Nitrogenase iron-molybdenum cofactor binding site: Protein conformational changes associated with cofactor binding

Magnuson, Jon; Paustian, Timothy D.; Shah, Vinod K.; Dean, Dennis R.; Roberts, Gary P.; Rees, Douglas C.; Howard, James B.

doi:10.1016/s0040-4020(97)00710-2

Cited by 12 publications

(14 citation statements)

References 38 publications

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“…Third, although the ␣-Cys 275 residue is part of domain II of the ␣ subunit and its relative position remains unaltered in the structures of the apo-and holo-MoFe proteins, the displacement of domain III in the apo-MoFe protein causes ␣-Cys 275 to be very exposed to solvent. These domain rearrangements are consistent with the observed changes in reactivity of ␣-Cys 275 towards the alkylating agent iodoacetamide at different stages of apo-MoFe protein maturation (49). The thiol group of ␣-Cys 275 is nonreactive in the ⌬nifH apo-MoFe protein, very reactive in the ⌬nifB apo-MoFe protein after the maturation induced by the Fe protein takes place, and nonreactive again in the mature MoFe protein after FeMo-co insertion.…”

Section: Mofe Protein Maturationsupporting

confidence: 85%

“…Some MoFe protein variants with substitutions at the cysteine residues that are the ligands to the P-cluster consistently lack P-clusters and FeMo-co (42). In any event, the consequence of the Fe protein-dependent maturation is a conformational change in the apo-MoFe protein that makes the FeMo-co site accessible and promotes the binding of NafY (1,49). Although the three-dimensional molecular structure of the ⌬nifH MoFe protein is not available, thiol reactivity experiments suggest that maturation by the Fe protein causes a rearrangement of the region around residue ␣-Cys 275 of the MoFe protein and leaves its thiol group exposed to solvent (49).…”

Section: Mofe Protein Maturationmentioning

confidence: 99%

“…In any event, the consequence of the Fe protein-dependent maturation is a conformational change in the apo-MoFe protein that makes the FeMo-co site accessible and promotes the binding of NafY (1,49). Although the three-dimensional molecular structure of the ⌬nifH MoFe protein is not available, thiol reactivity experiments suggest that maturation by the Fe protein causes a rearrangement of the region around residue ␣-Cys 275 of the MoFe protein and leaves its thiol group exposed to solvent (49). It is important to stress here that the thiolate of ␣-Cys 275 is the ligand to the distal Fe atom of FeMo-co in mature MoFe protein.…”

Section: Mofe Protein Maturationmentioning

confidence: 99%

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Maturation of Nitrogenase: a Biochemical Puzzle

2005

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Section: Mofe Protein Maturationsupporting

confidence: 85%

Section: Mofe Protein Maturationmentioning

confidence: 99%

Section: Mofe Protein Maturationmentioning

confidence: 99%

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Maturation of Nitrogenase: a Biochemical Puzzle

2005

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“…Selective alkylation studies have shown that NafY-stabilized apo-NifDK has the side chain of ␣-Cys 275 exposed to solvent (␣-Cys 275 serves as ligand to FeMo-co) (28). Fourth, it could also be possible that n-NafY increases the K D of the FeMo-co⅐apo-NifDK complex.…”

