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

Te chemistry of hydrogen sulfde (H 2 S) has been directed towards physiologically relevant hemeproteins, including myoglobin, hemoglobin, and other similar proteins. Despite substantial eforts, there remains a need to elucidate the mechanism and identify the species involved in the reaction between oxy-hemeproteins and H 2 S. 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 diferences in intensity between sulfMb and sulfHb chromophores in the 620nm charge transfer region. Tis behavior could be attributed to the incomplete reaction of tetrameric oxy-Hb with H 2 S, where not all heme groups form sulfheme. Te data also show that, for the reaction of oxy-myoglobin (oxy-Mb) and H 2 S, the 622nm charge transfer band increases in intensity from a pH of 6.6 to 5.0. Tis 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, His64 ε . Computational hybrid QM/MM methods support the conclusion, indicating that oxy-Mb His64 εδ (pH 5.0) reacts with H 2 S in the triplet state, favored by − 31.0kcal/mol over the singlet His64 ε (pH 6.6) species. Te phenomenon is facilitated by a hydrogen bonding network within the heme pocket, between His64 εδ , heme Fe(II)O 2 , and H 2 S. Te results establish an energetically favored quantitative mechanism to produce sulfMb ( − 69.1kcal/mol) from the reactions of oxy-Mb and H 2 S. Curiously, the mechanism between met-aquo Mb, H 2 O 2 , and H 2 S shows similar reaction pathways and leads to sulfheme formation ( − 135.3kcal/mol). Te 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.

Radioactive sulfde species demonstrated that a single sulfur atom is incorporated into the heme structure per mol of Mb to form sulfMb [47].Te insertion resulted in a covalent heme modifcation by the specifc 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.Te 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][50][51][52]. Te 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).Tus, 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 H 2 O 2 in the presence of H 2 S. Te 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 diferent pathways have been proposed [46,47,55,57,58] to explain sulfheme formation.In one scheme, Berzofsky et al. [46,47] suggested that the hydrosulfde ion (HS − ) may be the initial reactant species because ∼70% of H 2 S 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 fnal products [57,58].
QM/MM calculations showed that a concerted interaction among the heme, distal His64, H 2 O 2 , and H 2 S led to the formation of the thiyl radical, HS • , necessary for sulfheme formation [53,56,59].Te calculations suggest a sulfheme mechanism initiated by hydrogen bonding interactions among heme Fe(III)−H 2 O 2 , His64, and H 2 S. Figure 1S (supplementary information) depicts a favorable formation of the intermediate Cpd-0 [Fe(III)OOH] that reacts with hydrogen sulfde 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 • .Te reaction is subsequently followed by a reaction between hemeFe(IV) =O and the HS • radical, leading to a −135.3kcal/mol potential energy decrease which correlates with the sulfMb kinetics (2.5 ± 0.1 × 10 6 M −1 s −1 ) and stability.Although the mechanism to generate the heme ferryl compound II and a thiyl radical, HS • , is diferent from the model proposed by Nicholls [57], the calculated energetic data indeed supported their hypothesis of the reaction between hem-eFe(IV)=O and the HS • to produce sulfheme.Furthermore, contrary to the classical peroxidative reaction of heme proteins with H 2 O 2 [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 sulfde [18][19][20][21][22][23][24][25].
Early results show that the reaction between oxy-Mb and hydrogen sulfde, 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 H 2 S to heme compound 0 [Fe(III)-OOH, Cpd-0].Tus, Scheme 2 presents a 23.3 kcal/mol barrier coupled with the homolytic cleavage of an S-H bond, following the favorable (−11.2kcal/ 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.Te report presented here builds upon earlier research [62], examining the infuence of pH on the populations of hemeligand triplets and singlets, as well as the His64 proton relay within the heme pocket.Te results show that oxy-Mb interacting with H 2 S to produce the 5-member ring metaquo-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-tosinglet transition afects the 622 nm absorption intensity and the population of sulfheme.
Hydrogen sulfde solutions were prepared from sodium sulfde salts (Na 2 S) in the same bufer solution as the hemeproteins.Hydrogen sulfde stock solution (83 mM) was prepared in an amber vial with 0.020 g of Na 2 S•9 H 2 O salt (Alfa Aesar) previously purged with nitrogen and 1.0 mL of HEPES 0.0500M bufer at a pH of 7.4.In HEPES bufer at pH 7.4, there is an equilibrium between 80% SH − and 20% H 2 S, while in phosphate and succinic acid, at pH 5.0 and 6.6, the predominant species is H 2 S. At pH 7.4, sulfmyoglobin (sulfMb) and sulfhemoglobin (sulfHb) derivatives in the HEPES bufer were prepared by the reaction between oxy-Mb (55 μM) and oxy-Hb (55 μM) solutions with hydrogen sulfde (H 2 S) solution in a molar ratio of 1 : 3 (oxyhemeprotein H 2 S).Te hydrogen sulfde concentration was 165 μM.Te scenario allowed us to understand the diferences 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 bufer solution by mixing the oxy-Mb solution with H 2 S solution at the same pH in a 1 : 100 molar ratio, oxy-Mb : H 2 S. UV-Vis spectral data were collected on a SHIMADZU 2600 UV-Vis with a temperature controller set at a constant temperature of 25 °C.

