Mechanistics of pH-Dependent Sulfmyoglobin Formation: Spin Control and His64 Proton Relay

Rodriguez-Mackenzie, Angel D.; Santos-Velazquez, Lysmarie; Arbelo-Lopez, Héctor D.; Wymore, Troy; Lopez-Garriga, Juan

doi:https://doi.org/10.1155/2024/4244579

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Research Article | Open Access

Volume 2024 | Article ID 4244579 | https://doi.org/10.1155/2024/4244579

Mechanistics of pH-Dependent Sulfmyoglobin Formation: Spin Control and His64 Proton Relay

Angel D. Rodriguez-Mackenzie,¹Lysmarie Santos-Velazquez,¹Héctor D. Arbelo-Lopez,¹Troy Wymore,²and Juan Lopez-Garriga^1,3

Academic Editor: Jose Corchado

Received21 Nov 2022

Revised18 Jan 2024

Accepted02 Feb 2024

Published21 Feb 2024

Abstract

The chemistry of hydrogen sulfide (H₂S) has been directed towards physiologically relevant hemeproteins, including myoglobin, hemoglobin, and other similar proteins. Despite substantial efforts, there remains a need to elucidate the mechanism and identify the species involved in the reaction between oxy-hemeproteins and H₂S. Here, we summarize both our experimental data and computational modeling results revealing the mechanisms by which sulfmyoglobin (sulfMb) and sulfhemoglobin (sulfHb) are formed. Our experimental data at pH 7.4 reveal differences in intensity between sulfMb and sulfHb chromophores in the 620 nm charge transfer region. This behavior could be attributed to the incomplete reaction of tetrameric oxy-Hb with H₂S, where not all heme groups form sulfheme. The data also show that, for the reaction of oxy-myoglobin (oxy-Mb) and H₂S, the 622 nm charge transfer band increases in intensity from a pH of 6.6 to 5.0. This increase is attributed to the presence of the heme pocket distal His64_εδ, which is positively charged, resulting in an elevated yield of sulfMb formation compared to the mono-protonated tautomer, His64ε. Computational hybrid QM/MM methods support the conclusion, indicating that oxy-Mb His64_εδ (pH 5.0) reacts with H₂S in the triplet state, favored by −31.0 kcal/mol over the singlet His64ε (pH 6.6) species. The phenomenon is facilitated by a hydrogen bonding network within the heme pocket, between His64_εδ, heme Fe(II)O₂, and H₂S. The results establish an energetically favored quantitative mechanism to produce sulfMb (−69.1 kcal/mol) from the reactions of oxy-Mb and H₂S. Curiously, the mechanism between met-aquo Mb, H₂O₂, and H₂S shows similar reaction pathways and leads to sulfheme formation (−135.3 kcal/mol). The energetic barrier towards intermediate Cpd-0 is the limiting step in sulfheme formation for both systems. ‬Both mechanisms show that the thiyl radical, HS^•, is the species attacking the β-β double bond of heme pyrrole B, leading to the sulfheme structure.

1. Introduction

Hydrogen sulfide (H₂S) is a gas endogenously produced in mammalian tissues and organs by the enzymes cystathionine β-synthase (CBS), cystathionine ɣ-lyase (CSE), D-amino acid oxidase (DAO), and indirectly by 3-mercaptopyruvate sulfurtransferase (3-MST) [1–4]. H₂S concentrations and enzyme distribution in tissues are correlated with physiological function [1–4] due to the highly lipophilic nature of H₂S that facilitates easy penetration into the cell by simple diffusion [5]. H₂S plasma values can vary between 50 μM and 150 μM and are associated with several conditions, including the fibrosis phenomenon [6]. In addition, SARS-CoV-2 survivors show serum H₂S levels that are higher (≥150 μM) than those of nonsurvivors [7, 8]. Thus, the production of H₂S has been suggested to act as a defense mechanism against COVID-19 [8–10]. Overexpressed CSE in the pancreas increases H₂S levels, inducing the pathogenesis of diabetes in rats [11]. In contrast, a reduction in the enzymatic activity of CSE promoted hypertension in rats and women with preeclampsia [12, 13]. Reduced H₂S levels are also related to Parkinson’s, Alzheimer’s, and atherosclerosis [14, 15]. Furthermore, H₂S has been correlated with chronic diseases [16, 17], inflammation [18, 19], oxidative stress [20–25], oral diseases [26], glaucoma [27], subarachnoid hemorrhage [28], infertile men [29], arterial oxygen saturation [30], and skin diseases [31].

Human exposure to exogenous concentrations of H₂S can trigger sulfhemoglobinemia-induced cyanosis, a bluish skin color associated with the lack of oxygen [13, 32–41]. In addition, excessive amounts of drugs such as acetanilide, metoclopramide, phenacetin, dapsone, and sulfanilamide-containing medications produce the same physiological condition [32]. The phenomena have been observed in the newborn [33], gamma rays’ irradiation [34, 35], cancer, neurodegenerative diseases [36], and dimethyl sulfide (DMSO) dermal applications [37].

