Detoxification of hydrogen sulfide by synthetic heme-model compounds

Detoxification of hydrogen sulfide by synthetic heme-model compounds | 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 Detoxification of hydrogen sulfide by synthetic heme-model compounds Atsuki Nakagami, Qiyue Mao, Masaki Horitani, Masahito Kodera, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4591678/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Hydrogen sulfide is a lethal toxic gas that disrupts cellular respiration in the mitochondrial system. Currently, no antidote is available for the clinical treatment of hydrogen sulfide poisoning. In this study, we investigated the function of iron(III)porphyrin complexes as hydrogen sulfide scavengers in water and evaluated their potential use as therapeutic agents for hydrogen sulfide poisoning. The compounds, named met-hemoCD-P and met-hemoCD-I, are composed of iron(III)porphyrin complexed with per-methylated b-cyclodextrin dimers that contain a pyridine (met-hemoCD-P) or imidazole axial fifth ligand that is coordinated to Fe(III) (met-hemoCD-I). These compounds formed stable HS–Fe(III) complexes under physiological conditions, with binding constants of 1.2 x 10 5 and 2.5 x 10 6 M –1 for met-hemoCD-P and met-hemoCD-I, respectively. The binding constant of met-hemoCD-I was much greater than those reported for native met-hemoglobin and met-myoglobin. Electron paramagnetic resonance (EPR) spectroscopy and H 2 S quantification assays revealed that after SH – was coordinated to met-hemoCD-I, it was efficiently converted to nontoxic sulfite and sulfate ions via homolytic cleavage of the HS–Fe(III) bond followed by aerobic oxidation. Mouse animal experiments revealed that the survival rate was significantly improved when NaSH-treated mice were injected with met-hemoCD-I. After the injection, mitochondrial C c O function in brain and heart tissues recovered, and met-hemoCD-I injected was excreted in the urine without chemical decomposition. Hydrogen sulfide Heme Porphyrin Cyclodextrin Injectable antidote Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hydrogen sulfide (H 2 S) is a colorless, flammable, and hazardous gas with a rotten egg smell. The toxic effect of H 2 S is similar to that of hydrogen cyanide (HCN), which strongly binds to cytochrome c oxidase (C c O) in the mitochondrial respiratory chain; thus, H 2 S is classified as a cellular asphyxiant. 1–5 As its specific gravity (1.19) is greater than that of air, H 2 S tends to accumulate at lower altitudes, often causing poisoning accidents at sites such as in manholes, sewage systems, and mining operations. 1,6 Although there are fewer than 10 cases of industrial H 2 S poisoning per year in Japan, 220 cases with 208 deaths were reported in 2007 in Japan due to suicide by intentional H 2 S generation, which is known as the detergent suicide pandemic. 6,7 The following year, this method of suicide was observed in the United States and worldwide. 8,9 Frighteningly, residual H 2 S gas often spreads from the source or victim and causes secondary poisoning to the rescue personnel and/or individuals at the site. 8,9 No clinical antidote is currently available for H 2 S poisoning. Therefore, ready-to-use antidotes that can be stored for long durations and are immediately effective are greatly needed, especially for situations involving emergency rescue. The administration of heme proteins or artificial heme-model compounds may be a promising approach for the development of antidotes against poisoning caused by inhaled gases such as H 2 S, which reacts with metalloproteins (including hemoglobin) in red blood cells (RBCs) 10,11 . For this purpose, the compounds should exhibit higher binding affinities toward toxins than native hemes. Relying on this strategy, researchers have proposed potential antidotes for carbon monoxide (CO), hydrogen cyanide (HCN), and H 2 S poisoning using native and modified heme proteins as well as natural vitamin B 12 analogs. 4,12–18 However, few studies have established antidote systems using synthetic compounds. 12,19 Therefore, our group developed synthetic heme-model compounds composed of iron tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) complexes encapsulated by per- O -methylated b-cyclodextrin (CD) dimers. 20–22 Fig. 1 shows two representative CD dimers, Py3CD and Im3CD, that form inclusion complexes with Fe(III)TPPS to yield met-hemoCD-P and met-hemoCD-I, respectively. We have shown that reduced hemoCD-P in the ferrous state functions as an internal CO scavenger in vivo 21,23 and that met-hemoCD-I functions as a potential cyanide antidote. 24,25 When these heme model compounds were injected intravenously or intraperitoneally into mice or rats, they bound gaseous molecules in the circulation system. 21–25 Interestingly, these compounds were rapidly and quantitatively excreted in the urine through renal clearance; thus, they do not accumulate in the body. Therefore, compared to native protein-based scavenging systems, our system shows potential as an injectable antidote. In the present study, we investigated the potential of met-hemoCD-P and met-hemoCD-I as hydrogen sulfide scavengers. Using the ferric forms of these two complexes, we first present the basic reactivity toward hydrogen sulfide in aqueous solution in view of thermodynamic and kinetic parameters and spectroscopic characterizations. Then, the antidote effect against hydrogen sulfide-induced intoxication was tested in mice. Results Binding of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I In this study, we used sodium hydrogen sulfide (NaSH) as the sulfide source. In aqueous media, SH – equilibrates with H 2 S, and the p K a is 7. 26 The presence of S 2– could be negligible under aqueous conditions due to the low acidity of SH – (p K a = 19). 26 In this article, we use the term “hydrogen sulfide” to refer to both H 2 S and SH – species. The binding constants ( K ) of hydrogen sulfide to met-hemoCD-I and met-hemoCD-P were evaluated by UV‒vis spectroscopic titration. Upon the addition of NaSH, the spectra of met-hemoCD-P and met-hemoCD-I changed stepwise with clear isosbestic points (Fig. 2 A and B). The spectral changes were saturated at approximately one equivalent of added NaSH. In the absence of CD dimers, Fe(III)TPPS was decomposed by the addition of NaSH (Figure S1 A). Similar porphyrin decomposition was also observed for Fe(III)TPPS complexed with 2,3,6-tri- O -methyl-b-CD (TMe-b-CD), which lacks an axial fifth ligand (Figure S1 B). This result suggested that, in addition to protection by the CD cavity, pyridine or imidazole ligation in Py3CD or Im3CD contributed to the formation of a stable HS–Fe(III)porphyrin complex. The titration curves were well fitted to the 1:1 equilibrium model, affording K values of 1.2 x 10 5 M –1 and 2.5 x 10 6 M –1 for met-hemoCD-P and met-hemoCD-I, respectively, in phosphate buffer at pH 7.4. The kinetic parameters for the binding of hydrogen sulfide were determined by time-resolved UV‒vis spectral measurements (Fig. 2 C and D). Single-exponential curve fitting analysis was used to determine the apparent association rate constants ( k on app ). The second-order rate constants ( k on ) were determined via linear regression of the k on app values as a function of [NaSH] (Fig. 3 A and B). The parameters were strongly dependent on the pH of the solution. As listed in Table 1 , met-hemoCD-I exhibits higher K and k on values than those of met-hemoCD-P. The plot of k on and k off values versus pH (Fig. 3 C and D) clearly shows the high ability of met-hemoCD-I to act as a hydrogen sulfide scavenger at physiological pH (7.4). Hydrogen sulfide binds more quickly to met-hemoCD-I at neutral pH because the p K a (p K a H2O = 7.7) of the axial aqua ligand is higher than that of hemoCD-P (p K a H2O = 5.5). 22,24 Table 1 Binding constants ( K ) and rate constants ( k on , k off ) of met-hemoCD-P and met-hemoCD-I with hydrogen sulfide in 0.05 M phosphate buffer at different pH values and at 25°C. met-hemoCD-P met-hemoCD-I pH 10 –6 K (M –1 ) 10 –3 k on (M –1 s –1 ) 10 3 k off (s –1 ) 10 –6 K (M –1 ) 10 –3 k on (M –1 s –1 ) 10 3 k off (s –1 ) 6.0 9.0 ± 2.6 1.54 ± 0.12 0.17 ± 0.1 0.69 ± 0.2 2.23 ± 0.62 3.23 ± 1.0 7.0 0.29 ± 0.1 0.38 ± 0.03 1.31 ± 0.4 2.7 ± 0.8 1.77 ± 0.35 0.66 ± 0.2 7.4 0.12 ± 0.04 0.10 ± 0.01 0.84 ± 0.3 2.5 ± 0.7 1.48 ± 0.78 0.59 ± 0.2 8.0 0.03 ± 0.01 0.04 ± 0.01 1.53 ± 0.5 0.67 ± 0.2 0.72 ± 0.44 1.07 ± 0.3 Table 2 Binding constants ( K ) and rate constants ( k on , k off ) of met-hemoCD, met-Hb and met-Mb with hydrogen sulfide. K (M –1 ) k on (M –1 s –1 ) k off (s –1 ) met-hemoCD-P 1.2 x 10 5 1.0 x 10 2 8.4 x 10 –4 met-hemoCD-I 2.5 x 10 6 1.5 x 10 3 5.9 x 10 –4 met-Mb (horse) a 1.0 x 10 4 1.6 x 10 4 1.6 met-Hb (human) b,c 2.8 x 10 5 9.9 x 10 2 3.5 x 10 –3 met-Mb (sperm whale) b , c 9.6 x 10 4 4.6 x 10 3 4.8 x 10 –2 a Ref. 31. b Ref. 32. c The kinetic parameters were recalculated as a function of the hydrogen sulfide (H 2 S/SH – ) concentration. A continuous variation plot (Job plot) was constructed for the met-hemoCD-I and NaSH systems (Figure S2). The maximum complexation ratio observed at a 1:1 molar ratio clearly indicates that a 1:1 complex of met-hemoCD-I and NaSH was present; these results indicate that poly(sulfide) complexes such as HS(S) n –Fe(II), which are often proposed in biological systems, were not formed. 11 Additionally, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) revealed that the HS–Fe(III) complex in met-hemoCD-I formed (Figure S3). The molecular ion peak of met-hemoCD-I (MW = 3947.9) was detected at 1315.4 ( m / z ) as a tri-anionic species. With NaSH, the molecular ion peak of met-hemoCD-I was mainly detected at 995.1 ( m / z ), which could be assigned to HS-bound met-hemoCD-I (MW = 3981.0) in the terta-anionic form. These results revealed that a stable 1:1 complex of met-hemoCD-I with hydrogen sulfide (HS–Fe(III)porphyrin) formed in aqueous media at physiological pH. Oxidative degradation of hydrogen sulfide by met-hemoCD-I In the presence of excess NaSH (100 equivalents), met-hemoCD-I gradually degraded under aerobic conditions (Fig. 4 A). The degradation was significantly suppressed under anaerobic conditions with a Soret band at 434 nm, indicating that a ferrous hemoCD-I complex formed in the deoxy form. 27 To confirm the iron oxidation state, carbon monoxide (CO) gas was introduced into the solution after met-hemoCD-I was mixed with NaSH (Fig. 4 B). A sharp Soret band appeared at 422 nm, which is characteristic of the CO–Fe(II) complex. Therefore, homolytic bond cleavage of the HS–Fe(III) complex occurred, generating a sulfide radical (HS•) and Fe(II) complex of hemoCD-I. In the presence of molecular oxygen (O 2 ), the reduced Fe(II) complex formed the O 2 adduct, which was readily autoxidized to ferric met-hemoCD-I with the generation of superoxide (Fig. 4 C). The autoxidation rate of the O 2 adduct for hemoCD-I ( t 1/2 ~ 36 min at 37°C) is much faster than that for ferrous hemoCD-P ( t 1/2 ~ 5 h at 37°C). 25 Therefore, hydrogen sulfide should be efficiently converted to sulfite or sulfate ions in the presence of met-hemoCD-I under aerobic conditions. To further characterize the reaction of met-hemoCD-I with NaSH, electron paramagnetic resonance (EPR) spectra of met-hemoCD-I were obtained before and after the reaction of NaSH at physiological pH and 5 K (Fig. 4 D). Before the reaction with NaSH occurred, met-hemoCD-I showed EPR signals at g = 6.03, 2.30, and 2.00. The signals at g = 6.03 and 2.00 could be assigned to the characteristic signals of 5-coordinated high-spin iron(III)porphyrin with or without a weakly coordinated sixth ligand, such as H 2 O, while the signal at g = 2.30 could be assigned to the iron(III)porphyrin coordinated with a hydroxoligand. 28 Immediately after NaSH was added, characteristic signals at g = 2.41, 2.20, and 1.92 were generated due to the HS–Fe(III)–N(imidazole) 6-coordinated low-spin complex. 29–31 The EPR signals became almost silent when the solution was incubated for an hour and then frozen after NaSH was added, indicating that an EPR-inactive ferrous Fe(II) complex formed due to the homolysis of HS–Fe(III). Ferric high-spin species of met-hemoCD-I were detected after 2 and 3 hours of incubation. The time-course change in the EPR spectra confirmed that met-hemoCD-I was converted to the ferrous Fe(II) complex via HS–Fe(III) complex formation, as proposed in Fig. 4 C. The hydrogen sulfide species in aqueous solution decomposed rapidly in the presence of met-hemoCD-I (Fig. 5 A). The efficacy of hydrogen sulfide decomposition is correlated with binding parameters for met-hemoCD-I, met-hemoCD-P, and met-Hb, as summarized in Table 2 . Consistently, sulfite and sulfate ions were efficiently produced in the presence of met-hemoCD-I (Fig. 5 B). The control data under anaerobic conditions support the involvement of O 2 in met-hemoCD-I-assisted decomposition of hydrogen sulfide. Strength of met-hemoCD-I antidotes for hydrogen sulfide poisoning in mice : We then evaluated the strength of met-hemoCD-I antidotes for hydrogen sulfide in mice. As shown in the survival curve of the mice in Fig. 6 A, the intraperitoneal injection of NaSH (21 mg/kg) into the mice caused significant lethal toxicity. When 7 mM met-hemoCD-I aqueous solution (0.2 mL) was intraperitoneally injected prior to NaSH injection, the survival curve significantly improved, indicating the efficacy of NaSH detoxification. The antidote effect was also significant when met-hemoCD-I was injected immediately after the mice were poisoned with NaSH (Fig. 6 B). Therefore, met-hemoCD-I is effective before and after poisoning with NaSH. The lactate level in the blood was increased by NaSH but returned to normal by met-hemoCD-I (Fig. 7 A). Therefore, anaerobic metabolism caused by NaSH was recovered in mice by the injection of met-hemoCD-I. More directly, we investigated the activity of C c O in organs (Fig. 7 B–D). In brain and heart tissues, the NaSH-induced decrease in C c O activity returned to normal in the met-hemoCD-I-treated mice, whereas no significant change was observed in the liver. Overall, these analyses support that met-hemoCD-I injection protects against hydrogen sulfide-induced asphyxial death in mice. After met-hemoCD-I was injected, dark red urine was obtained from the mice within 60 min. The UV‒vis spectrum of the urine showed the characteristic Soret and Q bands of met-hemoCD-I (Fig. 8 ). The spectral simulation revealed that the urine contained 80% met-hemoCD-I and 20% CO-hemoCD-I. Ferrous hemoCD-I could be formed by a natural reduction system in the body 33 and/or the homolytic cleavage of the HS–Fe(III) bond in hemoCD-I formed during circulation. Ferrous hemoCD-I bound to endogenous CO in the circulation and was subsequently excreted. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopic analysis of urine revealed no changes in the Im3CD structure (Fig. 8 inset). The inclusion complex of met-hemoCD-I was dissociated upon laser irradiation according to MALDI-TOF mass spectrometry. These results indicate that injected met-hemoCD-I in mice was excreted in the urine without chemical changes. Injected met-hemoCD-I could react with hydrogen sulfide and metabolize the compound to sulfite and/or sulfate during circulation, then return to the met-form via the mechanism proposed in Fig. 4 C. Discussion In this work, we investigated the use of met-hemoCD-P and met-hemoCD-I as hydrogen sulfide receptors in aqueous solution and in vivo . Compared to native Hb and Mb, met-hemoCD-I showed a greater binding rate and affinity toward hydrogen sulfide; therefore, we concluded that met-hemoCD-I is an effective and ready-to-use antidote for hydrogen sulfide-induced poisoning. Here, we discuss the binding of hydrogen sulfide to our heme-model system and compare it with that of other potential antidotes. The binding of hydrogen sulfide to metal ions in proteins, including heme proteins, has been proposed in many studies. 10,11 However, compared to that of O 2 , CO, and cyanide, detailed characterizations of HS–metal complexes with thermodynamic and kinetic parameters have been rarely reported. Among the native systems, the interactions between ferric Hb and Mb with hydrogen sulfide have been characterized in detail. 31,32 As reported in these studies, the binding parameters strongly depend on the pH of the solutions. The binding is relatively fast at low pH, and under these conditions, hydrogen sulfide presents as H 2 S. Interestingly, the pH dependency is reversed for a small heme protein, MP-11, 34 in which the heme cofactor is exposed to the aqueous bulk phase. These data indicate that the preferable attacking species, H 2 S or SH – , should differ depending on the environment around the iron center of the heme. 35 In the hemoCD system, the binding parameters were also dependent on pH, similar to those of Hb and Mb, indicating that hydrophobic H 2 S tends to enter the iron(III) site located in the CD cavity. In contrast, we confirmed that a stable 1:1 complex of iron(III)porphyrin formed with SH – . The net charge of the iron(III)porphyrin ring becomes zero upon SH – ligation. The electronically neutralized porphyrin should be stable in the hydrophobic cavity provided by the CD dimer; thus, this force drives anion binding to iron(III)porphyrin–methylated CD complexes in water. 36 Hydrophobic H 2 S is easily accessible to the iron(III) center of met-hemoCD, followed by deprotonation to SH – to neutralize the net charge of iron(III)porphyrin. The pH-dependent binding character is explained by the state of hydrogen sulfide (H 2 S or SH – ) and the axial ligand species on the iron(III) center before binding. Met-hemoCD-I has an exchangeable aqua ligand at neutral pH due to its p K a H2O (7.7), whereas met-hemoCD-P (p K a H2O = 5.5) has a strongly coordinated hydroxo ligand on iron(III) at neutral pH. Therefore, met-hemoCD-I, which exhibits a higher p K a H2O than that of met-hemoCD-P, bound more quickly to hydrogen sulfide under physiological conditions; thus, met-hemoCD-I could be superior to met-hemoCD-P as a hydrogen sulfide receptor in vivo . The interaction between hydrogen sulfide and synthetic iron(III) porphyrins has been investigated through biomimetic chemistry. In the system that involves a picket-fence-type Fe/Cu binuclear porphyrin complex, electrocatalytic O 2 reduction on the gold electrode was inhibited at a high concentration of H 2 S in a reversible manner. 37 This result suggests the toxic mechanism by which H 2 S inhibits the native mitochondrial C c O system. Another study using picket-fence porphyrins in nonaqueous media revealed that stable HS–Fe(III)porphyrin complexes formed via a 1:1 reaction with the SH – anion. 38 A synthetic heme-peptide conjugate model was also synthesized as an MP-11 model and formed a 6-coordinated HS–Fe(III) low-spin species in water. 39 Atmospheric oxygen causes the porphyrins to significantly decompose, which is common in these model studies; thus, these HS–Fe(III)porphyrin complexes have been characterized under anaerobic conditions. Due to the difficulty in preparing HS–heme species, a stable synthetic model with the R 3 Si–S–Fe(III) complex has been proposed for detailed structural characterization. 40 To our knowledge, except for our previous study using the hemoCD system, 30 there are no synthetic models for the binding between hydrogen sulfide and iron-porphyrins under aerobic conditions in water. Importantly, in our model, once hydrogen sulfide bound to the met-hemoCDs, the porphyrin ring was significantly protected against oxidative degradation induced by SH – owing to the CD cavity and axial fifth coordination. Due to its high stability, the bound SH – anion could be efficiently converted to sulfite and sulfate ions via oxidation by atmospheric O 2 . This oxidative conversion from hydrogen sulfide to sulfite and sulfate ions has been similarly reported in the native Hb and Mb systems, 29,31 in which the heme cofactors are also protected in the hydrophobic heme pockets provided by apo-proteins. Therefore, due to the protection of the porphyrin ring by the CD dimer, we efficiently detoxified hydrogen sulfide under physiological conditions. Furthermore, in contrast to Mb and Hb, injected met-hemoCD is easily excreted in the urine due to its small molecular weight, which is another advantage of the present antidote system. Currently, no antidote is available to clinically treat H 2 S poisoning, but several approaches have been proposed to detoxify hydrogen sulfide in animals and clinical trials. As the toxic mechanism is almost identical to that of hydrogen cyanide, vitamin B 12 analogs (hydroxocobalamin and cobinamide), which are used as cyanide antidotes, are effective treatments for hydrogen sulfide poisoning. 4,17,41,42 Intravenously injected vitamin B 12 analogs can capture hydrogen sulfide during circulation, after which it is strongly captured by serum proteins and accumulates for a long period (over a month). 24,43 Therefore, patients that receive high doses of vitamin B 12 analogs may need to avoid strong light exposure due to the photosensitizing property of cobalamins. Oxidized Hb (met-Hb) could function as an antidote for hydrogen sulfide. Amyl nitrate (NaNO 2 ), which is an oxidizing agent that produces met-Hb in circulating RBCs, has been shown to exhibit an antidote effect on hydrogen sulfide. 44 However, this method cannot be easily adjusted to the met-Hb ratio (%) in RBCs. In another recent trial using met-Hb, a met-Hb-albumin cluster was injected and markedly improved the survival rate of mice. 18 Compared to these potential candidates, the hemoCD system is advantageous because ( 1 ) met-hemoCD-I shows greater binding affinity toward hydrogen sulfide than that of met-Hb and met-Mb, and ( 2 ) the hemoCD compound injected is quantitatively excreted in urine within several hours. Therefore, met-hemoCD-I can effectively capture H 2 S, and after detoxification, injected met-hemoCD-I easily disappears from the body through renal clearance. In a previous study, ferrous hemoCD-P injected into mice or rats was quantitatively detected in the CO-bound form in urine. 21,23,25 However, for hydrogen sulfide, HS-bound met-hemoCD-I was not detected in the urine. Instead, met-hemoCD-I was mainly detected without change because the bound hydrogen sulfide on met-hemoCD-I in the body could be smoothly converted to sulfite or sulfate ions via catalytic oxidation with O 2 , as demonstrated in this study. We recently reported an antidote system for CO and cyanide mixed intoxication using hemoCD-Twins, a mixture of hemoCD-P and hemoCD-I in the ferrous state. 25 It is well known that CO and HCN are generated as lethal toxic gases during building fires. Previous studies in mice demonstrated that CO and HCN exhibit a synergetic lethal effect, which was counteracted by hemoCD-Twins. Interestingly, when hemoCD-Twins was injected into mice, hemoCD-P remained ferrous and could capture CO, while hemoCD-I was oxidized during circulation, and met-hemoCD-I detoxified cyanide. In general, CO poisoning can be quickly diagnosed by pulse CO oximetry or blood gas analysis. On the other hand, HCN in blood is difficult to detect rapidly at the site of accidents. The dual antidote system with hemoCD-Twins is advantageous because it can be injected without determination of which gas the patient primarily inhaled. Additionally, in this study, met-hemoCD-I clearly detoxified hydrogen sulfide. Although hydrogen sulfide is rarely produced in fire accidents, hemoCD-Twins are the first choice for patients that are potentially poisoned by an unknown gas. As the injected hemoCD complex is smoothly excreted in urine, it can be employed without a risk of side effects due to accumulated compounds. We are attempting to apply this detoxification system using hemoCD-Twins in clinical practice. In conclusion, synthetic heme model compounds composed of iron(III)porphyrin and a per- O -methylated cyclodextrin dimer were found to bind to hydrogen sulfide and form a stable HS–Fe(III) complex. Furthermore, met-hemoCD-I catalytically and efficiently decomposed hydrogen sulfide into nontoxic sulfite/sulfate ions under physiological conditions. Mouse animal experiments revealed that met-hemoCD-I exhibits excellent properties as a novel antidote for hydrogen sulfide poisoning. We expect that this hemoCD-based system will serve as a ready-to-use, multifunctional gas poisoning antidote that can simultaneously remove CO, cyanide, and hydrogen sulfide via a single injection. Materials and Methods Materials. Met-HemoCD-P and met-hemoCD-I were synthesized in our laboratory as described in previous reports. 25 We used sodium hydrogen sulfide (NaSH) as a hydrogen sulfide source. NaSH was purchased from Stream Chem. Inc. Before the use of NaSH, its purity was determined by iodometric titration. 30 All other reagents were purchased and used as received. Preparation of the NaSH solution. Since NaSH is easily oxidized in water, we prepared a stock solution of NaSH before each application. Milli-Q water was deoxidized by nitrogen gas bubbling for 30 min. Then, NaSH was solubilized in deoxidized water and used as a stock solution. Once a NaSH solution was prepared, it was used within 3 h. The concentration of NaSH in solution, C [mM], was determined as follows: $$C \left[\text{m}\text{M}\right]=\left\{\left(x \left[\text{m}\text{g}\right]\times y\left[\%\right]\times {10}^{3}\right)/\left(56.063\left[\text{g}\bullet {\text{m}\text{o}\text{l}}^{-1}\right]\times z\left[\text{m}\text{L}\right]\right)\right\}$$ where x is the weight of NaSH, y is the purity of NaSH determined by iodometric titration as described above, and z is the volume of the stock solution. Instruments. UV‒vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer with a thermostatic cell holder. Kinetic studies were carried out using a JASCO FS-110 Fast Scan Spectrometer with a thermostatic cell holder. EPR measurements were obtained with a Bruker E500 spectrometer at the Institute for Molecular Science in continuous-wave (CW) mode operating at ~ 9.66 GHz and equipped with an Oxford Instruments ESR900 continuous helium flow cryostat. The experimental parameters were 5 mW microwave power, 100 kHz field modulation, and 10 G modulation amplitude. MALDI-TOF mass spectra were measured on Bruker Daltonics Autoflex speed spectrometers in linear mode. a-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix. ESI-TOF mass spectra were taken on a JEOL JMS-T100CS spectrometer. Spectroscopic measurements. The binding constants ( K ) of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I were determined by UV‒vis photometric titration. NaSH (up to 50 mM) was added to an aerobic solution of met-hemoCD-I or met-hemoCD-P (5 µM) at 25°C in 0.05 M phosphate buffer at pH 6.0, 7.0, 7.4 and 8.0. The absorbance changes versus [NaSH] were plotted, and the data were fitted to a theoretical curve of an equation for 1:1 complex formation to obtain K . The association rate constants ( k on ) for the binding of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I were obtained under pseudo-first-order conditions with excess NaSH at 25°C. An aerobic solution of met-hemoCD-P or met-hemoCD-I (5 µM) was mixed rapidly with various concentrations of NaSH (100–1400 µM before mixing) in 0.05 M phosphate buffer at pH 7.0, 7.4 or 8.0. The change in absorbance at 410 nm was monitored, and the data were fitted to a single or double exponential function to obtain the observed rates k obs . When the reaction between met-hemoCD and hydrogen sulfide was too fast ( k obs > 0.5), a double exponential fitting was required due to an unknown slow reaction component. In that case, the component of k fast occupied more than 80% of the total. In addition, that ratio was independent of pH. Therefore, we adopted k fast as the actual k obs . (reaction between met-hemoCDs and hydrogen sulfide), while k slow is possibly due to the reaction between met-hemoCDs and impurities derived from NaSH, such as polysulfides. 45 Finally, the k on values were obtained from linear regression of k obs as a function of the hydrogen sulfide concentration. Quantification of hydrogen sulfide. An aqueous solution of NaSH (1.0 mM) was mixed with met-hemoCD or met-Hb (50 µM) and incubated at 25°C. The residual hydrogen sulfide concentrations were determined using the reported “methylene blue” method with some modifications. 