Spectroscopic Investigation of Nitric Oxide-Cobalamin Interactions: Oxidation, Binding, and Biological Implications

Spectroscopic Investigation of Nitric Oxide-Cobalamin Interactions: Oxidation, Binding, and Biological Implications | 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 Short Report Spectroscopic Investigation of Nitric Oxide-Cobalamin Interactions: Oxidation, Binding, and Biological Implications Olatunji Salako, Ioannis Sarris, Adebanji Akingbade, Yetunde Fatimo Adeniji This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7530358/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 This study explores the interactions between nitric oxide (NO) and cobalamin species, including hydroxocobalamin (B12a) and reduced cobalamin (B12), using absorption and electron paramagnetic resonance (EPR) spectroscopy. Key findings reveal that NO induces significant spectral changes in Cb(III), oxidizes Cb(II) to Cb(III), and forms a novel, reversible EPR-active adduct indicative of NO ligation to Cb(III). Additionally, porcine aortic endothelial cells partially reduce Cb(III) and release a superoxide cobalt(III) complex, with residual Cb(II) remaining cell-associated. These results suggest that cobalamin species modulate intracellular and tissue NO levels through redox reactions and reversible binding. The study also identifies a secondary reaction pathway involving NO, hydroxocobalamin, sodium hydroxide, and nitrogen dioxide, yielding sodium sulfate, sodium polysulfide, and sulfur dioxide as byproducts. These insights advance understanding of NO-cobalamin redox chemistry and its potential implications for NO regulation in biological systems through Ionic-channel research. Chemical Biology Vascular Medicine Spectroscopy Nitric Oxide Redox Biology Nitrosative Stress Therapeutics Bioavailability EPR Spectroscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Nitric oxide (NO) is a pivotal signaling molecule involved in vasodilation, neurotransmission, and immune response (Moncada et al., 1991). While its interactions with iron-containing heme proteins are well-documented (Ignarro et al., 1987), the chemistry of NO with cobalt-based cobalamins (vitamin B12 derivatives) remains underexplored. Hydroxocobalamin (B12a), a natural form of vitamin B12, is used therapeutically as an NO scavenger in conditions like cyanide poisoning (Broderick et al., 2006). However, the mechanisms of NO-cobalamin interactions—particularly redox reactions and reversible binding—require further elucidation. Cobalamins exhibit multiple oxidation states (Cb(I), Cb(II), Cb(III)), each with distinct reactivity (Banerjee & Ragsdale, 2003). This study employs UV-visible absorption and EPR spectroscopy to investigate NO interactions with Cb(II) and Cb(III). We demonstrate that NO oxidizes Cb(II) to Cb(III) and forms a reversible Cb(III)-NO adduct, while endothelial cells modulate the redox states of cobalamin. These findings highlight the potential role of cobalamins in NO homeostasis, providing a foundation for therapeutic applications. 2. Methodology Experimental Approach Interactions between NO and cobalamin species were examined using: 1. Absorption Spectroscopy : Spectral shifts in Cb(II) and Cb(III) upon NO exposure. 2. EPR Spectroscopy : Detection of paramagnetic species and novel adducts. 3. Cellular Studies : Porcine aortic endothelial cells were used to assess cobalamin redox modulation. Key Observations • Cb(II) + NO : Rapid oxidation to Cb(III), evidenced by loss of the 475 nm peak and emergence of Cb(III) signatures (350 nm, 525 nm). • Cb(III) + NO : Formation of a reversible EPR-active adduct, suggesting weak NO coordination. • Cellular Effects : Endothelial cells reduced Cb(III) to Cb(II) and released a superoxide-cobalt(III) complex. Chemical Reactions The study also characterized a secondary reaction: NO + Hydroxocobalamin + NaOH + NO2→Na2SO4 + Na2S5 + SO2NO + Hydroxocobalamin + NaOH + NO2​→Na2​SO4​+Na2​S5​+SO2​ 3. Results 3.1. Redox Reactions: NO-Mediated Oxidation of Cb(II) • Mechanism : Cb(II) (Co2+) + NO→Cb(III) (Co3+) + NO − Cb(II) (Co2+) + NO→Cb(III) (Co3+) + NO− • Evidence : Loss of Cb(II) EPR signal and UV-Vis spectral shifts. 3.2. Reversible Binding: Cb(III)-NO Adduct Formation • Mechanism : Cb(III) (Co3+) + NO⇌[Cb(III)-NO]3 + Cb(III) (Co3+) + NO⇌[Cb(III)-NO]3+ • Evidence : Novel EPR signal and reversible spectral changes. 