Accumulation of 55Fe-Labeled Precursors of the Iron-Molybdenum Cofactor of Nitrogenase on NifH and NifX ofAzotobacter vinelandii

Rangaraj, Priya; Rüttimann-Johnson, Carmen; Shah, Vinod K.; Ludden, Paul W.

doi:10.1074/jbc.m100907200

Cited by 35 publications

(38 citation statements)

References 39 publications

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“…First, the ␥ protein binds FeMo-co (13). Second, NifX and VnfX from A. vinelandii are also able to bind FeMo-co (37), 2 as well as NifB-co (32,37). Third, VnfX is also able to bind a FeV-co precursor (vanadiumcontaining iron-sulfur cluster) lacking homocitrate (32).…”

Section: Resultsmentioning

confidence: 99%

Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

Rubio¹,

Rangaraj²,

Homer³

et al. 2002

Journal of Biological Chemistry

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Dinitrogenase is a heterotetrameric (␣ 2 ␤ 2 ) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition ␣ 2 ␤ 2 ␥ 2 , which can be activated in vitro by the addition of FeMo-co. The ␥ protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the ␥ subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now ␥. In vitro FeMo-co insertion experiments presented in this work demonstrate that ␥ stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.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 molybdenum-iron protein) is a 240-kDa ␣ 2 ␤ 2 tetramer of the nifD and nifK gene products (3). Each ␣␤ nitrogenase dimer contains an iron-molybdenum cofactor (FeMo-co) 1 and a P cluster (3, 4). Dinitrogenase reductase (also termed component II or iron protein) is a 60-kDa ␣ 2 dimer of the nifH gene product which contains a single 4Fe-4S center coordinated between the two subunits (5). NifH has at least three roles in the nitrogenase enzyme system (6): first, it serves as electron donor to nitrogenase; second, it participates in the biosynthesis of FeMo-co; and third, it is required for maturation of apodinitrogenase to a FeMo-co-activable form.

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Section: Resultsmentioning

confidence: 99%

Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

Rubio¹,

Rangaraj²,

Homer³

et al. 2002

Journal of Biological Chemistry

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“…The presence of the C-terminal NifX-like domain could indicate that this region in NifB is either involved in binding NifB-co precursors and forming the NifB-co or binding a preformed NifB-co for transfer to another protein in the FeMoco biosynthetic pathway [44] . Although it was evident from previous reports that NifX is capable of binding to NifB-co in one of the stages during FeMoco biosynthesis [30] , a direct proteinprotein interaction between the NifB and NifX proteins had not been demonstrated before. Our results support the idea that there is an interaction between NifB and NifX as found by measuring the β-galactosidase activity of E. coli cotransformants harboring the plasmids pMH6002 (λCI:NifB) and pMH5002 (RNAP:NifX).…”

Section: Interactionmentioning

confidence: 82%

“…Following this, the core is transferred from NifB to the NifEN complex. Based on previous studies and the detection of an interaction between NifB and NifX in our study, it could be possible that NifX facilitates the transfer of the core from NifB to NifEN [30] . It is possible that the addition of homocitrate takes place at this step, since one previous study has shown that the NifX may be involved in specifying the organic acid moiety of the FeMoco precursor [31] .…”

Section: Interaction Of Nifdk (Fusion) and Nifk With Nifxmentioning

confidence: 99%

“…In NifB, the NIFX SMBD is fused with a biotin-synthase-like, metal-dependent catalytic domain and in the archaeal MTH1172 protein, it is fused to a cation transporter [33] . Previous studies in Azotobacter vinelandii have identified certain important molecular interactions of the NifX protein with the other Nif proteins involved in the FeMoco biosynthesis/transport pathway; mainly it was shown that NifX binds to the NifB-co and also a FeMoco precursor from the NifEN complex [10,30] . Clearly, a protein-protein interaction based study of NifX with other FeMoco maturation proteins would enhance our insight into the role of NifX and may help to solve unanswered questions regarding its position in the FeMoco biosynthetic pathway.…”

