Gain‐of‐Function Engineering of the NifEN Scaffold Into a Self‐Assembling Nitrogenase
- 1Department of Molecular Biology and Biochemistry University of California Irvine California USA
- 2Department of Chemistry University of California Irvine California USA
Abstract
Biological nitrogen fixation by Mo‐nitrogenase is a complex process requiring coordinated electron transfer through specialized metal clusters. NifEN, an α2β2 heterotetrameric homolog of the nitrogenase catalytic component NifDK, naturally serves as a maturation scaffold but exhibits only trace catalytic activity due to its “imperfect” cluster environments. In this study, we systematically engineered NifEN to reconstruct the structural features of NifDK, specifically targeting the P→M cluster electron relay and the active‐site M‐cluster environment. Specifically, we developed a series of variants, culminating in NifENP/M_S2B, which incorporates a restored P‐cluster species and a refined M‐cluster coordination site and S2B‐associated belt region. Spectroscopic analysis confirmed the successful installation of the target clusters. Moreover, activity assays and nanoSIMS analysis demonstrated that NifENP/M_S2B supported ATP‐dependent N2 reduction both in vitro and in vivo. Structural modeling further identified remaining catalytic bottlenecks, particularly those in the S3A and S5A regions, highlighting the importance of precise cofactor belt organization for substrate reduction. Together, these findings establish NifEN as a self‐assembling, catalytically competent nitrogenase scaffold that bypasses the need for complex cofactor transfer steps, providing a streamlined platform for mechanistic studies and engineering efforts of biological nitrogen fixation.
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BibTeX
@article{yang2026gainoffunction,
title = {{Gain‐of‐Function Engineering of the NifEN Scaffold Into a Self‐Assembling Nitrogenase}},
author = {Yimo Yang and Chi Chung Lee and Steve Ortiz and Markus W. Ribbe and Yilin Hu},
journal = {Angewandte Chemie (International Ed. in English)},
year = {2026},
month = sep,
doi = {10.1002/anie.8683437},
url = {https://doi.org/10.1002/anie.8683437},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Gain‐of‐Function Engineering of the NifEN Scaffold Into a Self‐Assembling Nitrogenase
AU - Yimo Yang
AU - Chi Chung Lee
AU - Steve Ortiz
AU - Markus W. Ribbe
AU - Yilin Hu
PY - 2026
DA - 2026/09/21
JO - Angewandte Chemie (International Ed. in English)
DO - 10.1002/anie.8683437
UR - https://doi.org/10.1002/anie.8683437
AB - Biological nitrogen fixation by Mo‐nitrogenase is a complex process requiring coordinated electron transfer through specialized metal clusters. NifEN, an α2β2 heterotetrameric homolog of the nitrogenase catalytic component NifDK, naturally serves as a maturation scaffold but exhibits only trace catalytic activity due to its “imperfect” cluster environments. In this study, we systematically engineered NifEN to reconstruct the structural features of NifDK, specifically targeting the P→M cluster electron relay and the active‐site M‐cluster environment. Specifically, we developed a series of variants, culminating in NifENP/M_S2B, which incorporates a restored P‐cluster species and a refined M‐cluster coordination site and S2B‐associated belt region. Spectroscopic analysis confirmed the successful installation of the target clusters. Moreover, activity assays and nanoSIMS analysis demonstrated that NifENP/M_S2B supported ATP‐dependent N2 reduction both in vitro and in vivo. Structural modeling further identified remaining catalytic bottlenecks, particularly those in the S3A and S5A regions, highlighting the importance of precise cofactor belt organization for substrate reduction. Together, these findings establish NifEN as a self‐assembling, catalytically competent nitrogenase scaffold that bypasses the need for complex cofactor transfer steps, providing a streamlined platform for mechanistic studies and engineering efforts of biological nitrogen fixation.
SN - 1433-7851
SN - 1521-3773
ER -