Sulfhemoglobinemia [38–41] is associated with hemoglobin and myoglobin, presenting an oxygen reduction affinity of 135 and 2,500 times upon sulfhemoglobin (sulfHb) and sulfmyoglobin (sulfMb) formation, respectively [42]. Normal levels of physiological sulfhemoglobin are below 0.037 g/dL, whereas 0.5 g/dL in the blood is enough to present cyanosis symptoms [13, 43, 44]. A consensus on the relationship between pH, oxy-hemoglobin (oxy-Hb), oxy-myoglobin (oxy-Mb), and hydrogen sulfide to produce sulfHb and sulfMb has still not been reached. Experimentally, the reaction between H₂S and met-aquo-Hb[Fe(III)] or met-aquo-Mb [Fe(III)] in the presence of H₂O₂ and oxy-Hb[Fe(II)] and oxy-Mb[Fe(II)] under an O₂ environment generates sulfHb and sulfMb (reactions (1a) and (1b)), respectively [45–47].

Radioactive sulfide species demonstrated that a single sulfur atom is incorporated into the heme structure per mol of Mb to form sulfMb [47]. The insertion resulted in a covalent heme modification by the specific incorporation of the sulfur ring across the β-β double bond of heme pyrrole B, Figure 1. X-ray [48] and NMR [49] spectroscopy have supported the structural assignment. The chromophore shows a characteristic optical band in the 620 nm region. Slight displacements of this band depend on the isomer and ligation states [42, 46, 49–52]. The absorption region is characterized by a π to dπ (dyz, dxz) charge transfer transition associated with the sulfur ring attached to the pyrrole B and the heme iron [53].

Interestingly, an inverse relationship between increased sulfMb formation and a pH decrease has been shown in reaction (1a). Thus, it has been suggested that H2S, rather than its anion (HS-), is the reactive sulfur species in the sulfheme reaction mechanism [54, 55]. Furthermore, several hemeproteins were evaluated for their reaction with H₂O₂ in the presence of H₂S. The data revealed that the formation of the sulfheme derivative requires the presence of the His64 residue in the distal heme site [56]. All other amino acids tested at this position generate the classical heme-ferryl compound I radical [Fe(IV)=O^+•, Cpd-I] and heme ferryl compound II [hemeFe(IV)=O, Cpd-II]. Two different pathways have been proposed [46, 47, 55, 57, 58] to explain sulfheme formation. In one scheme, Berzofsky et al. [46, 47] suggested that the hydrosulfide ion (HS⁻) may be the initial reactant species because ∼70% of H₂S dissociates to HS⁻ (pKa of 7.40) at physiological pH. Yet, experiments indicate that sulfMb presence decreases by 250-fold at basic pH values, at which the predominant species is HS⁻ [55], suggesting that this is not the leading reactive species. On the other hand, Nicholls et al. [57] proposed that sulfMb can be formed stoichiometrically upon the reaction of Cpd-II with a thiyl radical, HS^•, and that a mixture of ferric sulfMb and small quantities of ferrous sulfMb were generated as final products [57, 58].

QM/MM calculations showed that a concerted interaction among the heme, distal His64, H₂O₂, and H₂S led to the formation of the thiyl radical, HS^•, necessary for sulfheme formation [53, 56, 59]. The calculations suggest a sulfheme mechanism initiated by hydrogen bonding interactions among heme Fe(III)−H₂O₂, His64, and H₂S. Figure 1S (supplementary information) depicts a favorable formation of the intermediate Cpd-0 [Fe(III)OOH] that reacts with hydrogen sulfide to produce, upon homolytic cleavage of both Cpd-0 and an S-H bond, a favorable heme ferryl compound II and a thiyl radical, HS^•. The reaction is subsequently followed by a reaction between hemeFe(IV)=O and the HS^• radical, leading to a −135.3 kcal/mol potential energy decrease which correlates with the sulfMb kinetics (2.5 ± 0.1 × 10⁶ M⁻¹s⁻¹) and stability. Although the mechanism to generate the heme ferryl compound II and a thiyl radical, HS^•, is different from the model proposed by Nicholls [57], the calculated energetic data indeed supported their hypothesis of the reaction between hemeFe(IV)=O and the HS^• to produce sulfheme. Furthermore, contrary to the classical peroxidative reaction of heme proteins with H₂O₂ [60, 61],the absence of the heme Cpd-I radical [Fe(IV)=O^+•] in sulfheme formation through reaction (1a) [53, 59] supports the antioxidant role of hydrogen sulfide [18–25].

