Hydrogen sulfide binds to the catalytic zinc ion in HDAC6 to modulate enzyme activity via an alternative deacetylation pathway

Hydrogen sulfide binds to the catalytic zinc ion in HDAC6 to modulate enzyme activity via an alternative deacetylation pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Hydrogen sulfide binds to the catalytic zinc ion in HDAC6 to modulate enzyme activity via an alternative deacetylation pathway Yi-Chun Zhu, Chen Sun, Yin-fang Wang, Sheng-Yang Cai, Kui-fang Xie, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-777997/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The present study aims to examine if a tiny gas molecule, hydrogen sulfide (H2S), directly binds to the catalytic zinc ion of histone deacetylase 6 (HDAC6) to regulate enzyme activity via a mechanism beyond the post-translational modifications (PTMs) theory. H2S was found to coordinate with the catalytic metal ions in a series of metalloenzymes centered by zinc, ferrum, calcium, manganese, or copper via sulfur-metal coordination bonds evidenced by formation of specific localized molecular orbitals (LMOs). In HDAC6, H2S diffused to the bottom of active-site tunnel to identify the catalytic zinc ion yielding an SZn bond involving interactions of atom/molecular orbitals to promote enzyme activity. The binding between HDAC6 and H2S was dependent on H2S concentrations as measured using surface plasmon resonance (SPR). Quantum chemical (QC) calculations further uncovered a new deacetylation pathway modulated by H2S. This alternative reaction was characterized with an H2S-mediated nucleophilic attack generating a novel product, thioacetic acid, which was identified in cell-free reactions, cultured cells, mouse organs and human serums. Such alternative reaction had a much lower energy barrier in the rate-limiting reaction step resulting in much higher reaction efficiency. The cryo-electron microscopy (cryo-EM) structure of human HDAC6 also revealed a rather wide active-site tunnel in CD2 which allowed substrate binding for a series of substrates in contrast to a narrow and twisted active-site tunnel in CD1. QC calculations also revealed the mechanism of H2S recycling inside the CD2-active-site. In cultured cells and a mouse model of hind-limb ischemia, H2S-induced increase in cell migration and angiogenesis was dependent on HDAC6. The CD2-active-site was essential in H2S-induced increase in HDAC6 activity and cell migration, as examined in the cells expressing a series of recombinant HDAC6 variants with mutations to inactivate either CD1 or CD2 active sites. Acetylomics regulated via the CSE/H2S-HDAC6 pathway were also displayed revealing 7 regulated proteins relevant to cell migration in addition to well-established -tubulin. In conclusion, H2S directly identifies multiple catalytic metal ions in the metalloenzyme family via interaction of molecular/atom orbitals. The biochemical role of such an H2S coordination was embodied in HDAC6 where H2S modulated an alternative deacetylation pathway coupled with generation of thioacetic acid to promote cell migration and angiogenesis, suggesting a mechanism of protein regulation beyond the current PTMs concept as well as a role of HDAC6 as an H2S senser. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Cell biology/Cell migration/Lamellipodia Biological sciences/Chemical biology/Computational chemistry Biological sciences/Physiology/Cardiovascular biology/Angiogenesis Histone deacetylase 6 Hydrogen sulfide Zinc-active-site Metalloenzymes Cell migration H2S senser Full Text Additional Declarations There is NO Competing Interest. Supplementary Files uncutgelimageforthemanuscript.pdf Gel source data D1300036976valreportfullP1.pdf PDB EM Validation Report D1300036976valreportfullP1.pdf SupplementaryVideos15forMolecularDynamicsSimulations.zip Supplementary Videos 1-5 for Molecular Dynamics Simulations uncutgelimageforthemanuscript.pdf SupplementaryVideos610forMolecularDynamicsSimulations.zip Supplementary Videos 6-10 for Molecular Dynamics Simulations VideoS1forFigure1acandS1.mp4 Video S1 VideoS2forFigureS2b.mp4 Video S2 uncutgelimageforthemanuscript.docx Uncut gel image for the manuscript Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-777997","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":428881180,"identity":"ca7e195b-7f21-441b-bedf-bb0120f7df55","order_by":0,"name":"Yi-Chun 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senser","lastPublishedDoi":"10.21203/rs.3.rs-777997/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-777997/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The present study aims to examine if a tiny gas molecule, hydrogen sulfide (H2S), directly binds to the catalytic zinc ion of histone deacetylase 6 (HDAC6) to regulate enzyme activity via a mechanism beyond the post-translational modifications (PTMs) theory. H2S was found to coordinate with the catalytic metal ions in a series of metalloenzymes centered by zinc, ferrum, calcium, manganese, or copper via sulfur-metal coordination bonds evidenced by formation of specific localized molecular orbitals (LMOs). In HDAC6, H2S diffused to the bottom of active-site tunnel to identify the catalytic zinc ion yielding an SZn bond involving interactions of atom/molecular orbitals to promote enzyme activity. The binding between HDAC6 and H2S was dependent on H2S concentrations as measured using surface plasmon resonance (SPR). Quantum chemical (QC) calculations further uncovered a new deacetylation pathway modulated by H2S. This alternative reaction was characterized with an H2S-mediated nucleophilic attack generating a novel product, thioacetic acid, which was identified in cell-free reactions, cultured cells, mouse organs and human serums. Such alternative reaction had a much lower energy barrier in the rate-limiting reaction step resulting in much higher reaction efficiency. The cryo-electron microscopy (cryo-EM) structure of human HDAC6 also revealed a rather wide active-site tunnel in CD2 which allowed substrate binding for a series of substrates in contrast to a narrow and twisted active-site tunnel in CD1. QC calculations also revealed the mechanism of H2S recycling inside the CD2-active-site. In cultured cells and a mouse model of hind-limb ischemia, H2S-induced increase in cell migration and angiogenesis was dependent on HDAC6. The CD2-active-site was essential in H2S-induced increase in HDAC6 activity and cell migration, as examined in the cells expressing a series of recombinant HDAC6 variants with mutations to inactivate either CD1 or CD2 active sites. Acetylomics regulated via the CSE/H2S-HDAC6 pathway were also displayed revealing 7 regulated proteins relevant to cell migration in addition to well-established -tubulin. In conclusion, H2S directly identifies multiple catalytic metal ions in the metalloenzyme family via interaction of molecular/atom orbitals. 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