: 94%

“…It is not clear whether incorporation of homocitrate directly requires NifH. Homocitrate incorporation follows molybdenum incorporation (57), which is dependent on NifH, and the incorporation of homocitrate into a molybdenum-containing precursor in the absence of NifH has not been reported.…”

Section: Role Of Nifen and Its Association With Nifhmentioning

confidence: 99%

“…Although we hypothesize that the molybdenum, iron-sulfur, and homocitrate precursors converge on NifEN, it is not clear whether NifEN alone provides a homocitrate binding site. Current evidence indicates that both NifEN and NifH must be present to achieve homocitrate incorporation into the FeMo-co precursor (15,36,57). Finally, assembled FeMo-co would be transferred to apo-NifDK via NafY, the product of a non-nif gene that also stabilizes the target apo-NifDK protein (67).…”

Section: Overview Of Femo-co Biosynthesismentioning

confidence: 99%

See 1 more Smart Citation

Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Rubio

Ludden

2008

Annu. Rev. Microbiol.

Self Cite

384

311

View full text Add to dashboard Cite

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

show abstract