Early results show that the reaction between oxy-Mb and hydrogen sulfide, in the presence of fully protonated His64_εδ, promotes an energetically favorable path (−31.0 kcal/mol) from a heme ligand singlet to a triplet state that stabilizes the heme compound-III [Fe(III)O-O⁻, Cpd-III], Scheme 1 [62].

Moreover, a synchronized higher energy process accompanies the transition from Cpd-III and H₂S to heme compound 0 [Fe(III)-OOH, Cpd-0]. Thus, Scheme 2 presents a 23.3 kcal/mol barrier coupled with the homolytic cleavage of an S-H bond, following the favorable (−11.2 kcal/mol) formation of transient Cpd-0 and thiyl radical (HS^•) [62]. However, despite this knowledge, the mechanism for sulfheme formation (reaction (1b)), intermediate structure, pH dependence, and the relation to its electronic charge transfer 620 nm band region have remained unresolved. The report presented here builds upon earlier research [62], examining the influence of pH on the populations of heme-ligand triplets and singlets, as well as the His64 proton relay within the heme pocket. The results show that oxy-Mb interacting with H₂S to produce the 5-member ring met-aquo-sulfMb (SC) from the Cpd-0 intermediate is energetically favorable by ∆E −69.1 kcal/mol. Experimental results support the pH-related behavior and how the triplet-to-singlet transition affects the 622 nm absorption intensity and the population of sulfheme.

2. Materials and Methods

2.1. Experimental Sulfmyoglobin (sulfMb) and Sulfhemoglobin (sulfHb) Formation

Myoglobin (Mb) from equine skeletal muscle and human hemoglobin obtained from Sigma-Aldrich were used without further purification. The hemeproteins deoxy-Mb (Soret (435 nm) and Q (560 nm)) and deoxy-Hb (Soret (430 nm) and Q (555 nm)) species [hemeFe(II)], in HEPES buffer 0.05M pH = 7.4, were obtained upon anaerobic reduction of the met-aquo-hemeproteins [hemeFe(III)] with sodium dithionite. All solutions were degassed and then purged with N_2(g) for three cycles of 15 minutes each. At pH 7.4, the Mb : Na₂S₂O₄ and Hb : Na₂S₂O₄ molar ratio was 1 : 2. At pH 5.0 and 6.6, sperm whale deoxy-myoglobin was prepared in phosphate and succinic acid buffer solution with an Mb : Na₂S₂O₄ molar ratio of 1 : 8 [42, 45–47]. All samples were prepared under anaerobic conditions. The oxy-Mb and oxy-Hb derivatives were prepared by saturating deoxy-Mb and deoxy-Hb solutions with ultrapure dioxygen gas for 30 minutes and 10 psi. They showed the characteristics of the electronic transitions at Soret (418 nm) and Q (542 nm and 580 nm) and Soret (415 nm) and Q (541 nm and 577 nm), respectively, [42, 45–47, 63, 64].

Hydrogen sulfide solutions were prepared from sodium sulfide salts (Na₂S) in the same buffer solution as the hemeproteins. Hydrogen sulfide stock solution (83 mM) was prepared in an amber vial with 0.020 g of Na₂S·9 H₂O salt (Alfa Aesar) previously purged with nitrogen and 1.0 mL of HEPES 0.0500M buffer at a pH of 7.4. In HEPES buffer at pH 7.4, there is an equilibrium between 80% SH⁻ and 20% H₂S, while in phosphate and succinic acid, at pH 5.0 and 6.6, the predominant species is H₂S. At pH 7.4, sulfmyoglobin (sulfMb) and sulfhemoglobin (sulfHb) derivatives in the HEPES buffer were prepared by the reaction between oxy-Mb (55 μM) and oxy-Hb (55 μM) solutions with hydrogen sulfide (H₂S) solution in a molar ratio of 1 : 3 (oxy-hemeprotein H₂S). The hydrogen sulfide concentration was 165 μM. The scenario allowed us to understand the differences between the monomeric sulfMb and the tetrameric sulfHb and the charge transfer band in the 620 nm region, characteristic of the sulfheme formation [42, 45–47, 53, 59, 65]. Sulfmyoglobin at pH 5.0 and 6.6 was prepared from sperm whale oxy-myoglobin in phosphate and succinic acid buffer solution by mixing the oxy-Mb solution with H₂S solution at the same pH in a 1 : 100 molar ratio, oxy-Mb : H₂S. UV-Vis spectral data were collected on a SHIMADZU 2600 UV-Vis with a temperature controller set at a constant temperature of 25°C.

