22 results on '"Jimenez-Vicente, Emilio"'
Search Results
2. CO as a substrate and inhibitor of H+ reduction for the Mo-, V-, and Fe-nitrogenase isozymes
- Author
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Harris, Derek F., Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Hoffman, Brian M., Dean, Dennis R., and Seefeldt, Lance C.
- Published
- 2020
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3. Electron Paramagnetic Resonance Characterization of Three Iron–Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus
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Wilcoxen, Jarett, Arragain, Simon, Scandurra, Alessandro A, Jimenez-Vicente, Emilio, Echavarri-Erasun, Carlos, Pollmann, Stephan, Britt, R David, and Rubio, Luis M
- Subjects
Inorganic Chemistry ,Chemical Sciences ,Bacterial Proteins ,Electron Spin Resonance Spectroscopy ,Iron Compounds ,Methanocaldococcus ,Molybdoferredoxin ,Nitrogenase ,S-Adenosylmethionine ,Substrate Specificity ,General Chemistry ,Chemical sciences ,Engineering - Abstract
NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed.
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- 2016
4. AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV-cofactor
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Perez-Gonzalez, Ana, Jimenez-Vicente, Emilio, Salinero-Lanzarote, Alvaro, Harris, Derek F., Seefeldt, Lance C., Dean, Dennis R., Perez-Gonzalez, Ana, Jimenez-Vicente, Emilio, Salinero-Lanzarote, Alvaro, Harris, Derek F., Seefeldt, Lance C., and Dean, Dennis R.
- Abstract
Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N-2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N-2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N-2. These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth.
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- 2022
5. A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster
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Van Stappen, Casey, Jimenez-Vicente, Emilio, Perez-Gonzalez, Ana, Yang, Zhi-Yong, Seefeldt, Lance C., DeBeer, Serena, Dean, Dennis R., Decamps, Laure, Van Stappen, Casey, Jimenez-Vicente, Emilio, Perez-Gonzalez, Ana, Yang, Zhi-Yong, Seefeldt, Lance C., DeBeer, Serena, Dean, Dennis R., and Decamps, Laure
- Abstract
Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD(2)K(2) polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD(2)K(2). P-cluster maturation involves the iron protein NifH(2) as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD(2)K(2) species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD(2)K(2) complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S](2+/+) clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.
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- 2022
6. AnfO controls fidelity of nitrogenase FeFe protein maturation by preventing misincorporation of FeV ‐cofactor
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Pérez‐González, Ana, primary, Jimenez‐Vicente, Emilio, additional, Salinero‐Lanzarote, Alvaro, additional, Harris, Derek F., additional, Seefeldt, Lance C., additional, and Dean, Dennis R., additional
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- 2022
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7. Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii
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Pérez-González, Ana, primary, Jimenez-Vicente, Emilio, additional, Gies-Elterlein, Jakob, additional, Salinero-Lanzarote, Alvaro, additional, Yang, Zhi-Yong, additional, Einsle, Oliver, additional, Seefeldt, Lance C., additional, and Dean, Dennis R., additional
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- 2021
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8. Specificity of NifEN and VnfEN for the Assembly of Nitrogenase Active Site Cofactors in Azotobacter vinelandii
- Author
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Perez-Gonzalez, Ana, Jimenez-Vicente, Emilio, Gies-Elterlein, Jakob, Salinero-Lanzarote, Alvaro, Yang, Zhi-Yong, Einsle, Oliver, Seefeldt, Lance C., Dean, Dennis R., Perez-Gonzalez, Ana, Jimenez-Vicente, Emilio, Gies-Elterlein, Jakob, Salinero-Lanzarote, Alvaro, Yang, Zhi-Yong, Einsle, Oliver, Seefeldt, Lance C., and Dean, Dennis R.
- Abstract
The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertiliza
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- 2021
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9. The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase
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Yang, Zhi-Yong, Jimenez-Vicente, Emilio, Kallas, Hayden, Lukoyanov, Dmitriy A., Yang, Hao, Del Campo, Julia S. Martin, Dean, Dennis R., Hoffman, Brian M., Seefeldt, Lance C., Yang, Zhi-Yong, Jimenez-Vicente, Emilio, Kallas, Hayden, Lukoyanov, Dmitriy A., Yang, Hao, Del Campo, Julia S. Martin, Dean, Dennis R., Hoffman, Brian M., and Seefeldt, Lance C.
- Abstract
The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V3+, 3Fe3+, 4Fe2+]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51V (I = 7/2) hyperfine splitting, aiso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V3+, 4Fe3+, 3Fe2+]. Our findings suggest that the V3+ does not change valency throughout the catalytic cycle.