46,47 The solution containing hydrogen sulfide and its scavenger was diluted to 2.5 mL with 0.05 M phosphate buffer at pH 7.4 to adjust the total hydrogen sulfide concentration to less than 50 µM. To the solution was added trifluoroacetic acid (0.5 mL). Then, solutions of N,N -dimethyl- p -phenylenediamine sulfate (200 mM stock solution, 30 µL) dissolved in HClaq (7.2 mM) and FeCl 3 (300 mM stock solution, 30 µL) dissolved in 1.2 mM HCl were added successively. The solutions were allowed to stand for 20 min at ambient room temperature. Finally, the absorbance at 663 nm was read, and the hydrogen sulfide concentration was determined based on the standard curve of methylene blue. Quantification of sulfate and sulfite. The relative production rate of sulfate and sulfite ions from hydrogen sulfide was determined by a simple turbidimetric method, in which the amount of sulfate (SO 4 2– ) was quantified by turbidity through the following reaction: 48 SO 4 2– + BaCl 2 → BaSO 4 ↓ + 2Cl – The average absorbance between 700 nm and 800 nm was used as turbidity, where met-hemoCD-I and other scavengers showed no absorbance in the area. The turbidity showed a linear correlation with the sulfate concentration from 0.5 mM to 4.0 mM upon the addition of 1.5 equivalents of BaCl 2 . We also confirmed that sulfite (SO 3 2– ) ions form a similar insoluble precipitate of BaSO 3 with BaCl 2 as follows: 49 SO 3 2– + BaCl 2 → BaSO 3 ↓ + 2Cl – Therefore, the relative production of sulfate and sulfite ions in solution was simply determined by reading the average absorbance between 700 nm and 800 nm after treatment of the solution with BaCl 2 . Animal experiments. All animal studies were performed under the approval of Doshisha University and carried out in accordance with the Guidelines for Animal Experiments of Doshisha University. We used female BALB/cCrSlc mice (Shimizu Laboratory Supplies, Co., Ltd.) weighing 20–22 g. The study is reported in accordance with ARRIVE guidelines. For acclimatization, the mice were housed under a 12 h/12 h light/dark cycle with free feedings under specific-pathogen-free (SPF) conditions for one week before the day in the experiments. For survival analysis of met-hemoCD-I in lethal hydrogen sulfide intoxication model mice, a solution of NaSH (21 mg/kg) in PBS (0.1 mL) was injected intraperitoneally. One minute after the injection of NaSH, a solution of met-hemoCD-I (7 mM) in PBS (0.2 mL) was injected intraperitoneally. For the predosing experiments, a solution of met-hemoCD-I (7 mM) in PBS (0.2 mL) was injected intraperitoneally. After 10 min, a solution of NaSH (21 mg/kg) in PBS (0.1 mL) was injected intraperitoneally. Survival rates were then monitored for one hour after hydrogen sulfide intoxication was induced. We determined the C c O activity and the concentration of lactate in the blood of lethal hydrogen sulfide intoxication model mice. Tissue samples (brain, heart, and liver) were collected from met-hemoCD-I-treated surviving mice one hour after hydrogen sulfide intoxication was induced. As a control, we collected tissue samples (brain, heart, and liver) from untreated mice immediately after death. The CcO activity in these organs was determined by the following method according to the literature. 50 Each tissue sample (~ 20 mg) was homogenized in 0.5 mL of sucrose muscle homogenization buffer (250 mM). Then, the suspension was centrifuged at 600 g for 10 min at 4°C. The supernatant (5 µL) was added to a 1 mL cuvette which contains 400 µL of Milli-Q water, 500 µL of potassium phosphate buffer (0.1 M, pH 7.0) and 50 µL of reduced cytochrome c (Product name; ab109911, abcam, UK). The absorbance at 550 nm was read for 3 min. The rate of activity (OD/min) was determined by calculating the slope between two points within the linear region. Finally, the CcO activity was determined by normalizing the rate activity by its protein amount in tissue using BCA assay (Thermo Fisher Scientific, Japan). The concentration of lactate in the blood was measured using LT-1730 Lactate Pro2 (Arkray). Statistical analysis. Statistical analyses were performed using GraphPad Prism, version 10.2.3 (GraphPad Software). All the data are presented as the means ± standard errors from at least three different experiments and were analyzed by Student's t test. Survival curves were analyzed using Kaplan‒Meier curves and the log-rank test. Differences with P values less than 0.05 were considered significant. Declarations Data availability. All relevant data are in the manuscript. Author contributions H.K. conceived the study. A.N. and Q.M. performed the main experiments and analyzed the data. M.H. performed the electron paramagnetic resonance spectroscopy experiments. M.K. provided useful suggestions for advancing this research. A.N. and H.K. wrote the manuscript. All authors participated in the revision of the manuscript. Funding This work was funded by JSPS KAKENHI (22H02097 and 24K01640), AMED (23ym0126814 and 24ym0126808j), and JST (JPMJSF2305, JPMJSP2129). Competing interests The authors declare no competing interests. Additional Information Supplementary Information The online version contains supplementary material available at https://doi.org/ Correspondence and requests for materials should be addressed to H.K. References Maldonado, C. S., Weir, A. & Rumbeiha, W. K. A comprehensive review of treatments for hydrogen sulfide poisoning: past, present, and future. Toxicol. Mech. Methods 33 , 183–196 (2023). Cuevasanta, E., Möller, M. N. & Alvarez, B. Biological chemistry of hydrogen sulfide and persulfides. Arch. Biochem. Biophys. 617 , 9–25 (2017). Olson, K. R. & Straub, K. D. The role of hydrogen sulfide in evolution and the evolution of hydrogen sulfide in metabolism and signaling. Physiology 31 , 60–72 (2016). Jiang, J., Chan, A., Ali, S. et al. Hydrogen sulfide–mechanisms of toxicity and development of an antidote. Sci. Rep. 6 , 20831 (2016). Blackstone, E., Morrison, M. & Roth, M. B. H 2 S induces a suspended animation-like state in mice. Science 22 , 518 (2005). Morii, D., Miyagatani, Y., Nakamae, N., Murao, M. & Taniyama, K. Japanese experience of hydrogen sulfide: the suicide craze in 2008. J. Occup. Med. Toxicol. 5 , 28 (2010). Iseki, K., Ozawa, A., Seino K., Goto, K. & Tase, C. The suicide pandemic of hydrogen sulfide poisoning in Japan. Asia Pac. J. Med. Toxicol. 3 , 13–17 (2013). Reedy, S. J. D., Schwartz, M. D. & Morgan, B. W. Suicide fads: Frequency and characteristics of hydrogen sulfide suicides in United States. West. J. Emerg. Med. 12, 300–304 (2011). Adkins, J. Hydrogen sulfide suicide: Latest technique hazardous to first responders and the public. Special Research Report in Regional Organized Crime Information Center. Available at https://npstc.org/documents/H2S%20Report%20for%204112.pdf Pietri, R., Román-Morales, E. & López-Garriga, J. Hydrogen sulfide and hemeproteins: Knowledge and mysteries. Antioxid. Redox Signal. 15 , 393–404 (2011). Domán, A., Dóka, É., Garai, D. et al. Interactions of reactive sulfur species with metalloproteins. Redox Biol. 60 , 102617 (2023). Parker, A. L. & Johnstone, T. C. Carbon monoxide poisoning: A problem uniquely suited to a medical inorganic chemistry solution. J. Inorg. Biochem. 251 , 112453 (2024). Azarov, I., Wang, L., Rose, J. J. et al. Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning. Sci. Transl. Med. 8 , 368ra173 (2016). Rose, J. J., Bocian, K. A., Xu, Q. et al. A neuroglobin-based high-affinity ligand trap reverses carbon monoxide–induced mitochondrial poisoning. J. Biol. Chem. 295 , 6357–6371 (2020). Xu, Q., Rose, J. J., Chen, X. et al. Cell-free and alkylated hemoproteins improve survival in mouse models of carbon monoxide poisoning. JCI Insight 7 , e153296 (2022). Chan, A., Jiang, J., Fridman, A. et al. Nitrocobinamide, a new cyanide antidote that can be administered by intramuscular injection. J. Med. Chem. 58 , 1750–1759 (2015). Fujita, Y., Fujino, Y., Onodera, M., Kikuchi, S., Kikkawa, T., Inoue, Y., Niitsu, H., Takahashi, K. & Endo, S. A fatal case of acute hydrogen sulfide poisoning caused by hydrogen sulfide: Hydroxocobalamin therapy for acute hydrogen sulfide poisoning. J. Anal. Toxicol. 35 , 119–123 (2011). Suzuki, Y., Taguchi, K., Okamoto, W., Enoki, Y. Komatsu, T. & Matsumoto, K. Methemoglobin-albumin clusters for the treatment of hydrogen sulfide intoxication. J. Control. Release 349 , 304–314 (2022). Droege, D. G. & Johnstone, T. C. A water-soluble iron-porphyrin complex capable of rescuing CO-poisoned red blood cells. Chem. Comm. 58 , 2722–2725 (2022). Kano, K., Kitagishi, H., Kodera, M. & Hirota, S. Dioxygen binding to a simple myoglobin model in aqueous solution. Angew. Chem. Int. Ed. 44 , 435–438 (2005). Kitagishi, H., Negi, S. Kiriyama, A., Honbo, A., Sugiura, Y., Kawaguchi, A. T. & Kano K. A diatomic molecule receptor that removes CO in a living organism. Angew. Chem. Int. Ed. 49 , 1312–1315 (2010). Kitagishi, H. & Kano, K. Synthetic heme protein models that function in aqueous solution. Chem. Commun . 57 , 148–173 (2021). Kitagishi, H., Minegishi, S., Yumura, A., Negi, S., Taketani, S., Amagase, Y., Mizukawa, Y., Urushidani, T., Sugiura, Y. & Kano, K., Feedback response to selective depletion of endogenous carbon monoxide in the blood. J. Am. Chem. Soc. 138 , 5417–5425 (2016). Watanabe, K., Kitagishi, H. & Kano, K. Supramolecular ferric porphyrins as cyanide receptors in aqueous solution. ACS Med. Chem. Lett. 2 , 943–947 (2011). Mao, Q., Zhao, X., Kiriyama, A., Negi, S., Fukuda, Y., Yoshioka, H., Kawaguchi, A. T., Motterlini, R., Foresti, R. & Kitagishi, H. A synthetic porphyrin as an effective dual antidote against carbon monoxide and cyanide poisoning. Proc. Natl. Acad. Sci. U.S.A. 120 , e2209924120 (2023). Li, Q. & Lancaster Jr., J. R. Chemical foundations of hydrogen sulfide biology. Nitric Oxide 35 , 21–34 (2013). Kano, K., Kitagishi, H., Mabuchi, T., Kodera, M. & Hirota, S. A myoglobin functional model composed of a ferrous porphyrin and a cyclodextrin dimer with an imidazole linker. Chem. Asian J. 1 , 358–366 (2006). Sono, M. & Dawson, J. H. Formation of low spin complexes of ferric cytochrome P-450-CAM with anionic ligands. J. Biol. Chem. 257 , 5469–5502 (1982). Vitvitsky, V., Yadav, P. K., Kurthen, A. & Banerjee, R. Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides. J. Biol. Chem. 290 , 8310–8320 (2015). Watanabe, K., Suzuki, T., Kitagishi, H. & Kano, K. Reaction between a haemoglobin model compound and hydrosulphide in aqueous solution. Chem. Commun. 51 , 4059–4061 (2015). Bostelaar, T., Vitvitsky, V., Kumutima, J. Hydrogen sulfide oxidation by myoglobin. J. Am. Chem. Soc. 138 , 8476–8488 (2016). Jensen, B. & Fago, A. Reactions of ferric hemoglobin and myoglobin with hydrogen sulfide under physiological conditions. J. Inorg. Biochem. 182 , 133–140 (2018). Noguchi, M., Mao, Q., Nakagami, A. & Kitagishi, H. Spontaneous reduction of iron(III)porphyrin to iron(II)porphyrin–CO complex in mouse circulation. Chem. Commun. 59 , 6211–6214 (2023). Boubeta, F. M., Bieza, S. A., Bringas, M., Estrin, D. A., Boechi, L. & Bari, S. E. Mechanism of sulfide binding by ferric hemeproteins. Inorg. Chem. 57 , 7591–7600 (2018). Boubeta, F. M., Bari, S. E., Estrin, D. A. & Boechi, L. Access and binding of H2S to hemeproteins: The case of Hbl of Lucina pectinata . J. Phys. Chem. B. 120 , 9642–9653 (2016). Kano, K., Kitagishi, H., Tamura, S. & Yamada, A. Anion binding to a ferric porphyrin complexed with per- O -methylated b-cyclodextrin in aqueous solution. J. Am. Chem. Soc. 126 , 15202–15210 (2004). Collman, J. P., Ghosh, S., Dey, A. & Decréau, R. A. Using a functional enzyme model to understand the chemistry behind hydrogen sulfide induced hibernation. Proc. Natl. Acad. Sci. U.S.A. 106 , 22090–22095 (2009). Hartle, M. D., Prell, J. S. & Pluth, M. D. Spectroscopic investigations into the binding of hydrogen sulfide to synthetic picket-fence porphyrins. Dalton Trans. 45 , 4843–4853 (2016). Zhao, Z., Wang, D., Wang, M., Sun, X., Wang, L., Huang, X., Ma, L. & Li, Z. Proximal environment controlling the reactivity between inorganic sulfide and heme-peptide model. RSC Adv. 6 , 78858–78864 (2016). Meininger, D. J., Caranto, J. D., Arman, H. D. & Tonzetich, Z. J. Studies of iron(III) porphyrins containing silanethiolate ligands. Inorg. Chem. 52 , 12468–12476 (2013). Ng, P. C., Hendry-Hofer, T., N. et al. Intramuscular cobinamide versus saline for treatment of severe hydrogen sulfide toxicity in swine. Clin. Toxicol. (Phila). 57 , 189–196 (2019). Haouzi, P., Chenuel, B. & Sonobe, T. High dose hydroxocobalamin administered after H 2 S exposure counteracts sulfide poisoning induced cardiac depression in sheep. Clin. Toxicol. (Phila). 53 , 28–36 (2015). Marques, H. M., Brown, K. L. & Jacobsen, D. W. Kinetics and activation parameters of the reaction of cyanide with free aquocobalamin and aquocobalamin bound to a haptocorrin from chicken serum. J. Biol. Chem. 263 , 12378−12383 (1988). Hoidal, C. R., Hall, A. H., Robinson, M. D., Kulig, K. & Rumack, B. H. Hydrogen sulfide poisoning from toxic inhalations of roofing asphalt fumes. Ann. Emerg. Med. 15 , 826–830 (1988). Greiner, R., Pálinkás, Z., Bäsell, K., Becher, D., Antelmann, H., Nagy, P. & Dick, T. P. Polysulfides link H 2 S to protein thiol oxidation. Antioxid. Redox Signal. 19 , 1749–1765 (2013). Lawrence, N. S., Davis, J. & Compton, R. G. Analytical strategies for the detection of sulfide: a review. Talanta 52 , 771-784 (2000). Morikawa, T., Kajimura, M., Nakamura, T., et al. Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway. Proc. Natl. Acad. Sci. U.S.A. 109 , 1293–1298 (2012). Coleman, R. L., Shults, W. D., Kelly, M. T. & Dean, J. A. Turbidimetry via parallel photometric analysis. Determination of sulfate. Anal. Chem. . 44 , 1031–1034 (1972). Müller, I. A., Brunner, B., Breuer, C., Coleman, M. & Bach, W. The oxygen isotope equilibrium fractionation between sulfite species and water. Geochim. Cosmochim. Acta 120 , 562–581 (2013). Spinazzi, M., Casarin, A., Pertegato, V., Salviati, L. & Angelini, C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat. Protoc. 7 , 1235-1246 (2012). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Sep, 2024 Reviews received at journal 13 Sep, 2024 Reviewers agreed at journal 28 Aug, 2024 Reviews received at journal 11 Jul, 2024 Reviewers agreed at journal 03 Jul, 2024 Reviewers invited by journal 02 Jul, 2024 Editor assigned by journal 29 Jun, 2024 Editor invited by journal 23 Jun, 2024 Submission checks completed at journal 20 Jun, 2024 First submitted to journal 17 Jun, 2024 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-4591678","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":323861430,"identity":"c5b77303-2d48-4ace-b551-98d617f882aa","order_by":0,"name":"Atsuki Nakagami","email":"","orcid":"","institution":"Doshisha University","correspondingAuthor":false,"prefix":"","firstName":"Atsuki","middleName":"","lastName":"Nakagami","suffix":""},{"id":323861432,"identity":"5b9fbdd2-f6c2-4f7d-80dc-044d3165438a","order_by":1,"name":"Qiyue Mao","email":"","orcid":"","institution":"Doshisha University","correspondingAuthor":false,"prefix":"","firstName":"Qiyue","middleName":"","lastName":"Mao","suffix":""},{"id":323861433,"identity":"bcf7b64d-8365-4b3b-9cac-7e2625e5ee46","order_by":2,"name":"Masaki Horitani","email":"","orcid":"","institution":"Saga University","correspondingAuthor":false,"prefix":"","firstName":"Masaki","middleName":"","lastName":"Horitani","suffix":""},{"id":323861434,"identity":"18d2fa88-e451-43a7-93f2-5739a10cab56","order_by":3,"name":"Masahito Kodera","email":"","orcid":"","institution":"Doshisha University","correspondingAuthor":false,"prefix":"","firstName":"Masahito","middleName":"","lastName":"Kodera","suffix":""},{"id":323861435,"identity":"374e0822-abe8-4a15-9655-b8489dcfb880","order_by":4,"name":"Hiroaki Kitagishi","email":"data:image/png;base64,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","orcid":"","institution":"Doshisha University","correspondingAuthor":true,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Kitagishi","suffix":""}],"badges":[],"createdAt":"2024-06-17 04:14:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4591678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4591678/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-80511-1","type":"published","date":"2024-12-10T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59999939,"identity":"0fe9cdae-8511-410c-83aa-7accc7f9879b","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":478082,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of cyclodextrin dimers Py3CD and Im3CD and met-hemoCD-P and met-hemoCD-I in combination with iron(III)porphyrin (Fe(III)TPPS) in aqueous saline solution.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/ef33ec4d297ea322dba33740.png"},{"id":60000304,"identity":"8c13a70e-d6ae-41f1-a1c9-814ca983bab4","added_by":"auto","created_at":"2024-07-10 10:33:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":460043,"visible":true,"origin":"","legend":"\u003cp\u003eSpectroscopic analysis of the binding of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) Changes in the UV‒vis absorption spectra of met-hemoCD-P (5 mM) and met-hemoCD-I (5 mM) in 0.05 M phosphate buffer solution at pH 7.4 and 25°C upon the addition of NaSH. The insets show plots of the absorbance changes versus [NaSH]. The solid lines are the best fit of the data to an equation for 1:1 complex formation to give the binding constants (\u003cem\u003eK\u003c/em\u003e). (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) UV‒vis absorption spectra collected over time in the reaction of met-hemoCD-P (5 mM) and met-hemoCD-I (5 mM) with excess NaSH in 0.05 M phosphate buffer solution at pH 7.4 and 25°C. The insets show traces at 410 nm (black) and the fitted curve (red) according to a single/double exponential function to derive the observed rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs.\u003c/sub\u003e).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/0199dae892434603f13cb482.png"},{"id":59999947,"identity":"b0a4c4ad-9715-4b9b-8923-19db55de1cd6","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":258935,"visible":true,"origin":"","legend":"\u003cp\u003eKinetic analysis of the binding between hydrogen sulfide and met-hemoCDs. (\u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e) Plots of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e for met-hemoCD-P and met-hemoCD-I as a function of [NaSH]. The solid lines represent linear least-square fitting of the data, which provided the \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e values. (\u003cstrong\u003eC\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e) Plots of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e for met-hemoCD-P and met-hemoCD-I as a function of pH.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/baf562a0c5895428e7a55a54.png"},{"id":60000303,"identity":"03128f22-fc2f-45df-b98f-2ec23c83d8d9","added_by":"auto","created_at":"2024-07-10 10:33:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":414750,"visible":true,"origin":"","legend":"\u003cp\u003eReaction of met-hemoCD-I with hydrogen sulfide in 0.05 M phosphate buffer solution at pH 7.4. (\u003cstrong\u003eA\u003c/strong\u003e) Changes in the UV‒vis absorption spectra of met-hemoCD-I (5 mM) after the addition of excess NaSH under aerobic and anaerobic conditions at 25°C. (\u003cstrong\u003eB\u003c/strong\u003e) UV‒vis absorption spectra of met-hemoCD-I (5 mM) before and after two molar equivalents of NaSH were added at 25°C. The solution was then bubbled with CO gas to confirm that CO-ferrous complexes formed. (\u003cstrong\u003eC\u003c/strong\u003e) Proposed pathway for the catalytic decomposition of hydrogen sulfide with met-hemoCD-I. (\u003cstrong\u003eD\u003c/strong\u003e) EPR spectral changes of met-hemoCD-I before and after NaSH (two equivalents) was added. The reagents were mixed and incubated at 25°C, and the spectra were measured in the frozen state at 5 K.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/eeea5d9259ee089bc6b8ce36.png"},{"id":59999940,"identity":"ace41ebd-c1bd-4db3-b3bf-6f24fd2fb38c","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53467,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation of hydrogen sulfide in 0.05 M phosphate buffer solution at pH 7.4. (\u003cstrong\u003eA\u003c/strong\u003e) Time course changes in the residual amount of hydrogen sulfide (initial: 1.0 mM) in solutions containing met-hemoCD-I, met-hemoCD-P, and met-Hb (50 mM each). The residual sulfide was detected by the methylene blue method (see Materials and Methods section). (\u003cstrong\u003eB\u003c/strong\u003e) Production of sulfite and sulfate ions during the reaction between hydrogen sulfide and met-hemoCD-I. The aqueous solution of hydrogen sulfide (9 mM) was mixed with met-hemoCD-I (0.45 mM) and incubated for 1 h. The sulfite and sulfate ions were detected by the addition of barium chloride and quantified by the solution turbidity (see Experimental section). Each bar represents the mean ± SE (\u003cem\u003en\u003c/em\u003e = 3). The asterisks denote statistical significance, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005. **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/c52546ae34589bf3656343ea.png"},{"id":59999943,"identity":"482b161e-4c7f-4318-b035-aac10a2c213b","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41212,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival curves for NaSH-treated mice treated with or without met-hemoCD-I. (\u003cstrong\u003eA\u003c/strong\u003e) Predosing. A solution of met-hemoCD-I (7 mM, 0.2 mL) in PBS was intraperitoneally injected into the mice, followed by an intraperitoneal injection of NaSH (21 mg/kg). (\u003cstrong\u003eB\u003c/strong\u003e) Postdosing. A solution of NaSH (21 mg/kg) in PBS was intraperitoneally injected into the mice, followed by an intraperitoneal injection of met-hemoCD-I (7 mM, 0.2 mL). The asterisks denote statistical significance, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/09eb56407ca92c6908120b9a.png"},{"id":59999944,"identity":"e6ca0744-def6-4ee4-8540-06641ddde4f2","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":120590,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of met-hemoCD-I antidotes on mice poisoned by NaSH. (\u003cstrong\u003eA\u003c/strong\u003e) Lactate levels in the blood of mice treated with NaSH followed by met-hemoCD-I. (\u003cstrong\u003eB\u003c/strong\u003e–\u003cstrong\u003eD\u003c/strong\u003e) Cytochrome \u003cem\u003ec\u003c/em\u003e oxidase (C\u003cem\u003ec\u003c/em\u003eO) activity in the brain (\u003cstrong\u003eB\u003c/strong\u003e), heart (\u003cstrong\u003eC\u003c/strong\u003e), and liver (\u003cstrong\u003eD\u003c/strong\u003e) of mice treated with NaSH followed by met-hemoCD-I. In the model, a solution of NaSH (21 mg/kg) in PBS was intraperitoneally injected into mice, followed by an intraperitoneal injection of met-hemoCD-I (7 mM, 0.2 mL). Each bar represents the mean ± SE (\u003cem\u003en\u003c/em\u003e ≧ 3). The asterisks denote statistical significance, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005. **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/1ad2c8b3c827fe0afaf8229e.png"},{"id":59999945,"identity":"4a9603d8-8d6b-460f-85ef-eda363af99f3","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":44427,"visible":true,"origin":"","legend":"\u003cp\u003eUV‒vis absorption spectrum of urine (black) excreted from mice treated with NaSH (21 mg/kg) followed by met-hemoCD-I (7 mM, 0.2 mL). The spectra of met-hemoCD-I (green) and CO-hemoCD-I (orange) are shown for comparison. The red dotted line shows the accumulated spectra of met-hemoCD-I (80%) and CO-hemoCD-I (20%), which were consistent with those of urine. The inset shows MALDI-TOF mass spectra of Im3CD and urine with subsequent addition of a-cyano-4-hydroxycinamic acid (positive mode). The calculated molecular weight (M) of Im3CD is 2961.4.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/04258f18440c7d01270701e0.png"},{"id":71552507,"identity":"fa6f8cd6-2ff9-472b-9969-429dd4bb54f6","added_by":"auto","created_at":"2024-12-16 16:06:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2381989,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/c7529f91-a5e5-46f1-a44d-a2c7b55ea445.pdf"},{"id":59999941,"identity":"34f1920e-9e20-4630-97bd-4cb9105f6810","added_by":"auto","created_at":"2024-07-10 10:25:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":784156,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4591678/v1/70dea53462732875a4451bea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detoxification of hydrogen sulfide by synthetic heme-model compounds","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS) is a colorless, flammable, and hazardous gas with a rotten egg smell. The toxic effect of H\u003csub\u003e2\u003c/sub\u003eS is similar to that of hydrogen cyanide (HCN), which strongly binds to cytochrome \u003cem\u003ec\u003c/em\u003e oxidase (C\u003cem\u003ec\u003c/em\u003eO) in the mitochondrial respiratory chain; thus, H\u003csub\u003e2\u003c/sub\u003eS is classified as a cellular asphyxiant.\u003csup\u003e1\u0026ndash;5\u003c/sup\u003e As its specific gravity (1.19) is greater than that of air, H\u003csub\u003e2\u003c/sub\u003eS tends to accumulate at lower altitudes, often causing poisoning accidents at sites such as in manholes, sewage systems, and mining operations.\u003csup\u003e1,6\u003c/sup\u003e Although there are fewer than 10 cases of industrial H\u003csub\u003e2\u003c/sub\u003eS poisoning per year in Japan, 220 cases with 208 deaths were reported in 2007 in Japan due to suicide by intentional H\u003csub\u003e2\u003c/sub\u003eS generation, which is known as the detergent suicide pandemic.\u003csup\u003e6,7\u003c/sup\u003e The following year, this method of suicide was observed in the United States and worldwide.\u003csup\u003e8,9\u003c/sup\u003e Frighteningly, residual H\u003csub\u003e2\u003c/sub\u003eS gas often spreads from the source or victim and causes secondary poisoning to the rescue personnel and/or individuals at the site.\u003csup\u003e8,9\u003c/sup\u003e No clinical antidote is currently available for H\u003csub\u003e2\u003c/sub\u003eS poisoning. Therefore, ready-to-use antidotes that can be stored for long durations and are immediately effective are greatly needed, especially for situations involving emergency rescue.\u003c/p\u003e \u003cp\u003eThe administration of heme proteins or artificial heme-model compounds may be a promising approach for the development of antidotes against poisoning caused by inhaled gases such as H\u003csub\u003e2\u003c/sub\u003eS, which reacts with metalloproteins (including hemoglobin) in red blood cells (RBCs)\u003csup\u003e10,11\u003c/sup\u003e. For this purpose, the compounds should exhibit higher binding affinities toward toxins than native hemes. Relying on this strategy, researchers have proposed potential antidotes for carbon monoxide (CO), hydrogen cyanide (HCN), and H\u003csub\u003e2\u003c/sub\u003eS poisoning using native and modified heme proteins as well as natural vitamin B\u003csub\u003e12\u003c/sub\u003e analogs.\u003csup\u003e4,12\u0026ndash;18\u003c/sup\u003e However, few studies have established antidote systems using synthetic compounds.\u003csup\u003e12,19\u003c/sup\u003e Therefore, our group developed synthetic heme-model compounds composed of iron tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) complexes encapsulated by per-\u003cem\u003eO\u003c/em\u003e-methylated b-cyclodextrin (CD) dimers.\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows two representative CD dimers, Py3CD and Im3CD, that form inclusion complexes with Fe(III)TPPS to yield met-hemoCD-P and met-hemoCD-I, respectively. We have shown that reduced hemoCD-P in the ferrous state functions as an internal CO scavenger \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e21,23\u003c/sup\u003e and that met-hemoCD-I functions as a potential cyanide antidote.\u003csup\u003e24,25\u003c/sup\u003e When these heme model compounds were injected intravenously or intraperitoneally into mice or rats, they bound gaseous molecules in the circulation system.\u003csup\u003e21\u0026ndash;25\u003c/sup\u003e Interestingly, these compounds were rapidly and quantitatively excreted in the urine through renal clearance; thus, they do not accumulate in the body. Therefore, compared to native protein-based scavenging systems, our system shows potential as an injectable antidote.