2.2. Theoretical Methods for Sulfmyoglobin Formation

The crystal structure coordinates of Equus caballus myoglobin 2VLY [66] were obtained from the RCSB protein data bank, which has a resolution of 1.6 Å. Models were constructed using CHARMM version 40b [67], describing the protein and solvent with CHARMM36 force field with CMAP correction [68] and the TIP3P water model [69], respectively. The description of the heme moiety used a CHARMM-modified force field [70]. The active site residue His64 was modeled in acidic pH with and protonated. All other residues were modeled in their canonical protonation states. Potassium and chloride ions were added to the solvated system at a concentration of 0.15 M with the Monte Carlo simulation method, resulting in a model size of 71 Å3 composed of 33,330 atoms. The models were all geometry optimized for 1000 steps using the steepest decent algorithm. The models were then simulated for 500 picoseconds (ps) at 300K, restraining the heavy atoms of the protein and heme with a harmonic force constant of 5 and 50 kcal/(mol Å), respectively. Finally, the SHAKE algorithm [71] was used to constrain all bonds to hydrogen to enable 2 fs time steps. These simulations’ primary objective was equilibrating the surrounding water molecules and those near the active site. Figure 2S (Supporting Information) shows the integrated model used for the calculations.

2.3. Refinement of Models Using PM6/MM Simulations

Hybrid QM/MM simulations were performed with pDynamo version 1.9 [72]. Electrostatic and Van der Waals interactions had an inner cutoff distance of 8 Å and an outer cutoff distance of 14 Å. The QM region consisted of the entire heme group, propionate and vinyl groups, residue His93, and the O₂ ligand for 92 atoms in the singlet state [73]. The models were geometry optimized with the SD algorithm for 1000 steps. The atomic velocities were scaled every 50 steps (1 fs time step) to simulate the models from 10 K to 300 over 3 ps. The models were then simulated for another 10 ps at 300 K. The final snapshot was used to model the interactions of H₂O and H₂S molecules with oxy-Mb using the Density Functional Theory (DFT) method. Visualization of the structures and analysis were performed with the VMD program [74].

2.4. DFT/MM Calculations Sulfmyoglobin Formation

Hybrid DFT/MM simulations were performed to unravel the sulfmyoglobin formation mechanism from the oxy-Mb reaction with H₂S with pDynamo interfaced with the ORCA program version 4.1 [75]. The models were truncated from the previous cubic model, deleting all water molecules and ions outside a 30 Å sphere centered on the Fe atom. All protein residues, water molecules, and ions containing an atom within 22 Å of the Fe atom were unrestrained; all others were fixed in position. Two water molecules that always entered and remained in the active site His64_εδ model during the PM6/MM simulations were replaced by H₂S molecules. The QM region consisted of the entire heme moiety, including propionate and vinyl groups, His64, His93, Val68, Leu29, Phe43, O₂, H₂S, and H₂O molecules. A total of 146 atoms were included in the MbHis64_εδ calculation. The hydrogen link atom approach was used to define the QM/MM boundary. Before substituting the H₂O molecule(s) with H₂S replacement, the models were geometry optimized for 300 steps using the SD algorithm and then by the conjugate gradient algorithm for 700 steps using the unrestricted DFT functional BP86 [76–78] with the D3 dispersion correction [79, 80]. The basis set was Ahlrichs triple-zeta with polarization TZVP and the Def2/J [81–83] resolution of identity approximation (RI). A Stuttgart-Dresden-Bonn relativistic effective core potential (ECP) was used for the iron atom [84, 85]. The calculations use the libint2 library to compute 2-electron integrals as specified by ORCA [86]. A total of thirty models were constructed by alternating multiplicity, H₂S/H₂O molecule positions, and availability of the Fe atom. After the replacement of H₂O by H₂S molecules, the models were geometry optimized for another 300 steps using the SD algorithm.

The energetic profile of Cpd-0 formation was calculated using the O=O bond length as the reaction coordinate over 20 windows in increments of 0.05 Å. Each window was geometry optimized with 50 steps of the SD algorithm using a force constant of 400 kcal/(mol Å) on the reaction coordinate. An accurate energy profile and the potential energy of the structures along the reaction coordinates were obtained using the BP86/MM method. It was calculated with the unrestricted hybrid functional B3LYP [87, 88] with TZVP and Def2/J auxiliary basis sets and RIJCOSX approximation for the Coulomb and Hartree–Fock (HF) exchange integrals. The functional was modified to have an HF exchange of 13%, providing more accurate energy values for iron-containing systems [89, 90]. The quasi-restricted orbitals obtained from the DFT-defined region were used to explore the effects of spin contamination with a restricted open-shell Kohn–Sham (ROKS) description of the QM region. When performing single energy points on ROKS, the RIJONX approximation for the Coulomb integrals was used. Spin density was calculated using the ORCA plot interactive suite of the ORCA 4.0.1 software. The conformations of interest were those at the local and global minima of the reaction coordinates. Corroboration of the triplet spin state system was performed by CASSCF using the TZVP basis set, Def2/JK auxiliary basis set, and RIJCOSX approximation. Our initial guess was generated using the resulting quasi-restricted orbitals (QROs) from our previous ROKS calculations. The active space consisted of iron’s 3d orbitals and six 2p orbitals of O₂, resulting in 11 orbitals and 14 electrons [91].