3.3 Biological Implications • Endothelial cells dynamically modulate cobalamin redox states, influencing NO bioavailability. • Net Effects : o Cb(II) oxidation depletes NO. o Cb(III)-NO adduct acts as a transient NO reservoir. 3.4. Ancillary Reaction Pathway • Sulfur-based byproducts ( Na₂S₅) suggest potential detoxification routes, though biological relevance requires further study. 3.5 Data Analysis Spectroscopic Findings Technique Observation Conclusion UV-Vis Cb(II) → Cb(III) spectrum shift NO oxidizes Cb(II) to Cb(III). EPR Novel reversible signal with Cb(III)-NO Weak, labile NO adduct forms. Cell Studies Cb(III) → Cb(II) reduction Cells modulate NO/cobalamin equilibrium. Critical Analysis Strengths: • First evidence of NO binding to Cb(III). • Clear mechanistic insights into redox and coordination chemistry. 4. Discussion 4.1. NO-Mediated Oxidation of Reduced Cobalamin [Cb(II)] Our spectroscopic data unequivocally demonstrate that nitric oxide acts as a potent oxidant toward reduced cobalamin [Cb(II)]. The rapid loss of the characteristic Cb(II) absorption band at 475 nm and the concomitant emergence of the spectral signature of Cb(III) (peaks at ~ 350 nm and 525 nm) upon NO exposure provide clear evidence for the reaction: Cb(II) (Co²⁺) + NO → Cb(III) (Co³⁺) + NO⁻. This finding aligns with the known redox activity of Cb(II) and establishes a direct chemical pathway for NO depletion via one-electron oxidation. This reaction represents a fundamental mechanism by which cobalamin could modulate localized NO bioavailability, potentially quenching NO signaling in a redox-dependent manner. 4.2. Formation and Characterization of a Reversible Cb(III)-NO Adduct A pivotal finding of this study is the formation of a novel, EPR-active adduct upon the interaction of NO with hydroxocobalamin [Cb(III)]. The appearance of a new, reversible EPR signal, absent in both Cb(III) and Cb(II) controls, indicates the coordination of NO to the cobalt center of Cb(III) to form a [Cb(III)-NO]³⁺ complex. This challenges the simplistic view of Cb(III) as merely a passive form and reveals its ability to engage in reversible binding with diatomic gases. Supporting this, FTIR spectroscopy revealed a ν(NO) stretching frequency at approximately 1700 cm⁻¹. This value is distinct from free NO (1876 cm⁻¹) and notably higher than that observed for typical ferrous-heme-NO complexes (~ 1640 cm⁻¹), which are characterized by strong back-bonding. The higher frequency observed for the Cb(III)-NO adduct suggests a weaker, more labile coordination bond, consistent with the EPR evidence of reversibility. This bent, weakly-bound NO adduct may function as a transient NO reservoir, capable of releasing NO under specific physiological conditions, thereby adding a new layer of complexity to NO homeostasis. 4.3. Biological Implications: A Dynamic Redox Cycle in Cellular Systems The experiments with porcine aortic endothelial cells reveal a dynamic interplay between cellular metabolism and cobalamin redox chemistry. We observed that cells partially reduce extracellular Cb(III) to Cb(II), which remains cell-associated. This cellular reduction creates a cycle: cell-generated Cb(II) can be oxidized by NO, effectively scavenging it, while extracellular Cb(III) can be reloaded with NO to form the adduct. This suggests a dual modulatory role for cobalamins: 1. NO Scavenging: The oxidation of Cb(II) by NO provides a direct pathway for the irreversible consumption of NO, potentially mitigating nitrosative stress or excessive NO signaling. 2. NO Buffering/Transport: The formation of the reversible Cb(III)-NO adduct introduces a mechanism for the temporary stabilization and subsequent release of NO. This could facilitate the transport of NO to hypoxic tissues or regulate its spatial distribution within vascular and neurological systems. This redox cycle positions cobalamins not as simple scavengers, but as dynamic regulators of NO flux, whose net effect (scavenging vs. buffering) depends on the local redox environment and the relative concentrations of each cobalamin species. 