Real-time Crystallographic Capture of Fe–NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex

Real-time Crystallographic Capture of Fe–NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex | 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 Article Real-time Crystallographic Capture of Fe–NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex Jaeheung Cho, Seungwon Sun, Sangho So, Dohyun Moon, Mu-Hyun Baik This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6722143/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 interplay between nitric oxide (NO • ) and iron (Fe) centers in metalloenzymes is central to many biological functions, with light-induced Fe − NO bond dissociation emerging as a key regulatory mechanism. Here, we present direct structural evidence for the stepwise dissociation of the Fe − NO bond in a crystalline nonheme iron nitrosyl complex, captured through in-situ photocrystallography under visible light. Marked Fe − NO bond elongation reveals real-time snapshots of excited-state fission at atomic resolution. Solution-phase studies confirm the generality of the observed photoreactivity. Multiconfigurational CASSCF calculations show the ground state as a resonance among Fe(I), Fe(II), and Fe(III) configurations, with a photoactive Fe I –NO + contribution enabling NO • release via metal-to-ligand charge transfer. Strategic placement of electron-withdrawing chlorides at the para-position of the pyridyl moiety further amplifies this character, promoting efficient NO • dissociation. These findings delineate a detailed mechanism of Fe–NO bond cleavage and provide rare structural insights into transient photoinduced processes central to NO signaling and metalloenzyme function. Physical sciences/Chemistry/Inorganic chemistry/Bioinorganic chemistry Physical sciences/Chemistry/Coordination chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nitric oxide (NO • ) is a key signaling molecule that regulates a variety of physiological and pathological processes in mammals, including vasodilatation, neurotransmission, and immune responses 1 – 7 . Endogenously, NO • is produced through the enzymatic conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS) 8 , 9 . Within physiological signaling pathways, NO • receptors often contain heme Fe centers that can form Fe − NO adducts 3 . For instance, NO • diffuses across the cell membrane to bind to the active site of soluble guanylate cyclase (sGC), leading to the production of cyclic guanosine monophosphate (cGMP), which facilitates vasorelaxation 10 , 11 . Beyond its physiological roles in mammals, NO • also modulates catalysis in microbial systems. In particular, NO • regulates the activity of Fe-containing nitrile hydratase (Fe-NHase), an enzyme that catalyzes the hydration of organic nitriles to their corresponding amides–an essential transformation in nitrile metabolism 12 . This regulation occurs via the reversible interaction of NO • and the nonheme Fe center of Fe-NHase, in which light-induced cleavage of the Fe–NO bond and rebinding of NO • to the Fe center modulates enzymatic activity 13 , 14 . In addition to its biological significance, the photoreactivity of nitrosyl complexes has garnered considerable attention in biomimetic and synthetic chemistry. The photolability of the metal–NO bond offers a promising strategy for the exogenous delivery of unstable NO • with spatiotemporal precision, particularly for therapeutic applications. To investigate the light sensitivity of NO-containing metal complexes, a wide range of mononuclear nitrosyl species incorporating diverse metal centers and supporting ligands have been synthesized and characterized using various physicochemical techniques 15 – 31 . The photodissociation behavior in organic solvents has also been extensively studied 15 , 16 , 21 . Notably, model complexes of Fe-NHase featuring sulfinates (− SO 2 ) and thiolates (− S), which modulate NO • binding affinity in metalloenzymes, exhibit high photolysis rates 23 – 26 . Analogous nitrosyl complexes with Mn and Co centers have also demonstrated significant NO • release capabilities 15 – 19 , 30 . In contrast, Ru − NO complexes typically display lower photoreactivity, reflecting their greater thermodynamic stability compared to first-row transition metal analogues 32 , 33 . Recent theoretical studies attribute light-induced NO • dissociation to electronic excitation from bonding or non-bonding molecular orbitals to antibonding orbitals, typically mediated by metal-to-ligand charge transfer (MLCT) or ligand-to-metal charge transfer (LMCT) transitions 30 , 31 , 34 – 39 . Despite extensive research on photolabile nitrosyl complexes and their dissociation mechanisms, a precise understanding of the Fe − NO bonding remains elusive. This challenge arises from the highly covalent nature of the Fe − NO bond and the redox non-innocence of the NO • ligand 40 , 41 . Moreover, although numerous studies have investigated photolabile metal-nitrosyl species, none have directly captured the photodissociation process through experimentally resolved geometric changes–such as bond lengths and angles–at atomic resolution. In this study, we report the first crystallographic observation of stepwise Fe–NO bond dissociation under visible light irradiation. Using in-situ single-crystal X-ray diffraction, we monitored the structural evolution of the iron nitrosyl complex, [Fe(Cl−TBDAP)(NO)(H 2 O)] 2+ ( 2 ), in real time during photolysis. The data reveal a consistent elongation of the Fe − NO bond accompanied by a concurrent shortening of the N–O bond. These structural changes provide high-resolution insight into the excited state dynamics of the Fe–NO photodissociation and offers new perspective on the bonding within the Fe–N–O unit. Results and discussion Synthesis and characterization of 1 and 2 The Fe(II) precursor complex, [Fe II (Cl−TBDAP)(CH 3 CN)(OTf)] + ( 1 ) (Cl−TBDAP = N , N’ -di- tert -butyl-2,11-diaza[3.3](2,6)- p -chloro-pyridinophane), was synthesized by reacting anhydrous FeCl 2 with one equivalent of the Cl−TBDAP ligand and two equivalents of AgOTf (Fig. 1 a). Complex 1 was characterized by ultraviolet-visible (UV-vis) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and single crystal X-ray diffractometry (SC-XRD) (Supplementary Figs. S1–S3 and Tables S1–S2). Exposure of 1 to excess nitric oxide gas in acetone at − 40°C yielded a reddish-brown solution of 2 (Fig. 1 a). The UV-vis spectrum of 2 displayed absorption bands at λ max = 408 ( ε = 1027 M − 1 cm − 1 ), 515 ( ε = 351 M − 1 cm − 1 ), and 741 nm ( ε = 138 M − 1 cm − 1 ) (Fig. 1 b) 31 . Complex 2 was stable at − 40°C in the absence of light, allowing for the growth of single crystals suitable for structural, spectroscopic, and photochemical studies. Cold-spray ionization mass spectrometry (CSI-MS) of 2 revealed prominent ion peaks at mass-to-charge ratio ( m/z ) of 282.0 and 654.9, consistent with the calculated values for [Fe(Cl−TBDAP)(NO)(CH 3 COCH 3 )] 2+ ( m/z = 282.1) and [Fe(Cl−TBDAP)(NO)(OTf)] + ( m/z = 655.1), respectively (Fig. 1 c and Supplementary Fig. S5). The single-crystal X-ray structure of 2 revealed a distorted octahedral coordination geometry, with four N atoms from the Cl−TBDAP ligand, a bound NO group, and a water molecule completing the coordination sphere (Fig. 1 d). The Fe − NO and N − O bond lengths were determined to be 1.762(4) and 1.157(4) Å, respectively, with a bent Fe − N−O angle of 152.8(4)° (Supplementary Table S2) 42 – 44 . These values closely match those reported for the related complex [Fe(TBDAP)(NO)(H 2 O)] 2+ , (1.754 Å for Fe − NO; 1.153 Å for N − O; 152.9° for Fe − N−O) 31 . The attenuated total reflectance infrared (ATR-IR) spectrum of crystalline 2 exhibited an N − O stretching vibration band at 1766 cm − 1 ; consistent with the reported range of 1720–1800 cm − 1 for high-spin {FeNO} 7 complexes and further supporting the assignment of a bound nitrosyl ligand as established by crystallography (Fig. 1 b, inset and Supplementary Fig. S4) 31 , 42 – 47 . The X-band electron paramagnetic resonance (EPR) spectrum of 2 in frozen acetone at 100 K displayed g values of 3.71 and 1.99, consistent with an S = 3/2 spin state of 2 (Supplementary Fig. S6) 42 – 45 . This assignment is further supported by a low-temperature magnetic moment of 4.26 µ B , determined by the 1 H NMR Evans method. Collectively, the characterization data obtained from SC-XRD, ATR-IR, EPR, and magnetic susceptibility are consistent with those of typical six-coordinate high-spin {FeNO} 7 complexes. Accordingly, 2 is best formulated as [Fe(Cl−TBDAP)(NO)(H 2 O)] 2+ , possessing a formal Fe III −NO − ground state ( S = 3/2), in which high-spin Fe III ( S = 5/2) and NO − ( S = 1) are antiferromagnetically coupled 31 , 42 – 47 . Photodissociation of 2 in solution The light-induced Fe − NO bond dissociation in 2 was investigated under white light irradiation (λ irr = 385‒740 nm, 500 mW, xenon lamp) (Fig. 2 a). The kinetics of photodissociation were monitored by following the decay of the absorption band at 515 nm. In the absence of light, the acetone solution of 2 was stable at − 40°C with no detectable spectral changes (Fig. 2 b, inset, red). Upon irradiation, however, a rapid decrease in the 515 nm absorption band was observed (Fig. 2 b, inset, black), indicative of Fe − NO bond cleavage. Notably, partial rebinding of NO • to the Fe center occurred when the xenon lamp was turned off. The Fe − NO dissociation followed first-order kinetics, with the observed rate constant ( k obs ) increasing proportionally with light intensity (Supplementary Fig. S9) 16 . The enhanced photoreactivity of 2 is attributed to the electron-withdrawing Cl substituent on the TBDAP ligand. This substitution increases the population of the Fe I −NO + configuration, facilitating the metal-to-ligand charge transfer (MLCT) absorption that promotes NO • release ( vide infra ) 31 . Consistent with this electronic effect, the redox potential of 2 is elevated relative to its parent complex [Fe(TBDAP)(NO)(H 2 O)] 2+ , resulting in a photodissociation rate that is 3.2 times higher (Supplementary Figs. S10 and S11). Following photolysis, the final UV-vis spectrum of the solution exhibited an absorption profile similar to that of the Fe(II) precursor 1 , consistent with the loss