Section: Introductionmentioning

confidence: 99%

“…As understood from research conducted thus far, a stepwise overview of the events involved in the biosynthesis and assembly of the FeMoco includes: (i) the formation of an Fe/S core of the FeMoco (designated NifB-co) by NifB [18,19] (ii) transfer of the NifB-co to the α 2 β 2 tetrameric scaffold NifEN protein [20,21] (iii) maturation of the FeMoco and apo-MoFe protein by the combined action of the Fe protein and MgATP [17,22,23] and further processing of the NifB-co on NifEN by an unknown mechanism, forming the completed FeMoco (iv) the final transfer of the fully formed FeMoco to a nafYencoded protein, called γ  which is a FeMoco carrier that aids in the insertion of the FeMoco into the apoMoFe [24,25] . In addition to these major events, a few other important steps are also involved in the entire process.The NifU and NifS proteins participate in the initial Fe/S mobilization [26,27] , the NifQ protein is involved in an early stage of the metal core formation and found to be essential in Mo-limiting conditions [28] , the NifV protein contributes the homocitrate component of the FeMoco [29] and the NifX protein probably functions as an 'escort' protein that delivers the FeMoco or its precursors from one assembly site to another or plays a role in specifying the organic acid moiety of FeMoco [30,31] . Although a wealth of data is available to support the functions of the NifU, NifS, NifB, NifEN, NifH and NifV, more research is required to explain the role of the NifX protein.…”

Section: Introductionmentioning

confidence: 99%

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The NifX Protein is Involved in the Final Stages of FeMo-cofactor Transport to the MoFe Protein

Lahiri¹,

Cole²,

Pulakat³

et al. 2007

American J. of Biochemistry and Biotechnology

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Abstract:The nitrogenase enzyme catalyzes the reduction of dinitrogen to ammonia and is composed of the Fe and MoFe proteins. The iron molybdenum cofactor (FeMo-co) of the MoFe protein is the site of active substrate reduction. The NifX protein has also been suggested to have a role in the FeMo-co synthesis, although its exact role is still open to investigation. We attempted to understand the role of NifX by determining the specific interactions it may have with other Nif proteins involved in FeMo-co synthesis, such as NifD, NifK, NifN, NifDK and NifH. Using the BacterioMatch Two-Hybrid System, a translationally fused construct of NifX with the N-terminal α-RNAP of the pTRG target vector was made and its interaction was tested with the NifDK fusion protein, translationally fused to the λCI of the pBT vector. The strength of the interaction, as determined by measuring the β-galactosidase activity, demonstrated that direct protein-protein interaction exists between NifDK and NifX proteins; the extent of interaction between NifK and NifX proteins was much higher than between NifD and NifX, when individually tested; also, reduced interaction was found between NifH and NifX.

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Nitrogenase Catalysis & AssemblyBased in part on the article Nitrogenases & the Iron–Molybdenum Cofactor by Paul W. Ludden which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

Schmid

Ribbe

2005

Encyclopedia of Inorganic Chemistry

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Nitrogenases are metalloenzymes that catalyze biological N 2 fixation, in which dinitrogen is reduced to ammonia. The best‐characterized molybdenum nitrogenase is composed of the iron (Fe) protein that contains a [4Fe–4S] cluster and the molybdenum–iron (MoFe) protein that contains an [8Fe–7S] cluster (P cluster) and a [Mo–7Fe–9S–homocitrate] cluster (FeMo cofactor). The catalysis of molybdenum nitrogenase occurs in a series of steps. First, the reduced Fe protein binds 2MgATP, undergoes a conformational change, and forms a complex with the MoFe protein. Then, coupled with the hydrolysis of 2MgATP, one electron is transferred from the Fe protein to the MoFe protein within the complex. This is followed by complex dissociation, which allows the enzyme to start the next cycle of electron transfer. Once a sufficient amount of electrons is accumulated on the MoFe protein, substrate reduction takes place. The assembly of the molybdenum nitrogenase Fe protein involves the nifH and at least the nifS, nifU , and nifM gene products that are implicated in the [4Fe–4S] cluster assembly, while the assembly of the MoFe protein requires at least 15 nif gene products and involves the biosynthesis and insertion of the FeMo cofactor into the MoFe protein.

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Accumulation of 55Fe-Labeled Precursors of the Iron-Molybdenum Cofactor of Nitrogenase on NifH and NifX ofAzotobacter vinelandii

Abstract: Iron

Cited by 35 publications

References 39 publications

Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

Cloning and Mutational Analysis of the γ Gene fromAzotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization

The NifX Protein is Involved in the Final Stages of FeMo-cofactor Transport to the MoFe Protein

Nitrogenase Catalysis & AssemblyBased in part on the article Nitrogenases & the Iron–Molybdenum Cofactor by Paul W. Ludden which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

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