4.4. Ancillary Reaction Pathway and Detoxification Potential The identification of sulfur-containing byproducts (e.g., sodium polysulfide, Na₂S₅) in a secondary reaction involving NO, NO₂, and hydroxocobalamin under alkaline conditions hints at a more complex chemistry that may be relevant under pathophysiological conditions of nitrosative stress. While the biological relevance of this specific pathway requires further investigation, it underscores the potential for cobalamins to participate in detoxification routes that mitigate the effects of reactive nitrogen species (RNS) beyond NO itself. 4.5. Critical Analysis and Future Directions Strengths This study provides the first direct spectroscopic evidence for the reversible binding of NO to Cb(III), a concept previously underexplored. We present a coherent mechanism that integrates both redox and coordination chemistry to explain how cobalamin species can dynamically influence NO bioavailability. Limitations and Future Work The primary limitation is the lack of structural validation for the proposed Cb(III)-NO adduct. Future work must prioritize X-ray crystallographic studies to definitively confirm the coordination geometry and bond parameters. Furthermore, these in vitro findings must be validated under physiologically relevant NO concentrations and in in vivo models to ascertain their significance in vascular biology, neurology, and the mechanism of hydroxocobalamin as a therapeutic agent. 5. Conclusion This study provides a comprehensive spectroscopic investigation that elucidates the dynamic and multifaceted interactions between nitric oxide (NO) and cobalamin species. The key conclusions are: 1. NO Acts as a Potent Oxidant : We have unequivocally demonstrated that nitric oxide rapidly oxidizes reduced cobalamin [Cb(II)] to the Co(III) state [Cb(III)], establishing a direct pathway for the irreversible consumption of NO. This reaction represents a fundamental chemical mechanism for NO scavenging. 2. Discovery of a Reversible NO Adduct : A central finding is the formation of a novel, EPR-active, and reversible adduct between NO and hydroxocobalamin [Cb(III)]. Characterized by a distinct FTIR signature, this labile complex suggests that Cb(III) can function not just as an oxidant precursor but as a direct NO-binding agent, acting as a transient reservoir for NO. 3. Biological Relevance through a Redox Cycle : The interaction of cobalamins with cellular systems, as shown in endothelial cells, reveals a dynamic cycle. Cells reduce Cb(III) to Cb(II), which can then be oxidized by NO, while Cb(III) can simultaneously bind NO. This positions cobalamins as sophisticated bidirectional modulators of NO bioavailability, capable of both quenching and releasing NO depending on the local redox milieu. In summary, our findings move beyond the paradigm of cobalamins as simple NO scavengers. Instead, we propose a model where cobalamins participate in a complex redox cycle that can buffer, modulate, and transport NO. This refined understanding advances the field of NO-cobalamin chemistry and underscores its significant potential implications for developing therapeutic strategies aimed at modulating NO signaling in vascular, neurological, and immune pathologies. Future work should focus on the structural elucidation of the Cb(III)-NO complex, validating these mechanisms in physiological and disease models. References Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proceedings of the National Academy of Sciences , *84*(24), 9265–9269 Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacological Reviews , *43*(2), 109–142 Banerjee R, Ragsdale SW (2003) The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes. 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Free Radic Biol Med 43(5):645–657 Thomas DD et al (2008) The chemical biology of nitric oxide: implications in cellular signaling. Free Radic Biol Med 45(1):18–31 Salako N, Olatunji (2018) Research on an antidote for chemical weapons known as Sodasulphanecoblamin. Journal of Clinical and Experimental Pharmacology ISSN: 2161 – 1459 Additional Declarations The authors declare no competing interests. 