of NO • and formation of a reduced Fe species (Supplementary Fig. S7). The presence of Fe(II) was further confirmed by ESI-MS measurements (Supplementary Fig. S8). To verify the release of free NO • , the gas was captured using [Co(TPP)] (TPP = 5,10,15,20-tetraphenyl-21H,23H-porphine). After 30 minutes of light exposure, the characteristic UV-vis absorption bands of [Co(TPP)] at 410 and 528 nm shifted to 414 and 538 nm, respectively, indicating the formation of [Co(NO)(TPP)] (Supplementary Fig. S12) 26 , 27 , 30 , 31 . Photodissociation of 2 in solid The photodissociation behavior of the Fe − NO bond in the solid state was investigated using single-crystal in-situ photocrystallography. Diffraction data were collected serially from a single crystal of 2 , with one dataset acquired every 130 seconds over a total irradiation period of 75 minutes under visible light. This time-resolved approach enabled the continuous monitoring of changes in the Fe − NO and N − O bond lengths (Supplementary Fig. S14 and Table S3). Upon irradiation, the Fe − NO bond length progressively increased from 1.762(4) to 1.805(10) Å, while the N − O bond length decreased from 1.157(4) to 1.103(9) Å. Concurrently, the Fe − N−O bond angle exhibited a slight bend, shifting from 152.8(4) to 150.9(10)°. The observed 0.054 Å shortening of the N − O bond is particularly notable and is comparable to the ~ 0.05 Å bond difference between free NO • (1.15 Å) and NO − (~ 1.20 Å), indicating a significant change in bonding character 3 . The time-dependent changes in Fe − NO elongation and N − O contraction displayed opposite trends with saturation behavior (Fig. 3 b). Despite the elongation of the Fe − NO bond to 1.805(10) Å, NO • remained coordinated within the crystal lattice. This retention is likely due to steric confinement around the Fe − NO moiety. In the crystal structure, the NO ligand is surrounded by two triflate (OTf − ) counterions and one acetone molecule, positioned at an average distance of 3.22 Å. Additionally, another molecule of complex 2 is located above the NO ligand at a distance of 3.35 Å. A hydrogen-bonding network involving two OTf − anions and two coordinated H 2 O molecules further contributes to the rigidity of the lattice, as illustrated by the ORTEP and space-filling models (Supplementary Fig. S13). These steric and non-covalent interactions collectively hinder the complete dissociation and diffusion NO • from the crystal matrix. Changes in the N − O stretching frequency upon photodissociation were evaluated by ATR-IR spectroscopy. A pristine crystal of 2 and a photoactivated crystal irradiated for 60 minutes were compared. After light exposure, the N–O stretching band shifted from 1766 cm − 1 to 1771 cm − 1 , while the C − O stretching vibration of acetone at 1700 cm − 1 remained unchanged (Fig. 3 c). The 5 cm − 1 blue shift in the N–O vibration correlates with the 0.054 Å decrease in the N–O bond length and is comparable to previous reports on nickel nitrosyl complexes, where a 0.04 Å contraction in N − O bond length led to a 6 cm − 1 shift in stretching frequency (e.g., PhB( Ad Im) 3 NiNO: ν NO = 1689 cm − 1 , N–O: 1.183 Å; HB( CF₃ mIm) 3 NiNO: ν NO = 1695 cm − 1 , N–O: 1.14 Å) 48 . Computational studies To elucidate the electronic structure and the mechanism of NO • release, complete active space self-consistent field (CASSCF) calculations were performed. In order to describe the bonding nature of the {FeNO} 7 unit accurately, an active space was selected that includes all electrons and orbitals associated with the Fe and NO moieties, as well as key ligand-based (See supporting information for details). The resulting CAS wavefunction was decomposed into five Fe d-orbitals and two NO-π* orbitals, as illustrated in Fig. 4 . The projection of 5.49 electrons into the Fe d-dominated molecular orbitals indicates that the Fe center is best described as Fe III in a d 5 configuration. Simultaneously, 0.88 and 0.80 electrons were found in the NO-π y * and NO-π z * orbitals, respectively, supporting an assignment of the NO ligand as anionic (NO – ). The ground state is predominantly described by the configuration (Fe-d x²–y² ) ↑ (Fe-d xy ) ↑ (Fe-d z² ) ↑ (Fe-d yz ) ↑ (Fe-d xz ) ↑ (NO-π* y ) ↓ (NO-π* z ) ↓ , which contributes approximately 45% to the total wavefunction (Supplementary Table S5). This configuration corresponds to a high-spin Fe III center ( S = 5/2) antiferromagnetically coupled to a triplet NO – moiety ( S = 1), consistent with an overall S = 3/2 ground state and in agreement with experimental characterization described above. These results support the Fe III –NO – formulation of complex 2 49 . To further investigate the excited-state behavior of 2 , state-averaged CASSCF calculations were conducted. In line with previous work 31 , the Fe I –NO + resonance form, which is only a minor contributor to the ground state, was found to be the key photoactive configuration. The lowest excited states, including Q 1 , predominantly exhibit Fe II –NO • character and arise via a MLCT transition from Fe I –NO + . These transitions involve electron promotion from the Fe d-dominated Fe–NO bonding orbitals to the NO-π* dominated Fe–NO antibonding orbitals, thereby weakening the Fe–NO bond and promoting NO • release (Supplementary Table S6 and Fig. S17). The effect of substituent electrics was also explored. In the parent complex [Fe(TBDAP)(NO)(H 2 O)] 2+ , 0.91 and 0.80 electrons were localized in the NO-π y * and NO-π z * orbitals, respectively. By contrast, in complex 2 , para-chloride substitution reduces the electron density on these orbitals and increases the Fe I –NO + character (Supplementary Fig. S18), thereby accelerating NO • release. These findings strongly support the conclusion that NO • dissociation proceeds via an MLCT originating from a minor Fe I –NO + ground state resonance contributor. Finally, geometry optimization of the lowest-lying excited state (Q 1 ) was performed at the CASSCF level. During this process, the Fe–NO complex was observed to dissociate into two fragments: free NO • and the remaining Fe complex. The two products stabilized separately, and the calculation ultimately failed to converge to a single wavefunction encompassing both fragments. This result provides compelling computational evidence that photoexcitation leads to complete and spontaneous NO • release and physical separation from the Fe center (Fig. 5 ). Conclusions In this study, we conducted a comprehensive investigation of the stepwise photodissociation of Fe − NO bond in the crystalline mononuclear nonheme high-spin {FeNO} 7 complex, [Fe(Cl−TBDAP)(NO)(H 2 O)] 2+ ( 2 ), using in-situ single-crystal photocrystallography. Sequential X-ray data collection under visible light irradiation enabled real-time monitoring of structural changes within the crystal lattice, providing direct experimental evidence of bond dissociation dynamics. The photoreaction was characterized by progressive elongation of the Fe − NO bond and a simultaneous contraction of the N − O bond, revealing specific geometric signatures of excited-state reactivity. The correlation between bond length changes and irradiation time underscore the photolabile nature of 2 in solid state. Importantly, the influence of the para-substituted Cl group on the photochemical properties of 2 was elucidated. As electron-withdrawing substituent, the Cl group decreases electron density in the NO-π* orbitals, thereby enhancing the contribution of the Fe I −NO + resonance form. The adjustment in electronic structure increases the susceptibility of the Fe − NO bond to photodissociation in solution, facilitating NO • release via a metal-to-ligand charge transfer process. State-averaged CASSCF calculations complemented the experimental findings by providing detailed insights into the electronic structure and excited-state behavior of the Fe − NO unit. These results confirm that NO • release proceeds through an MLCT transition from the minor Fe I –NO + component of the ground-state wavefunction. Together, the integration of high-resolution structural data and multiconfigurational electronic structure analysis offers a molecular-level understanding of Fe–NO photoreactivity. This work advances the fundamental knowledge of Fe–NO bonding and photodissociation, with broader implications for biological NO • regulation and the rational design of synthetic nitrosyl complexes for light-controlled NO • delivery. Declarations Data availability The data supporting the findings of this study are available within the paper and its Supplementary Information. Crystallographic data for the 1 -(OTf), 2 -(OTf) 2 ⸱CH 3 COCH, and photocrystallography have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 2452720, CCDC 2452919, and CCDC 2452920–2452969, respectively. These data can be obtained free of charge from the CCDC (http://www.ccdc.cam.ac.uk/data_request/cif). Source data are provided with this paper. Acknowledgements The research was supported by the National Research Foundation funded by the Ministry of Science, ICT and Future Planning (RS-2024-00333606) and the Ministry of Health and Welfare (RS-2023-00217242) of Korea. We thank the Institute for Basic Science (IBS-R10-A1) in Korea for financial support. This material is based upon work supported by the National Science Foundation under Grant No. 1651686. The synchrotron photocrystallography was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2021R1A2C1003080). Author contributions S. Sun, S. So, M.-H. B., and J. C. conceived and designed the experiments. S. Sun carried out synthesis and experimental work. D. M. conducted the SC-XRD experiment. S. So performed the computational calculations. All authors contributed to data analysis. S. Sun, S. So, M.-H. B., and J. C. co-wrote the paper. Competing interests The authors declare no competing interests. References Culotta E, Koshland DE (1992) NO news is good news. Science 258:1862–1865 Toledo JC Jr, Augusto O (2012) Connecting the chemical and biological properties of nitric oxide. Chem Res Toxicol 25:975–989 Lehnert N, Kim E, Dong HT, Harland JB, Hunt AP, Manickas EC, Oakley KM, Pham J, Reed GC, Alfaro VS (2021) The biologically relevant coordination chemistry of iron and nitric oxide: electronic structure and reactivity. Chem Rev 121:14682–14905 Farah C, Michel LYM, Balligand J-L (2018) Nitric oxide signalling in cardiovascular health and disease. Nat Rev Cardiol 15:292–316 Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526 Snyder SH (1992) Nitric oxide: first in a new class of neurotransmitters. Science 257:494–496 Wei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, Xu D, Muller W, Moncada S, Liew F (1995) Y. