Dual Regulation of Nitric Oxide by Cobalamin: Spectroscopic and Structural Evidence for Scavenging and Reversible Nitrosyl Formation

Dual Regulation of Nitric Oxide by Cobalamin: Spectroscopic and Structural Evidence for Scavenging and Reversible Nitrosyl Formation | 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 Systematic Review Dual Regulation of Nitric Oxide by Cobalamin: Spectroscopic and Structural Evidence for Scavenging and Reversible Nitrosyl Formation Olatunji Salako, Ioannis Sarris, Idayat Shalewa Salako, Vincent Chukwuemeka Eze This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8911642/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nitric oxide (NO) is a critical signaling molecule in vascular biology. Still, its interactions with cobalt-containing corrinoids, such as cobalamin (Vitamin B₁₂), are poorly understood compared to its well-known iron-heme chemistry. Using a multi-spectroscopic approach, we demonstrate that cobalamin species dynamically regulate NO bioavailability through two distinct mechanisms. First, NO rapidly and irreversibly oxidizes reduced cobalamin [Cb(II)] to Cb(III) via a one-electron transfer, establishing a potent NO-scavenging pathway shown in Fig. 1(graphical abstract ). Second, the oxidized hydroxocobalamin [Cb(III)] forms a reversible cobalt-nitrosyl adduct ([Cb(III)–NO]³⁺), characterized by a ν(NO) FTIR band at 1700 cm⁻¹ and a labile EPR signal, indicating its role as a transient NO reservoir. In porcine aortic endothelial cells, we observed a dynamic redox cycle in which extracellular Cb(III) is partially reduced to Cb(II), enabling the continuous modulation of NO flux. These findings reveal that cobalamins function as bifunctional redox modulators—both scavenging and storing NO—providing a new mechanistic framework for their therapeutic potential in nitrosative stress and vascular pathologies. Nitric oxide Cobalamin EPR spectroscopy Redox Chemistry NO scavenging Cobalt–nitrosyl adduct Endothelial modulation Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-8911642","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":598887255,"identity":"c776c25d-3520-4f57-9e95-6064ca4f2933","order_by":0,"name":"Olatunji Salako","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYHACxgMMBxjkgHQD8XpAWoxJ15JIvHp+/sMPDvw4Y5O+4XZzm8QPhtp8glokZ6QZHOy5kZa74c7BNskehuOWBK0zuMFgcIDnw+HcDTcS227wMBwzIGiLwfnjHw7++XA43QCo5eYforQcyDE4zHPjcAJIy20ehhrCWiRn5BQcljmTZjjzRmL7bxmDA4S18PMf3/jwzTEbeb4b6Y8N31TUEdaC7s7DpOpgYKgjXcsoGAWjYBQMewAARl5HuKeb2CkAAAAASUVORK5CYII=","orcid":"","institution":"University of West Attica","correspondingAuthor":true,"prefix":"","firstName":"Olatunji","middleName":"","lastName":"Salako","suffix":""},{"id":598887256,"identity":"db48401b-78fe-4995-9db0-6f059472d8fd","order_by":1,"name":"Ioannis Sarris","email":"","orcid":"","institution":"University of West Attica","correspondingAuthor":false,"prefix":"","firstName":"Ioannis","middleName":"","lastName":"Sarris","suffix":""},{"id":598887257,"identity":"e1b1e718-6247-4c70-90aa-2a04f0a4a74d","order_by":2,"name":"Idayat Shalewa Salako","email":"","orcid":"","institution":"Center for Countermeasures against Chemical and Biological Warfare Agents","correspondingAuthor":false,"prefix":"","firstName":"Idayat","middleName":"Shalewa","lastName":"Salako","suffix":""},{"id":598887258,"identity":"27374ce2-c091-4504-b03d-c1d4808e882e","order_by":3,"name":"Vincent Chukwuemeka Eze","email":"","orcid":"","institution":"Michael Okpara University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"Chukwuemeka","lastName":"Eze","suffix":""}],"badges":[],"createdAt":"2026-02-18 18:24:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8911642/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8911642/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105521612,"identity":"5f1419a3-c63c-48bd-b676-548258c25ab4","added_by":"auto","created_at":"2026-03-27 02:40:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1066699,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptonSpectroscopicInvestigationofNitricOxide.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8911642/v1_covered_55a56761-8ccd-4b83-95d4-62f7a4aa2629.pdf"},{"id":103925934,"identity":"f81b3544-62c6-4208-ae08-1ee4ad4ea585","added_by":"auto","created_at":"2026-03-04 15:26:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17450,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8911642/v1/bbdb19828f48604cefb43188.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual Regulation of Nitric Oxide by Cobalamin: Spectroscopic and Structural Evidence for Scavenging and Reversible Nitrosyl Formation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nitric oxide, Cobalamin, EPR spectroscopy, Redox Chemistry, NO scavenging, Cobalt–nitrosyl adduct, Endothelial modulation","lastPublishedDoi":"10.21203/rs.3.rs-8911642/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8911642/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNitric oxide (NO) is a critical signaling molecule in vascular biology. Still, its interactions with cobalt-containing corrinoids, such as cobalamin (Vitamin B₁₂), are poorly understood compared to its well-known iron-heme chemistry. Using a multi-spectroscopic approach, we demonstrate that cobalamin species dynamically regulate NO bioavailability through two distinct mechanisms. First, NO rapidly and irreversibly oxidizes reduced cobalamin [Cb(II)] to Cb(III) via a one-electron transfer, establishing a potent NO-scavenging pathway shown in Fig.\u0026nbsp;1(graphical abstract ). Second, the oxidized hydroxocobalamin [Cb(III)] forms a reversible cobalt-nitrosyl adduct ([Cb(III)\u0026ndash;NO]\u0026sup3;⁺), characterized by a ν(NO) FTIR band at 1700 cm⁻\u0026sup1; and a labile EPR signal, indicating its role as a transient NO reservoir. In porcine aortic endothelial cells, we observed a dynamic redox cycle in which extracellular Cb(III) is partially reduced to Cb(II), enabling the continuous modulation of NO flux. These findings reveal that cobalamins function as bifunctional redox modulators\u0026mdash;both scavenging and storing NO\u0026mdash;providing a new mechanistic framework for their therapeutic potential in nitrosative stress and vascular pathologies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Dual Regulation of Nitric Oxide by Cobalamin: Spectroscopic and Structural Evidence for Scavenging and Reversible Nitrosyl Formation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 15:25:26","doi":"10.21203/rs.3.rs-8911642/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"228e3b7c-46aa-49ba-8039-3662bd478ffc","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-27T02:39:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 15:25:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8911642","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8911642","identity":"rs-8911642","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}