3. Results and Discussion

3.1. Insight of pH-Dependent Sulfmyoglobin Formation

The pH dependence is crucial in deciding the histidine amino acid tautomeric population and its role in protein function [92–94]. For example, myoglobin, at neutral pH, His64_ε (HisE7), is predominantly mono-protonated on its nitrogen (), and its swinging motion opens and closes the heme pocket to ligand migration [95–97]. Thus, the heme-O₂-His64_ε hydrogen bond interaction is thermodynamically favorable. On the other hand, the sulfMb species, sulfur-derived heme, requires the proper orientation of the His64 residue in the distal heme site [56]. Moreover, in the case of reaction (1a), an inverse relationship exists between an increased production yield and a decreased pH [54, 55]. Figure 2 displays the visible spectra illustrating the time-dependent sulfMb formation from the interaction of oxy-Mb with H₂S (reaction (1b)) under conditions of His64_ε (pH 6.60) and His64_εδ (pH 5.00). The consumption of H₂S by oxy-Mb results in a reduction in intensity of the electronic transitions at 543 nm and 580 nm. In contrast, the charge transfer band at 622 nm increases as the sulfMb population rises, in agreement with the literature [42, 45–47, 53].

(a)

(b)

(c)

(d)

A comparison between Figures 2(a) and 2(b) (pH 5.0) and Figures 2(c) and 2(d) (pH 6.6) shows that His64_εδ drastically promotes sulfMb formation relative to the His64_ε tautomer. At a more acidic pH, the 622 nm band increases in intensity, reaching its maximum absorbance from dark blue to bright blue in 30 minutes (Figure 2(b)), while the process at the higher pH takes 350 minutes (Figure 2(d)). The intensity increase reaches its maximum absorbance approximately ten-fold faster at lower than higher pH. Likewise, the transitions at 543 nm and 580 nm exhibit distinct behaviors at various pH levels, as evidenced by the alterations in red-to-orange color intensity (Figures 2(c) and 2(d)). Within this pH window, the probability of full protonation of distal His64 increases with more acidic environments, further enabling proton exchange within the heme distal cavity.

Early work [62], conducted at a millisecond scale using stopped-flow spectrophotometry, revealed that, at the more acidic pH of 5.0 during the reaction of oxy-Mb with H₂S, there is a transition of the Soret band from 414 nm to 416 nm, reaching a maximum absorbance value at 100 ms. The 2 nm red shift was suggested to be caused by the presence of heme Cpd-0 [Fe(III)OOH]. Hence, the findings indicate that protonation of the delta nitrogen of His64_εδ (as defined in Scheme 2) enables the migration of the epsilon-located hydrogen, thereby facilitating reaction (1b) and augmenting the intensity of the 622 nm charge transfer transition. The stronger hydrogen bond between the nitrogen epsilon-located hydrogen and compound III dissipates emerging charged groups near the active site, facilitating Compound I formation, as discussed in the next section.

3.2. Insight into Sulfmyoglobin Formation Mechanism

The visible spectroscopic data between oxy-Mb and H₂S reactions were supported using two distinct models: (i) neutral (with His64_ε, pH 6.6) and (ii) positively charged histidine (His64_εδ, pH 5.0). Oxy-Mb with the His64_ε tautomer and H₂S favored the open shell singlet biradical state over the triplet state by approximately 7.0 kcal/mol. In contrast, oxy-Mb His64_εδ interaction with H₂S reveals that the triplet state is favored by 31.0 kcal/mol over the singlet biradical state (Scheme 1). In this paramagnetic triplet state, an unpaired electron occupies the central Fe d_XZ orbital, while the second unpaired electron is shared between the oxygen atoms bonded to heme. This electronic configuration is consistent with the heme compound III [Fe(III)O-O^•−, Cpd-III] also known as superoxy-Mbor the Weiss configuration [62] shown in Figure 3(a). Advancing along the reaction coordinate in Figure 3 (- bond distance) of the heme Fe(III)O-O^•− complex, the distal oxygen atom () forms hydrogen bond interaction with the nearby S-H moiety of H₂S. As a result, H₂S is homolytically cleaved, donating a hydrogen atom to the distal oxygen () to produce Cpd-0 [Fe(III)OOH] and a hydrosulfide radical (HS^•), Figure 3(b). There is a calculated energy barrier of 23.3 kcal/mol for the process. In this intermediate state, all electrons surrounding the ligand are paired, resulting in a null spin density for the O-H bond in Cpd-0, metastable by 11.2 kcal/mol, Figure 3(b). A proton transfer from His64_εδ is energetically favorable by 5 kcal/mol, resulting in the formation of a water molecule and the hypervalent Cpd-I radical [Fe(IV)=O^+•] intermediate, as illustrated in Figure 3(c).