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375:408–411 Kwon NS, Nathan CF, Gilker C, Griffith OW, Matthews DE, Stuehr DJ (1990) L-citrulline production from L-arginine by macrophage nitric oxide synthase. the ureido oxygen derives from dioxygen. J Biol Chem 265:13442–13445 Rosen GM, Tsai P, Pou S (2002) Mechanism of free-radical generation by nitric oxide synthase. Chem Rev 102:1191–1200 Liu R, Kang Y, Chen L (2021) Activation mechanism of human soluble guanylate cyclase by stimulators and activators. Nat Commun 12:5492 Kang Y, Liu R, Wu J-X, Chen L (2019) Structural insights into the mechanism of human soluble guanylate cyclase. Nature 574:206–210 Endo I, Nojiri M, Tsujimura M, Nakasako M, Nagashima S, Yohda M, Odaka M (2001) Fe-type nitrile hydratase. J Inorg Biochem 83:247–253 Odaka M, Fujii K, Hoshino M, Noguchi T, Tsujimura M, Nagashima S, Yohda M, Nagamune T, Inoue Y, Endo I (1997) Activity regulation of photoreactive nitrile hydratase by nitric oxide. J Am Chem Soc 119:3785–3791 Endo I, Odaka M, Yohda M (1999) An enzyme controlled by light: the molecular mechanism of photoreactivity in nitrile hydratase. Trends Biotechnol 17:244–248 Ghosh K, Eroy-Reveles AA, Avila B, Holman TR, Olmstead MM, Mascharak PK (2004) Reactions of NO with Mn(II) and Mn(III) centers coordinated to carboxamido nitrogen: synthesis of a manganese nitrosyl with photolabile NO. Inorg Chem 43:2988–2997 Eroy-Reveles AA, Leung Y, Beavers CM, Olmstead MM, Mascharak PK (2008) Near-infrared light activated release of nitric oxide from designed photoactive manganese nitrosyls: strategy, design, and potential as NO donors. J Am Chem Soc 130:4447–4458 Hoffman-Luca CG, Eroy-Reveles AA, Alvarenga J, Mascharak PK (2009) Syntheses, structures, and photochemistry of manganese nitrosyls derived from designed schiff base ligands: potential NO donors that can be activated by near-infrared light. Inorg Chem 48:9104–9111 Hitomi Y, Iwamoto Y, Kodera M (2014) Electronic tuning of nitric oxide release from manganese nitrosyl complexes by visible light irradiation: enhancement of nitric oxide release efficiency by the nitro-substituted quinoline ligand. Dalton Trans 43:2161–2167 Iwamoto Y, Kodera M, Hitomi Y (2015) Uncaging a catalytic hydrogen peroxide generator through the photo-induced release of nitric oxide from a {MnNO} 6 complex. Chem Commun 51:9539–9542 Patra AK, Afshar R, Olmstead MM, Mascharak PK (2002) The first non-heme iron(III) complex with a ligated carboxamido group that exhibits photolability of a bound NO ligand. Angew Chem Int Ed 41:2512–2515 Patra AK, Rowland JM, Marlin DS, Bill E, Olmstead MM, Mascharak PK (2003) Iron nitrosyls of a pentadentate ligand containing a single carboxamide group: syntheses, structures, electronic properties, and photolability of NO. Inorg Chem 42:6812–6823 Afshar RK, Patra AK, Olmstead MM, Mascharak PK (2004) Syntheses, structures, and reactivities of {Fe – NO} 6 nitrosyls derived from polypyridine-carboxamide ligands: photoactive NO-donors and reagents for S-nitrosylation of alkyl thiols. Inorg Chem 43:5736–5743 Szaciłowski K, Chmura A, Stasicka Z (2005) Interplay between iron complexes, nitric oxide and sulfur ligands: structure, (photo)reactivity and biological importance. Coord Chem Rev 249:2408–2436 Schweitzer D, Ellison JJ, Shoner SC, Lovell S, Kovacs JA (1998) A synthetic model for the NO-inactivated form of nitrile hydratase. J Am Chem Soc 120:10996–10997 Rose MJ, Betterley NM, Mascharak PK (2009) Thiolate S-oxygenation controls nitric oxide (NO) photolability of a synthetic iron nitrile hydratase (Fe-NHase) model derived from mixed carboxamide/thiolate ligand. J Am Chem Soc 131:8340–8341 Dey A, Confer AM, Vilbert AC, Moënne-Loccoz P, Lancaster KM, Goldberg (2018) D. P. A nonheme sulfur-ligated {FeNO} 6 complex and comparison with redox-interconvertible {FeNO} 7 and {FeNO} 8 analogues. Angew Chem Int Ed 57:13465–13469 McQuilken AC, Matsumura H, Dürr M, Confer AM, Sheckelton JP, Siegler MA, McQueen TM, Ivanović-Burmazović I, Moënne-Loccoz P (2016) Goldberg, D. P. Photoinitiated reactivity of a thiolate-ligated, spin-crossover nonheme {FeNO} 7 complex with dioxygen. J Am Chem Soc 138:3107–3117 McQuilken AC, Ha Y, Sutherlin KD, Siegler MA, Hodgson KO, Hedman B, Solomon EI, Jameson GN, Goldberg DP (2013) Preparation of non-heme {FeNO} 7 models of cysteine dioxygenase: sulfur versus nitrogen ligation and photorelease of nitric oxide. J Am Chem Soc 135:14024–14027 Chiang C-K, Chu K-T, Lin C-C, Xie S-R, Liu Y-C, Demeshko S, Lee G-H, Meyer F, Tsai M-L, Chiang M-H, Lee C-M (2020) Photoinduced NO and HNO production from mononuclear {FeNO} 6 complex bearing a pendant thiol. J Am Chem Soc 142:8649–8661 Shin S, Choe J, Park Y, Jeong D, Song H, You Y, Seo D, Cho J (2019) Artificial control of cell signaling using a photocleavable cobalt(III)–nitrosyl complex. Angew Chem Int Ed 58:10126–10131 Choe J, Kim SJ, Kim J-H, Baik M-H, Lee J, Cho J (2023) Photodynamic treatment of acute vascular occlusion by using an iron–nitrosyl complex. Chem 9:1309–1317 Fry NL, Heilman BJ, Mascharak PK (2011) Dye-tethered ruthenium nitrosyls containing planar dicarboxamide tetradentate N4 ligands: effects of in-plane ligand twist on NO photolability. Inorg Chem 50:317–324 Rose MJ, Mascharak PK (2008) Photoactive ruthenium nitrosyls: effects of light and potential application as NO donors. Coord Chem Rev 252:2093–2114 Greene SN, Richards NGJ (2004) Theoretical investigations of the electronic structure and spectroscopy of mononuclear, non-heme {Fe – NO} 6 complexes. Inorg Chem 43:7030–7041 Fry NL, Zhao XP, Mascharak PK (2011) Density functional theory studies on a designed photoactive {FeNO} 6 nitrosyl and the corresponding photoinactive {FeNO} 7 species: insight into the origin of NO photolability. Inorg Chim Acta 367:194–198 Merkle AC, Fry NL, Mascharak PK, Lehnert N (2011) Mechanism of NO photodissociation in photolabile manganese–NO complexes with pentadentate N5 ligands. Inorg Chem 50:12192–12203 Fry NL, Mascharak PK (2012) Photolability of NO in designed metal nitrosyls with carboxamido-N donors: a theoretical attempt to unravel the mechanism. Dalton Trans 41:4726–4735 Zheng W, Wu S, Zhao S, Geng Y, Jin J, Su Z, Fu Q (2012) Carbonyl amine/schiff base ligands in manganese complexes: theoretical study on the mechanism, capability of NO release. Inorg Chem 51:3972–3980 Freitag L, González L (2014) Theoretical spectroscopy and photodynamics of a ruthenium nitrosyl complex. Inorg Chem 53:6415–6426 Lewandowska H (2013) Nitrosyl complexes in inorganic chemistry, biochemistry and medicine I. Struct Bond 115–165. 10.1007/430_2013_109 Sun S, Choe J, Cho J (2024) Photo-triggered NO release of nitrosyl complexes bearing first-row transition metals and therapeutic applications. Chem Sci 15:20155–20170 Berto TC, Speelman AL, Zheng S, Lehnert N (2013) Mono- and dinuclear non-heme iron–nitrosyl complexes: models for key intermediates in bacterial nitric oxide reductases. Coord Chem Rev 257:244–259 Li J, Banerjee A, Pawlak PL, Brennessel WW, Chavez FA (2014) Highest recorded N–O stretching frequency for 6-coordinate {Fe-NO} 7 complexes: an iron nitrosyl model for His 3 active sites. Inorg Chem 53:5414–5416 Berto TC, Hoffman MB, Murata Y, Landenberger KB, Alp EE, Zhao J, Lehnert N (2011) Structural and electronic characterization of non-heme Fe(II)–nitrosyls as biomimetic models of the Fe B center of bacterial nitric oxide reductase. J Am Chem Soc 133:16714–16717 Dey A, Gordon JB, Albert T, Sabuncu S, Siegler MA, MacMillan SN, Lancaster KM, Moënne-Loccoz P, Goldberg D (2021) P. A nonheme mononuclear {FeNO} 7 complex that produces N 2 O in the absence of an exogenous reductant. Angew Chem Int Ed 60:21558–21564 Dong HT, Speelman AL, Kozemchak CE, Sil D, Krebs C, Lehnert N (2019) The Fe 2 (NO) 2 diamond core: a unique structural motif in non-heme iron–NO chemistry. Angew Chem Int Ed 58:17695–17699 Dong HT, Camarena S, Sil D, Lengel MO, Zhao J, Hu MY, Alp EE, Krebs C, Lehnert N (2022) What is the right level of activation of a high-spin {FeNO} 7 complex to enable direct N–N coupling? mechanistic insight into flavodiiron NO reductases. J Am Chem Soc 144:16395–16409 Scott JS, Schneider JE, Tewelde EG, Gardner JG, Anferov SW, Filatov AS, Anderson JS (2023) Combining donor strength and oxidative stability in scorpionates: a strongly donating fluorinated mesoionic tris(imidazol-5-ylidene)borate ligand. Inorg Chem 62:21224–21232 Radoń M, Broclawik E, Pierloot K (2010) Electronic structure of selected {FeNO}. complexes heme non-heme architectures: density Funct multireference ab initio study J Phys Chem B 114:1518–1528 Additional Declarations There is NO Competing Interest. Supplementary Files SI.pdf Real-time Crystallographic Capture of Fe–NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex GA.png Graphical Abstract 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6722143","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":462505756,"identity":"47888b6f-55b0-466c-973b-4ce464dab9e4","order_by":0,"name":"Jaeheung Cho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACxgYGNjCDH4gPQMTYiNQi2QDSkkCEFrgKA7AVxGhhbj987MHHtjt2m2/kHjz48weDPH8DW9oHvA7rSUs3nNn2LHnbjbyEwzwJDIYzDrAdnoHfLzlm0rxth5PNbuQYHAY6jHEDA3szXocx9r//Jv0XqMV4Ro7BwR8JDPaEtczIYZNmbDtsZyCRY3AA6LDEDQxshwloeWYm2XPucILEmTcGh3nSJJJnHGZLxqvFsD/5mcSPssP2/O05xh9/2NjY9re3GePX0gChE6G0BDDc8WpgYJCH0vYE1I2CUTAKRsFIBgDOsUommtnLhQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2712-4295","institution":"Ulsan National Institute of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Jaeheung","middleName":"","lastName":"Cho","suffix":""},{"id":462505757,"identity":"46bc7466-0048-41e9-a372-36bf986307b2","order_by":1,"name":"Seungwon Sun","email":"","orcid":"https://orcid.org/0000-0002-0874-6398","institution":"Ulsan National Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Seungwon","middleName":"","lastName":"Sun","suffix":""},{"id":462505758,"identity":"12f43e53-5581-4a77-b188-771ec7af2088","order_by":2,"name":"Sangho