Teoretical Methods for Sulfmyoglobin Formation.
Te 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 feld with CMAP correction [68] and the TIP3P water model [69], respectively.Te description of the heme moiety used a CHARMM-modifed force feld [70].Te active site residue His64 was modeled in acidic pH with N ε and N δ 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.Te models were all geometry optimized for 1000 steps using the steepest decent algorithm.Te 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.Tese 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.

Refnement 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 cutof distance of 8 Å and an outer cutof distance of 14 Å.Te QM region consisted of the entire heme group, propionate and vinyl groups, residue His93, and the O 2 ligand for 92 atoms in the singlet state [73].Te models were geometry optimized with the SD algorithm for 1000 steps.Te atomic velocities were scaled every 50 steps (1 fs time step) to simulate the models from 10 K to 300 over 3 ps.Te models were then simulated for another 10 ps at 300 K. Te fnal snapshot was used to model the interactions of H 2 O and H 2 S molecules with oxy-Mb using the Density Functional Teory (DFT) method.Visualization of the structures and analysis were performed with the VMD program [74].

DFT/MM Calculations Sulfmyoglobin Formation.
Hybrid DFT/MM simulations were performed to unravel the sulfmyoglobin formation mechanism from the oxy-Mb reaction with H 2 S with pDynamo interfaced with the ORCA program version 4.1 [75].Te 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 fxed in position.Two water molecules that always entered and remained in the active site His64  calculation.Te hydrogen link atom approach was used to defne the QM/MM boundary.Before substituting the H 2 O molecule(s) with H 2 S 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].Te basis set was Ahlrichs triple-zeta with polarization TZVP and the Def2/J [81][82][83] resolution of identity approximation (RI).A Stuttgart-Dresden-Bonn relativistic efective core potential (ECP) was used for the iron atom [84,85].Te calculations use the libint2 library to compute 2-electron integrals as specifed by ORCA [86].A total of thirty models were constructed by alternating multiplicity, H 2 S/H 2 O molecule positions, and availability of the Fe atom.After the replacement of H 2 O by H 2 S molecules, the models were geometry optimized for another 300 steps using the SD algorithm.Te energetic profle 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 profle 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.Te functional was modifed to have an HF exchange of 13%, providing more accurate energy values for iron-containing systems [89,90].Te quasi-restricted orbitals obtained from the DFT-defned region were used to explore the efects 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.Te 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.Te active space consisted of iron's 3d orbitals and six 2p orbitals of O 2 , resulting in 11 orbitals and 14 electrons [91].

Insight of pH-Dependent Sulfmyoglobin Formation.
Te pH dependence is crucial in deciding the histidine amino acid tautomeric population and its role in protein function [92][93][94].For example, myoglobin, at neutral pH, His64 ε (HisE7), is predominantly mono-protonated on its nitrogen (N ε ), and its swinging motion opens and closes the heme pocket to ligand migration [95][96][97].Tus, the heme-O 2 -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 timedependent sulfMb formation from the interaction of oxy-Mb with H 2 S (reaction (1b)) under conditions of His64 ε (pH 6.60) and His64 εδ (pH 5.00).Te consumption of H 2 S 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][46][47]53].
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)).Te 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 redto-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-fow spectrophotometry, revealed that, at the more acidic pH of 5.0 during the reaction of oxy-Mb with H 2 S, there is a transition of the Soret band from 414 nm to 416 nm, reaching a maximum absorbance value at 100 ms.Te 2 nm red shift was suggested to be caused by the presence of heme Cpd-0 [Fe(III)OOH].Hence, the fndings indicate that protonation of the delta nitrogen of His64 εδ (as defned 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.Te 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.

Insight into Sulfmyoglobin Formation Mechanism.
Te visible spectroscopic data between oxy-Mb and H 2 S 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 H 2 S 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 H 2 S 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.Tis electronic confguration is consistent with the heme compound III [Fe(III)O-O •− , Cpd-III] also known as superoxy-Mbor the Weiss confguration [62] shown Journal of Chemistry in Figure 3(a).Advancing along the reaction coordinate in Figure 3  proton to His64 εδ .Tis process is energetically favorable by −7.8 kcal/mol, resulting in the formation of the met-aquo-sulfMbS c species and the SH − anion (Figure 3(f )).Concurrently, steric efects from the departing water molecule result in the integration of spin density into the heme group via sulfheme isomeric structure formation.Te S C isomeric structure, Figure 3(f ), is favored over the S A arrangement by only −2.8 kcal/mol.As shown in Figure 5, geometrical fexibility and increased conjugation in the heme porphyrin π system, facilitated by the 5-membered ring, may contribute to the structural preference.Te preference for this conformation is consistent with our previous work, with a met-aquo Mb analog and hydrogen sulfde (reaction (1a)) [53,59].Te orientation of the water molecule allows it to function as a bridge between the hydrosulfde and His64 εδ .Te 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 metaquo-sulfMb.Overall, the formation of the water molecule during the breaking of Cpd-0 O-O bond reveals a concerted proton relay.Te pH-dependent triplet state and the His64 εδ proton relay are responsible for these specifc reaction pathways and the stability of the fnal products.Also, the mechanism presented in Figure 3 is analogous to the oxy-heme open-shell singlet biradical state.Te calculated potential energy profles do indicate some diferences, given that in oxy-Mb His64 εδ interaction with H 2 S, the triplet state is favored by 31.0 kcal/mol, while hydrogen sulfde stabilizes the complex by 7.0 kcal/mol in the singlet scenario.

