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Cited by 7Metalloenzymes and iron-sulfur proteinsAmmonia Synthesis and Nitrogen ReductionInorganic Chemistry and Materials

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Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

Open access
Barahona, Emma, Collantes-García, Juan Andrés, Rosa-Núñez, Elena, Xiong, Jin, Jiang, Xi, Jiménez-Vicente, Emilio, Echávarri-Erasun, Carlos, Guo, Yisong, Rubio, Luis M., González-Guerrero, Manuel·2024·Journal of Biological Chemistry
DOI 10.1016/j.jbc.2024.107900PMC PMC11605450

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Abstract

ABSTRACT Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe 3 S 4 ] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe 4 -S 4 ] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.

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Crossref · 10.1016/j.jbc.2024.107900

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Figures

Figure 1 NifU and NifS copurify withSNifQ from E. coli extracts expressing nifQ, nifU, and nifS.Top panel shows the Coomassie staining of an SDS-PAGE of cell free extract (CFE), flowthrough (FT), wash (W1-W6) and elution (E1-E3) fractions of the extracts passed through a Streptactin column. The remaining panels show immunoblots of the same fractions developed with anti-NifQ, anti-NifU, or anti-NifS antibodies. Images show a representative assay (n = 2). Uncropped immunoblots and gels are shown in Fig. S5.
Figure 1 NifU and NifS copurify withSNifQ from E. coli extracts expressing nifQ, nifU, and nifS.Top panel shows the Coomassie staining of an SDS-PAGE of cell free extract (CFE), flowthrough (FT), wash (W1-W6) and elution (E1-E3) fractions of the extracts passed through a Streptactin column. The remaining panels show immunoblots of the same fractions developed with anti-NifQ, anti-NifU, or anti-NifS antibodies. Images show a representative assay (n = 2). Uncropped immunoblots and gels are shown in Fig. S5.CC BY · PubMed Central
Figure 2 AS-NifUS interacts with apo-NifQH.Top panel shows the Coomassie staining of an SDS-PAGE of flowthrough (FT), wash (W1-W12) and elution (E1-E2) fractions of a mixture solution containing AS-NifUS, SNifS, and apo-NifQH passed through a Ni2+ column. The remaining panels are the immunoblots of the same fractions developed with anti-NifQ, anti-NifU, or anti-NifS antibodies. Images show a representative assay (n = 3). Uncropped immunoblots and gels are shown in Fig. S6.
Figure 2 AS-NifUS interacts with apo-NifQH.Top panel shows the Coomassie staining of an SDS-PAGE of flowthrough (FT), wash (W1-W12) and elution (E1-E2) fractions of a mixture solution containing AS-NifUS, SNifS, and apo-NifQH passed through a Ni2+ column. The remaining panels are the immunoblots of the same fractions developed with anti-NifQ, anti-NifU, or anti-NifS antibodies. Images show a representative assay (n = 3). Uncropped immunoblots and gels are shown in Fig. S6.CC BY · PubMed Central
Figure 3 Holo-NifQHdoes not co-elute with AS-NifUS. Top panel shows the Coomassie staining of an SDS-PAGE of holo-NifQH pull-down assay using AS-NifUS as bait. FT1 represents the flow-through fraction obtained after loading AS-NifUS onto the Strep-column. W2 represents the second wash fraction. FT2 represents the flow-through fraction obtained after loading holo-NifQH onto the column. W3 represents the third wash fraction after passing holo-NifQH over an AS-NifUS-charged column. E1, 2, 3, four and five represent elution fractions. Images show a representative assay (n = 4). Uncropped immunoblots and gels are shown in Fig. S7.
Figure 3 Holo-NifQHdoes not co-elute with AS-NifUS. Top panel shows the Coomassie staining of an SDS-PAGE of holo-NifQH pull-down assay using AS-NifUS as bait. FT1 represents the flow-through fraction obtained after loading AS-NifUS onto the Strep-column. W2 represents the second wash fraction. FT2 represents the flow-through fraction obtained after loading holo-NifQH onto the column. W3 represents the third wash fraction after passing holo-NifQH over an AS-NifUS-charged column. E1, 2, 3, four and five represent elution fractions. Images show a representative assay (n = 4). Uncropped immunoblots and gels are shown in Fig. S7.CC BY · PubMed Central
Figure 4 NifUS transfers iron to apo-NifQH.A, top panel shows the Coomassie staining of an SDS-PAGE of flowthrough (FT), wash (W1-W7) and elution (E1) fractions of a mixture solution containing R-NifUS and apo-NifQH passed through a Strep-tactin column. The remaining panels are the immunoblots of the same fractions developed with anti-NifQ or an anti-NifU antibody. Uncropped immunoblots and gels are shown in Fig. S8. B, iron content per monomer of isolated proteins prior to interaction, and in the FT fraction obtained from passing through a Strep-tactin-column a solution in which apo-NifQH was incubated for 5 min with R-NifUS. Bars represent the average ± SD (n = 3). Different letters indicate statistically significant differences (p < 0.01). The dots represent each individual data point.
Figure 4 NifUS transfers iron to apo-NifQH.A, top panel shows the Coomassie staining of an SDS-PAGE of flowthrough (FT), wash (W1-W7) and elution (E1) fractions of a mixture solution containing R-NifUS and apo-NifQH passed through a Strep-tactin column. The remaining panels are the immunoblots of the same fractions developed with anti-NifQ or an anti-NifU antibody. Uncropped immunoblots and gels are shown in Fig. S8. B, iron content per monomer of isolated proteins prior to interaction, and in the FT fraction obtained from passing through a Strep-tactin-column a solution in which apo-NifQH was incubated for 5 min with R-NifUS. Bars represent the average ± SD (n = 3). Different letters indicate statistically significant differences (p < 0.01). The dots represent each individual data point.CC BY · PubMed Central