So","email":"","orcid":"","institution":"Korea Advanced Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Sangho","middleName":"","lastName":"So","suffix":""},{"id":462505759,"identity":"07e5fece-fb06-4267-9b0c-f5559070065a","order_by":3,"name":"Dohyun Moon","email":"","orcid":"https://orcid.org/0000-0002-6903-0270","institution":"Pohang Accelerator Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Dohyun","middleName":"","lastName":"Moon","suffix":""},{"id":462505760,"identity":"7e77e060-b635-4d2f-ab00-c5b9ec183774","order_by":4,"name":"Mu-Hyun Baik","email":"","orcid":"https://orcid.org/0000-0002-8832-8187","institution":"Center for Catalytic Hydrocarbon Functionalizations, Institute for Basic Science (IBS)","correspondingAuthor":false,"prefix":"","firstName":"Mu-Hyun","middleName":"","lastName":"Baik","suffix":""}],"badges":[],"createdAt":"2025-05-22 07:11:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6722143/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6722143/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103566805,"identity":"d837fb4b-c984-4502-98c2-ac868082ffac","added_by":"auto","created_at":"2026-02-27 07:26:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":476087,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Synthetic procedure of Fe precursor and nitrosyl complex (b) UV-vis absorption spectra of \u003cstrong\u003e1\u003c/strong\u003e (black) and \u003cstrong\u003e2\u003c/strong\u003e (red) in acetone at −40 °C. The inset shows FTIR spectra of \u003cstrong\u003e1\u003c/strong\u003e (black) and \u003cstrong\u003e2\u003c/strong\u003e (red). Asterisk (*) marks the C−O vibration due to an acetone molecule in the crystal. (c) CSI-MS spectrum of \u003cstrong\u003e2\u003c/strong\u003e in acetone at −30 °C. The peaks at \u003cem\u003em/z\u003c/em\u003e 282.0 and 654.9 correspond to [Fe(Cl−TBDAP)(NO)(CH\u003csub\u003e3\u003c/sub\u003eCOCH\u003csub\u003e3\u003c/sub\u003e)]\u003csup\u003e2+\u003c/sup\u003e and [Fe(Cl−TBDAP)(NO)(OTf)]\u003csup\u003e+\u003c/sup\u003e, respectively. The inset shows the simulated (blue) and experimental (red) isotope distribution patterns for the peak at \u003cem\u003em/z\u003c/em\u003e = 282.0. (d) ORTEP diagram of \u003cstrong\u003e2\u003c/strong\u003e with the thermal ellipsoids drawn at the 30% probability. Hydrogen atoms are omitted for clarity, except for the hydrogens of water. Fe, scarlet; O, red; N, blue; C, dark gray; Cl, green; H, white. Selected bond lengths (Å) and angle (°): Fe–N1 1.762(4), Fe–N2 2.307(4), Fe–N3 2.067(4), Fe–N4 2.309(4), Fe–N5 2.043(4), Fe–O2 2.062(3), N1–O1 1.157(4), and Fe–N1–O1 152.8(3).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/2c7658624b104a9f00e7bc15.png"},{"id":103566821,"identity":"d4fe73d9-fd89-4b76-8664-7999a231e7a7","added_by":"auto","created_at":"2026-02-27 07:26:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":246871,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photolysis reaction of \u003cstrong\u003e2\u003c/strong\u003e upon irradiation with white light (λ\u003csub\u003eirr\u003c/sub\u003e = 385‒740 nm, 500 mW, xenon lamp) (b) UV-vis spectral changes of \u003cstrong\u003e2\u003c/strong\u003e (2.0 mM) upon irradiation in acetone at −40 °C. The inset shows the time courses of the absorption band at 515 nm in the presence (black) and absence (red) of light.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/7e688b0edf214aea58372d95.png"},{"id":103566786,"identity":"972304da-d378-4b7c-9d58-5029d38d37fb","added_by":"auto","created_at":"2026-02-27 07:26:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":453744,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Crystallographic description of the photoactivation of \u003cstrong\u003e2\u003c/strong\u003e at 233 K. (b) Time courses of Fe−NO (upper) and N−O (lower) bond length changes against irradiation time. The data points were collected every 130 seconds. (c) ATR-IR band shift of \u003cstrong\u003e2\u003c/strong\u003e after irradiation. The IR band of the N−O bond shifts from 1766 cm\u003csup\u003e−1\u003c/sup\u003e (black) to 1771 cm\u003csup\u003e−1\u003c/sup\u003e (red). Asterisk (*) marks the C−O vibration due to an acetone molecule in the crystal.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/f93b07d9dd860e8259af6637.png"},{"id":103566839,"identity":"cfa83313-3fb8-4e2e-944d-792692b5b56d","added_by":"auto","created_at":"2026-02-27 07:26:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":561632,"visible":true,"origin":"","legend":"\u003cp\u003eGround-state electronic structure of \u003cstrong\u003e2\u003c/strong\u003e computed at the CASSCF(11,9)/def2-TZVP level of theory with a single root. The CAS wavefunction is projected into the Foster-Boys localized orbital manifold for visualization. Non-essential hydrogen atoms are omitted for clarity, and an isodensity value of 0.05 e/Å\u003csup\u003e3\u003c/sup\u003e is used for the orbital plots. Average occupation numbers are given in blue.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/1a1aad54dcd0797da4556538.png"},{"id":103566848,"identity":"fa1f518d-f513-4725-a5a6-8fb3cd06e86b","added_by":"auto","created_at":"2026-02-27 07:26:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212250,"visible":true,"origin":"","legend":"\u003cp\u003eTo confirm that the Q\u003csub\u003e1\u003c/sub\u003e state induces photolability, geometry optimization of the Q\u003csub\u003e1\u003c/sub\u003e state was performed using CASSCF(11,9)/SVP level of theory with 10 roots. During the optimization process, the energy of the Q\u003csub\u003e1\u003c/sub\u003e state decreased as the Fe–NO bond elongated. As a result, the Fe–NO complex dissociates into two separate molecules.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/8c20cb507a94963d8ef6cf6d.png"},{"id":103567005,"identity":"7c2e259c-303c-42f3-b663-268874bc6036","added_by":"auto","created_at":"2026-02-27 07:27:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2663828,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/19688437-84ca-4e27-9f13-7a4fc9bced41.pdf"},{"id":103566659,"identity":"e2e1204e-d695-435c-a1b3-982d6230fa41","added_by":"auto","created_at":"2026-02-27 07:25:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10369619,"visible":true,"origin":"","legend":"Real-time Crystallographic Capture of Fe\u0026#x2013;NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex","description":"","filename":"SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/e69784dd77b524a05632aac2.pdf"},{"id":103566769,"identity":"4c347934-8557-4ec8-a842-7c69b0ef0d1f","added_by":"auto","created_at":"2026-02-27 07:26:07","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":667336,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-6722143/v1/d9f03003d74ce6dabcc25fad.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Real-time Crystallographic Capture of Fe–NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNitric oxide (NO\u003csup\u003e\u0026bull;\u003c/sup\u003e) is a key signaling molecule that regulates a variety of physiological and pathological processes in mammals, including vasodilatation, neurotransmission, and immune responses\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Endogenously, NO\u003csup\u003e\u0026bull;\u003c/sup\u003e is produced through the enzymatic conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Within physiological signaling pathways, NO\u003csup\u003e\u0026bull;\u003c/sup\u003e receptors often contain heme Fe centers that can form Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO adducts\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For instance, NO\u003csup\u003e\u0026bull;\u003c/sup\u003e diffuses across the cell membrane to bind to the active site of soluble guanylate cyclase (sGC), leading to the production of cyclic guanosine monophosphate (cGMP), which facilitates vasorelaxation\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Beyond its physiological roles in mammals, NO\u003csup\u003e\u0026bull;\u003c/sup\u003e also modulates catalysis in microbial systems. In particular, NO\u003csup\u003e\u0026bull;\u003c/sup\u003e regulates the activity of Fe-containing nitrile hydratase (Fe-NHase), an enzyme that catalyzes the hydration of organic nitriles to their corresponding amides\u0026ndash;an essential transformation in nitrile metabolism\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This regulation occurs via the reversible interaction of NO\u003csup\u003e\u0026bull;\u003c/sup\u003e and the nonheme Fe center of Fe-NHase, in which light-induced cleavage of the Fe\u0026ndash;NO bond and rebinding of NO\u003csup\u003e\u0026bull;\u003c/sup\u003e to the Fe center modulates enzymatic activity\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to its biological significance, the photoreactivity of nitrosyl complexes has garnered considerable attention in biomimetic and synthetic chemistry. The photolability of the metal\u0026ndash;NO bond offers a promising strategy for the exogenous delivery of unstable NO\u003csup\u003e\u0026bull;\u003c/sup\u003e with spatiotemporal precision, particularly for therapeutic applications. To investigate the light sensitivity of NO-containing metal complexes, a wide range of mononuclear nitrosyl species incorporating diverse metal centers and supporting ligands have been synthesized and characterized using various physicochemical techniques\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28 CR29 CR30\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The photodissociation behavior in organic solvents has also been extensively studied\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Notably, model complexes of Fe-NHase featuring sulfinates (\u0026minus;\u0026thinsp;SO\u003csub\u003e2\u003c/sub\u003e) and thiolates (\u0026minus;\u0026thinsp;S), which modulate NO\u003csup\u003e\u0026bull;\u003c/sup\u003e binding affinity in metalloenzymes, exhibit high photolysis rates\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Analogous nitrosyl complexes with Mn and Co centers have also demonstrated significant NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release capabilities\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In contrast, Ru\u0026thinsp;\u0026minus;\u0026thinsp;NO complexes typically display lower photoreactivity, reflecting their greater thermodynamic stability compared to first-row transition metal analogues\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Recent theoretical studies attribute light-induced NO\u003csup\u003e\u0026bull;\u003c/sup\u003e dissociation to electronic excitation from bonding or non-bonding molecular orbitals to antibonding orbitals, typically mediated by metal-to-ligand charge transfer (MLCT) or ligand-to-metal charge transfer (LMCT) transitions\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite extensive research on photolabile nitrosyl complexes and their dissociation mechanisms, a precise understanding of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bonding remains elusive. This challenge arises from the highly covalent nature of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond and the redox non-innocence of the NO\u003csup\u003e\u0026bull;\u003c/sup\u003e ligand\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Moreover, although numerous studies have investigated photolabile metal-nitrosyl species, none have directly captured the photodissociation process through experimentally resolved geometric changes\u0026ndash;such as bond lengths and angles\u0026ndash;at atomic resolution.