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The identities and functional relevance of gas channels leading to the buried [NiFe] active site remain elusive, hindering rational enzyme engineering. Here, we introduce an artificial intelligence\u2013guided\n                    <jats:italic>de novo<\/jats:italic>\n                    protein binder design strategy to map hydrophobic O\n                    <jats:sub>2<\/jats:sub>\n                    diffusion pathways in\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    hydrogenase\u20102 (Hyd\u20102). By integrating RFdiffusion, ProteinMPNN, and AlphaFold, \u223c100,000 candidate binders were computationally screened, yielding two high\u2010affinity binders, L1 and L2. Biophysical and electrochemical analyses show that L1 selectively occludes the primary O\n                    <jats:sub>2<\/jats:sub>\n                    ingress channel, enhancing the enzyme's oxygen tolerance by more than threefold, whereas L2, which targets another putative channel, has a negligible effect, indicating that this pathway contributes minimally to O\n                    <jats:sub>2<\/jats:sub>\n                    ingress under the tested conditions. Furthermore, we determined the L1\u2010Hyd\u20102 complex structure by cryo\u2010electron microscopy and revealed the key interaction residues and interface conformation. Integrated structural and computational analyses provide mechanistic insights into the system's oxygen tolerance and high\u2010affinity L1\u2010Hyd\u20102 interaction. All findings provide the first direct experimental evidence for hierarchical O\n                    <jats:sub>2<\/jats:sub>\n                    diffusion channels in [NiFe]\u2010hydrogenases and establish binder\u2010mediated channel occlusion as a generalizable, mutation\u2010free strategy for elucidating and modulating gas transport in metalloenzymes.\n                  <\/jats:p>","DOI":"10.1002\/anie.6395554","type":"journal-article","created":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T06:34:51Z","timestamp":1787812491000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["<i>De Novo<\/i>\n                    Design of a Protein Binder to Probe Gas Channel and Enhance the Oxygen Tolerance of [NiFe]\u2010Hydrogenase"],"prefix":"10.1002","volume":"65","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2648-2637","authenticated-orcid":false,"given":"Xuan","family":"Sun","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences &amp; Biotechnology, the Joint International Research Laboratory of Metabolic &amp; Developmental Sciences Shanghai Jiao Tong University  Shanghai P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-2709-5546","authenticated-orcid":false,"given":"Qi","family":"Xiao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5796-8851","authenticated-orcid":false,"given":"Nan","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7103-8781","authenticated-orcid":false,"given":"Wenjin","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hang","family":"Luo","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9269-5273","authenticated-orcid":false,"given":"Yanxin","family":"Gao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leyan","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, School of Life Sciences &amp; Biotechnology, the Joint International Research Laboratory of Metabolic &amp; Developmental Sciences Shanghai Jiao Tong University  Shanghai P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yilin","family":"Fan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-4614-1492","authenticated-orcid":false,"given":"Peiyu","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Science, Li Ka Shing Medical School Hong Kong University  Hong Kong P. R. China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yucen","family":"Huang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences Nankai University Tianjin P. R. 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