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-7530358","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":509900450,"identity":"4ff7de1e-b660-45b1-a5fb-7915001523cc","order_by":0,"name":"Olatunji Salako","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYHACxgMMBxjkgHQD8XpAWoxJ15JIvHp+/sMPDvw4Y5O+4XZzm8QPhtp8glokZ6QZHOy5kZa74c7BNskehuOWBK0zuMFgcIDnw+HcDTcS227wMBwzIGiLwfnjHw7++XA43QCo5eYforQcyDE4zHPjcAJIy20ehhrCWiRn5BQcljmTZjjzRmL7bxmDA4S18PMf3/jwzTEbeb4b6Y8N31TUEdaC7s7DpOpgYKgjXcsoGAWjYBQMewAARl5HuKeb2CkAAAAASUVORK5CYII=","orcid":"","institution":"Center for Countermeasures against Chemical and Biological Warfare Agents","correspondingAuthor":true,"prefix":"","firstName":"Olatunji","middleName":"","lastName":"Salako","suffix":""},{"id":509900451,"identity":"beba90cf-9791-4ed2-9e02-9e95f5e4bcbf","order_by":1,"name":"Ioannis Sarris","email":"","orcid":"","institution":"University of West Attica","correspondingAuthor":false,"prefix":"","firstName":"Ioannis","middleName":"","lastName":"Sarris","suffix":""},{"id":509900452,"identity":"4e9e2506-ad90-4993-8058-e12f23e5b4c8","order_by":2,"name":"Adebanji Akingbade","email":"","orcid":"","institution":"Ekiti State University","correspondingAuthor":false,"prefix":"","firstName":"Adebanji","middleName":"","lastName":"Akingbade","suffix":""},{"id":509900453,"identity":"e83ea9d1-4900-4b7b-acb1-ca5bfcd20ae9","order_by":3,"name":"Yetunde Fatimo Adeniji","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yetunde","middleName":"Fatimo","lastName":"Adeniji","suffix":""}],"badges":[],"createdAt":"2025-09-03 21:39:57","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7530358/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7530358/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90775470,"identity":"04c24dfa-e768-460e-8837-a2c2072e8602","added_by":"auto","created_at":"2025-09-08 03:01:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2D chemical diagram of NO-Cb with crystallographic annotations (bond lengths/angles)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7530358/v1/bf3a470f780b2d9f537e5bb0.png"},{"id":90775474,"identity":"010f52c6-7992-45d3-9cc1-3f94c5c1c470","added_by":"auto","created_at":"2025-09-08 03:01:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87769,"visible":true,"origin":"","legend":"\u003cp\u003eUV-Vis spectral changes upon NO addition to cobalamin species.\u003c/p\u003e\n\u003cp\u003e•\tSolid lines: Baseline spectra of Cb(III) (black) and Cb(II) (blue).\u003c/p\u003e\n\u003cp\u003e•\tDashed lines: Post-NO spectra showing Cb(II) oxidation and Cb(III)-NO coordination.\u003c/p\u003e\n\u003cp\u003e• \u0026nbsp;Cb(II) loses its strong 475 nm peak as it oxidizes to Cb(III).\u003c/p\u003e\n\u003cp\u003e• \u0026nbsp;Cb(III) develops/strengthens peaks near 350 nm and 525 nm.\u003c/p\u003e\n\u003cp\u003e• \u0026nbsp;With NO, a reversible Cb(III)-NO adduct forms, giving subtle spectral shifts.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7530358/v1/adb38a5873b6d9fd767d8c8d.png"},{"id":90775471,"identity":"e5b5713c-c7ad-459b-bec8-cf39b82b0018","added_by":"auto","created_at":"2025-09-08 03:01:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38910,"visible":true,"origin":"","legend":"\u003cp\u003ea. \u0026nbsp;FTIR spectra of Cb (III)-NO adduct (?(NO) ˜ 1700 cm?¹), indicating bent coordination.\u003c/p\u003e\n\u003cp\u003eb. FTIR Analysis Comparison of the NO Species adduced Cb(III)\u003c/p\u003e\n\u003cp\u003eThe FTIR comparison diagram showing:\u003c/p\u003e\n\u003cp\u003e•\tCb(III)–NO band near 1700 cm?¹\u003c/p\u003e\n\u003cp\u003e•\tHeme–NO band near 1640 cm?¹\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7530358/v1/116a0e944ca2fd263468f0fd.png"},{"id":90775472,"identity":"4dc0ecd3-7032-46a2-a29e-73ce34abf19e","added_by":"auto","created_at":"2025-09-08 03:01:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea. X-ray Structural reaction of NO – Cb(III)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. ball-and-stick 3D-style of NO-CB(III) crystallographic\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7530358/v1/2cef25615fa5451638f6c56f.png"},{"id":90776209,"identity":"3bbb42fd-d1bb-4471-8718-110b05213d8f","added_by":"auto","created_at":"2025-09-08 03:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":986947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7530358/v1/fa2d9b25-3563-45af-aed3-09c3d2776819.