\u003c/p\u003e \u003cp\u003eIn this study, we report the first crystallographic observation of stepwise Fe\u0026ndash;NO bond dissociation under visible light irradiation. Using in-situ single-crystal X-ray diffraction, we monitored the structural evolution of the iron nitrosyl complex, [Fe(Cl\u0026minus;TBDAP)(NO)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e2\u003c/b\u003e), in real time during photolysis. The data reveal a consistent elongation of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond accompanied by a concurrent shortening of the N\u0026ndash;O bond. These structural changes provide high-resolution insight into the excited state dynamics of the Fe\u0026ndash;NO photodissociation and offers new perspective on the bonding within the Fe\u0026ndash;N\u0026ndash;O unit.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis and characterization of 1 and 2\u003c/h2\u003e \u003cp\u003eThe Fe(II) precursor complex, [Fe\u003csup\u003eII\u003c/sup\u003e(Cl\u0026minus;TBDAP)(CH\u003csub\u003e3\u003c/sub\u003eCN)(OTf)]\u003csup\u003e+\u003c/sup\u003e (\u003cb\u003e1\u003c/b\u003e) (Cl\u0026minus;TBDAP\u0026thinsp;=\u0026thinsp;\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u0026rsquo;\u003c/em\u003e-di-\u003cem\u003etert\u003c/em\u003e-butyl-2,11-diaza[3.3](2,6)-\u003cem\u003ep\u003c/em\u003e-chloro-pyridinophane), was synthesized by reacting anhydrous FeCl\u003csub\u003e2\u003c/sub\u003e with one equivalent of the Cl\u0026minus;TBDAP ligand and two equivalents of AgOTf (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Complex \u003cb\u003e1\u003c/b\u003e was characterized by ultraviolet-visible (UV-vis) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and single crystal X-ray diffractometry (SC-XRD) (Supplementary Figs. S1\u0026ndash;S3 and Tables S1\u0026ndash;S2). Exposure of \u003cb\u003e1\u003c/b\u003e to excess nitric oxide gas in acetone at \u0026minus;\u0026thinsp;40\u0026deg;C yielded a reddish-brown solution of \u003cb\u003e2\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The UV-vis spectrum of \u003cb\u003e2\u003c/b\u003e displayed absorption bands at \u003cem\u003eλ\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;408 (\u003cem\u003eε\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1027 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), 515 (\u003cem\u003eε\u003c/em\u003e\u0026thinsp;=\u0026thinsp;351 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and 741 nm (\u003cem\u003eε\u003c/em\u003e\u0026thinsp;=\u0026thinsp;138 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Complex \u003cb\u003e2\u003c/b\u003e was stable at \u0026minus;\u0026thinsp;40\u0026deg;C in the absence of light, allowing for the growth of single crystals suitable for structural, spectroscopic, and photochemical studies. Cold-spray ionization mass spectrometry (CSI-MS) of \u003cb\u003e2\u003c/b\u003e revealed prominent ion peaks at mass-to-charge ratio (\u003cem\u003em/z\u003c/em\u003e) of 282.0 and 654.9, consistent with the calculated values for [Fe(Cl\u0026minus;TBDAP)(NO)(CH\u003csub\u003e3\u003c/sub\u003eCOCH\u003csub\u003e3\u003c/sub\u003e)]\u003csup\u003e2+\u003c/sup\u003e (\u003cem\u003em/z\u003c/em\u003e\u0026thinsp;=\u0026thinsp;282.1) and [Fe(Cl\u0026minus;TBDAP)(NO)(OTf)]\u003csup\u003e+\u003c/sup\u003e (\u003cem\u003em/z\u003c/em\u003e\u0026thinsp;=\u0026thinsp;655.1), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Supplementary Fig. S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe single-crystal X-ray structure of \u003cb\u003e2\u003c/b\u003e revealed a distorted octahedral coordination geometry, with four N atoms from the Cl\u0026minus;TBDAP ligand, a bound NO group, and a water molecule completing the coordination sphere (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO and N\u0026thinsp;\u0026minus;\u0026thinsp;O bond lengths were determined to be 1.762(4) and 1.157(4) \u0026Aring;, respectively, with a bent Fe\u0026thinsp;\u0026minus;\u0026thinsp;N\u0026minus;O angle of 152.8(4)\u0026deg; (Supplementary Table S2)\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. These values closely match those reported for the related complex [Fe(TBDAP)(NO)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e, (1.754 \u0026Aring; for Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO; 1.153 \u0026Aring; for N\u0026thinsp;\u0026minus;\u0026thinsp;O; 152.9\u0026deg; for Fe\u0026thinsp;\u0026minus;\u0026thinsp;N\u0026minus;O)\u003csup\u003e31\u003c/sup\u003e. The attenuated total reflectance infrared (ATR-IR) spectrum of crystalline \u003cb\u003e2\u003c/b\u003e exhibited an N\u0026thinsp;\u0026minus;\u0026thinsp;O stretching vibration band at 1766 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; consistent with the reported range of 1720\u0026ndash;1800 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for high-spin {FeNO}\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e complexes and further supporting the assignment of a bound nitrosyl ligand as established by crystallography (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, inset and Supplementary Fig. S4)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe X-band electron paramagnetic resonance (EPR) spectrum of \u003cb\u003e2\u003c/b\u003e in frozen acetone at 100 K displayed \u003cem\u003eg\u003c/em\u003e values of 3.71 and 1.99, consistent with an \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3/2 spin state of \u003cb\u003e2\u003c/b\u003e (Supplementary Fig. S6)\u003csup\u003e\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. This assignment is further supported by a low-temperature magnetic moment of 4.26 \u003cem\u003e\u0026micro;\u003c/em\u003e\u003csub\u003eB\u003c/sub\u003e, determined by the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR Evans method. Collectively, the characterization data obtained from SC-XRD, ATR-IR, EPR, and magnetic susceptibility are consistent with those of typical six-coordinate high-spin {FeNO}\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e complexes. Accordingly, \u003cb\u003e2\u003c/b\u003e is best formulated as [Fe(Cl\u0026minus;TBDAP)(NO)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e, possessing a formal Fe\u003csup\u003eIII\u003c/sup\u003e\u0026minus;NO\u003csup\u003e\u0026minus;\u003c/sup\u003e ground state (\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3/2), in which high-spin Fe\u003csup\u003eIII\u003c/sup\u003e (\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5/2) and NO\u003csup\u003e\u0026minus;\u003c/sup\u003e (\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1) are antiferromagnetically coupled\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan additionalcitationids=\"CR43 CR44 CR45 CR46\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhotodissociation of 2 in solution\u003c/h3\u003e\n\u003cp\u003eThe light-induced Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond dissociation in \u003cb\u003e2\u003c/b\u003e was investigated under white light irradiation (λ\u003csub\u003eirr\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;385‒740 nm, 500 mW, xenon lamp) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The kinetics of photodissociation were monitored by following the decay of the absorption band at 515 nm. In the absence of light, the acetone solution of \u003cb\u003e2\u003c/b\u003e was stable at \u0026minus;\u0026thinsp;40\u0026deg;C with no detectable spectral changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, inset, red). Upon irradiation, however, a rapid decrease in the 515 nm absorption band was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, inset, black), indicative of Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond cleavage. Notably, partial rebinding of NO\u003csup\u003e\u0026bull;\u003c/sup\u003e to the Fe center occurred when the xenon lamp was turned off. The Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO dissociation followed first-order kinetics, with the observed rate constant (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e) increasing proportionally with light intensity (Supplementary Fig. S9)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The enhanced photoreactivity of \u003cb\u003e2\u003c/b\u003e is attributed to the electron-withdrawing Cl substituent on the TBDAP ligand. This substitution increases the population of the Fe\u003csup\u003eI\u003c/sup\u003e\u0026minus;NO\u003csup\u003e+\u003c/sup\u003e configuration, facilitating the metal-to-ligand charge transfer (MLCT) absorption that promotes NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release (\u003cem\u003evide infra\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Consistent with this electronic effect, the redox potential of \u003cb\u003e2\u003c/b\u003e is elevated relative to its parent complex [Fe(TBDAP)(NO)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e, resulting in a photodissociation rate that is 3.2 times higher (Supplementary Figs. S10 and S11).