{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:25:18Z","timestamp":1787059518056,"version":"build-2736575974"},"reference-count":45,"publisher":"American Chemical Society (ACS)","issue":"16","license":[{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T00:00:00Z","timestamp":1785715200000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["461338801"],"award-info":[{"award-number":["461338801"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["GRK 2341"],"award-info":[{"award-number":["GRK 2341"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,18]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>[FeFe]-hydrogenases are very active biocatalysts for H2 conversion. However, their active site is vulnerable to irreversible degradation initiated by O2 binding at the catalytic iron ion (Fed) of the active center. CbA5H, the [FeFe]-hydrogenase from Clostridium beijerinckii, exhibits stability toward oxygen (O2) due to its ability to reversibly enter an inactive state termed Hinact upon contact with O2. We previously proposed that the close distance of approximately 3.1 \u00c5 between the thiol of a nearby cysteine (C367) and Fed, based on a 2.9 \u00c5 crystal structure of CbA5H in the Hinact state, enables their binding to each other. This binding therefore was suggested to shield Fed from O2 damage. However, there is currently a lack of evidence to support this hypothesis. Furthermore, density functional theory (DFT) calculations based on a homologous model favored hydroxide as the binding ligand of Fed over the thiol of C367. In this study, we present the crystal structure of CbA5H in the Hinact state at an improved resolution of 2.15 \u00c5. The structure reveals a direct binding between the thiol of C367 and Fed with a distance of approximately 2.77 \u00c5, which is well supported by our DFT calculations based on the new crystallographic data. It is noteworthy that the 2.77 \u00c5 bond distance is strikingly long when compared with other iron\u2013sulfur bonds. This finding may provide a crucial foundation for understanding the rapid reversibility of the Hinact state.<\/jats:p>","DOI":"10.1021\/acs.biochem.6c00395","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:34:56Z","timestamp":1785854096000},"page":"2611-2616","source":"Crossref","is-referenced-by-count":0,"title":["Direct Binding\nof Cysteine-367 Thiolate to the Active\nSite of the [FeFe]-Hydrogenase from\n                    <i>Clostridium beijerinckii<\/i>\n                    in the O2-Stable State"],"prefix":"10.1021","volume":"65","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5158-2253","authenticated-orcid":true,"given":"Jifu","family":"Duan","sequence":"first","affiliation":[{"name":"Ruhr University Bochum , , Universita\u0308tsstrasse 150 , ,","place":["Bochum, Germany, 44801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0691-7517","authenticated-orcid":true,"given":"Federica","family":"Arrigoni","sequence":"additional","affiliation":[{"name":"University of Milano-Bicocca , , Piazza della Scienza 2 , ,","place":["Milan, Italy, 20126"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andreas","family":"Rutz","sequence":"additional","affiliation":[{"name":"Ruhr University Bochum , , Universita\u0308tsstrasse 150 , ,","place":["Bochum, Germany, 44801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4874-372X","authenticated-orcid":true,"given":"Eckhard","family":"Hofmann","sequence":"additional","affiliation":[{"name":"Ruhr University Bochum , , Universita\u0308tsstrasse 150 , ,","place":["Bochum, Germany, 44801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9628-7875","authenticated-orcid":true,"given":"Claudio","family":"Greco","sequence":"additional","affiliation":[{"name":"University of Milano-Bicocca , , Piazza della Scienza 1 , ,","place":["Milan, Italy, 20126"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1206-5234","authenticated-orcid":true,"given":"Thomas","family":"Happe","sequence":"additional","affiliation":[{"name":"Ruhr University Bochum , , Universita\u0308tsstrasse 150 , ,","place":["Bochum, Germany, 44801"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,3]]},"reference":[{"key":"2026081808121415200_cit1","doi-asserted-by":"publisher","first-page":"1214","DOI":"10.1139\/m80-203","article-title":"Purification\nand properties of the\nperiplasmic hydrogenase from Desulfovibrio desulfuricans","volume":"26","author":"Glick","year":"1980","journal-title":"Can. 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Rev."