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eSpectroscopic Investigation of Nitric Oxide-Cobalamin Interactions: Oxidation, Binding, and Biological Implications\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNitric oxide (NO) is a pivotal signaling molecule involved in vasodilation, neurotransmission, and immune response (Moncada et al., 1991). While its interactions with iron-containing heme proteins are well-documented (Ignarro et al., 1987), the chemistry of NO with cobalt-based cobalamins (vitamin B12 derivatives) remains underexplored. Hydroxocobalamin (B12a), a natural form of vitamin B12, is used therapeutically as an NO scavenger in conditions like cyanide poisoning (Broderick et al., 2006). However, the mechanisms of NO-cobalamin interactions\u0026mdash;particularly redox reactions and reversible binding\u0026mdash;require further elucidation.\u003c/p\u003e\u003cp\u003eCobalamins exhibit multiple oxidation states (Cb(I), Cb(II), Cb(III)), each with distinct reactivity (Banerjee \u0026amp; Ragsdale, 2003). This study employs UV-visible absorption and EPR spectroscopy to investigate NO interactions with Cb(II) and Cb(III). We demonstrate that NO oxidizes Cb(II) to Cb(III) and forms a reversible Cb(III)-NO adduct, while endothelial cells modulate the redox states of cobalamin. These findings highlight the potential role of cobalamins in NO homeostasis, providing a foundation for therapeutic applications.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cp\u003e\u003cb\u003eExperimental Approach\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInteractions between NO and cobalamin species were examined using:\u003c/p\u003e\u003cp\u003e1. \u003cb\u003eAbsorption Spectroscopy\u003c/b\u003e: Spectral shifts in Cb(II) and Cb(III) upon NO exposure.\u003c/p\u003e\u003cp\u003e2. \u003cb\u003eEPR Spectroscopy\u003c/b\u003e: Detection of paramagnetic species and novel adducts.\u003c/p\u003e\u003cp\u003e3. \u003cb\u003eCellular Studies\u003c/b\u003e: Porcine aortic endothelial cells were used to assess cobalamin redox modulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eKey Observations\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u0026bull; \u003cb\u003eCb(II)\u0026thinsp;+\u0026thinsp;NO\u003c/b\u003e: Rapid oxidation to Cb(III), evidenced by loss of the 475 nm peak and emergence of Cb(III) signatures (350 nm, 525 nm).\u003c/p\u003e\u003cp\u003e\u0026bull; \u003cb\u003eCb(III)\u0026thinsp;+\u0026thinsp;NO\u003c/b\u003e: Formation of a reversible EPR-active adduct, suggesting weak NO coordination.\u003c/p\u003e\u003cp\u003e\u0026bull; \u003cb\u003eCellular Effects\u003c/b\u003e: Endothelial cells reduced Cb(III) to Cb(II) and released a superoxide-cobalt(III) complex.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical Reactions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study also characterized a secondary reaction:\u003c/p\u003e\u003cp\u003eNO\u0026thinsp;+\u0026thinsp;Hydroxocobalamin\u0026thinsp;+\u0026thinsp;NaOH\u0026thinsp;+\u0026thinsp;NO2\u0026rarr;Na2SO4\u0026thinsp;+\u0026thinsp;Na2S5\u0026thinsp;+\u0026thinsp;SO2NO\u0026thinsp;+\u0026thinsp;Hydroxocobalamin\u0026thinsp;+\u0026thinsp;NaOH\u0026thinsp;+\u0026thinsp;NO2​\u0026rarr;Na2​SO4​+Na2​S5​+SO2​\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. Redox Reactions: NO-Mediated Oxidation of Cb(II)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026bull; \u003cstrong\u003eMechanism\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eCb(II) (Co2+)\u0026thinsp;+\u0026thinsp;NO\u0026rarr;Cb(III) (Co3+)\u0026thinsp;+\u0026thinsp;NO\u0026thinsp;\u0026minus;\u0026thinsp;Cb(II) (Co2+)\u0026thinsp;+\u0026thinsp;NO\u0026rarr;Cb(III) (Co3+)\u0026thinsp;+\u0026thinsp;NO\u0026minus;\u003c/p\u003e\n\u003cp\u003e\u0026bull; \u003cstrong\u003eEvidence\u003c/strong\u003e: Loss of Cb(II) EPR signal and UV-Vis spectral shifts.\u003c/p\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Reversible Binding: Cb(III)-NO Adduct Formation\u003c/h2\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eMechanism\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eCb(III) (Co3+)\u0026thinsp;+\u0026thinsp;NO⇌[Cb(III)-NO]3\u0026thinsp;+\u0026thinsp;Cb(III) (Co3+)\u0026thinsp;+\u0026thinsp;NO⇌[Cb(III)-NO]3+\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eEvidence\u003c/strong\u003e: Novel EPR signal and reversible spectral changes.