Mechanism of Reduction of Ferric Porphyrins by Sulfide: Identification

Mar 15, 2017 - Cai and Holm prepared a similar iron hydrosulfide complex by reacting H2S gas with [Fe(OEP]2O. Although preliminary NMR data suggested ...
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Mechanism of Reduction of Ferric Porphyrins by Sulfide: Identification of a Low Spin FeIII−SH Intermediate Kaustuv Mittra,# Asmita Singha,# and Abhishek Dey* Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Kolkata, India 700032 S Supporting Information *

ABSTRACT: The reaction of FeIII porphyrin complexes bearing distal hydrogen bonding residues with sulfide/hydrosulfide is kinetically monitored to reveal the presence of an intermediate and a kH/kD of 3.0. This intermediate is trapped at low temperatures and investigated with resonance Raman and electron paramagnetic resonance spectroscopy. The results, corroborated by density functional theory calculations, indicate that this species is a sixcoordinate low spin hydrosulfide bound ferric porphyrin. The homolytic cleavage of the FeIII−SH bond resulting in the formation of a ferrous porphyrin and hydrosulfide radical (trapped with 5,5-dimethyl-1-pyrrilone-N-oxide) is found to be the overall rate-determining step of the reaction.

1. INTRODUCTION Hydrogen sulfide (H2S) is a naturally occurring signaling molecule that plays a major role in the cardiovascular, neuronal, and immune systems.1,2 H2S is a physiologically significant dilator in cerebral circulation.3−5 It is reported to relax blood vessels and lower blood pressure by opening ATP-sensitive K+ channels in vascular smooth muscles.6,7 Produced endogenously from cystathionine-β-synthase (CBS), cystathionine-γlyase (CSE), and cysteine amino transferase (CAT) in combination with 3-mercaptosulfurtransferase (3MST), H2S exists primarily as its anionic hydrosulfide (HS−, pka1 of H2S = 7.0) under normal physiological conditions. H2S has also been shown to induce hibernation in rats and pigeons, which has been related to the reversible inhibition of cytochrome c oxidase, a heme enzyme responsible for the reduction of O2 to H2O in the mitochondria by an enhanced level of H2S in the blood.8−10 Despite having some important physiological functions, H2S can be a toxin.11 Increasing levels of H2S in the body can be a result of overexpression of cystathionine-γ-lyase, which is a key enzyme in the trans-sulfuration pathway.12 Excess H2S can result in a refractory circulatory shock resulting from a direct inhibition of mitochondrial cytochrome c preventing mitochondrial ATP production.13 Methemoglobin (where the iron in heme is oxidized to its ferric state) can form very strong Fe−S bonds with H2S.14 In fact methemoglobin has been proposed to treat cases of H2S poisoning.15,16 H2S can attack the porphyrin ring in heme to form sulfhemoglobin resulting in a physiological condition called sulfhemoglobinemia.17−19 Sulfhemoglobin is a stable, green-pigmented molecule which cannot carry oxygen. Formation of sulfhemoglobin is irreversible and can last the lifetime of the red blood cell.20 A hydrosulfide bound ferric porphyrin species (FeIII−SH) is often © 2017 American Chemical Society

invoked to be an intermediate involved in the formation of sulfhemoglobin. Keilin in 1933 presented data that demonstrated that a new red product could be formed by the anaerobic reaction of H2S with methemoglobin (pKa = 7.0).21 This is commonly believed to be a hydrosulfide adduct of heme.22 One of the earliest known iron-hydrosulfide porphyrin comp lexes [5,10,15,20-t etr akis(4-methoxyphenyl)porphyrinato] (hydrosuphido) iron(III) (Fe(TAP)SH) was isolated by Scheidt and co-workers.23 It was reported it to be a five-coodrinate (5C) low spin (LS) FeIII complex which forms a dimer in the presence of oxygen. Recently, Scheidt and coworkers established the binding of hydrosulfide (HS−) to a FeII porphyrin.22 In general it is well-known that sulfide can reduce FeIII porphyrin to FeII porphyrins and itself get oxidized to elemental sulfur in organic solvents. 8,24 However, the mechanism of this reaction still remains to be explored in detail. An inner sphere mechanism will entail formation of a hydrosulfide (HS−) bound ferric porphyrin. Cai and Holm prepared a similar iron hydrosulfide complex by reacting H2S gas with [Fe(OEP]2O. Although preliminary NMR data suggested it to be a transient high-spin FeIII species which decays to FeII within 5 min,25 to date the existence of the Fe− SH bond has only been indirectly demonstrated in any of these proposed hydrosulfide complexes/intermediates of ferric porphyrins so far.26,27 In this manuscript, we have used two iron porphyrins (FeEs4 and FePf, Figure 1) with hydrogen bonding (H-bonding) residues in the distal pocket to investigate the reaction mechanism of reduction of ferric porphyrins with sulfide/ hydrosulfide. The H-bonding interactions aim at stabilizing Received: December 5, 2016 Published: March 15, 2017 3916

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Figure 1. Models investigated in this manuscript.