- Published
- 2021
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10. The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase
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Biochemistry, Yang, Zhi-Yong, Jimenez-Vicente, Emilio, Kallas, Hayden, Lukoyanov, Dmitriy A., Yang, Hao, Del Campo, Julia S. Martin, Dean, Dennis R., Hoffman, Brian M., Seefeldt, Lance C., Biochemistry, Yang, Zhi-Yong, Jimenez-Vicente, Emilio, Kallas, Hayden, Lukoyanov, Dmitriy A., Yang, Hao, Del Campo, Julia S. Martin, Dean, Dennis R., Hoffman, Brian M., and Seefeldt, Lance C.
- Abstract
The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2 EPR signal (Kramers state) having g values of ∼4.3 and 3.8, along with suggestions that it contains metal-ions with valencies [1V3+, 3Fe3+, 4Fe2+]. In the present work, genetic, biochemical, and spectroscopic approaches were combined to reveal that the EPR signals previously assigned to FeV-cofactor do not correlate with active VFe-protein, and thus cannot arise from the resting-state of catalytically relevant FeV-cofactor. It, instead, appears resting-state FeV-cofactor is either diamagnetic, S = 0, or non-Kramers, integer-spin (S = 1, 2 etc.). When VFe-protein is freeze-trapped during high-flux turnover with its natural electron-donating partner Fe protein, conditions which populate reduced states of the FeV-cofactor, a new rhombic S = 1/2 EPR signal from such a reduced state is observed, with g = [2.18, 2.12, 2.09] and showing well-defined 51V (I = 7/2) hyperfine splitting, aiso = 110 MHz. These findings indicate a different assignment for the electronic structure of the resting state of FeV-cofactor: S = 0 (or integer-spin non-Kramers state) with metal-ion valencies, [1V3+, 4Fe3+, 3Fe2+]. Our findings suggest that the V3+ does not change valency throughout the catalytic cycle.
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- 2021
11. The electronic structure of FeV-cofactor in vanadium-dependent nitrogenase
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Yang, Zhi-Yong, primary, Jimenez-Vicente, Emilio, additional, Kallas, Hayden, additional, Lukoyanov, Dmitriy A., additional, Yang, Hao, additional, Martin del Campo, Julia S., additional, Dean, Dennis R., additional, Hoffman, Brian M., additional, and Seefeldt, Lance C., additional
- Published
- 2021
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12. The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters
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Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Del Campo, Julia S. Martin, Cash, Valerie L., Seefeldt, Lance C., Dean, Dennis R., Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Del Campo, Julia S. Martin, Cash, Valerie L., Seefeldt, Lance C., and Dean, Dennis R.
- Abstract
The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an (22) heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N-2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N-2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity-based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one -unit with a FeMo-cofactor and mature P-cluster and the other -unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.
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- 2019
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13. The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters
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Biochemistry, Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Del Campo, Julia S. Martin, Cash, Valerie L., Seefeldt, Lance C., Dean, Dennis R., Biochemistry, Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Del Campo, Julia S. Martin, Cash, Valerie L., Seefeldt, Lance C., and Dean, Dennis R.
- Abstract
The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an (22) heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N-2 reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N-2-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity-based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii. These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one -unit with a FeMo-cofactor and mature P-cluster and the other -unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.
- Published
- 2019
14. Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation
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Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Ray, W. Keith, Echavarri-Erasun, Carlos, Cash, Valerie L., Rubio, Luis M., Seefeldt, Lance C., Dean, Dennis R., Biochemistry, and American Society for Biochemistry and Molecular Biology
- Subjects
Azotobacter vinelandii ,Molybdoferredoxin ,Protein Conformation ,protein assembly ,nitrogenase ,Catalysis ,Electron Transport ,Chemistry ,electron paramagnetic resonance (EPR) ,P-cluster ,Bacterial Proteins ,FeMo-cofactor ,MoFe protein ,nitrogen fixation ,Catalytic Domain ,protein purification ,reductase ,Enzymology - Abstract
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as assembly factors. Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein-assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein-NafY, MoFe protein-NifY, and MoFe protein-NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states. Fralin Life Science Institute; Agricultural Experiment Station Hatch Program; McIntire-Stennis Program at Virginia Tech Mass spectrometry resources used in this work are maintained in part through funding by the Fralin Life Science Institute, the Agricultural Experiment Station Hatch Program, and the McIntire-Stennis Program at Virginia Tech and are managed by Richard F. Helm.
- Published
- 2018
15. The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters
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Jimenez-Vicente, Emilio, primary, Yang, Zhi-Yong, additional, Martin del Campo, Julia S., additional, Cash, Valerie L., additional, Seefeldt, Lance C., additional, and Dean, Dennis R., additional
- Published
- 2019
- Full Text
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16. Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation
- Author
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Biochemistry, Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Ray, W. Keith, Echavarri-Erasun, Carlos, Cash, Valerie L., Rubio, Luis M., Seefeldt, Lance C., Dean, Dennis R., Biochemistry, Jimenez-Vicente, Emilio, Yang, Zhi-Yong, Ray, W. Keith, Echavarri-Erasun, Carlos, Cash, Valerie L., Rubio, Luis M., Seefeldt, Lance C., and Dean, Dennis R.