\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the potential of met-hemoCD-P and met-hemoCD-I as hydrogen sulfide scavengers. Using the ferric forms of these two complexes, we first present the basic reactivity toward hydrogen sulfide in aqueous solution in view of thermodynamic and kinetic parameters and spectroscopic characterizations. Then, the antidote effect against hydrogen sulfide-induced intoxication was tested in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cstrong\u003eBinding of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I\u003c/strong\u003e \u003cp\u003eIn this study, we used sodium hydrogen sulfide (NaSH) as the sulfide source. In aqueous media, SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e equilibrates with H\u003csub\u003e2\u003c/sub\u003eS, and the p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e is 7.\u003csup\u003e26\u003c/sup\u003e The presence of S\u003csup\u003e2\u0026ndash;\u003c/sup\u003e could be negligible under aqueous conditions due to the low acidity of SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e (p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e = 19).\u003csup\u003e26\u003c/sup\u003e In this article, we use the term \u0026ldquo;hydrogen sulfide\u0026rdquo; to refer to both H\u003csub\u003e2\u003c/sub\u003eS and SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e species. The binding constants (\u003cem\u003eK\u003c/em\u003e) of hydrogen sulfide to met-hemoCD-I and met-hemoCD-P were evaluated by UV‒vis spectroscopic titration. Upon the addition of NaSH, the spectra of met-hemoCD-P and met-hemoCD-I changed stepwise with clear isosbestic points (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B). The spectral changes were saturated at approximately one equivalent of added NaSH. In the absence of CD dimers, Fe(III)TPPS was decomposed by the addition of NaSH (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Similar porphyrin decomposition was also observed for Fe(III)TPPS complexed with 2,3,6-tri-\u003cem\u003eO\u003c/em\u003e-methyl-b-CD (TMe-b-CD), which lacks an axial fifth ligand (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). This result suggested that, in addition to protection by the CD cavity, pyridine or imidazole ligation in Py3CD or Im3CD contributed to the formation of a stable HS\u0026ndash;Fe(III)porphyrin complex.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe titration curves were well fitted to the 1:1 equilibrium model, affording \u003cem\u003eK\u003c/em\u003e values of 1.2 x 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and 2.5 x 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for met-hemoCD-P and met-hemoCD-I, respectively, in phosphate buffer at pH 7.4. The kinetic parameters for the binding of hydrogen sulfide were determined by time-resolved UV‒vis spectral measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Single-exponential curve fitting analysis was used to determine the apparent association rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e\u003csup\u003eapp\u003c/sup\u003e). The second-order rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e) were determined via linear regression of the \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e\u003csup\u003eapp\u003c/sup\u003e values as a function of [NaSH] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). The parameters were strongly dependent on the pH of the solution. As listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, met-hemoCD-I exhibits higher \u003cem\u003eK\u003c/em\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e values than those of met-hemoCD-P. The plot of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e and \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e values versus pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D) clearly shows the high ability of met-hemoCD-I to act as a hydrogen sulfide scavenger at physiological pH (7.4). Hydrogen sulfide binds more quickly to met-hemoCD-I at neutral pH because the p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e (p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003csup\u003eH2O\u003c/sup\u003e = 7.7) of the axial aqua ligand is higher than that of hemoCD-P (p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003csup\u003eH2O\u003c/sup\u003e = 5.5).\u003csup\u003e22,24\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding constants (\u003cem\u003eK\u003c/em\u003e) and rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e) of met-hemoCD-P and met-hemoCD-I with hydrogen sulfide in 0.05 M phosphate buffer at different pH values and at 25\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003emet-hemoCD-P\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003emet-hemoCD-I\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e\u0026ndash;6\u003c/sup\u003e\u003cem\u003eK\u003c/em\u003e (M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e\u0026ndash;3\u003c/sup\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e (M\u003csup\u003e\u0026ndash;1\u003c/sup\u003es\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e (s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003csup\u003e\u0026ndash;6\u003c/sup\u003e\u003cem\u003eK\u003c/em\u003e (M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003csup\u003e\u0026ndash;3\u003c/sup\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e (M\u003csup\u003e\u0026ndash;1\u003c/sup\u003es\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e (s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding constants (\u003cem\u003eK\u003c/em\u003e) and rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e) of met-hemoCD, met-Hb and met-Mb with hydrogen sulfide.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eK\u003c/em\u003e (M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e (M\u003csup\u003e\u0026ndash;1\u003c/sup\u003es\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ek\u003c/em\u003e\u003csub\u003eoff\u003c/sub\u003e (s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emet-hemoCD-P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.2 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0 x 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.4 x 10\u003csup\u003e\u0026ndash;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emet-hemoCD-I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.5 x 10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.9 x 10\u003csup\u003e\u0026ndash;4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emet-Mb (horse)\u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.6 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emet-Hb (human)\u003csup\u003e\u003cem\u003eb,c\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.8 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.9 x 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5 x 10\u003csup\u003e\u0026ndash;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emet-Mb (sperm whale)\u003csup\u003e\u003cem\u003eb\u003c/em\u003e,\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.6 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.8 x 10\u003csup\u003e\u0026ndash;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003ea\u003c/em\u003e \u003c/sup\u003eRef. 31. \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003eRef. 32. \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003eThe kinetic parameters were recalculated as a function of the hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS/SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e) concentration.\u003c/p\u003e \u003cp\u003eA continuous variation plot (Job plot) was constructed for the met-hemoCD-I and NaSH systems (Figure S2). The maximum complexation ratio observed at a 1:1 molar ratio clearly indicates that a 1:1 complex of met-hemoCD-I and NaSH was present; these results indicate that poly(sulfide) complexes such as HS(S)\u003csub\u003en\u003c/sub\u003e\u0026ndash;Fe(II), which are often proposed in biological systems, were not formed.\u003csup\u003e11\u003c/sup\u003e Additionally, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) revealed that the HS\u0026ndash;Fe(III) complex in met-hemoCD-I formed (Figure S3). The molecular ion peak of met-hemoCD-I (MW\u0026thinsp;=\u0026thinsp;3947.9) was detected at 1315.4 (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e) as a tri-anionic species. With NaSH, the molecular ion peak of met-hemoCD-I was mainly detected at 995.1 (\u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e), which could be assigned to HS-bound met-hemoCD-I (MW\u0026thinsp;=\u0026thinsp;3981.0) in the terta-anionic form. These results revealed that a stable 1:1 complex of met-hemoCD-I with hydrogen sulfide (HS\u0026ndash;Fe(III)porphyrin) formed in aqueous media at physiological pH.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOxidative degradation of hydrogen sulfide by met-hemoCD-I\u003c/strong\u003e \u003cp\u003eIn the presence of excess NaSH (100 equivalents), met-hemoCD-I gradually degraded under aerobic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The degradation was significantly suppressed under anaerobic conditions with a Soret band at 434 nm, indicating that a ferrous hemoCD-I complex formed in the deoxy form.\u003csup\u003e27\u003c/sup\u003e To confirm the iron oxidation state, carbon monoxide (CO) gas was introduced into the solution after met-hemoCD-I was mixed with NaSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A sharp Soret band appeared at 422 nm, which is characteristic of the CO\u0026ndash;Fe(II) complex. Therefore, homolytic bond cleavage of the HS\u0026ndash;Fe(III) complex occurred, generating a sulfide radical (HS\u0026bull;) and Fe(II) complex of hemoCD-I. In the presence of molecular oxygen (O\u003csub\u003e2\u003c/sub\u003e), the reduced Fe(II) complex formed the O\u003csub\u003e2\u003c/sub\u003e adduct, which was readily autoxidized to ferric met-hemoCD-I with the generation of superoxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The autoxidation rate of the O\u003csub\u003e2\u003c/sub\u003e adduct for hemoCD-I (\u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e ~ 36 min at 37\u0026deg;C) is much faster than that for ferrous hemoCD-P (\u003cem\u003et\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e ~ 5 h at 37\u0026deg;C).\u003csup\u003e25\u003c/sup\u003e Therefore, hydrogen sulfide should be efficiently converted to sulfite or sulfate ions in the presence of met-hemoCD-I under aerobic conditions.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the reaction of met-hemoCD-I with NaSH, electron paramagnetic resonance (EPR) spectra of met-hemoCD-I were obtained before and after the reaction of NaSH at physiological pH and 5 K (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Before the reaction with NaSH occurred, met-hemoCD-I showed EPR signals at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.03, 2.30, and 2.00. The signals at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.03 and 2.00 could be assigned to the characteristic signals of 5-coordinated high-spin iron(III)porphyrin with or without a weakly coordinated sixth ligand, such as H\u003csub\u003e2\u003c/sub\u003eO, while the signal at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.30 could be assigned to the iron(III)porphyrin coordinated with a hydroxoligand.\u003csup\u003e28\u003c/sup\u003e Immediately after NaSH was added, characteristic signals at \u003cem\u003eg\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.41, 2.20, and 1.92 were generated due to the HS\u0026ndash;Fe(III)\u0026ndash;N(imidazole) 6-coordinated low-spin complex.\u003csup\u003e29\u0026ndash;31\u003c/sup\u003e The EPR signals became almost silent when the solution was incubated for an hour and then frozen after NaSH was added, indicating that an EPR-inactive ferrous Fe(II) complex formed due to the homolysis of HS\u0026ndash;Fe(III). Ferric high-spin species of met-hemoCD-I were detected after 2 and 3 hours of incubation. The time-course change in the EPR spectra confirmed that met-hemoCD-I was converted to the ferrous Fe(II) complex via HS\u0026ndash;Fe(III) complex formation, as proposed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003eThe hydrogen sulfide species in aqueous solution decomposed rapidly in the presence of met-hemoCD-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The efficacy of hydrogen sulfide decomposition is correlated with binding parameters for met-hemoCD-I, met-hemoCD-P, and met-Hb, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Consistently, sulfite and sulfate ions were efficiently produced in the presence of met-hemoCD-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The control data under anaerobic conditions support the involvement of O\u003csub\u003e2\u003c/sub\u003e in met-hemoCD-I-assisted decomposition of hydrogen sulfide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStrength of met-hemoCD-I antidotes for hydrogen sulfide poisoning in mice\u003c/b\u003e: We then evaluated the strength of met-hemoCD-I antidotes for hydrogen sulfide in mice. As shown in the survival curve of the mice in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the intraperitoneal injection of NaSH (21 mg/kg) into the mice caused significant lethal toxicity. When 7 mM met-hemoCD-I aqueous solution (0.2 mL) was intraperitoneally injected prior to NaSH injection, the survival curve significantly improved, indicating the efficacy of NaSH detoxification. The antidote effect was also significant when met-hemoCD-I was injected immediately after the mice were poisoned with NaSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Therefore, met-hemoCD-I is effective before and after poisoning with NaSH.\u003c/p\u003e \u003cp\u003eThe lactate level in the blood was increased by NaSH but returned to normal by met-hemoCD-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Therefore, anaerobic metabolism caused by NaSH was recovered in mice by the injection of met-hemoCD-I. More directly, we investigated the activity of C\u003cem\u003ec\u003c/em\u003eO in organs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026ndash;D). In brain and heart tissues, the NaSH-induced decrease in C\u003cem\u003ec\u003c/em\u003eO activity returned to normal in the met-hemoCD-I-treated mice, whereas no significant change was observed in the liver. Overall, these analyses support that met-hemoCD-I injection protects against hydrogen sulfide-induced asphyxial death in mice.