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Portions of the paper were developed from the thesis of L.P.-T.Wethank Luis Fernandez Pacios for NifDKstructure models, Marcel Veldhuizen for help withE.coli fermentations, and Alvaro Salinero and Diana Coroian fortechnical support. This work was supported in part or in whole by the Bill & Melinda Gates Foundation (INV-005889) to L.M.R. Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to theAuthorAccepted Manuscript version that might arise from this submission.This work was also supported by grant PID2021-128802OB-100 funded by MICIU/AEI/s1910.13039/501100011033 and by ERDF/EU. L.C.S. and Z.-Y.Y. were supported by US Department of Energy, Office of Science, Basic Energy Sciences (BES) award DE-SC0010687 to L.C.S. L.P.-T. was recipient of FPU16/02284 funded by Ministerio de Educacion, Cultura y Deporte. N.M.-S. is a recipient of PRE2022-102872 funded by MICIU/AEI/10.13039/501100011033 and by ESF+. A.P.-G. is a recipient of RYC2021-031246-I funded by MICIU/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR.

Analysis of institutional authors

Paya-Tormo, LuciaAuthorEchavarri-Erasun, CarlosCorresponding AuthorMakarovsky-Saavedra, NataliaAuthorPerez-Gonzalez, AnaAuthorRubio, Luis MCorresponding Author

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December 3, 2024
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Iron- molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

Publicated to:Proceedings Of The National Academy Of Sciences Of The United States Of America. 121 (46): e2406198121- - 2024-11-12 121(46), DOI: 10.1073/pnas.2406198121

Authors: Payá-Tormo, L; Echavarri-Erasun, C; Makarovsky-Saavedra, N; Pérez-González, A; Yang, ZY; Guo, YS; Seefeldt, LC; Rubio, LM

Affiliations

Carnegie Mellon Univ, Dept Chem, Pittsburgh, PA 15213 USA - Author
CSIC, Inst Nacl Invest & Tecnol Agr & Alimentaria, Madrid 28223, Spain - Author
Univ Politecn Madrid, Ctr Biotecnol & Genom Plantas, Madrid 28223, Spain - Author
Univ Politecn Madrid, Escuela Tecn Super Ingn Agron Alimentaria & Biosis, Dept Biotecnol Biol Vegetal, Madrid 28040, Spain - Author
Utah State Univ, Dept Chem & Biochem, Logan, UT 84322 USA - Author
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Abstract

The maturation and installation of the active site metal cluster (FeMo-co, Fe7S9CMo-R-homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe8S9C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDK(RS) contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK(RS)). Apo-NifDK(RS) could be activated for N-2 reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK(RS) plus E. coli produced NifB(RS) and NifH(RS) were sufficient to yield active NifDK(RS) when incubated with the necessary substrates (homocitrate, molybdate, and S-adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifH(RS) and NifB(RS) proteins were independently shown to be functional. The reconstituted NifDK(RS) demonstrated reduction of N-2, protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH(RS), NifB(RS), and apo-NifDK(RS).

Keywords

Amino acid sequenceArticleAzotobacterAzotobacter vinelandiiBacterial proteinBacterial proteinsBiosynthesisCoenzymeCoenzymesComponentDrug synthesisElectron spin resonanceEnzyme activityEnzymologyEscherichia coliEvolutionFemo-cFemo-coFixatioFluorometryGas chromatographyGene expressionGeneticsHomocitrateHydrogen bondIn vitro studyIn-vitro synthesisMass fragmentographyMass spectrometryMetabolismMetalloproteinMetalloproteinsMolybdenum cofactorMolybdenum cofactorsMolybdenum iron proteinMolybdoferredoxinMolybdopterinNifb protein, bacteriaNifenNitrogen fixation evolutionNitrogenaseNonhumanP-clusterProteinProtein purificationPteridine derivativePteridinesPurificationRoseiflexusSize exclusion chromatography

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal Proceedings Of The National Academy Of Sciences Of The United States Of America due to its progression and the good impact it has achieved in recent years, according to the agency WoS (JCR), it has become a reference in its field. In the year of publication of the work, 2024 there are still no calculated indicators, but in 2023, it was in position 13/134, thus managing to position itself as a Q1 (Primer Cuartil), in the category Multidisciplinary Sciences. Notably, the journal is positioned above the 90th percentile.

Independientemente del impacto esperado determinado por el canal de difusión, es importante destacar el impacto real observado de la propia aportación.

Según las diferentes agencias de indexación, el número de citas acumuladas por esta publicación hasta la fecha 2025-07-07:

  • Scopus: 1

Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2025-07-07:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 13.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 13 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 50.75.
  • The number of mentions on the social network X (formerly Twitter): 6 (Altmetric).
  • The number of mentions in news outlets: 5 (Altmetric).

It is essential to present evidence supporting full alignment with institutional principles and guidelines on Open Science and the Conservation and Dissemination of Intellectual Heritage. A clear example of this is:

  • The work has been submitted to a journal whose editorial policy allows open Open Access publication.

Leadership analysis of institutional authors

This work has been carried out with international collaboration, specifically with researchers from: United States of America.

There is a significant leadership presence as some of the institution’s authors appear as the first or last signer, detailed as follows: First Author (PAYA TORMO, LUCIA) and Last Author (RUBIO HERRERO, LUIS MANUEL).

the authors responsible for correspondence tasks have been ECHAVARRI ERASUN, CARLOS and RUBIO HERRERO, LUIS MANUEL.