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

• 成果类型: Article
• 署名作者: Paya-Tormo, Lucia; Echavarri-Erasun, Carlos; Makarovsky-Saavedra, Natalia; Perez-Gonzalez, Ana; Yang, Zhi-Yong; Guo, Yisong; Seefeldt, Lance C.; Rubio, Luis M.
• 署名单位: Universidad Politecnica de Madrid; Consejo Superior de Investigaciones Cientificas (CSIC); Instituto Nacional Investigacion Tecnologia Agraria Alimentaria (INIA); Universidad Politecnica de Madrid; Utah System of Higher Education; Utah State University; Carnegie Mellon University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10418
• DOI: 10.1073/pnas.2406198121
• 发表日期: 2024-11-12
• 关键词: in-vitro synthesis; p-cluster; azotobacter; purification; EVOLUTION; biosynthesis; homocitrate; protein; component; Fixation

摘要

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).