The breaking of the O-O bond of Cpd-0, seen in Figure 4(a), reveals that the spin density is distributed primarily over the β-β double bond of the C₃ and C₂ carbon of the heme pyrrole B. Spin density is also found over the porphyrin central iron atom. This configuration of delocalized electrons over the heme and iron is consistent with the heme ferryl Cpd-I. The short-lived heme Fe(IV)=O^+• radical is extinguished by the immediate integration of the HS^• radical into the heme group, resulting in the formation of the heme ferryl Cpd-II, Fe(IV)=O, and the episulfide group attached to the β-β double bond of heme pyrrole B, Figure 4(b). The process is accompanied by an energy drop of 32.7 kcal/mol (Figure 3(d)). The formation of the 3-member ring isomer (), seen in Figure 4(c), unravels a series of proton exchanges between the sulfur, a water molecule, and His64_εδ. The structure is energetically favored over the opened-ring episulfide conformation by 20.8 kcal/mol (Figure 3(e)).

(a)

(b)

(c)

As the three-member ring closes (Figure 4(c)), a proton is transferred from ring-opened episulfide to the bridging water molecule, which simultaneously transfers another proton to His64_εδ. This process is energetically favorable by −7.8 kcal/mol, resulting in the formation of the met-aquo-sulfMb species and the SH⁻ anion (Figure 3(f)). Concurrently, steric effects from the departing water molecule result in the integration of spin density into the heme group via sulfheme isomeric structure formation. The isomeric structure, Figure 3(f), is favored over the arrangement by only −2.8 kcal/mol. As shown in Figure 5, geometrical flexibility and increased conjugation in the heme porphyrin π system, facilitated by the 5-membered ring, may contribute to the structural preference. The preference for this conformation is consistent with our previous work, with a met-aquo Mb analog and hydrogen sulfide (reaction (1a)) [53, 59]. The orientation of the water molecule allows it to function as a bridge between the hydrosulfide and His64_εδ.

(a)

(b)

The mutual extinction of the short-lifetime intermediates, Cpd-I and thiyl radical (HS^•), leads to a potential energy drop of −69.1 kcal/mol from Cpd-0 to met-aquo-sulfMb. Overall, the formation of the water molecule during the breaking of Cpd-0 O-O bond reveals a concerted proton relay. The pH-dependent triplet state and the His64_εδ proton relay are responsible for these specific reaction pathways and the stability of the final products. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬Also, the mechanism presented in Figure 3 is analogous to the oxy-heme open-shell singlet biradical state. The calculated potential energy profiles do indicate some differences, given that in oxy-Mb His64_εδ interaction with H₂S, the triplet state is favored by 31.0 kcal/mol, while hydrogen sulfide stabilizes the complex by 7.0 kcal/mol in the singlet scenario.

3.3. Sulfheme Pathways: Analogous but Exclusive

Interestingly, the sulfheme formation mechanisms, presented in Figure 1S (reaction (1a)) and Figure 3 (reaction (1b)), respectively, show different heme intermediates and energy barriers towards heme Cpd-0. Furthermore, both reactions show a diverse mechanism to produce the thiyl radical, HS^•. In the former case, the reaction between Cpd-0 (Heme Fe(III)-OOH) and H₂S produces the radical. In the latter scenario, the reaction between Cpd-III (Fe(III)O-O^•− and H₂S leads to the species. Yet in both cases, favorable potential energy pathways of −135.3 kcal/mol and −69.1 kcal/mol come from the thiyl radical species attacking the β-β double bond of heme pyrrole B to produce the sulfheme structure (). Although the mechanism to generate HS^• differs from the model proposed by Nicholls [57], the calculated energetic data supported its presence. Thus, the heme iron oxidation state [FeIII) or Fe(II)], coordinated ligand (H₂O₂ or O₂), singlet or triplet spin states, and pH define the number of electrons available for the reaction processes. Furthermore, the hydrogen bond interactions between heme Fe(III)-H₂O₂ or heme Fe(II)-O₂, heme distal His64, and H₂S also regulate the process.‬‬‬‬‬‬‬‬‬‬‬‬