\u003c/p\u003e \u003cp\u003eAfter met-hemoCD-I was injected, dark red urine was obtained from the mice within 60 min. The UV‒vis spectrum of the urine showed the characteristic Soret and Q bands of met-hemoCD-I (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The spectral simulation revealed that the urine contained 80% met-hemoCD-I and 20% CO-hemoCD-I. Ferrous hemoCD-I could be formed by a natural reduction system in the body\u003csup\u003e33\u003c/sup\u003e and/or the homolytic cleavage of the HS\u0026ndash;Fe(III) bond in hemoCD-I formed during circulation. Ferrous hemoCD-I bound to endogenous CO in the circulation and was subsequently excreted. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopic analysis of urine revealed no changes in the Im3CD structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e inset). The inclusion complex of met-hemoCD-I was dissociated upon laser irradiation according to MALDI-TOF mass spectrometry. These results indicate that injected met-hemoCD-I in mice was excreted in the urine without chemical changes. Injected met-hemoCD-I could react with hydrogen sulfide and metabolize the compound to sulfite and/or sulfate during circulation, then return to the met-form via the mechanism proposed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we investigated the use of met-hemoCD-P and met-hemoCD-I as hydrogen sulfide receptors in aqueous solution and \u003cem\u003ein vivo\u003c/em\u003e. Compared to native Hb and Mb, met-hemoCD-I showed a greater binding rate and affinity toward hydrogen sulfide; therefore, we concluded that met-hemoCD-I is an effective and ready-to-use antidote for hydrogen sulfide-induced poisoning. Here, we discuss the binding of hydrogen sulfide to our heme-model system and compare it with that of other potential antidotes.\u003c/p\u003e \u003cp\u003eThe binding of hydrogen sulfide to metal ions in proteins, including heme proteins, has been proposed in many studies.\u003csup\u003e10,11\u003c/sup\u003e However, compared to that of O\u003csub\u003e2\u003c/sub\u003e, CO, and cyanide, detailed characterizations of HS\u0026ndash;metal complexes with thermodynamic and kinetic parameters have been rarely reported. Among the native systems, the interactions between ferric Hb and Mb with hydrogen sulfide have been characterized in detail.\u003csup\u003e31,32\u003c/sup\u003e As reported in these studies, the binding parameters strongly depend on the pH of the solutions. The binding is relatively fast at low pH, and under these conditions, hydrogen sulfide presents as H\u003csub\u003e2\u003c/sub\u003eS. Interestingly, the pH dependency is reversed for a small heme protein, MP-11,\u003csup\u003e34\u003c/sup\u003e in which the heme cofactor is exposed to the aqueous bulk phase. These data indicate that the preferable attacking species, H\u003csub\u003e2\u003c/sub\u003eS or SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e, should differ depending on the environment around the iron center of the heme.\u003csup\u003e35\u003c/sup\u003e In the hemoCD system, the binding parameters were also dependent on pH, similar to those of Hb and Mb, indicating that hydrophobic H\u003csub\u003e2\u003c/sub\u003eS tends to enter the iron(III) site located in the CD cavity. In contrast, we confirmed that a stable 1:1 complex of iron(III)porphyrin formed with SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e. The net charge of the iron(III)porphyrin ring becomes zero upon SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e ligation. The electronically neutralized porphyrin should be stable in the hydrophobic cavity provided by the CD dimer; thus, this force drives anion binding to iron(III)porphyrin\u0026ndash;methylated CD complexes in water.\u003csup\u003e36\u003c/sup\u003e Hydrophobic H\u003csub\u003e2\u003c/sub\u003eS is easily accessible to the iron(III) center of met-hemoCD, followed by deprotonation to SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e to neutralize the net charge of iron(III)porphyrin. The pH-dependent binding character is explained by the state of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS or SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e) and the axial ligand species on the iron(III) center before binding. Met-hemoCD-I has an exchangeable aqua ligand at neutral pH due to its p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003csup\u003eH2O\u003c/sup\u003e (7.7), whereas met-hemoCD-P (p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003csup\u003eH2O\u003c/sup\u003e = 5.5) has a strongly coordinated hydroxo ligand on iron(III) at neutral pH. Therefore, met-hemoCD-I, which exhibits a higher p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003csup\u003eH2O\u003c/sup\u003e than that of met-hemoCD-P, bound more quickly to hydrogen sulfide under physiological conditions; thus, met-hemoCD-I could be superior to met-hemoCD-P as a hydrogen sulfide receptor \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe interaction between hydrogen sulfide and synthetic iron(III) porphyrins has been investigated through biomimetic chemistry. In the system that involves a picket-fence-type Fe/Cu binuclear porphyrin complex, electrocatalytic O\u003csub\u003e2\u003c/sub\u003e reduction on the gold electrode was inhibited at a high concentration of H\u003csub\u003e2\u003c/sub\u003eS in a reversible manner.\u003csup\u003e37\u003c/sup\u003e This result suggests the toxic mechanism by which H\u003csub\u003e2\u003c/sub\u003eS inhibits the native mitochondrial C\u003cem\u003ec\u003c/em\u003eO system. Another study using picket-fence porphyrins in nonaqueous media revealed that stable HS\u0026ndash;Fe(III)porphyrin complexes formed via a 1:1 reaction with the SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e anion.\u003csup\u003e38\u003c/sup\u003e A synthetic heme-peptide conjugate model was also synthesized as an MP-11 model and formed a 6-coordinated HS\u0026ndash;Fe(III) low-spin species in water.\u003csup\u003e39\u003c/sup\u003e Atmospheric oxygen causes the porphyrins to significantly decompose, which is common in these model studies; thus, these HS\u0026ndash;Fe(III)porphyrin complexes have been characterized under anaerobic conditions. Due to the difficulty in preparing HS\u0026ndash;heme species, a stable synthetic model with the R\u003csub\u003e3\u003c/sub\u003eSi\u0026ndash;S\u0026ndash;Fe(III) complex has been proposed for detailed structural characterization.\u003csup\u003e40\u003c/sup\u003e To our knowledge, except for our previous study using the hemoCD system,\u003csup\u003e30\u003c/sup\u003e there are no synthetic models for the binding between hydrogen sulfide and iron-porphyrins under aerobic conditions in water. Importantly, in our model, once hydrogen sulfide bound to the met-hemoCDs, the porphyrin ring was significantly protected against oxidative degradation induced by SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e owing to the CD cavity and axial fifth coordination. Due to its high stability, the bound SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e anion could be efficiently converted to sulfite and sulfate ions via oxidation by atmospheric O\u003csub\u003e2\u003c/sub\u003e. This oxidative conversion from hydrogen sulfide to sulfite and sulfate ions has been similarly reported in the native Hb and Mb systems,\u003csup\u003e29,31\u003c/sup\u003e in which the heme cofactors are also protected in the hydrophobic heme pockets provided by apo-proteins. Therefore, due to the protection of the porphyrin ring by the CD dimer, we efficiently detoxified hydrogen sulfide under physiological conditions. Furthermore, in contrast to Mb and Hb, injected met-hemoCD is easily excreted in the urine due to its small molecular weight, which is another advantage of the present antidote system.\u003c/p\u003e \u003cp\u003eCurrently, no antidote is available to clinically treat H\u003csub\u003e2\u003c/sub\u003eS poisoning, but several approaches have been proposed to detoxify hydrogen sulfide in animals and clinical trials. As the toxic mechanism is almost identical to that of hydrogen cyanide, vitamin B\u003csub\u003e12\u003c/sub\u003e analogs (hydroxocobalamin and cobinamide), which are used as cyanide antidotes, are effective treatments for hydrogen sulfide poisoning.\u003csup\u003e4,17,41,42\u003c/sup\u003e Intravenously injected vitamin B\u003csub\u003e12\u003c/sub\u003e analogs can capture hydrogen sulfide during circulation, after which it is strongly captured by serum proteins and accumulates for a long period (over a month).\u003csup\u003e24,43\u003c/sup\u003e Therefore, patients that receive high doses of vitamin B\u003csub\u003e12\u003c/sub\u003e analogs may need to avoid strong light exposure due to the photosensitizing property of cobalamins. Oxidized Hb (met-Hb) could function as an antidote for hydrogen sulfide. Amyl nitrate (NaNO\u003csub\u003e2\u003c/sub\u003e), which is an oxidizing agent that produces met-Hb in circulating RBCs, has been shown to exhibit an antidote effect on hydrogen sulfide.\u003csup\u003e44\u003c/sup\u003e However, this method cannot be easily adjusted to the met-Hb ratio (%) in RBCs. In another recent trial using met-Hb, a met-Hb-albumin cluster was injected and markedly improved the survival rate of mice.\u003csup\u003e18\u003c/sup\u003e Compared to these potential candidates, the hemoCD system is advantageous because (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) met-hemoCD-I shows greater binding affinity toward hydrogen sulfide than that of met-Hb and met-Mb, and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) the hemoCD compound injected is quantitatively excreted in urine within several hours. Therefore, met-hemoCD-I can effectively capture H\u003csub\u003e2\u003c/sub\u003eS, and after detoxification, injected met-hemoCD-I easily disappears from the body through renal clearance. In a previous study, ferrous hemoCD-P injected into mice or rats was quantitatively detected in the CO-bound form in urine.\u003csup\u003e21,23,25\u003c/sup\u003e However, for hydrogen sulfide, HS-bound met-hemoCD-I was not detected in the urine. Instead, met-hemoCD-I was mainly detected without change because the bound hydrogen sulfide on met-hemoCD-I in the body could be smoothly converted to sulfite or sulfate ions via catalytic oxidation with O\u003csub\u003e2\u003c/sub\u003e, as demonstrated in this study.\u003c/p\u003e \u003cp\u003eWe recently reported an antidote system for CO and cyanide mixed intoxication using hemoCD-Twins, a mixture of hemoCD-P and hemoCD-I in the ferrous state.\u003csup\u003e25\u003c/sup\u003e It is well known that CO and HCN are generated as lethal toxic gases during building fires. Previous studies in mice demonstrated that CO and HCN exhibit a synergetic lethal effect, which was counteracted by hemoCD-Twins. Interestingly, when hemoCD-Twins was injected into mice, hemoCD-P remained ferrous and could capture CO, while hemoCD-I was oxidized during circulation, and met-hemoCD-I detoxified cyanide. In general, CO poisoning can be quickly diagnosed by pulse CO oximetry or blood gas analysis. On the other hand, HCN in blood is difficult to detect rapidly at the site of accidents. The dual antidote system with hemoCD-Twins is advantageous because it can be injected without determination of which gas the patient primarily inhaled. Additionally, in this study, met-hemoCD-I clearly detoxified hydrogen sulfide. Although hydrogen sulfide is rarely produced in fire accidents, hemoCD-Twins are the first choice for patients that are potentially poisoned by an unknown gas. As the injected hemoCD complex is smoothly excreted in urine, it can be employed without a risk of side effects due to accumulated compounds. We are attempting to apply this detoxification system using hemoCD-Twins in clinical practice.\u003c/p\u003e \u003cp\u003eIn conclusion, synthetic heme model compounds composed of iron(III)porphyrin and a per-\u003cem\u003eO\u003c/em\u003e-methylated cyclodextrin dimer were found to bind to hydrogen sulfide and form a stable HS\u0026ndash;Fe(III) complex. Furthermore, met-hemoCD-I catalytically and efficiently decomposed hydrogen sulfide into nontoxic sulfite/sulfate ions under physiological conditions. Mouse animal experiments revealed that met-hemoCD-I exhibits excellent properties as a novel antidote for hydrogen sulfide poisoning. We expect that this hemoCD-based system will serve as a ready-to-use, multifunctional gas poisoning antidote that can simultaneously remove CO, cyanide, and hydrogen sulfide via a single injection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eMaterials.\u003c/b\u003e Met-HemoCD-P and met-hemoCD-I were synthesized in our laboratory as described in previous reports.\u003csup\u003e25\u003c/sup\u003e We used sodium hydrogen sulfide (NaSH) as a hydrogen sulfide source. NaSH was purchased from Stream Chem. Inc. Before the use of NaSH, its purity was determined by iodometric titration.\u003csup\u003e30\u003c/sup\u003e All other reagents were purchased and used as received.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of the NaSH solution.\u003c/b\u003e Since NaSH is easily oxidized in water, we prepared a stock solution of NaSH before each application. Milli-Q water was deoxidized by nitrogen gas bubbling for 30 min. Then, NaSH was solubilized in deoxidized water and used as a stock solution. Once a NaSH solution was prepared, it was used within 3 h. The concentration of NaSH in solution, \u003cem\u003eC\u003c/em\u003e [mM], was determined as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$C \\left[\\text{m}\\text{M}\\right]=\\left\\{\\left(x \\left[\\text{m}\\text{g}\\right]\\times y\\left[\\%\\right]\\times {10}^{3}\\right)/\\left(56.063\\left[\\text{g}\\bullet {\\text{m}\\text{o}\\text{l}}^{-1}\\right]\\times z\\left[\\text{m}\\text{L}\\right]\\right)\\right\\}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003ex\u003c/em\u003e is the weight of NaSH, \u003cem\u003ey\u003c/em\u003e is the purity of NaSH determined by iodometric titration as described above, and \u003cem\u003ez\u003c/em\u003e is the volume of the stock solution.