Figure 5 NifQ requires physical interaction from NifU to receive iron. Iron content per monomer of pure isolated proteins or from proteins separated by a 2-kDa pore-size cutoff dialysis membrane after five or 120 min incubation. Bars represent average iron content ± SD (n = 3). Different letters indicate statistically significant differences after being compared by ANOVA followed by Bonferroni’s multiple comparison test (p < 0.01). The dots represent each individual data point.
Figure 5 NifQ requires physical interaction from NifU to receive iron. Iron content per monomer of pure isolated proteins or from proteins separated by a 2-kDa pore-size cutoff dialysis membrane after five or 120 min incubation. Bars represent average iron content ± SD (n = 3). Different letters indicate statistically significant differences after being compared by ANOVA followed by Bonferroni’s multiple comparison test (p < 0.01). The dots represent each individual data point.CC BY · PubMed Central
Figure 6 NifU is the primary donor of [Fe-S] clusters to NifQ in vivo.A, His-NifQ purification using A. vinelandii UW3000 (wild type strain) and MGGAV1 (ΔnifU strain) cells. Elutions were tested for HNifQ presence Coomassie stained SDS gels. W1 to W3 indicates the three washes, and E1 to E6 the elution fractions, Final is the concentrated protein combined from E1 to E6. B, Ponceau staining and immunoblot of the purified concentrated HNifQ obtained from UW3000 or MGGAV1. The table shows the iron or molybdenum molar ratios of the purified NifQS. C, visible molar extinction coefficients of pure NifQ isolated from UW3000 (green) or MGGAV1 (red).
Figure 6 NifU is the primary donor of [Fe-S] clusters to NifQ in vivo.A, His-NifQ purification using A. vinelandii UW3000 (wild type strain) and MGGAV1 (ΔnifU strain) cells. Elutions were tested for HNifQ presence Coomassie stained SDS gels. W1 to W3 indicates the three washes, and E1 to E6 the elution fractions, Final is the concentrated protein combined from E1 to E6. B, Ponceau staining and immunoblot of the purified concentrated HNifQ obtained from UW3000 or MGGAV1. The table shows the iron or molybdenum molar ratios of the purified NifQS. C, visible molar extinction coefficients of pure NifQ isolated from UW3000 (green) or MGGAV1 (red).CC BY · PubMed Central
Figure 7 NifQ receives a [Fe4-S4] cluster from NifU.A, Molar extinction coefficients in UV-Visible spectra. B, Molar extinction coefficients in UV-Visible spectra of flowthrough fraction after 5-min apo-NifQH/R-NifUS interaction. C, X-band cw-EPR spectra in the field range of 300 to 400 mT at 15 K under power-unsaturated conditions of the following NifQ and NifU samples: 1) apo-NifQH, 2) R-NifUS, 3) flowthrough fraction after 5 min interaction of apo-NifQH and R-NifUS, 4) a second biological replicate of the spectrum of flowthrough fraction after 5 min interaction of apo-NifQH and R-NifUS, and 5) AS-NifQH. Black lines present experimental spectra, red lines present total simulation, while colored dashed lines present individual simulation components: Species I (green), Species II (blue), and Species III (orange).
Figure 7 NifQ receives a [Fe4-S4] cluster from NifU.A, Molar extinction coefficients in UV-Visible spectra. B, Molar extinction coefficients in UV-Visible spectra of flowthrough fraction after 5-min apo-NifQH/R-NifUS interaction. C, X-band cw-EPR spectra in the field range of 300 to 400 mT at 15 K under power-unsaturated conditions of the following NifQ and NifU samples: 1) apo-NifQH, 2) R-NifUS, 3) flowthrough fraction after 5 min interaction of apo-NifQH and R-NifUS, 4) a second biological replicate of the spectrum of flowthrough fraction after 5 min interaction of apo-NifQH and R-NifUS, and 5) AS-NifQH. Black lines present experimental spectra, red lines present total simulation, while colored dashed lines present individual simulation components: Species I (green), Species II (blue), and Species III (orange).CC BY · PubMed Central
Figure 8 NifU provides [Fe4-S4] clusters to NifH, NifB, and NifQ for FeMo-co biosynthesis. [Fe4-S4]-containing NifU transfers this group to NifH, NifB, and NifQ. The latter likely interacts with an unknown protein that would mediate the substitution of a Fe for a Mo in the cluster. This group will be transferred together with the NifB-co synthesized by NifB with electrons likely provided by FdxN, and homocitrate to NifEN, where in a process assisted by NifH, FeMo-co will be produced. Structural models for NifU, NifQ, and FdxN were generated using AlphaFold (29) and the Protein Data Bank accession 1CP2 for NifH and 6Y1X for NifB. All structures were visualized with PyMOL (Schörindger, Inc.).
Figure 8 NifU provides [Fe4-S4] clusters to NifH, NifB, and NifQ for FeMo-co biosynthesis. [Fe4-S4]-containing NifU transfers this group to NifH, NifB, and NifQ. The latter likely interacts with an unknown protein that would mediate the substitution of a Fe for a Mo in the cluster. This group will be transferred together with the NifB-co synthesized by NifB with electrons likely provided by FdxN, and homocitrate to NifEN, where in a process assisted by NifH, FeMo-co will be produced. Structural models for NifU, NifQ, and FdxN were generated using AlphaFold (29) and the Protein Data Bank accession 1CP2 for NifH and 6Y1X for NifB. All structures were visualized with PyMOL (Schörindger, Inc.).CC BY · PubMed Central