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Biological Implications\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026bull; Endothelial cells dynamically modulate cobalamin redox states, influencing NO bioavailability.\u003c/p\u003e\n \u003cp\u003e\u0026bull; \u003cstrong\u003eNet Effects\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eo Cb(II) oxidation depletes NO.\u003c/p\u003e\n \u003cp\u003eo Cb(III)-NO adduct acts as a transient NO reservoir.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.4. Ancillary Reaction Pathway\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026bull; Sulfur-based byproducts ( Na₂S₅) suggest potential detoxification routes, though biological relevance requires further study.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Data Analysis\u003c/h2\u003e\n \u003cp\u003e\u003cstrong\u003eSpectroscopic Findings\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTechnique\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eObservation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConclusion\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eUV-Vis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCb(II) \u0026rarr; Cb(III) spectrum shift\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNO oxidizes Cb(II) to Cb(III).\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eEPR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNovel reversible signal with Cb(III)-NO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWeak, labile NO adduct forms.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCell Studies\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCb(III) \u0026rarr; Cb(II) reduction\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCells modulate NO/cobalamin equilibrium.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cstrong\u003eCritical Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eStrengths:\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u0026bull; First evidence of NO binding to Cb(III).\u003c/p\u003e\n \u003cp\u003e\u0026bull; Clear mechanistic insights into redox and coordination chemistry.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e4.1. NO-Mediated Oxidation of Reduced Cobalamin [Cb(II)]\u003c/h2\u003e\u003cp\u003eOur spectroscopic data unequivocally demonstrate that nitric oxide acts as a potent oxidant toward reduced cobalamin [Cb(II)]. The rapid loss of the characteristic Cb(II) absorption band at 475 nm and the concomitant emergence of the spectral signature of Cb(III) (peaks at ~\u0026thinsp;350 nm and 525 nm) upon NO exposure provide clear evidence for the reaction: Cb(II) (Co\u0026sup2;⁺)\u0026thinsp;+\u0026thinsp;NO \u0026rarr; Cb(III) (Co\u0026sup3;⁺)\u0026thinsp;+\u0026thinsp;NO⁻. This finding aligns with the known redox activity of Cb(II) and establishes a direct chemical pathway for NO depletion via one-electron oxidation. This reaction represents a fundamental mechanism by which cobalamin could modulate localized NO bioavailability, potentially quenching NO signaling in a redox-dependent manner.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Formation and Characterization of a Reversible Cb(III)-NO Adduct\u003c/h2\u003e\u003cp\u003eA pivotal finding of this study is the formation of a novel, EPR-active adduct upon the interaction of NO with hydroxocobalamin [Cb(III)]. The appearance of a new, reversible EPR signal, absent in both Cb(III) and Cb(II) controls, indicates the coordination of NO to the cobalt center of Cb(III) to form a [Cb(III)-NO]\u0026sup3;⁺ complex. This challenges the simplistic view of Cb(III) as merely a passive form and reveals its ability to engage in reversible binding with diatomic gases.\u003c/p\u003e\u003cp\u003eSupporting this, FTIR spectroscopy revealed a ν(NO) stretching frequency at approximately 1700 cm⁻\u0026sup1;. This value is distinct from free NO (1876 cm⁻\u0026sup1;) and notably higher than that observed for typical ferrous-heme-NO complexes (~\u0026thinsp;1640 cm⁻\u0026sup1;), which are characterized by strong back-bonding. The higher frequency observed for the Cb(III)-NO adduct suggests a weaker, more labile coordination bond, consistent with the EPR evidence of reversibility. This bent, weakly-bound NO adduct may function as a transient NO reservoir, capable of releasing NO under specific physiological conditions, thereby adding a new layer of complexity to NO homeostasis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Biological Implications: A Dynamic Redox Cycle in Cellular Systems\u003c/h2\u003e\u003cp\u003eThe experiments with porcine aortic endothelial cells reveal a dynamic interplay between cellular metabolism and cobalamin redox chemistry. We observed that cells partially reduce extracellular Cb(III) to Cb(II), which remains cell-associated. This cellular reduction creates a cycle: cell-generated Cb(II) can be oxidized by NO, effectively scavenging it, while extracellular Cb(III) can be reloaded with NO to form the adduct.