Heterologous expression of a fully active Azotobacter vinelandii nitrogenase Fe protein in Escherichia coli

Journal Article · 19 December 2023 · mBio (Online)

DOI: https://doi.org/10.1128/mbio.02572-23 · OSTI ID:2204441

Solomon, Joseph B. ^[1]; Liu, Yiling A. ^[2]; Górecki, Kamil ^[2]; Quechol, Robert ^[2]; Lee, Chi Chung ^[2]; Jasniewski, Andrew J. ^[2]; Hu, Yilin  ^[2]; Ribbe, Markus W.  ^[1]; Johnson, ed., Michael David Leslie

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1. Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA, Department of Chemistry, University of California, Irvine, California, USA,
2. Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA

ABSTRACT The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii ( Av NifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active Av NifH protein upon co-expression of this protein with Av IscS/U and Av NifM in E. coli . Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed Av NifH protein has a high [Fe ₄ S ₄ ] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that Av NifM could serve as a chaperone and assist the maturation of a cluster-replete Av NifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the “clean” genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

• Research Organization: Univ. of California, Irvine, CA (United States)
• Sponsoring Organization: USDOE; USDOE Office of Science (SC)
• Grant/Contract Number: SC0016510; CHE-1651398
• OSTI ID: 2204441
• Alternate ID(s): OSTI ID: 2472186
• Journal Information: mBio (Online), Journal Name: mBio (Online) Vol. 14 Journal Issue: 6; ISSN 2150-7511
• Publisher: American Society for MicrobiologyCopyright Statement
• Country of Publication: United States
• Language: English