},{"key":"2026081808121415200_cit3","doi-asserted-by":"publisher","first-page":"1853","DOI":"10.1126\/science.282.5395.1853","article-title":"X-ray crystal\nstructure\nof the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution","volume":"282","author":"Peters","year":"1998","journal-title":"Science"},{"key":"2026081808121415200_cit4","doi-asserted-by":"publisher","first-page":"13","DOI":"10.1016\/S0969-2126(99)80005-7","article-title":"Desulfovibrio desulfuricans iron hydrogenase:\nthe structure shows unusual coordination to an active site Fe binuclear\ncenter","volume":"7","author":"Nicolet","year":"1999","journal-title":"Structure"},{"key":"2026081808121415200_cit5","doi-asserted-by":"publisher","first-page":"572","DOI":"10.1046\/j.1432-1327.1998.2580572.x","article-title":"A low-spin\niron\nwith CN and CO as intrinsic ligands forms the core of the active site\nin [Fe]-hydrogenases","volume":"258","author":"Pierik","year":"1998","journal-title":"Eur. J. Biochem."},{"key":"2026081808121415200_cit6","doi-asserted-by":"publisher","first-page":"6592","DOI":"10.1039\/b905841a","article-title":"14N HYSCORE investigation\nof the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in\nthe dithiol bridge","volume":"11","author":"Silakov","year":"2009","journal-title":"Phys. Chem. Chem. Phys."},{"key":"2026081808121415200_cit7","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1038\/nature12239","article-title":"Biomimetic\nassembly and activation of [FeFe]-hydrogenases","volume":"499","author":"Berggren","year":"2013","journal-title":"Nature"},{"key":"2026081808121415200_cit8","doi-asserted-by":"publisher","first-page":"607","DOI":"10.1038\/nchembio.1311","article-title":"Spontaneous activation of [FeFe]-hydrogenases\nby an inorganic [2Fe] active site mimic","volume":"9","author":"Esselborn","year":"2013","journal-title":"Nat.\nChem. Biol."},{"key":"2026081808121415200_cit9","doi-asserted-by":"publisher","first-page":"83","DOI":"10.1021\/jacs.6b11409","article-title":"Identification of a Catalytic Iron-Hydride\nat the H-Cluster of [FeFe]-Hydrogenase","volume":"139","author":"Mulder","year":"2017","journal-title":"J. Am.\nChem. Soc."},{"key":"2026081808121415200_cit10","doi-asserted-by":"publisher","first-page":"4306","DOI":"10.1021\/jacs.7b00686","article-title":"Direct\nObservation of\nan Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance\nVibrational Spectroscopy","volume":"139","author":"Reijerse","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808121415200_cit11","doi-asserted-by":"publisher","first-page":"16115","DOI":"10.1038\/ncomms16115","article-title":"Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases","volume":"8","author":"Winkler","year":"2017","journal-title":"Nat. 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Chem., Int.\nEd."},{"key":"2026081808121415200_cit15","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202216903","article-title":"Cyanide Binding\nto [FeFe]-Hydrogenase Stabilizes the Alternative\nConfiguration of the Proton Transfer Pathway","volume":"62","author":"Duan","year":"2023","journal-title":"Angew. Chem., Int. Ed."},{"key":"2026081808121415200_cit16","doi-asserted-by":"publisher","first-page":"2826","DOI":"10.1039\/d2sc06098a","article-title":"Binding of exogenous cyanide reveals new active-site\nstates in [FeFe] hydrogenases","volume":"14","author":"Martini","year":"2023","journal-title":"Chem. Sci."},{"key":"2026081808121415200_cit17","doi-asserted-by":"publisher","first-page":"38341","DOI":"10.1074\/jbc.M111.254664","article-title":"Mechanism\nof proton transfer in [FeFe]-hydrogenase from Clostridium pasteurianum","volume":"286","author":"Cornish","year":"2011","journal-title":"J. Biol. 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U.S.A."},{"key":"2026081808121415200_cit21","doi-asserted-by":"publisher","first-page":"20588","DOI":"10.1021\/ja209165r","article-title":"Regioselectivity of H cluster oxidation","volume":"133","author":"Bruska","year":"2011","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808121415200_cit22","doi-asserted-by":"publisher","first-page":"1809","DOI":"10.1021\/ja510169s","article-title":"[FeFe]-hydrogenase oxygen\ninactivation is initiated at the H cluster 2Fe subcluster","volume":"137","author":"Swanson","year":"2015","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808121415200_cit23","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1038\/nchem.2592","article-title":"Mechanism of O2 diffusion and reduction in FeFe hydrogenases","volume":"9","author":"Kubas","year":"2017","journal-title":"Nat. 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Soc."