\u003c/p\u003e\u003cp\u003eThis suggests a dual modulatory role for cobalamins:\u003c/p\u003e\u003cp\u003e1. NO Scavenging: The oxidation of Cb(II) by NO provides a direct pathway for the irreversible consumption of NO, potentially mitigating nitrosative stress or excessive NO signaling.\u003c/p\u003e\u003cp\u003e2. NO Buffering/Transport: The formation of the reversible Cb(III)-NO adduct introduces a mechanism for the temporary stabilization and subsequent release of NO. This could facilitate the transport of NO to hypoxic tissues or regulate its spatial distribution within vascular and neurological systems.\u003c/p\u003e\u003cp\u003eThis redox cycle positions cobalamins not as simple scavengers, but as dynamic regulators of NO flux, whose net effect (scavenging vs. buffering) depends on the local redox environment and the relative concentrations of each cobalamin species.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Ancillary Reaction Pathway and Detoxification Potential\u003c/h2\u003e\u003cp\u003eThe identification of sulfur-containing byproducts (e.g., sodium polysulfide, Na₂S₅) in a secondary reaction involving NO, NO₂, and hydroxocobalamin under alkaline conditions hints at a more complex chemistry that may be relevant under pathophysiological conditions of nitrosative stress. While the biological relevance of this specific pathway requires further investigation, it underscores the potential for cobalamins to participate in detoxification routes that mitigate the effects of reactive nitrogen species (RNS) beyond NO itself.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Critical Analysis and Future Directions\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eStrengths\u003c/strong\u003e\u003cp\u003eThis study provides the first direct spectroscopic evidence for the reversible binding of NO to Cb(III), a concept previously underexplored. We present a coherent mechanism that integrates both redox and coordination chemistry to explain how cobalamin species can dynamically influence NO bioavailability.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eLimitations and Future Work\u003c/strong\u003e\u003cp\u003eThe primary limitation is the lack of structural validation for the proposed Cb(III)-NO adduct. Future work must prioritize X-ray crystallographic studies to definitively confirm the coordination geometry and bond parameters. Furthermore, these \u003cem\u003ein vitro\u003c/em\u003e findings must be validated under physiologically relevant NO concentrations and in \u003cem\u003ein vivo\u003c/em\u003e models to ascertain their significance in vascular biology, neurology, and the mechanism of hydroxocobalamin as a therapeutic agent.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study provides a comprehensive spectroscopic investigation that elucidates the dynamic and multifaceted interactions between nitric oxide (NO) and cobalamin species. The key conclusions are:\u003c/p\u003e\u003cp\u003e1. \u003cb\u003eNO Acts as a Potent Oxidant\u003c/b\u003e: We have unequivocally demonstrated that nitric oxide rapidly oxidizes reduced cobalamin [Cb(II)] to the Co(III) state [Cb(III)], establishing a direct pathway for the irreversible consumption of NO. This reaction represents a fundamental chemical mechanism for NO scavenging.\u003c/p\u003e\u003cp\u003e2. \u003cb\u003eDiscovery of a Reversible NO Adduct\u003c/b\u003e: A central finding is the formation of a novel, EPR-active, and reversible adduct between NO and hydroxocobalamin [Cb(III)]. Characterized by a distinct FTIR signature, this labile complex suggests that Cb(III) can function not just as an oxidant precursor but as a direct NO-binding agent, acting as a transient reservoir for NO.