- Abstract
Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as assembly factors. Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein-assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein-NafY, MoFe protein-NifY, and MoFe protein-NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states.
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- 2018
17. Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation
- Author
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Jimenez-Vicente, Emilio, primary, Yang, Zhi-Yong, additional, Ray, W. Keith, additional, Echavarri-Erasun, Carlos, additional, Cash, Valerie L., additional, Rubio, Luis M., additional, Seefeldt, Lance C., additional, and Dean, Dennis R., additional
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- 2018
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18. Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB
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Arragain, Simon, Jimenez-Vicente, Emilio, Scandurra, Alessandro A., Buren, Stefan, Rubio, Luis M., Echavarri-Erasun, Carlos, Arragain, Simon, Jimenez-Vicente, Emilio, Scandurra, Alessandro A., Buren, Stefan, Rubio, Luis M., and Echavarri-Erasun, Carlos
- Abstract
One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (singledomain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (Delta nifB) mutant restoring the Nif(+) phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.
- Published
- 2017
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19. Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB
- Author
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Biochemistry, Arragain, Simon, Jimenez-Vicente, Emilio, Scandurra, Alessandro A., Buren, Stefan, Rubio, Luis M., Echavarri-Erasun, Carlos, Biochemistry, Arragain, Simon, Jimenez-Vicente, Emilio, Scandurra, Alessandro A., Buren, Stefan, Rubio, Luis M., and Echavarri-Erasun, Carlos
- Abstract
One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (singledomain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (Delta nifB) mutant restoring the Nif(+) phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles.
- Published
- 2017
20. Electron Paramagnetic Resonance Characterization of Three Iron–Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB fromMethanocaldococcus infernus
- Author
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Wilcoxen, Jarett, primary, Arragain, Simon, additional, Scandurra, Alessandro A., additional, Jimenez-Vicente, Emilio, additional, Echavarri-Erasun, Carlos, additional, Pollmann, Stephan, additional, Britt, R. David, additional, and Rubio, Luis M., additional
- Published
- 2016
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21. EXAFS reveals two Mo environments in the nitrogenase iron–molybdenum cofactor biosynthetic protein NifQ
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George, Simon J., primary, Hernandez, Jose A., additional, Jimenez-Vicente, Emilio, additional, Echavarri-Erasun, Carlos, additional, and Rubio, Luis M., additional
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- 2016
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22. Molecular Microbiology
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Ana Pérez‐González, Emilio Jimenez‐Vicente, Alvaro Salinero‐Lanzarote, Derek F. Harris, Lance C. Seefeldt, Dennis R. Dean, Bill & Melinda Gates Foundation, Pérez-González, Ana, Jimenez-Vicente, Emilio, Salinero-Lanzarote, Alvaro, Harris, Derek F, Seefeldt, Lance C, Dean, Dennis R, Pérez-González, Ana [0000-0002-7724-9985], Jimenez-Vicente, Emilio [0000-0002-2843-5132], Salinero-Lanzarote, Alvaro [0000-0001-5980-460X], Harris, Derek F [0000-0003-4277-2976], Seefeldt, Lance C [0000-0002-6457-9504], and Dean, Dennis R [0000-0001-8960-6196]
- Subjects
Molybdoferredoxin ,Azotobacter vinelandii ,Bacterial Proteins ,Metalloprotein ,Nitrogen fixation ,Catalytic Domain ,FeFe protein ,FeV-cofactor ,Nitrogenase ,Molecular Biology ,Microbiology - Abstract
Azotobacter vinelandii produces three genetically distinct, but structurally and mechanistically similar nitrogenase isozymes designated as Mo-dependent, V-dependent, or Fe-only based on the heterometal contained within their associated active site cofactors. These catalytic cofactors, which provide the site for N2 binding and reduction, are, respectively, designated as FeMo-cofactor, FeV-cofactor, and FeFe-cofactor. Fe-only nitrogenase is a poor catalyst for N2 fixation, when compared to the Mo-dependent and V-dependent nitrogenases and is only produced when neither Mo nor V is available. Under conditions favoring the production of Fe-only nitrogenase a gene product designated AnfO preserves the fidelity of Fe-only nitrogenase by preventing the misincorporation of FeV-cofactor, which results in the accumulation of a hybrid enzyme that cannot reduce N2 . These results are interpreted to indicate that AnfO controls the fidelity of Fe-only nitrogenase maturation during the physiological transition from conditions that favor V-dependent nitrogenase utilization to Fe-only nitrogenase utilization to support diazotrophic growth., The work performed on the laboratory of DRD was supported by Bill and Melinda Gates Foundation Grants BNF Cereals Phase II (OPP1143172) and BNF Cereals Phase III (INV-005889). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.
- Published
- 2022
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