},{"key":"2026081808121415200_cit26","doi-asserted-by":"publisher","first-page":"5897","DOI":"10.1021\/acs.biochem.6b00780","article-title":"Oxygen stability in\nthe new [FeFe]-hydrogenase from Clostridium beijerinckii SM10 (CbA5H)","volume":"55","author":"Morra","year":"2016","journal-title":"Biochemistry"},{"key":"2026081808121415200_cit27","doi-asserted-by":"publisher","first-page":"12409","DOI":"10.1021\/jacs.0c04964","article-title":"Investigation\nof the unusual ability\nof the [FeFe] hydrogenase from Clostridium beijerinckii to access\nan O2-protected state","volume":"142","author":"Corrigan","year":"2020","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808121415200_cit28","doi-asserted-by":"publisher","first-page":"756","DOI":"10.1038\/s41467-020-20861-2","article-title":"A safety cap protects\nhydrogenase from oxygen attack","volume":"12","author":"Winkler","year":"2021","journal-title":"Nat. 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Chem."},{"key":"2026081808121415200_cit31","doi-asserted-by":"publisher","first-page":"9346","DOI":"10.1021\/jacs.8b04339","article-title":"Sulfide\nProtects [FeFe] Hydrogenases\nFrom O2","volume":"140","author":"Rodriguez-Macia","year":"2018","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808121415200_cit32","doi-asserted-by":"publisher","first-page":"15170","DOI":"10.1021\/jacs.4c18483","article-title":"Protein Dynamics\nAffect O2-Stability of Group B [FeFe]-Hydrogenase from\nThermosediminibacter\noceani","volume":"147","author":"Ghosh","year":"2025","journal-title":"J. Am. Chem. Soc."},{"key":"2026081808121415200_cit33","doi-asserted-by":"publisher","first-page":"7001","DOI":"10.1021\/acscatal.4c01352","article-title":"Catalytic\nbias and redox-driven inactivation of the group B FeFe hydrogenase\nCpIII","volume":"14","author":"Fasano","year":"2024","journal-title":"ACS Catal."},{"key":"2026081808121415200_cit34","doi-asserted-by":"publisher","first-page":"2455","DOI":"10.1021\/acs.biochem.5c00066","article-title":"H-cluster\nIntermediates and Catalytic Properties of Clostridium pasteurianum [FeFe]-Hydrogenase III","volume":"64","author":"Kisgeropoulos","year":"2025","journal-title":"Biochemistry"},{"key":"2026081808121415200_cit35","doi-asserted-by":"publisher","first-page":"15162","DOI":"10.1021\/acscatal.1c04838","article-title":"Mechanism\nof hydrogen sulfide-dependent inhibition of FeFe hydrogenase","volume":"11","author":"Felbek","year":"2021","journal-title":"ACS Catal."},{"key":"2026081808121415200_cit36","doi-asserted-by":"publisher","first-page":"11033","DOI":"10.1021\/jacs.2c13458","article-title":"Evidence of\natypical structural flexibility\nof the active site surrounding of an [FeFe] hydrogenase from Clostridium beijerinkii","volume":"145","author":"Corrigan","year":"2023","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026081808121415200_cit37","doi-asserted-by":"publisher","first-page":"696","DOI":"10.1038\/nchem.2306","article-title":"Significantly\nshorter Fe\u2013S\nbond in cytochrome P450-I is consistent with greater reactivity relative\nto chloroperoxidase","volume":"7","author":"Krest","year":"2015","journal-title":"Nat. Chem."},{"key":"2026081808121415200_cit38","doi-asserted-by":"publisher","first-page":"5394","DOI":"10.1039\/d5sc07952g","article-title":"A visualizable and widely applicable\nsteric repulsion descriptor for guiding experimental chemistry","volume":"17","author":"Just","year":"2026","journal-title":"Chem. Sci."},{"key":"2026081808121415200_cit39","doi-asserted-by":"publisher","first-page":"527","DOI":"10.1021\/cr020615+","article-title":"Synthetic analogues of the active sites of iron\u2013\nsulfur proteins","volume":"104","author":"Venkateswara Rao","year":"2004","journal-title":"Chem. Rev."},{"key":"2026081808121415200_cit40","doi-asserted-by":"publisher","first-page":"16563","DOI":"10.1073\/pnas.0507069102","article-title":"X-ray absorption spectroscopy of\nchloroperoxidase compound I: Insight into the reactive intermediate\nof P450 chemistry","volume":"102","author":"Stone","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2026081808121415200_cit41","doi-asserted-by":"publisher","first-page":"7328","DOI":"10.1002\/chem.200500156","article-title":"Mononuclear,\ndinuclear, and pentanuclear\n[{N, S (thiolate)} Iron (II)] complexes: nuclearity control, incorporation\nof hydroxide bridging ligands, and magnetic behavior","volume":"11","author":"Krishnamurthy","year":"2005","journal-title":"Chem.\u2500Eur. J."},{"key":"2026081808121415200_cit42","doi-asserted-by":"publisher","first-page":"681","DOI":"10.1107\/S2052252520005916","article-title":"Paired refinement under\nthe control of PAIREF","volume":"7","author":"Maly","year":"2020","journal-title":"IUCrJ"},{"key":"2026081808121415200_cit43","doi-asserted-by":"publisher","first-page":"226","DOI":"10.1107\/S2053230X21006129","article-title":"PAIREF: paired\nrefinement\nalso for Phenix users","volume":"77","author":"Maly","year":"2021","journal-title":"Acta Crystallogr., Sect.\nF: Struct. Biol. Cryst. 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