Table 1 summarizes the intermediates and energy of met-aquo-Mb [Fe(III)] + H₂O₂ and H₂S (reaction (1a)) and oxy-Mb[Fe(II)] in the presence of H₂S (reaction (1b)) to produce sulfheme structure S_C, Figures 1S and 3. ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬In reaction (1a), the energetic barrier for the hydrogen bonding ferric hydroperoxyl moiety to heme compound 0 [Fe(III)-OOH, Cpd-0] is only 1.9 kcal/mol (Figures 1S-A and 1S-B). While in reaction 1b (Figures 3(a) and 3(b)), transition from heme Cpd-III to heme Cpd-0 is accompanied by the synchronized transformation to higher energy (23.3 kcal/mol), transient state [Fe(III)O-O^−•]. The homolytic cleavage of H₂S follows the generation of a favorable (−11.2 kcal/mol) intermediate, Cpd-0, and a transient thiyl radical (HS^•) (Figure 3(b)). Furthermore, reaction (1a) (Figures 1S–1C) shows a favorable (−48.6 kcal/mol) hydrogen transfer route from H₂S to Fe(III)−OOH, heme compound 0 complex, resulting in the homolytic cleavage of the O−O and S−H bonds to form the heme ferryl compound II [Fe(IV)=O], a reactive thiyl radical (HS^•), and a water molecule.

Subsequent addition of HS^• to the pyrrole B carbon of heme compound II, [heme Fe(IV)=O], continues to lower the reaction energy (−16.3 kcal/mol) (Figures 1S–1D) to a metastable ring-opened episulfide (). The process allows the formation (−73.3 kcal/mol) of met-aquo-heme Fe(III) three-membered thiochlorinring () (Figures 1S–1E). The five-membered thiochlorin structure is formed () with a small barrier of 2.9 kcal/mol (Figures 1S–1E). The mechanism progression generates a favorable (−135.3 kcal/mol) pathway for sulfheme. However, in reaction (1b) (Figure 3(c)), the proton transfer from His64 to the Fe(III)−OOH is associated with a 5 kcal/mol barrier and a transient hypervalent heme Cpd-I radical. This short-lived radical conformation is extinguished by the immediate integration of the thiyl radical into the heme group. The process leads to a potential energy change of −32.7 kcal/mol upon formation of the Cpd-II and the ring-opened episulfide (Figure 3(d)). The presence of the 3-member ring, , is energetically favored over the opened-ring episulfide by −20.8 kcal/mol (Figure 3(e)). A favorable potential energy change of −7.8 kcal/mol is calculated to form met-aquo-sulfMb species and the SH⁻ anion. The isomeric five-member ring structure is favored over the arrangement by only −2.8 kcal/mol. There is a favorable potential energy decrease, −69.1 kcal/mol, from Cpd-0 to sulfMb (). A primary conclusion from these results is that these reactions primarily differ mechanistically and energetically towards Cpd-0 formation. Yet, the sulfheme product and the intermediate states, except Cpd-I formation, are essentially the same [53, 59, 62].‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

3.4. SulfMb and SulfHb Formation: Insight of the Charge Transfer Band Intensity

Figure 6 shows sulfMb and sulfHb formation from the reaction between oxy-Mb and oxy-Hb with H₂S under the same experimental conditions of 0.05 mM HEPES buffer solution, pH 7.4, and five hours of reaction time. Both Q electronic transitions at 540 nm and 580 nm regions present similar intensities assigned to π to excitations [42, 45, 50, 57]. At the same time, the 620 nm region is characterized by π to dπ (dyz, dxz) charge transfer transitions [53].

(a)

(b)

Therefore, the absorption ratios of the 620 nm and 580 nm regions (A₆₂₀/A₅₈₀) have been used to estimate the sulfheme reaction yields. In addition, electron paramagnetic resonance (EPR) measurements have been used to determine sulfheme absolute yield [42, 46, 55, 98–100]. Figure 6 indicates that sulfHb presents smaller (A₆₂₃/A₅₇₆) ratios than sulfMb (A₆₁₈/A₅₈₂). Factors producing this behavior include (i) sulfheme synthesis techniques, (ii) reaction time, (iii) sulfheme ligand species, (iv) autoxidation processes, and (v) pH. However, the 623 nm sulfHb intensity (Figure 6(b)) is also influenced by the tetrameric nature of oxy-Hb [42, 46, 53, 55, 98–100]. Experimentally, 80% yields have been obtained for pure sulfHb formation. Under these circumstances, the process is initiated from the reaction between met-aquo-Hb and H₂O₂, followed by the complete sulfheme product oxidation using potassium ferricyanide, K₃Fe(CN)₆, to obtain the hydroxyl sulfheme [hemeFe(III)-OH] form at pH 8.5. The species undergoes reduction to deoxy-sulfheme, and when exposed to high molecular oxygen pressure, oxy-sulfHb is produced [42, 46, 53, 55, 98–101]. However, as time evolves, the autoxidation process leads to hydroxyl-, deoxy-, and oxy-sulfheme resulting in the intensity decrease and a broad 623 nm band. Moreover, not all monomeric subunits of Hb generate sulfheme, suggesting a mixture of heme and sulfheme within the Hb tetrameric structure. The generation of different sulfHb centers, from one to four, also limits, relative to sulfHb, the intensity of the 623 nm region. These results have been beneficial in explaining the 10% to 15% sulfHb yield from a patient-derived sample [98–100]. Therefore, the lower 623 nm intensity of sulfHb suggests that the approach can be further used, with caution, to comprehend and directly detect the landscape of these sulfheme derivatives in human tissues and red blood cells. Otherwise, the result could be interpreted as lacking sulfHb formation, contrary to the literature observations [13, 32–41].