Sulfheme Pathways: Analogous but Exclusive.
Interestingly, the sulfheme formation mechanisms, presented in Figure 1S (reaction (1a)) and Figure 3 (reaction (1b)), respectively, show diferent 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 H 2 S produces the radical.In the latter scenario, the reaction between Cpd-III (Fe(III)O-O •− and H 2 S 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 (S C ).Although the mechanism to generate HS • difers from the model proposed by Nicholls [57], the calculated energetic data supported its presence.Tus, the heme iron oxidation state [FeIII) or Fe(II)], coordinated ligand (H 2 O 2 or O 2 ), singlet or triplet spin states, and pH defne the number of electrons available for the reaction processes.Furthermore, the hydrogen bond interactions between heme Fe(III)-H 2 O 2 or heme Fe(II)-O 2 , heme distal His64, and H 2 S also regulate the process.

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 H 2 S under the same experimental conditions of 0.05 mM HEPES bufer solution, pH 7.4, and fve hours of reaction time.Both Q electronic transitions at 540 nm and 580 nm regions present similar intensities assigned to π to π * 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].

Conclusions
Experimentally, the reaction between oxy-Mb and H 2 S 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 .10

Journal of Chemistry
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.Tus, 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 diferences 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.processes.Te results support the hypothesis that the energy barrier towards the heme Cpd-0 formation is responsible for the energetic diference between these two unique reactions to produce sulfMb.Likewise, both mechanisms suggest, albeit with diferent 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 fve-member ring sulfheme structure.However, the experimental determination of whether the fnal stable products in sulfMb and sulfHb are exclusively met-aquo-sulfMb and aquo-sulfHb or a mixture of various sulfheme-ligated species, including hydrogen sulfde as a ligand bound to the heme group, remains a subject of controversy [56,63,102,103].

Figure 2 :Figure 3 :Figure 4 :
Figure 2: UV-Vis spectra of the time evolution for the reaction between oxy-Mb and H 2 S (reaction (1b)) at pH 5.0 (a, b) and 6.6 (c, d) for 80 and 450 minutes, respectively.
Heme Fe(III)-OOH, Cpd-0 Transition and formation +1.9 (kcal/mol) Heme Fe(III)-OOH, Cpd-0, and HS • Transition +23.3 kcal/mol formation Heme Fe(IV)�O Cpd-II and HS • −48.6 kcal/mol Heme Fe(IV)�O Cpd-II and ring-opened episulfde (S 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.Tis short-lived radical conformation is extinguished by the immediate integration of the thiyl radical into the heme group.Te process leads to a potential energy change of −32.7 kcal/mol upon formation of the Cpd-II and the ring-opened episulfde (Figure 3(d)).Te presence of the 3-member ring, S A , is energetically favored over the opened-ring episulfde by −20.8 kcal/mol (Figure 3(e)).A favorable potential energy change of −7.8 kcal/mol is calculated to form met-aquo-sulfMbS c species and the SH − anion.Te S C isomeric fve-member ring structure is favored over the S A arrangement by only −2.8 kcal/mol.Tere is a favorable potential energy decrease, −69.1 kcal/mol, from Cpd-0 to sulfMb (S C ).A primary conclusion from these results is that these reactions primarily difer 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].
εδ model during the PM6/MM simulations were replaced by H 2 S molecules.Te QM region consisted of the entire heme moiety, including propionate and vinyl groups, His64, His93, Val68, Leu29, Phe43, O 2 , H 2 S, and H 2 O molecules.A total of 146 atoms were included in the MbHis64 εδ

Table 1 :
Intermediates and energy of met-aquo-Mb [Fe(III)] + H 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 H 2 S (reaction (1b)) in aqueous media, leading to the formation of sulfMb.Te data indicated that pH changes and the protonation state of the distal His 64 regulate the Cpd-III triplet population that drives the intermediates (Table1) to favorable energetics (−69.1 kcal/mol) towards sulfMb (S C ) formation.Te hydrogen bonding network between His64 εδ , heme Fe(II)O 2 , and H 2 S is responsible for the phenomenon.Furthermore, Table1presents the energetics of reaction intermediates for the interaction between met-aquo-Mb [Fe(III)] and H 2 O 2 in the presence of H 2 S, resulting in sulfMb formation (reaction (1a)).Notably, the process exhibits a favorable sulfheme formation (S C ) with an energy of −135.3 kcal/mol.Furthermore, Table1summarizes intermediate pathways for oxy-Mb and H 2 S (reaction (1b)) and met-aquo Mb, hydrogen peroxide, and H 2 S (reaction (1a))