{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T16:05:26Z","timestamp":1790006726779,"version":"4.0.1"},"reference-count":44,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T00:00:00Z","timestamp":1789948800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T00:00:00Z","timestamp":1789948800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Angew Chem Int Ed"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Biological nitrogen fixation by Mo\u2010nitrogenase is a complex process requiring coordinated electron transfer through specialized metal clusters. NifEN, an \u03b1\n                    <jats:sub>2<\/jats:sub>\n                    \u03b2\n                    <jats:sub>2<\/jats:sub>\n                    heterotetrameric homolog of the nitrogenase catalytic component NifDK, naturally serves as a maturation scaffold but exhibits only trace catalytic activity due to its \u201cimperfect\u201d cluster environments. In this study, we systematically engineered NifEN to reconstruct the structural features of NifDK, specifically targeting the P\u2192M cluster electron relay and the active\u2010site M\u2010cluster environment. Specifically, we developed a series of variants, culminating in NifEN\n                    <jats:sup>P\/M_S2B<\/jats:sup>\n                    , which incorporates a restored P\u2010cluster species and a refined M\u2010cluster coordination site and S2B\u2010associated belt region. Spectroscopic analysis confirmed the successful installation of the target clusters. Moreover, activity assays and nanoSIMS analysis demonstrated that NifEN\n                    <jats:sup>P\/M_S2B<\/jats:sup>\n                    supported ATP\u2010dependent N\n                    <jats:sub>2<\/jats:sub>\n                    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\u2010assembling, 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.\n                  <\/jats:p>","DOI":"10.1002\/anie.8683437","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T15:33:41Z","timestamp":1790004821000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Gain\u2010of\u2010Function Engineering of the NifEN Scaffold Into a Self\u2010Assembling Nitrogenase"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2117-8890","authenticated-orcid":false,"given":"Yimo","family":"Yang","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and Biochemistry University of California  Irvine California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5266-6686","authenticated-orcid":false,"given":"Chi Chung","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Biochemistry University of California  Irvine California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-3601-0626","authenticated-orcid":false,"given":"Steve","family":"Ortiz","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Biochemistry University of California  Irvine California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7366-1526","authenticated-orcid":false,"given":"Markus W.","family":"Ribbe","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Biochemistry University of California  Irvine California USA"},{"name":"Department of Chemistry University of California  Irvine California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9088-2865","authenticated-orcid":false,"given":"Yilin","family":"Hu","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Biochemistry University of California  Irvine California USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,21]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2025.11.009"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.2c00612"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.9b00704"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/cr950057h"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1021\/cr950055x"},{"key":"e_1_2_9_7_1","doi-asserted-by":"crossref","first-page":"971","DOI":"10.1098\/rsta.2004.1539","article-title":"Structural Basis of Biological Nitrogen Fixation","volume":"363","author":"Rees D. 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