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInstruments.\u003c/b\u003e UV‒vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer with a thermostatic cell holder. Kinetic studies were carried out using a JASCO FS-110 Fast Scan Spectrometer with a thermostatic cell holder. EPR measurements were obtained with a Bruker E500 spectrometer at the Institute for Molecular Science in continuous-wave (CW) mode operating at ~\u0026thinsp;9.66 GHz and equipped with an Oxford Instruments ESR900 continuous helium flow cryostat. The experimental parameters were 5 mW microwave power, 100 kHz field modulation, and 10 G modulation amplitude. MALDI-TOF mass spectra were measured on Bruker Daltonics Autoflex speed spectrometers in linear mode. a-Cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix. ESI-TOF mass spectra were taken on a JEOL JMS-T100CS spectrometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpectroscopic measurements.\u003c/b\u003e The binding constants (\u003cem\u003eK\u003c/em\u003e) of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I were determined by UV‒vis photometric titration. NaSH (up to 50 mM) was added to an aerobic solution of met-hemoCD-I or met-hemoCD-P (5 \u0026micro;M) at 25\u0026deg;C in 0.05 M phosphate buffer at pH 6.0, 7.0, 7.4 and 8.0. The absorbance changes versus [NaSH] were plotted, and the data were fitted to a theoretical curve of an equation for 1:1 complex formation to obtain \u003cem\u003eK\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe association rate constants (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e) for the binding of hydrogen sulfide to met-hemoCD-P and met-hemoCD-I were obtained under pseudo-first-order conditions with excess NaSH at 25\u0026deg;C. An aerobic solution of met-hemoCD-P or met-hemoCD-I (5 \u0026micro;M) was mixed rapidly with various concentrations of NaSH (100\u0026ndash;1400 \u0026micro;M before mixing) in 0.05 M phosphate buffer at pH 7.0, 7.4 or 8.0. The change in absorbance at 410 nm was monitored, and the data were fitted to a single or double exponential function to obtain the observed rates \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e. When the reaction between met-hemoCD and hydrogen sulfide was too fast (\u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e \u0026gt; 0.5), a double exponential fitting was required due to an unknown slow reaction component. In that case, the component of \u003cem\u003ek\u003c/em\u003e\u003csub\u003efast\u003c/sub\u003e occupied more than 80% of the total. In addition, that ratio was independent of pH. Therefore, we adopted \u003cem\u003ek\u003c/em\u003e\u003csub\u003efast\u003c/sub\u003e as the actual \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e. (reaction between met-hemoCDs and hydrogen sulfide), while \u003cem\u003ek\u003c/em\u003e\u003csub\u003eslow\u003c/sub\u003e is possibly due to the reaction between met-hemoCDs and impurities derived from NaSH, such as polysulfides.\u003csup\u003e45\u003c/sup\u003e Finally, the \u003cem\u003ek\u003c/em\u003e\u003csub\u003eon\u003c/sub\u003e values were obtained from linear regression of \u003cem\u003ek\u003c/em\u003e\u003csub\u003eobs\u003c/sub\u003e as a function of the hydrogen sulfide concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification of hydrogen sulfide.\u003c/b\u003e An aqueous solution of NaSH (1.0 mM) was mixed with met-hemoCD or met-Hb (50 \u0026micro;M) and incubated at 25\u0026deg;C. The residual hydrogen sulfide concentrations were determined using the reported \u0026ldquo;methylene blue\u0026rdquo; method with some modifications.\u003csup\u003e46,47\u003c/sup\u003e The solution containing hydrogen sulfide and its scavenger was diluted to 2.5 mL with 0.05 M phosphate buffer at pH 7.4 to adjust the total hydrogen sulfide concentration to less than 50 \u0026micro;M. To the solution was added trifluoroacetic acid (0.5 mL). Then, solutions of \u003cem\u003eN,N\u003c/em\u003e-dimethyl-\u003cem\u003ep\u003c/em\u003e-phenylenediamine sulfate (200 mM stock solution, 30 \u0026micro;L) dissolved in HClaq (7.2 mM) and FeCl\u003csub\u003e3\u003c/sub\u003e (300 mM stock solution, 30 \u0026micro;L) dissolved in 1.2 mM HCl were added successively. The solutions were allowed to stand for 20 min at ambient room temperature. Finally, the absorbance at 663 nm was read, and the hydrogen sulfide concentration was determined based on the standard curve of methylene blue.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification of sulfate and sulfite.\u003c/b\u003e The relative production rate of sulfate and sulfite ions from hydrogen sulfide was determined by a simple turbidimetric method, in which the amount of sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026ndash;\u003c/sup\u003e) was quantified by turbidity through the following reaction:\u003csup\u003e48\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026ndash;\u003c/sup\u003e + BaCl\u003csub\u003e2\u003c/sub\u003e \u0026rarr; BaSO\u003csub\u003e4\u003c/sub\u003e\u0026darr; + 2Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe average absorbance between 700 nm and 800 nm was used as turbidity, where met-hemoCD-I and other scavengers showed no absorbance in the area. The turbidity showed a linear correlation with the sulfate concentration from 0.5 mM to 4.0 mM upon the addition of 1.5 equivalents of BaCl\u003csub\u003e2\u003c/sub\u003e. We also confirmed that sulfite (SO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026ndash;\u003c/sup\u003e) ions form a similar insoluble precipitate of BaSO\u003csub\u003e3\u003c/sub\u003e with BaCl\u003csub\u003e2\u003c/sub\u003e as follows:\u003csup\u003e49\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026ndash;\u003c/sup\u003e + BaCl\u003csub\u003e2\u003c/sub\u003e \u0026rarr; BaSO\u003csub\u003e3\u003c/sub\u003e\u0026darr; + 2Cl\u003csup\u003e\u0026ndash;\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTherefore, the relative production of sulfate and sulfite ions in solution was simply determined by reading the average absorbance between 700 nm and 800 nm after treatment of the solution with BaCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e\u003cb\u003eAnimal experiments.\u003c/b\u003e All animal studies were performed under the approval of Doshisha University and carried out in accordance with the Guidelines for Animal Experiments of Doshisha University. We used female BALB/cCrSlc mice (Shimizu Laboratory Supplies, Co., Ltd.) weighing 20\u0026ndash;22 g. The study is reported in accordance with ARRIVE guidelines. For acclimatization, the mice were housed under a 12 h/12 h light/dark cycle with free feedings under specific-pathogen-free (SPF) conditions for one week before the day in the experiments.\u003c/p\u003e \u003cp\u003eFor survival analysis of met-hemoCD-I in lethal hydrogen sulfide intoxication model mice, a solution of NaSH (21 mg/kg) in PBS (0.1 mL) was injected intraperitoneally. One minute after the injection of NaSH, a solution of met-hemoCD-I (7 mM) in PBS (0.2 mL) was injected intraperitoneally. For the predosing experiments, a solution of met-hemoCD-I (7 mM) in PBS (0.2 mL) was injected intraperitoneally. After 10 min, a solution of NaSH (21 mg/kg) in PBS (0.1 mL) was injected intraperitoneally. Survival rates were then monitored for one hour after hydrogen sulfide intoxication was induced.\u003c/p\u003e \u003cp\u003eWe determined the C\u003cem\u003ec\u003c/em\u003eO activity and the concentration of lactate in the blood of lethal hydrogen sulfide intoxication model mice. Tissue samples (brain, heart, and liver) were collected from met-hemoCD-I-treated surviving mice one hour after hydrogen sulfide intoxication was induced. As a control, we collected tissue samples (brain, heart, and liver) from untreated mice immediately after death. The CcO activity in these organs was determined by the following method according to the literature.\u003csup\u003e50\u003c/sup\u003e Each tissue sample (~\u0026thinsp;20 mg) was homogenized in 0.5 mL of sucrose muscle homogenization buffer (250 mM). Then, the suspension was centrifuged at 600 g for 10 min at 4\u0026deg;C. The supernatant (5 \u0026micro;L) was added to a 1 mL cuvette which contains 400 \u0026micro;L of Milli-Q water, 500 \u0026micro;L of potassium phosphate buffer (0.1 M, pH 7.0) and 50 \u0026micro;L of reduced cytochrome \u003cem\u003ec\u003c/em\u003e (Product name; ab109911, abcam, UK). The absorbance at 550 nm was read for 3 min. The rate of activity (OD/min) was determined by calculating the slope between two points within the linear region. Finally, the CcO activity was determined by normalizing the rate activity by its protein amount in tissue using BCA assay (Thermo Fisher Scientific, Japan). The concentration of lactate in the blood was measured using LT-1730 Lactate Pro2 (Arkray).\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Statistical analyses were performed using GraphPad Prism, version 10.2.3 (GraphPad Software). All the data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors from at least three different experiments and were analyzed by Student's t test. Survival curves were analyzed using Kaplan‒Meier curves and the log-rank test. Differences with \u003cem\u003eP\u003c/em\u003e values less than 0.05 were considered significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability.\u003c/strong\u003e All relevant data are in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.K. conceived the study. A.N. and Q.M. performed the main experiments and analyzed the data. M.H. performed the electron paramagnetic resonance spectroscopy experiments. M.K. provided useful suggestions for advancing this research. A.N. and H.K. wrote the manuscript. All authors participated in the revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by JSPS KAKENHI (22H02097 and 24K01640), AMED (23ym0126814 and 24ym0126808j), and JST (JPMJSF2305, JPMJSP2129).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eThe online version contains supplementary material available at https://doi.org/\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to H.K.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMaldonado, C. S., Weir, A. \u0026amp; Rumbeiha, W. K. A comprehensive review of treatments for hydrogen sulfide poisoning: past, present, and future. \u003cem\u003eToxicol. Mech. Methods\u003c/em\u003e\u003cstrong\u003e33\u003c/strong\u003e, 183\u0026ndash;196 (2023). \u003c/li\u003e\n\u003cli\u003eCuevasanta, E., M\u0026ouml;ller, M. N. \u0026amp; Alvarez, B. Biological chemistry of hydrogen sulfide and persulfides. \u003cem\u003eArch. Biochem. Biophys.\u003c/em\u003e\u003cstrong\u003e617\u003c/strong\u003e, 9\u0026ndash;25 (2017).\u003c/li\u003e\n\u003cli\u003eOlson, K. R. \u0026amp; Straub, K. D. The role of hydrogen sulfide in evolution and the evolution of hydrogen sulfide in metabolism and signaling. \u003cem\u003ePhysiology\u003c/em\u003e\u003cstrong\u003e31\u003c/strong\u003e, 60\u0026ndash;72 (2016).\u003c/li\u003e\n\u003cli\u003eJiang, J., Chan, A., Ali, S. \u003cem\u003eet al.\u003c/em\u003e Hydrogen sulfide\u0026ndash;mechanisms of toxicity and development of an antidote. \u003cem\u003eSci. Rep.\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 20831 (2016).\u003c/li\u003e\n\u003cli\u003eBlackstone, E., Morrison, M. \u0026amp; Roth, M. B. H\u003csub\u003e2\u003c/sub\u003eS induces a suspended animation-like state in mice. \u003cem\u003eScience\u003c/em\u003e\u003cstrong\u003e22\u003c/strong\u003e, 518 (2005).\u003c/li\u003e\n\u003cli\u003eMorii, D., Miyagatani, Y., Nakamae, N., Murao, M. \u0026amp; Taniyama, K. Japanese experience of hydrogen sulfide: the suicide craze in 2008. \u003cem\u003eJ. Occup. Med. Toxicol.\u003c/em\u003e\u003cstrong\u003e5\u003c/strong\u003e, 28 (2010).\u003c/li\u003e\n\u003cli\u003eIseki, K., Ozawa, A., Seino K., Goto, K. \u0026amp; Tase, C. The suicide pandemic of hydrogen sulfide poisoning in Japan. \u003cem\u003eAsia Pac. J. Med. Toxicol.\u003c/em\u003e\u003cstrong\u003e3\u003c/strong\u003e, 13\u0026ndash;17 (2013).\u003c/li\u003e\n\u003cli\u003eReedy, S. J. D., Schwartz, M. D. \u0026amp; Morgan, B. W. Suicide fads: Frequency and characteristics of hydrogen sulfide suicides in United States. West. J. Emerg. Med. 12, 300\u0026ndash;304 (2011).\u003c/li\u003e\n\u003cli\u003eAdkins, J. Hydrogen sulfide suicide: Latest technique hazardous to first responders and the public. Special Research Report in Regional Organized Crime Information Center. Available at https://npstc.org/documents/H2S%20Report%20for%204112.pdf\u003c/li\u003e\n\u003cli\u003ePietri, R., Rom\u0026aacute;n-Morales, E. \u0026amp; L\u0026oacute;pez-Garriga, J. Hydrogen sulfide and hemeproteins: Knowledge and mysteries. \u003cem\u003eAntioxid. Redox Signal.\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 393\u0026ndash;404 (2011).\u003c/li\u003e\n\u003cli\u003eDom\u0026aacute;n, A., D\u0026oacute;ka, \u0026Eacute;., Garai, D. \u003cem\u003eet al. \u003c/em\u003eInteractions of reactive sulfur species with metalloproteins. \u003cem\u003eRedox Biol.\u003c/em\u003e\u003cstrong\u003e60\u003c/strong\u003e, 102617 (2023).\u003c/li\u003e\n\u003cli\u003eParker, A. L. \u0026amp; Johnstone, T. C. Carbon monoxide poisoning: A problem uniquely suited to a medical inorganic chemistry solution. \u003cem\u003eJ. Inorg. Biochem.\u003c/em\u003e\u003cstrong\u003e251\u003c/strong\u003e, 112453 (2024). \u003c/li\u003e\n\u003cli\u003eAzarov, I., Wang, L., Rose, J. J. \u003cem\u003eet al.\u003c/em\u003e Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning. \u003cem\u003eSci. Transl. Med. \u003c/em\u003e\u003cstrong\u003e8\u003c/strong\u003e, 368ra173 (2016).\u003c/li\u003e\n\u003cli\u003eRose, J. J., Bocian, K. A., Xu, Q. \u003cem\u003eet al.\u003c/em\u003e A neuroglobin-based high-affinity ligand trap reverses carbon monoxide\u0026ndash;induced mitochondrial poisoning. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e\u003cstrong\u003e295\u003c/strong\u003e, 6357\u0026ndash;6371 (2020).\u003c/li\u003e\n\u003cli\u003eXu, Q., Rose, J. J., Chen, X. \u003cem\u003eet al.\u003c/em\u003e Cell-free and alkylated hemoproteins improve survival in mouse models of carbon monoxide poisoning. \u003cem\u003eJCI Insight\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, e153296 (2022).