4. Conclusions

‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬Experimentally, the reaction between oxy-Mb and H₂S at pH 5.00 and 6.60 showed that the distal doubly protonated tautomer His64_εδ in the heme active site promotes faster sulfMb formation than the mono-protonated analog, His64_ε. At more acidic pH, the 622 nm charge transfer band, characteristic of sulfMb, increases in intensity, reaching its maximum absorbance in 30 minutes, while the process at the higher pH takes 350 minutes. Thus, as pH increases, the transformation from the triplet state to the singlet state is preferred, explaining the intensity decrease of the 622 nm transition. Also, results show that in sulfMb and sulfHb, the differences between the 618 nm and 623 nm transition intensities could be attributed to partial sulfheme presence in the heme groups of the oxy-Hb tetrameric protein. Further research is required to understand the sulfheme derivative formation in red blood cells. Moreover, employing hybrid QM/MM methods aids in elucidating the mechanism and energetics governing the reaction between oxy-Mb and H₂S (reaction (1b)) in aqueous media, leading to the formation of sulfMb. The data indicated that pH changes and the protonation state of the distal His 64 regulate the Cpd-III triplet population that drives the intermediates (Table 1) to favorable energetics (−69.1 kcal/mol) towards sulfMb () formation. The hydrogen bonding network between His64_εδ, heme Fe(II)O₂, and H₂S is responsible for the phenomenon. Furthermore, Table 1 presents the energetics of reaction intermediates for the interaction between met-aquo-Mb [Fe(III)] and H₂O₂ in the presence of H₂S, resulting in sulfMb formation (reaction (1a)). Notably, the process exhibits a favorable sulfheme formation () with an energy of −135.3 kcal/mol. Furthermore, Table 1 summarizes intermediate pathways for oxy-Mb and H₂S (reaction (1b)) and met-aquo Mb, hydrogen peroxide, and H₂S (reaction (1a)) processes. The results support the hypothesis that the energy barrier towards the heme Cpd-0 formation is responsible for the energetic difference between these two unique reactions to produce sulfMb. Likewise, both mechanisms suggest, albeit with different energetics, that the formation of the thiyl radical HS^• is the key species driving the attack on the β-β double bond of heme pyrrole B, leading to a five-member ring sulfheme structure. However, the experimental determination of whether the final stable products in sulfMb and sulfHb are exclusively met-aquo-sulfMb and aquo-sulfHb or a mixture of various sulfheme-ligated species, including hydrogen sulfide as a ligand bound to the heme group, remains a subject of controversy [56, 63, 102, 103].

Data Availability

The data used to support the findings of this study are included within the article. Additional information is available upon request to Ángel D. Rodriguez-Mackenzie and Lysmarie Santos Velazquez.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This research was supported by PR-INBRE, funded by NIH/NIGMS, under grant no. P20GM103475. In addition, we thank the support of the Alfred P. Sloan Foundation Graduate Scholarship Program.

Supplementary Materials

Figure 1S: The energy barrier from ferric hydroperoxyl moiety (Figure 1S-A) to heme compound 0 (Figure 1S-B) is only 1.9 kcal/mol. There is an energetically favorable hydrogen transfer, −48.6 kcal/mol, from H₂S to Fe(III)−OOH (Figures 1S-1C). The heme compound 0 complex enables the homolytic cleavage of the O−O and S−H bonds to form heme ferryl compound-II, a reactive thiyl radical (HS^•), and a water molecule [104]. Figures 1S–1D show the subsequent addition of HS^• to the pyrrole B carbon of heme compound-II, which continues to lower the reaction energy (−16.3 kcal/mol) resulting in a metastable ring-opened episulfide (SB). The reaction is followed by the formation (−73.3 kcal/mol) of met-aquo-heme Fe(III) three-membered thiochlorin ring (SA), Figures 1S–1E. Finally, the five-membered thiochlorin structure is formed (SC) with a small 2.9 kcal/mol barrier, Figure 1S–1F. Figure 2S: Sperm Whale Mb solvated in a 71Å³ water cube. Including the entire heme group, O₂ ligand, two H₂S molecules, distal amino acid residues; His64, Val68, Phe43, Arg29, and proximal His93. (Supplementary Materials)

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Copyright © 2024 Angel D. Rodriguez-Mackenzie et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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