Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

Payá-Tormo, Lucía; Echavarri-Erasun, Carlos; Makarovsky-Saavedra, Natalia; Pérez-González, Ana; Yang, Zhi-Yong; Guo, Yisong; Seefeldt, Lance C.; Rubio, Luis M.

doi:10.1073/pnas.2406198121

Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN

Journal Article · Wed Nov 06 04:00:00 EST 2024 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2406198121· OSTI ID:2476412

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Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid e Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria/Consejo Superior de Investigaciones Científicas, Madrid 28223, Spain, Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid 28040, Spain
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid e Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria/Consejo Superior de Investigaciones Científicas, Madrid 28223, Spain
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322
Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213

The maturation and installation of the active site metal cluster (FeMo-co, Fe ₇ S ₉ CMo- R -homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe ₈ S ₉ C]) 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 .

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Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0010687

OSTI ID:: 2476412

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 46 Vol. 121; ISSN 0027-8424

Publisher:: Proceedings of the National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

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