Section: Discussionmentioning

confidence: 99%

A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

Hernández

Phillips

Erbil

et al. 2011

Journal of Biological Chemistry

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NafY participates in the final steps of nitrogenase maturation, having a dual role as iron-molybdenum cofactor (FeMoco) carrier and as chaperone to the FeMo-co-deficient apoNifDK (apo-dinitrogenase). NafY contains an N-terminal domain of unknown function (n-NafY) and a C-terminal domain (core-NafY) necessary for FeMo-co binding. We show here that n-NafY and core-NafY have very weak interactions in intact NafY. The NMR structure of n-NafY reveals that it belongs to the sterile ␣-motif (SAM) family of domains, which are frequently involved in protein-protein interactions. The presence of a SAM domain in NafY was unexpected and could not be inferred from its amino acid sequence. Although SAM domains are very commonly found in eukaryotic proteins, they have rarely been identified in prokaryotes. The n-NafY SAM domain binds apo-NifDK. As opposed to full-length NafY, nNafY impaired FeMo-co insertion when present in molar excess relative to FeMo-co and apo-NifDK. The implications of these observations are discussed to offer a plausible mechanism of FeMo-co insertion. NafY domain structure, molecular tumbling, and interdomain motion, as well as NafY interaction with apo-NifDK are consistent with the function of NafY in FeMo-co delivery to apo-NifDK. Complex metalloclusters are generated from simple [Fe-S] clusters by incorporating additional metals and organic/inorganic ligands. In the case of the FeMo-co of nitrogenase, simple [Fe-S] clusters are the substrates of the NifB protein, which uses them to generate NifB-co, a complex [Fe-S] cluster with a proposed [6Fe-

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“…NafY serves as a "molecular prop" whose function is to maintain the conformation of the FeMo-co-deficient apodinitrogenase competent for FeMo-co insertion (7,9,13,14). FeMo-co is found buried, and thus inaccessible, within the protein in the mature ␣ 2 ␤ 2 dinitrogenase.…”

mentioning

confidence: 99%

Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

Rubio

Singer

Ludden

2004

Journal of Biological Chemistry

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The formation of an active dinitrogenase requires the synthesis and the insertion of the iron-molybdenum cofactor (FeMo-co) into a presynthesized apodinitrogenase. In Azotobacter vinelandii, NafY (also known as ␥ protein) has been proposed to be a FeMo-co insertase because of its ability to bind FeMo-co and apodinitrogenase. Here we report the purification and biochemical characterization of NafY and reach the following conclusions. First, NafY is a 26-kDa monomeric protein that binds one molecule of FeMo-co with very high affinity (K d Ϸ 60 nM); second, the NafY-FeMo-co complex exhibits a S ‫؍‬ 3/2 EPR signal with features similar to the signals for extracted FeMo-co and the M center of dinitrogenase; third, site-directed mutagenesis of nafY indicates that the His 121 residue of NafY is involved in cofactor binding; and fourth, NafY binding to apodinitrogenase or to FeMo-co does not require the presence of any additional protein. In addition, we have obtained evidence that suggests the ability of NafY to bind NifB-co, an FeS cluster of unknown structure that is a biosynthetic precursor to FeMo-co.Nitrogenase catalyzes the reduction of nitrogen gas to ammonium in an ATP-and reductant-dependent reaction. It is one of the best characterized metalloenzymes and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2). Dinitrogenase (also termed component I or iron-molybdenum protein) is a 240-kDa ␣ 2 ␤ 2 tetramer of the nifD and nifK gene products (3, 4). Each ␣␤ nitrogenase dimer contains an iron-molybdenum cofactor (FeMo-co) 1 and a P cluster (4, 5). Dinitrogenase reductase (also termed component II or iron protein) is a 60-kDa ␣ 2 dimer of the nifH gene product that contains a single 4Fe-4S center coordinated between the two subunits (6). In contrast to the ␣ 2 ␤ 2 subunit composition of the mature dinitrogenase, apodinitrogenase (FeMo-co-deficient) from Klebsiella pneumoniae or Azotobacter vinelandii strains with mutations in nifB, nifN, or nifE have a hexameric composition (␣ 2

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Nitrogenase iron-molybdenum cofactor binding site: Protein conformational changes associated with cofactor binding

Cited by 12 publications

References 38 publications

Maturation of Nitrogenase: a Biochemical Puzzle

Maturation of Nitrogenase: a Biochemical Puzzle

A Sterile α-Motif Domain in NafY Targets Apo-NifDK for Iron-Molybdenum Cofactor Delivery via a Tethered Domain

Purification and Characterization of NafY (Apodinitrogenase γ Subunit) from Azotobacter vinelandii

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