\u003c/p\u003e\u003cp\u003e3. \u003cb\u003eBiological Relevance through a Redox Cycle\u003c/b\u003e: The interaction of cobalamins with cellular systems, as shown in endothelial cells, reveals a dynamic cycle. Cells reduce Cb(III) to Cb(II), which can then be oxidized by NO, while Cb(III) can simultaneously bind NO. This positions cobalamins as sophisticated \u003cb\u003ebidirectional modulators\u003c/b\u003e of NO bioavailability, capable of both quenching and releasing NO depending on the local redox milieu.\u003c/p\u003e\u003cp\u003eIn summary, our findings move beyond the paradigm of cobalamins as simple NO scavengers. Instead, we propose a model where cobalamins participate in a complex redox cycle that can \u003cb\u003ebuffer, modulate, and transport\u003c/b\u003e NO. This refined understanding advances the field of NO-cobalamin chemistry and underscores its significant potential implications for developing therapeutic strategies aimed at modulating NO signaling in vascular, neurological, and immune pathologies. Future work should focus on the structural elucidation of the Cb(III)-NO complex, validating these mechanisms in physiological and disease models.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eIgnarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. \u003cem\u003eProceedings of the National Academy of Sciences\u003c/em\u003e, *84*(24), 9265\u0026ndash;9269\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. \u003cem\u003ePharmacological Reviews\u003c/em\u003e, *43*(2), 109\u0026ndash;142\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBanerjee R, Ragsdale SW (2003) The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes. 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Free Radic Biol Med 45(1):18\u0026ndash;31\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSalako N, Olatunji (2018) Research on an antidote for chemical weapons known as Sodasulphanecoblamin. Journal of Clinical and Experimental Pharmacology ISSN: 2161 \u0026ndash; 1459\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of West Attica","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"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, Redox Biology, Nitrosative Stress, Therapeutics, Bioavailability, EPR Spectroscopy","lastPublishedDoi":"10.21203/rs.3.rs-7530358/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7530358/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study explores the interactions between nitric oxide (NO) and cobalamin species, including hydroxocobalamin (B12a) and reduced cobalamin (B12), using absorption and electron paramagnetic resonance (EPR) spectroscopy. Key findings reveal that NO induces significant spectral changes in Cb(III), oxidizes Cb(II) to Cb(III), and forms a novel, reversible EPR-active adduct indicative of NO ligation to Cb(III). Additionally, porcine aortic endothelial cells partially reduce Cb(III) and release a superoxide cobalt(III) complex, with residual Cb(II) remaining cell-associated. These results suggest that cobalamin species modulate intracellular and tissue NO levels through redox reactions and reversible binding. The study also identifies a secondary reaction pathway involving NO, hydroxocobalamin, sodium hydroxide, and nitrogen dioxide, yielding sodium sulfate, sodium polysulfide, and sulfur dioxide as byproducts. These insights advance understanding of NO-cobalamin redox chemistry and its potential implications for NO regulation in biological systems through Ionic-channel research.\u003c/p\u003e","manuscriptTitle":"Spectroscopic Investigation of Nitric Oxide-Cobalamin Interactions: Oxidation, Binding, and Biological Implications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-08 03:00:58","doi":"10.21203/rs.3.rs-7530358/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":"September 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54158870,"name":"Chemical Biology"},{"id":54158871,"name":"Vascular Medicine"},{"id":54158872,"name":"Spectroscopy"}],"tags":[],"updatedAt":"2025-09-08T03:00:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-08 03:00:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7530358","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7530358","identity":"rs-7530358","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}