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing photolysis, the final UV-vis spectrum of the solution exhibited an absorption profile similar to that of the Fe(II) precursor \u003cb\u003e1\u003c/b\u003e, consistent with the loss of NO\u003csup\u003e\u0026bull;\u003c/sup\u003e and formation of a reduced Fe species (Supplementary Fig. S7). The presence of Fe(II) was further confirmed by ESI-MS measurements (Supplementary Fig. S8). To verify the release of free NO\u003csup\u003e\u0026bull;\u003c/sup\u003e, the gas was captured using [Co(TPP)] (TPP\u0026thinsp;=\u0026thinsp;5,10,15,20-tetraphenyl-21H,23H-porphine). After 30 minutes of light exposure, the characteristic UV-vis absorption bands of [Co(TPP)] at 410 and 528 nm shifted to 414 and 538 nm, respectively, indicating the formation of [Co(NO)(TPP)] (Supplementary Fig. S12)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003ePhotodissociation of 2 in solid\u003c/h3\u003e\n\u003cp\u003eThe photodissociation behavior of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond in the solid state was investigated using single-crystal in-situ photocrystallography. Diffraction data were collected serially from a single crystal of \u003cb\u003e2\u003c/b\u003e, with one dataset acquired every 130 seconds over a total irradiation period of 75 minutes under visible light. This time-resolved approach enabled the continuous monitoring of changes in the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO and N\u0026thinsp;\u0026minus;\u0026thinsp;O bond lengths (Supplementary Fig. S14 and Table S3). Upon irradiation, the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond length progressively increased from 1.762(4) to 1.805(10) \u0026Aring;, while the N\u0026thinsp;\u0026minus;\u0026thinsp;O bond length decreased from 1.157(4) to 1.103(9) \u0026Aring;. Concurrently, the Fe\u0026thinsp;\u0026minus;\u0026thinsp;N\u0026minus;O bond angle exhibited a slight bend, shifting from 152.8(4) to 150.9(10)\u0026deg;. The observed 0.054 \u0026Aring; shortening of the N\u0026thinsp;\u0026minus;\u0026thinsp;O bond is particularly notable and is comparable to the ~\u0026thinsp;0.05 \u0026Aring; bond difference between free NO\u003csup\u003e\u0026bull;\u003c/sup\u003e (1.15 \u0026Aring;) and NO\u003csup\u003e\u0026minus;\u003c/sup\u003e (~\u0026thinsp;1.20 \u0026Aring;), indicating a significant change in bonding character\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The time-dependent changes in Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO elongation and N\u0026thinsp;\u0026minus;\u0026thinsp;O contraction displayed opposite trends with saturation behavior (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite the elongation of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond to 1.805(10) \u0026Aring;, NO\u003csup\u003e\u0026bull;\u003c/sup\u003e remained coordinated within the crystal lattice. This retention is likely due to steric confinement around the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO moiety. In the crystal structure, the NO ligand is surrounded by two triflate (OTf\u003csup\u003e\u0026minus;\u003c/sup\u003e) counterions and one acetone molecule, positioned at an average distance of 3.22 \u0026Aring;. Additionally, another molecule of complex \u003cb\u003e2\u003c/b\u003e is located above the NO ligand at a distance of 3.35 \u0026Aring;. A hydrogen-bonding network involving two OTf\u003csup\u003e\u0026minus;\u003c/sup\u003e anions and two coordinated H\u003csub\u003e2\u003c/sub\u003eO molecules further contributes to the rigidity of the lattice, as illustrated by the ORTEP and space-filling models (Supplementary Fig. S13). These steric and non-covalent interactions collectively hinder the complete dissociation and diffusion NO\u003csup\u003e\u0026bull;\u003c/sup\u003e from the crystal matrix.\u003c/p\u003e \u003cp\u003eChanges in the N\u0026thinsp;\u0026minus;\u0026thinsp;O stretching frequency upon photodissociation were evaluated by ATR-IR spectroscopy. A pristine crystal of \u003cb\u003e2\u003c/b\u003e and a photoactivated crystal irradiated for 60 minutes were compared. After light exposure, the N\u0026ndash;O stretching band shifted from 1766 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 1771 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while the C\u0026thinsp;\u0026minus;\u0026thinsp;O stretching vibration of acetone at 1700 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The 5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e blue shift in the N\u0026ndash;O vibration correlates with the 0.054 \u0026Aring; decrease in the N\u0026ndash;O bond length and is comparable to previous reports on nickel nitrosyl complexes, where a 0.04 \u0026Aring; contraction in N\u0026thinsp;\u0026minus;\u0026thinsp;O bond length led to a 6 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shift in stretching frequency (e.g., PhB(\u003csup\u003eAd\u003c/sup\u003eIm)\u003csub\u003e3\u003c/sub\u003eNiNO: ν\u003csub\u003eNO\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1689 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, N\u0026ndash;O: 1.183 \u0026Aring;; HB(\u003csup\u003eCF₃\u003c/sup\u003emIm)\u003csub\u003e3\u003c/sub\u003eNiNO: ν\u003csub\u003eNO\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1695 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, N\u0026ndash;O: 1.14 \u0026Aring;)\u003csup\u003e48\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eComputational studies\u003c/h3\u003e\n\u003cp\u003eTo elucidate the electronic structure and the mechanism of NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release, complete active space self-consistent field (CASSCF) calculations were performed. In order to describe the bonding nature of the {FeNO}\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e unit accurately, an active space was selected that includes all electrons and orbitals associated with the Fe and NO moieties, as well as key ligand-based (See supporting information for details). The resulting CAS wavefunction was decomposed into five Fe d-orbitals and two NO-π* orbitals, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The projection of 5.49 electrons into the Fe d-dominated molecular orbitals indicates that the Fe center is best described as Fe\u003csup\u003eIII\u003c/sup\u003e in a d\u003csup\u003e5\u003c/sup\u003e configuration. Simultaneously, 0.88 and 0.80 electrons were found in the NO-π\u003csub\u003ey\u003c/sub\u003e* and NO-π\u003csub\u003ez\u003c/sub\u003e* orbitals, respectively, supporting an assignment of the NO ligand as anionic (NO\u003csup\u003e\u0026ndash;\u003c/sup\u003e). The ground state is predominantly described by the configuration (Fe-d\u003csub\u003ex\u0026sup2;\u0026ndash;y\u0026sup2;\u003c/sub\u003e)\u003csup\u003e\u0026uarr;\u003c/sup\u003e(Fe-d\u003csub\u003exy\u003c/sub\u003e)\u003csup\u003e\u0026uarr;\u003c/sup\u003e(Fe-d\u003csub\u003ez\u0026sup2;\u003c/sub\u003e)\u003csup\u003e\u0026uarr;\u003c/sup\u003e(Fe-d\u003csub\u003eyz\u003c/sub\u003e)\u003csup\u003e\u0026uarr;\u003c/sup\u003e(Fe-d\u003csub\u003exz\u003c/sub\u003e)\u003csup\u003e\u0026uarr;\u003c/sup\u003e(NO-π*\u003csub\u003ey\u003c/sub\u003e)\u003csup\u003e\u0026darr;\u003c/sup\u003e(NO-π*\u003csub\u003ez\u003c/sub\u003e)\u003csup\u003e\u0026darr;\u003c/sup\u003e, which contributes approximately 45% to the total wavefunction (Supplementary Table S5). This configuration corresponds to a high-spin Fe\u003csup\u003eIII\u003c/sup\u003e center (\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5/2) antiferromagnetically coupled to a triplet NO\u003csup\u003e\u0026ndash;\u003c/sup\u003e moiety (\u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), consistent with an overall \u003cem\u003eS\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3/2 ground state and in agreement with experimental characterization described above. These results support the Fe\u003csup\u003eIII\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e\u0026ndash;\u003c/sup\u003e formulation of complex \u003cb\u003e2\u003c/b\u003e\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the excited-state behavior of \u003cb\u003e2\u003c/b\u003e, state-averaged CASSCF calculations were conducted. In line with previous work\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, the Fe\u003csup\u003eI\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e+\u003c/sup\u003e resonance form, which is only a minor contributor to the ground state, was found to be the key photoactive configuration. The lowest excited states, including Q\u003csub\u003e1\u003c/sub\u003e, predominantly exhibit Fe\u003csup\u003eII\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e\u0026bull;\u003c/sup\u003e character and arise via a MLCT transition from Fe\u003csup\u003eI\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e+\u003c/sup\u003e. These transitions involve electron promotion from the Fe d-dominated Fe\u0026ndash;NO bonding orbitals to the NO-π* dominated Fe\u0026ndash;NO antibonding orbitals, thereby weakening the Fe\u0026ndash;NO bond and promoting NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release (Supplementary Table S6 and Fig. S17).\u003c/p\u003e \u003cp\u003eThe effect of substituent electrics was also explored. In the parent complex [Fe(TBDAP)(NO)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e, 0.91 and 0.80 electrons were localized in the NO-π\u003csub\u003ey\u003c/sub\u003e* and NO-π\u003csub\u003ez\u003c/sub\u003e* orbitals, respectively. By contrast, in complex \u003cb\u003e2\u003c/b\u003e, para-chloride substitution reduces the electron density on these orbitals and increases the Fe\u003csup\u003eI\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e+\u003c/sup\u003e character (Supplementary Fig. S18), thereby accelerating NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release. These findings strongly support the conclusion that NO\u003csup\u003e\u0026bull;\u003c/sup\u003e dissociation proceeds via an MLCT originating from a minor Fe\u003csup\u003eI\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e+\u003c/sup\u003e ground state resonance contributor.