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Metadata

Authors
Barahona, E., Collantes-García, J. A., Rosa-Núñez, E., Xiong, J., Jiang, X., Jiménez-Vicente, E., Echávarri-Erasun, C., Guo, Y., Rubio, L. M., & González-Guerrero, M.
Affiliations
  • Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (ES)
  • Carnegie Mellon University (US)
Corresponding author
Rubio, Luis M.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2024-11
Volume
300
Issue
11
Pages
107900
ISSN
0021-9258 (ISSN-L)
DOI
10.1016/j.jbc.2024.107900
PMC
PMC11605450
OpenAlex
W4403660587
Cited by
7
References
40
Citations by year
2026: 5 · 2025: 1
FWCI
0.40
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Hispana (acceptedVersion, cc-by-nc-nd)
  • UPM Digital Archive (Technical University of Madrid) (acceptedVersion, cc-by-nc-nd)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
  • BURJC Digital (King Juan Carlos University) (submittedVersion, cc-by)
  • Hispana (submittedVersion, cc-by)
Funders
  • Foundation for the National Institutes of Health
  • State Agency of Research (PRE2022-101253, PRE2018-084895, SEV-2016-0672)
  • Bill and Melinda Gates Foundation (INV-005889, PID2021-124060OB-100)
  • National Institutes of Health
  • European Regional Development Fund
  • Universidad Politécnica de Madrid
  • Ministerio de Ciencia e Innovación
  • Consejo Superior de Investigaciones Científicas
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Metalloenzymes and iron-sulfur proteins
  • Physical Sciences › Chemical Engineering › Catalysis › Ammonia Synthesis and Nitrogen Reduction
  • Physical Sciences › Chemistry › Inorganic Chemistry › Inorganic Chemistry and Materials
Keywords
Azotobacter vinelandii, nitrogenase, NifU, IscU, protein-protein interaction
MeSH terms
Molybdenum Cofactors, Bacterial Proteins, Coenzymes, Iron, Metalloproteins, Molybdenum, Molybdoferredoxin, Nitrogenase, Pteridines, Azotobacter vinelandii

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CoChat. (2026). Citation check: Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase. https://app.cochat.ai/papers/10.1016/j.jbc.2024.107900
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Barahona, E., Collantes-García, J. A., Rosa-Núñez, E., Xiong, J., Jiang, X., Jiménez-Vicente, E., Echávarri-Erasun, C., Guo, Y., Rubio, L. M., & González-Guerrero, M. (2024). Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase. Journal of Biological Chemistry. https://doi.org/10.1016/j.jbc.2024.107900

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