\u003c/li\u003e\n\u003cli\u003eChan, A., Jiang, J., Fridman, A. \u003cem\u003eet al. \u003c/em\u003eNitrocobinamide, a new cyanide antidote that can be administered by intramuscular injection. \u003cem\u003eJ. Med. Chem.\u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 1750\u0026ndash;1759 (2015).\u003c/li\u003e\n\u003cli\u003eFujita, Y., Fujino, Y., Onodera, M., Kikuchi, S., Kikkawa, T., Inoue, Y., Niitsu, H., Takahashi, K. \u0026amp; Endo, S. A fatal case of acute hydrogen sulfide poisoning caused by hydrogen sulfide: Hydroxocobalamin therapy for acute hydrogen sulfide poisoning. \u003cem\u003eJ. Anal. Toxicol.\u003c/em\u003e\u003cstrong\u003e35\u003c/strong\u003e, 119\u0026ndash;123 (2011).\u003c/li\u003e\n\u003cli\u003eSuzuki, Y., Taguchi, K., Okamoto, W., Enoki, Y. Komatsu, T. \u0026amp; Matsumoto, K. Methemoglobin-albumin clusters for the treatment of hydrogen sulfide intoxication.\u003cem\u003e J. Control. Release\u003c/em\u003e\u003cstrong\u003e349\u003c/strong\u003e, 304\u0026ndash;314 (2022).\u003c/li\u003e\n\u003cli\u003eDroege, D. G. \u0026amp; Johnstone, T. C. A water-soluble iron-porphyrin complex capable of rescuing CO-poisoned red blood cells. \u003cem\u003eChem. Comm.\u003c/em\u003e\u003cstrong\u003e58\u003c/strong\u003e, 2722\u0026ndash;2725 (2022). \u003c/li\u003e\n\u003cli\u003eKano, K., Kitagishi, H., Kodera, M. \u0026amp; Hirota, S. Dioxygen binding to a simple myoglobin model in aqueous solution. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e\u003cstrong\u003e44\u003c/strong\u003e, 435\u0026ndash;438 (2005).\u003c/li\u003e\n\u003cli\u003eKitagishi, H., Negi, S. Kiriyama, A., Honbo, A., Sugiura, Y., Kawaguchi, A. T. \u0026amp; Kano K. A diatomic molecule receptor that removes CO in a living organism. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e\u003cstrong\u003e49\u003c/strong\u003e, 1312\u0026ndash;1315 (2010).\u003c/li\u003e\n\u003cli\u003eKitagishi, H. \u0026amp; Kano, K. Synthetic heme protein models that function in aqueous solution. \u003cem\u003eChem. Commun\u003c/em\u003e. \u003cstrong\u003e57\u003c/strong\u003e, 148\u0026ndash;173 (2021).\u003c/li\u003e\n\u003cli\u003eKitagishi, H., Minegishi, S., Yumura, A., Negi, S., Taketani, S., Amagase, Y., Mizukawa, Y., Urushidani, T., Sugiura, Y. \u0026amp; Kano, K., Feedback response to selective depletion of endogenous carbon monoxide in the blood. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e138\u003c/strong\u003e, 5417\u0026ndash;5425 (2016).\u003c/li\u003e\n\u003cli\u003eWatanabe, K., Kitagishi, H. \u0026amp; Kano, K. Supramolecular ferric porphyrins as cyanide receptors in aqueous solution. \u003cem\u003eACS Med. Chem. Lett.\u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e, 943\u0026ndash;947 (2011).\u003c/li\u003e\n\u003cli\u003eMao, Q., Zhao, X., Kiriyama, A., Negi, S., Fukuda, Y., Yoshioka, H., Kawaguchi, A. T., Motterlini, R., Foresti, R. \u0026amp; Kitagishi, H. A synthetic porphyrin as an effective dual antidote against carbon monoxide and cyanide poisoning. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e\u003cstrong\u003e120\u003c/strong\u003e, e2209924120 (2023).\u003c/li\u003e\n\u003cli\u003eLi, Q. \u0026amp; Lancaster Jr., J. R. Chemical foundations of hydrogen sulfide biology. \u003cem\u003eNitric Oxide\u003c/em\u003e\u003cstrong\u003e35\u003c/strong\u003e, 21\u0026ndash;34 (2013).\u003c/li\u003e\n\u003cli\u003eKano, K., Kitagishi, H., Mabuchi, T., Kodera, M. \u0026amp; Hirota, S. A myoglobin functional model composed of a ferrous porphyrin and a cyclodextrin dimer with an imidazole linker. \u003cem\u003eChem. Asian J. \u003c/em\u003e\u003cstrong\u003e1\u003c/strong\u003e, 358\u0026ndash;366 (2006). \u003c/li\u003e\n\u003cli\u003eSono, M. \u0026amp; Dawson, J. H. Formation of low spin complexes of ferric cytochrome P-450-CAM with anionic ligands. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e\u003cstrong\u003e257\u003c/strong\u003e, 5469\u0026ndash;5502 (1982).\u003c/li\u003e\n\u003cli\u003eVitvitsky, V., Yadav, P. K., Kurthen, A. \u0026amp; Banerjee, R. Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e\u003cstrong\u003e290\u003c/strong\u003e, 8310\u0026ndash;8320 (2015).\u003c/li\u003e\n\u003cli\u003eWatanabe, K., Suzuki, T., Kitagishi, H. \u0026amp; Kano, K. Reaction between a haemoglobin model compound and hydrosulphide in aqueous solution. \u003cem\u003eChem. Commun.\u003c/em\u003e\u003cstrong\u003e51\u003c/strong\u003e, 4059\u0026ndash;4061 (2015).\u003c/li\u003e\n\u003cli\u003eBostelaar, T., Vitvitsky, V., Kumutima, J. Hydrogen sulfide oxidation by myoglobin. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e\u003cstrong\u003e138\u003c/strong\u003e, 8476\u0026ndash;8488 (2016).\u003c/li\u003e\n\u003cli\u003eJensen, B. \u0026amp; Fago, A. Reactions of ferric hemoglobin and myoglobin with hydrogen sulfide under physiological conditions. \u003cem\u003eJ. Inorg. Biochem.\u003c/em\u003e\u003cstrong\u003e182\u003c/strong\u003e, 133\u0026ndash;140 (2018).\u003c/li\u003e\n\u003cli\u003eNoguchi, M., Mao, Q., Nakagami, A. \u0026amp; Kitagishi, H. Spontaneous reduction of iron(III)porphyrin to iron(II)porphyrin\u0026ndash;CO complex in mouse circulation. \u003cem\u003eChem. Commun.\u003c/em\u003e\u003cstrong\u003e59\u003c/strong\u003e, 6211\u0026ndash;6214 (2023).\u003c/li\u003e\n\u003cli\u003eBoubeta, F. M., Bieza, S. A., Bringas, M., Estrin, D. A., Boechi, L. \u0026amp; Bari, S. E. Mechanism of sulfide binding by ferric hemeproteins. \u003cem\u003eInorg. Chem.\u003c/em\u003e\u003cstrong\u003e57\u003c/strong\u003e, 7591\u0026ndash;7600 (2018). \u003c/li\u003e\n\u003cli\u003eBoubeta, F. M., Bari, S. E., Estrin, D. A. \u0026amp; Boechi, L. Access and binding of H2S to hemeproteins: The case of Hbl of \u003cem\u003eLucina pectinata\u003c/em\u003e. \u003cem\u003eJ. Phys. Chem. B. \u003c/em\u003e\u003cstrong\u003e120\u003c/strong\u003e, 9642\u0026ndash;9653 (2016). \u003c/li\u003e\n\u003cli\u003eKano, K., Kitagishi, H., Tamura, S. \u0026amp; Yamada, A. Anion binding to a ferric porphyrin complexed with per-\u003cem\u003eO\u003c/em\u003e-methylated b-cyclodextrin in aqueous solution. \u003cem\u003eJ. Am. Chem. Soc. \u003c/em\u003e\u003cstrong\u003e126\u003c/strong\u003e, 15202\u0026ndash;15210 (2004).\u003c/li\u003e\n\u003cli\u003eCollman, J. P., Ghosh, S., Dey, A. \u0026amp; Decr\u0026eacute;au, R. A. Using a functional enzyme model to understand the chemistry behind hydrogen sulfide induced hibernation.\u003cem\u003e Proc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e\u003cstrong\u003e106\u003c/strong\u003e, 22090\u0026ndash;22095 (2009).\u003c/li\u003e\n\u003cli\u003eHartle, M. D., Prell, J. S. \u0026amp; Pluth, M. D. Spectroscopic investigations into the binding of hydrogen sulfide to synthetic picket-fence porphyrins. \u003cem\u003eDalton Trans.\u003c/em\u003e\u003cstrong\u003e45\u003c/strong\u003e, 4843\u0026ndash;4853 (2016).\u003c/li\u003e\n\u003cli\u003eZhao, Z., Wang, D., Wang, M., Sun, X., Wang, L., Huang, X., Ma, L. \u0026amp; Li, Z. Proximal environment controlling the reactivity between inorganic sulfide and heme-peptide model. \u003cem\u003eRSC Adv.\u003c/em\u003e\u003cstrong\u003e6\u003c/strong\u003e, 78858\u0026ndash;78864 (2016).\u003c/li\u003e\n\u003cli\u003eMeininger, D. J., Caranto, J. D., Arman, H. D. \u0026amp; Tonzetich, Z. J. Studies of iron(III) porphyrins containing silanethiolate ligands. \u003cem\u003eInorg. Chem.\u003c/em\u003e\u003cstrong\u003e52\u003c/strong\u003e, 12468\u0026ndash;12476 (2013).\u003c/li\u003e\n\u003cli\u003eNg, P. C., Hendry-Hofer, T., N. \u003cem\u003eet al.\u003c/em\u003e Intramuscular cobinamide versus saline for treatment of severe hydrogen sulfide toxicity in swine. \u003cem\u003eClin. Toxicol. (Phila).\u003c/em\u003e\u003cstrong\u003e57\u003c/strong\u003e, 189\u0026ndash;196 (2019).\u003c/li\u003e\n\u003cli\u003eHaouzi, P., Chenuel, B. \u0026amp; Sonobe, T. High dose hydroxocobalamin administered after H\u003csub\u003e2\u003c/sub\u003eS exposure counteracts sulfide poisoning induced cardiac depression in sheep.\u003cem\u003e Clin. Toxicol. (Phila).\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 28\u0026ndash;36 (2015).\u003c/li\u003e\n\u003cli\u003eMarques, H. M., Brown, K. L. \u0026amp; Jacobsen, D. W. Kinetics and activation parameters of the reaction of cyanide with free aquocobalamin and aquocobalamin bound to a haptocorrin from chicken serum. \u003cem\u003eJ. Biol. Chem. \u003c/em\u003e\u003cstrong\u003e263\u003c/strong\u003e, 12378\u0026minus;12383 (1988).\u003c/li\u003e\n\u003cli\u003eHoidal, C. R., Hall, A. H., Robinson, M. D., Kulig, K. \u0026amp; Rumack, B. H. Hydrogen sulfide poisoning from toxic inhalations of roofing asphalt fumes.\u003cem\u003e Ann. Emerg. Med.\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 826\u0026ndash;830 (1988).\u003c/li\u003e\n\u003cli\u003eGreiner, R., P\u0026aacute;link\u0026aacute;s, Z., B\u0026auml;sell, K., Becher, D., Antelmann, H., Nagy, P. \u0026amp; Dick, T. P. Polysulfides link H\u003csub\u003e2\u003c/sub\u003eS to protein thiol oxidation. \u003cem\u003eAntioxid. Redox Signal.\u003c/em\u003e\u003cstrong\u003e19\u003c/strong\u003e, 1749\u0026ndash;1765 (2013).\u003c/li\u003e\n\u003cli\u003eLawrence, N. S., Davis, J. \u0026amp; Compton, R. G. Analytical strategies for the detection of sulfide: a review. \u003cem\u003eTalanta\u003c/em\u003e\u003cstrong\u003e52\u003c/strong\u003e, 771-784 (2000).\u003c/li\u003e\n\u003cli\u003eMorikawa, T., Kajimura, M., Nakamura, T., \u003cem\u003eet al.\u003c/em\u003e Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway. \u003cem\u003eProc. Natl. Acad. Sci. U.S.A.\u003c/em\u003e\u003cstrong\u003e109\u003c/strong\u003e, 1293\u0026ndash;1298 (2012).\u003c/li\u003e\n\u003cli\u003eColeman, R. L., Shults, W. D., Kelly, M. T. \u0026amp; Dean, J. A. Turbidimetry via parallel photometric analysis. Determination of sulfate. \u003cem\u003eAnal. Chem.\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e44\u003c/strong\u003e, 1031\u0026ndash;1034 (1972).\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller, I. A., Brunner, B., Breuer, C., Coleman, M. \u0026amp; Bach, W. The oxygen isotope equilibrium fractionation between sulfite species and water. \u003cem\u003eGeochim. Cosmochim. Acta\u003c/em\u003e\u003cstrong\u003e120\u003c/strong\u003e, 562\u0026ndash;581 (2013).\u003c/li\u003e\n\u003cli\u003eSpinazzi, M., Casarin, A., Pertegato, V., Salviati, L. \u0026amp; Angelini, C. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. \u003cem\u003eNat. Protoc.\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, 1235-1246 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hydrogen sulfide, Heme, Porphyrin, Cyclodextrin, Injectable antidote","lastPublishedDoi":"10.21203/rs.3.rs-4591678/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4591678/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogen sulfide is a lethal toxic gas that disrupts cellular respiration in the mitochondrial system. Currently, no antidote is available for the clinical treatment of hydrogen sulfide poisoning. In this study, we investigated the function of iron(III)porphyrin complexes as hydrogen sulfide scavengers in water and evaluated their potential use as therapeutic agents for hydrogen sulfide poisoning. The compounds, named met-hemoCD-P and met-hemoCD-I, are composed of iron(III)porphyrin complexed with per-methylated b-cyclodextrin dimers that contain a pyridine (met-hemoCD-P) or imidazole axial fifth ligand that is coordinated to Fe(III) (met-hemoCD-I). These compounds formed stable HS\u0026ndash;Fe(III) complexes under physiological conditions, with binding constants of 1.2 x 10\u003csup\u003e5\u003c/sup\u003e and 2.5 x 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for met-hemoCD-P and met-hemoCD-I, respectively. The binding constant of met-hemoCD-I was much greater than those reported for native met-hemoglobin and met-myoglobin. Electron paramagnetic resonance (EPR) spectroscopy and H\u003csub\u003e2\u003c/sub\u003eS quantification assays revealed that after SH\u003csup\u003e\u0026ndash;\u003c/sup\u003e was coordinated to met-hemoCD-I, it was efficiently converted to nontoxic sulfite and sulfate ions via homolytic cleavage of the HS\u0026ndash;Fe(III) bond followed by aerobic oxidation. Mouse animal experiments revealed that the survival rate was significantly improved when NaSH-treated mice were injected with met-hemoCD-I. After the injection, mitochondrial C\u003cem\u003ec\u003c/em\u003eO function in brain and heart tissues recovered, and met-hemoCD-I injected was excreted in the urine without chemical decomposition.\u003c/p\u003e","manuscriptTitle":"Detoxification of hydrogen sulfide by synthetic heme-model compounds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-10 10:25:22","doi":"10.21203/rs.3.rs-4591678/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-17T05:49:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-13T17:54:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135388003558536566256374270735104332679","date":"2024-08-28T15:06:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T01:08:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307243294985510741400046098955720134500","date":"2024-07-03T17:02:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T01:57:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-29T05:50:08+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-23T18:16:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-20T08:39:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-17T04:11:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b702b87b-f53a-4351-88a9-8ce7dfc2d25b","owner":[],"postedDate":"July 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T16:03:36+00:00","versionOfRecord":{"articleIdentity":"rs-4591678","link":"https://doi.org/10.1038/s41598-024-80511-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-10 15:58:01","publishedOnDateReadable":"December 10th, 2024"},"versionCreatedAt":"2024-07-10 10:25:22","video":"","vorDoi":"10.1038/s41598-024-80511-1","vorDoiUrl":"https://doi.org/10.1038/s41598-024-80511-1","workflowStages":[]},"version":"v1","identity":"rs-4591678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4591678","identity":"rs-4591678","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}