\u003c/p\u003e \u003cp\u003eFinally, geometry optimization of the lowest-lying excited state (Q\u003csub\u003e1\u003c/sub\u003e) was performed at the CASSCF level. During this process, the Fe\u0026ndash;NO complex was observed to dissociate into two fragments: free NO\u003csup\u003e\u0026bull;\u003c/sup\u003e and the remaining Fe complex. The two products stabilized separately, and the calculation ultimately failed to converge to a single wavefunction encompassing both fragments. This result provides compelling computational evidence that photoexcitation leads to complete and spontaneous NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release and physical separation from the Fe center (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we conducted a comprehensive investigation of the stepwise photodissociation of Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond in the crystalline mononuclear nonheme high-spin {FeNO}\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e complex, [Fe(Cl\u0026minus;TBDAP)(NO)(H\u003csub\u003e2\u003c/sub\u003eO)]\u003csup\u003e2+\u003c/sup\u003e (\u003cb\u003e2\u003c/b\u003e), using in-situ single-crystal photocrystallography. Sequential X-ray data collection under visible light irradiation enabled real-time monitoring of structural changes within the crystal lattice, providing direct experimental evidence of bond dissociation dynamics. The photoreaction was characterized by progressive elongation of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond and a simultaneous contraction of the N\u0026thinsp;\u0026minus;\u0026thinsp;O bond, revealing specific geometric signatures of excited-state reactivity. The correlation between bond length changes and irradiation time underscore the photolabile nature of \u003cb\u003e2\u003c/b\u003e in solid state.\u003c/p\u003e \u003cp\u003eImportantly, the influence of the para-substituted Cl group on the photochemical properties of \u003cb\u003e2\u003c/b\u003e was elucidated. As electron-withdrawing substituent, the Cl group decreases electron density in the NO-π* orbitals, thereby enhancing the contribution of the Fe\u003csup\u003eI\u003c/sup\u003e\u0026minus;NO\u003csup\u003e+\u003c/sup\u003e resonance form. The adjustment in electronic structure increases the susceptibility of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond to photodissociation in solution, facilitating NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release via a metal-to-ligand charge transfer process.\u003c/p\u003e \u003cp\u003eState-averaged CASSCF calculations complemented the experimental findings by providing detailed insights into the electronic structure and excited-state behavior of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO unit. These results confirm that NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release proceeds through an MLCT transition from the minor Fe\u003csup\u003eI\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e+\u003c/sup\u003e component of the ground-state wavefunction. Together, the integration of high-resolution structural data and multiconfigurational electronic structure analysis offers a molecular-level understanding of Fe\u0026ndash;NO photoreactivity. This work advances the fundamental knowledge of Fe\u0026ndash;NO bonding and photodissociation, with broader implications for biological NO\u003csup\u003e\u0026bull;\u003c/sup\u003e regulation and the rational design of synthetic nitrosyl complexes for light-controlled NO\u003csup\u003e\u0026bull;\u003c/sup\u003e delivery.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the paper and its Supplementary Information. Crystallographic data for the \u003cstrong\u003e1\u003c/strong\u003e-(OTf), \u003cstrong\u003e2\u003c/strong\u003e-(OTf)\u003csub\u003e2\u003c/sub\u003e⸱CH\u003csub\u003e3\u003c/sub\u003eCOCH, and photocrystallography have been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition numbers CCDC 2452720, CCDC 2452919, and CCDC 2452920\u0026ndash;2452969, respectively. These data can be obtained free of charge from the CCDC (http://www.ccdc.cam.ac.uk/data_request/cif). Source data are provided with this paper.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by the National Research Foundation funded by the Ministry of Science, ICT and Future Planning (RS-2024-00333606) and the Ministry of Health and Welfare (RS-2023-00217242) of Korea. We thank the Institute for Basic Science (IBS-R10-A1) in Korea for financial support. This material is based upon work supported by the National Science Foundation under Grant No. 1651686. The synchrotron photocrystallography was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2021R1A2C1003080).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. Sun, S. So, M.-H. B., and J. C. conceived and designed the experiments. S. Sun carried out synthesis and experimental work. D. M. conducted the SC-XRD experiment. S. So performed the computational calculations. All authors contributed to data analysis. S. Sun, S. So, M.-H. B., and J. C. co-wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cbr\u003e \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCulotta E, Koshland DE (1992) NO news is good news. Science 258:1862\u0026ndash;1865\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToledo JC Jr, Augusto O (2012) Connecting the chemical and biological properties of nitric oxide. Chem Res Toxicol 25:975\u0026ndash;989\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLehnert N, Kim E, Dong HT, Harland JB, Hunt AP, Manickas EC, Oakley KM, Pham J, Reed GC, Alfaro VS (2021) The biologically relevant coordination chemistry of iron and nitric oxide: electronic structure and reactivity. Chem Rev 121:14682\u0026ndash;14905\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarah C, Michel LYM, Balligand J-L (2018) Nitric oxide signalling in cardiovascular health and disease. Nat Rev Cardiol 15:292\u0026ndash;316\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524\u0026ndash;526\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSnyder SH (1992) Nitric oxide: first in a new class of neurotransmitters. Science 257:494\u0026ndash;496\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei XQ, Charles IG, Smith A, Ure J, Feng GJ, Huang FP, Xu D, Muller W, Moncada S, Liew F (1995) Y. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375:408\u0026ndash;411\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwon NS, Nathan CF, Gilker C, Griffith OW, Matthews DE, Stuehr DJ (1990) L-citrulline production from L-arginine by macrophage nitric oxide synthase. the ureido oxygen derives from dioxygen. J Biol Chem 265:13442\u0026ndash;13445\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosen GM, Tsai P, Pou S (2002) Mechanism of free-radical generation by nitric oxide synthase. Chem Rev 102:1191\u0026ndash;1200\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu R, Kang Y, Chen L (2021) Activation mechanism of human soluble guanylate cyclase by stimulators and activators. Nat Commun 12:5492\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang Y, Liu R, Wu J-X, Chen L (2019) Structural insights into the mechanism of human soluble guanylate cyclase. Nature 574:206\u0026ndash;210\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEndo I, Nojiri M, Tsujimura M, Nakasako M, Nagashima S, Yohda M, Odaka M (2001) Fe-type nitrile hydratase. J Inorg Biochem 83:247\u0026ndash;253\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOdaka M, Fujii K, Hoshino M, Noguchi T, Tsujimura M, Nagashima S, Yohda M, Nagamune T, Inoue Y, Endo I (1997) Activity regulation of photoreactive nitrile hydratase by nitric oxide. J Am Chem Soc 119:3785\u0026ndash;3791\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEndo I, Odaka M, Yohda M (1999) An enzyme controlled by light: the molecular mechanism of photoreactivity in nitrile hydratase. Trends Biotechnol 17:244\u0026ndash;248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhosh K, Eroy-Reveles AA, Avila B, Holman TR, Olmstead MM, Mascharak PK (2004) Reactions of NO with Mn(II) and Mn(III) centers coordinated to carboxamido nitrogen: synthesis of a manganese nitrosyl with photolabile NO. Inorg Chem 43:2988\u0026ndash;2997\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEroy-Reveles AA, Leung Y, Beavers CM, Olmstead MM, Mascharak PK (2008) Near-infrared light activated release of nitric oxide from designed photoactive manganese nitrosyls: strategy, design, and potential as NO donors. J Am Chem Soc 130:4447\u0026ndash;4458\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffman-Luca CG, Eroy-Reveles AA, Alvarenga J, Mascharak PK (2009) Syntheses, structures, and photochemistry of manganese nitrosyls derived from designed schiff base ligands: potential NO donors that can be activated by near-infrared light. Inorg Chem 48:9104\u0026ndash;9111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHitomi Y, Iwamoto Y, Kodera M (2014) Electronic tuning of nitric oxide release from manganese nitrosyl complexes by visible light irradiation: enhancement of nitric oxide release efficiency by the nitro-substituted quinoline ligand. Dalton Trans 43:2161\u0026ndash;2167\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwamoto Y, Kodera M, Hitomi Y (2015) Uncaging a catalytic hydrogen peroxide generator through the photo-induced release of nitric oxide from a {MnNO}\u003csup\u003e6\u003c/sup\u003e complex. Chem Commun 51:9539\u0026ndash;9542\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatra AK, Afshar R, Olmstead MM, Mascharak PK (2002) The first non-heme iron(III) complex with a ligated carboxamido group that exhibits photolability of a bound NO ligand. Angew Chem Int Ed 41:2512\u0026ndash;2515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatra AK, Rowland JM, Marlin DS, Bill E, Olmstead MM, Mascharak PK (2003) Iron nitrosyls of a pentadentate ligand containing a single carboxamide group: syntheses, structures, electronic properties, and photolability of NO. Inorg Chem 42:6812\u0026ndash;6823\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfshar RK, Patra AK, Olmstead MM, Mascharak PK (2004) Syntheses, structures, and reactivities of {Fe\u0026thinsp;\u0026ndash;\u0026thinsp;NO}\u003csup\u003e6\u003c/sup\u003e nitrosyls derived from polypyridine-carboxamide ligands: photoactive NO-donors and reagents for S-nitrosylation of alkyl thiols. Inorg Chem 43:5736\u0026ndash;5743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzaciłowski K, Chmura A, Stasicka Z (2005) Interplay between iron complexes, nitric oxide and sulfur ligands: structure, (photo)reactivity and biological importance. Coord Chem Rev 249:2408\u0026ndash;2436\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchweitzer D, Ellison JJ, Shoner SC, Lovell S, Kovacs JA (1998) A synthetic model for the NO-inactivated form of nitrile hydratase. J Am Chem Soc 120:10996\u0026ndash;10997\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRose MJ, Betterley NM, Mascharak PK (2009) Thiolate S-oxygenation controls nitric oxide (NO) photolability of a synthetic iron nitrile hydratase (Fe-NHase) model derived from mixed carboxamide/thiolate ligand. J Am Chem Soc 131:8340\u0026ndash;8341\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey A, Confer AM, Vilbert AC, Mo\u0026euml;nne-Loccoz P, Lancaster KM, Goldberg (2018) D. P. A nonheme sulfur-ligated {FeNO}\u003csup\u003e6\u003c/sup\u003e complex and comparison with redox-interconvertible {FeNO}\u003csup\u003e7\u003c/sup\u003e and {FeNO}\u003csup\u003e8\u003c/sup\u003e analogues. Angew Chem Int Ed 57:13465\u0026ndash;13469\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcQuilken AC, Matsumura H, D\u0026uuml;rr M, Confer AM, Sheckelton JP, Siegler MA, McQueen TM, Ivanović-Burmazović I, Mo\u0026euml;nne-Loccoz P (2016) Goldberg, D. P. Photoinitiated reactivity of a thiolate-ligated, spin-crossover nonheme {FeNO}\u003csup\u003e7\u003c/sup\u003e complex with dioxygen. J Am Chem Soc 138:3107\u0026ndash;3117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcQuilken AC, Ha Y, Sutherlin KD, Siegler MA, Hodgson KO, Hedman B, Solomon EI, Jameson GN, Goldberg DP (2013) Preparation of non-heme {FeNO}\u003csup\u003e7\u003c/sup\u003e models of cysteine dioxygenase: sulfur versus nitrogen ligation and photorelease of nitric oxide. J Am Chem Soc 135:14024\u0026ndash;14027\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChiang C-K, Chu K-T, Lin C-C, Xie S-R, Liu Y-C, Demeshko S, Lee G-H, Meyer F, Tsai M-L, Chiang M-H, Lee C-M (2020) Photoinduced NO and HNO production from mononuclear {FeNO}\u003csup\u003e6\u003c/sup\u003e complex bearing a pendant thiol. J Am Chem Soc 142:8649\u0026ndash;8661\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin S, Choe J, Park Y, Jeong D, Song H, You Y, Seo D, Cho J (2019) Artificial control of cell signaling using a photocleavable cobalt(III)\u0026ndash;nitrosyl complex. Angew Chem Int Ed 58:10126\u0026ndash;10131\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoe J, Kim SJ, Kim J-H, Baik M-H, Lee J, Cho J (2023) Photodynamic treatment of acute vascular occlusion by using an iron\u0026ndash;nitrosyl complex. Chem 9:1309\u0026ndash;1317\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFry NL, Heilman BJ, Mascharak PK (2011) Dye-tethered ruthenium nitrosyls containing planar dicarboxamide tetradentate N4 ligands: effects of in-plane ligand twist on NO photolability. Inorg Chem 50:317\u0026ndash;324\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRose MJ, Mascharak PK (2008) Photoactive ruthenium nitrosyls: effects of light and potential application as NO donors. Coord Chem Rev 252:2093\u0026ndash;2114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreene SN, Richards NGJ (2004) Theoretical investigations of the electronic structure and spectroscopy of mononuclear, non-heme {Fe\u0026thinsp;\u0026ndash;\u0026thinsp;NO}\u003csup\u003e6\u003c/sup\u003e complexes. Inorg Chem 43:7030\u0026ndash;7041\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFry NL, Zhao XP, Mascharak PK (2011) Density functional theory studies on a designed photoactive {FeNO}\u003csup\u003e6\u003c/sup\u003e nitrosyl and the corresponding photoinactive {FeNO}\u003csup\u003e7\u003c/sup\u003e species: insight into the origin of NO photolability. Inorg Chim Acta 367:194\u0026ndash;198\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerkle AC, Fry NL, Mascharak PK, Lehnert N (2011) Mechanism of NO photodissociation in photolabile manganese\u0026ndash;NO complexes with pentadentate N5 ligands. Inorg Chem 50:12192\u0026ndash;12203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFry NL, Mascharak PK (2012) Photolability of NO in designed metal nitrosyls with carboxamido-N donors: a theoretical attempt to unravel the mechanism. Dalton Trans 41:4726\u0026ndash;4735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W, Wu S, Zhao S, Geng Y, Jin J, Su Z, Fu Q (2012) Carbonyl amine/schiff base ligands in manganese complexes: theoretical study on the mechanism, capability of NO release. Inorg Chem 51:3972\u0026ndash;3980\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreitag L, Gonz\u0026aacute;lez L (2014) Theoretical spectroscopy and photodynamics of a ruthenium nitrosyl complex. Inorg Chem 53:6415\u0026ndash;6426\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewandowska H (2013) Nitrosyl complexes in inorganic chemistry, biochemistry and medicine I. Struct Bond 115\u0026ndash;165. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/430_2013_109\u003c/span\u003e\u003cspan address=\"10.1007/430_2013_109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun S, Choe J, Cho J (2024) Photo-triggered NO release of nitrosyl complexes bearing first-row transition metals and therapeutic applications. Chem Sci 15:20155\u0026ndash;20170\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerto TC, Speelman AL, Zheng S, Lehnert N (2013) Mono- and dinuclear non-heme iron\u0026ndash;nitrosyl complexes: models for key intermediates in bacterial nitric oxide reductases. Coord Chem Rev 257:244\u0026ndash;259\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Banerjee A, Pawlak PL, Brennessel WW, Chavez FA (2014) Highest recorded N\u0026ndash;O stretching frequency for 6-coordinate {Fe-NO}\u003csup\u003e7\u003c/sup\u003e complexes: an iron nitrosyl model for His\u003csub\u003e3\u003c/sub\u003e active sites. Inorg Chem 53:5414\u0026ndash;5416\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerto TC, Hoffman MB, Murata Y, Landenberger KB, Alp EE, Zhao J, Lehnert N (2011) Structural and electronic characterization of non-heme Fe(II)\u0026ndash;nitrosyls as biomimetic models of the Fe\u003csub\u003eB\u003c/sub\u003e center of bacterial nitric oxide reductase. J Am Chem Soc 133:16714\u0026ndash;16717\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey A, Gordon JB, Albert T, Sabuncu S, Siegler MA, MacMillan SN, Lancaster KM, Mo\u0026euml;nne-Loccoz P, Goldberg D (2021) P. A nonheme mononuclear {FeNO}\u003csup\u003e7\u003c/sup\u003e complex that produces N\u003csub\u003e2\u003c/sub\u003eO in the absence of an exogenous reductant. Angew Chem Int Ed 60:21558\u0026ndash;21564\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong HT, Speelman AL, Kozemchak CE, Sil D, Krebs C, Lehnert N (2019) The Fe\u003csub\u003e2\u003c/sub\u003e(NO)\u003csub\u003e2\u003c/sub\u003e diamond core: a unique structural motif in non-heme iron\u0026ndash;NO chemistry. Angew Chem Int Ed 58:17695\u0026ndash;17699\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong HT, Camarena S, Sil D, Lengel MO, Zhao J, Hu MY, Alp EE, Krebs C, Lehnert N (2022) What is the right level of activation of a high-spin {FeNO}\u003csup\u003e7\u003c/sup\u003e complex to enable direct N\u0026ndash;N coupling? mechanistic insight into flavodiiron NO reductases. J Am Chem Soc 144:16395\u0026ndash;16409\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott JS, Schneider JE, Tewelde EG, Gardner JG, Anferov SW, Filatov AS, Anderson JS (2023) Combining donor strength and oxidative stability in scorpionates: a strongly donating fluorinated mesoionic tris(imidazol-5-ylidene)borate ligand. Inorg Chem 62:21224\u0026ndash;21232\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadoń M, Broclawik E, Pierloot K (2010) Electronic structure of selected {FeNO}. complexes heme non-heme architectures: density Funct multireference ab initio study J Phys Chem B 114:1518\u0026ndash;1528\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6722143/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6722143/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe interplay between nitric oxide (NO\u003csup\u003e\u0026bull;\u003c/sup\u003e) and iron (Fe) centers in metalloenzymes is central to many biological functions, with light-induced Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond dissociation emerging as a key regulatory mechanism. Here, we present direct structural evidence for the stepwise dissociation of the Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond in a crystalline nonheme iron nitrosyl complex, captured through in-situ photocrystallography under visible light. Marked Fe\u0026thinsp;\u0026minus;\u0026thinsp;NO bond elongation reveals real-time snapshots of excited-state fission at atomic resolution. Solution-phase studies confirm the generality of the observed photoreactivity. Multiconfigurational CASSCF calculations show the ground state as a resonance among Fe(I), Fe(II), and Fe(III) configurations, with a photoactive Fe\u003csup\u003eI\u003c/sup\u003e\u0026ndash;NO\u003csup\u003e+\u003c/sup\u003e contribution enabling NO\u003csup\u003e\u0026bull;\u003c/sup\u003e release via metal-to-ligand charge transfer. Strategic placement of electron-withdrawing chlorides at the para-position of the pyridyl moiety further amplifies this character, promoting efficient NO\u003csup\u003e\u0026bull;\u003c/sup\u003e dissociation. These findings delineate a detailed mechanism of Fe\u0026ndash;NO bond cleavage and provide rare structural insights into transient photoinduced processes central to NO signaling and metalloenzyme function.\u003c/p\u003e","manuscriptTitle":"Real-time Crystallographic Capture of Fe–NO Bond Photodissociation in a Nonheme Iron Nitrosyl Complex","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 07:23:18","doi":"10.21203/rs.3.rs-6722143/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3894ce7b-a5f1-4dfc-b7cf-fcf8008a3bb8","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49108733,"name":"Physical sciences/Chemistry/Inorganic chemistry/Bioinorganic chemistry"},{"id":49108734,"name":"Physical sciences/Chemistry/Coordination chemistry"}],"tags":[],"updatedAt":"2026-02-27T07:23:22+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 07:23:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6722143","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6722143","identity":"rs-6722143","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}