Table 1. DFT Calculated and Experimental (FePf) Vibrational Frequencies (cm−1) and Their H/D Isotope Shifts calculated frequency (isotope shift) BP 86

observed shift in FePf (isotope shift)

CH3OH

CD3OH

CH3OD

CD3OD

CH3OH

CD3OH

CH3OD

CD3OD

assignment

394 383 543 1007 1065 1360 1568

394 383 543 960 858 1360 1568

389 383 432 1002 858 1360 1568

389 383 428 953 772 1359 1568

394 380 539 896 1107 1365 1560

394 380 539 896 600−900 1365 1560

378 380 429 792 600−900 1365 1560

387 380 412 792 600−900 1365 1560

Fe−S stretching coupled with Fe−O(H)-Me bending ν8 Fe−OMe(H) stretching C−O stretching (MeOH) H−O−CH3 inplane bending ν4 ν2

recorded in an Agilent UV−vis spectrophotometer. GC-MS data were collected on an Agilent instrument. 2.1. Experimental Details. All solvents used in this investigation were dried and degassed (three cycles of freeze−pump−thaw) before use. All the preparation and reactions were performed inside a N2 glovebox or in airtight sample holders. To prepare 1 mM solution of the metallo-porphyrins, a 1.167 mg portion of α4-FeEs4 or 1.066 mg of α4-FePf complex was dissolved in 1 mL of dry degassed dichloromethane (DCM) solvent. A 2.4 mg portion of Na2S was dissolved in a minimum volume of methanol and diluted with dry degassed DCM to make the final volume 1 mL, so that the final strength of the solution was 10 mM. The NaSH solutions were prepared in the same way. Next 200 μL of this 1 mM FeEs4 and FePf solution was taken in two EPR tubes. To one of these EPR tubes, 1 equiv (generally ∼20 μL solution) of Na2S/NaSH was added at −80 °C. EPR and rR of both the tubes were recorded. The UV−vis experiments to monitor the kinetics of formation of the intermediate was done by adding 1 equiv of Na2S (dissolved in methanol) to a 2 mL 10 μM DCM solution of FeEs4. For GC-MS experiments 10 mM of FeEs4 was reduced by Na2S in DCM solvent as above. The solvent (DCM-MeOH) was evaporated, and an equal volume of CCl4 added to dissolve the precipitated sulfur and keep the concentration to 10 mM. 2.2. Computational Details. All calculations were performed at the IACS Inorganic HPC cluster using Gaussian 03 software.31 Both BP8632 and B3LYP33,34 functionals were used to obtain the optimized geometries and frequencies (Table 1). A mixed basis set with 6-311 g* on the Fe, N, and S atoms and 6-31g* on the C and H atoms were used for optimization and frequency calculations (Table 1). No negative frequencies were found for the structures reported. The potential energy scans and energy calculations (single point and solvent corrections) were done on a unsubstituted iron-porphyrin model obtained by replacing the phenyl rings of tetraphenylporphyrin (TPP) with hydrogens (Figure 11). The BP86 functional and 6311+g* basis set on all atoms was used. The PCM model was used for solvent correction, and dichloromethane was used as a solvent.

potential intermediates, if any, formed during the course of the reaction. While FeEs4 can act as both H-bond donor and acceptor, FePf can only act as a H-bond donor. However, the H-bonds between the amide groups present in FePf and an axial hydroxide (HO−) ligand have been found to be weak because of the large distance between the bound −OH and the amide N.28 On the other hand, because of its larger size, sulfur derived ligands can form H-bonds with the amides of FePf. Additionally, the triazole environment (FeEs4) is more hydrophilic than the picket fence environment (FePf) due to the pivolyl substitution in the latter.28 An intermediate is indeed isolated at cryogenic temperature. The isolated Fe−SH intermediate is identified using resonance Raman (rR) and electron paramagnetic resonance (EPR) spectroscopy to be a six-coordinated (6C) LS methanol (solvent) bound MeOH− FeIII−SH species. The reduction of this intermediate proceeds via homolytic cleavage of FeIII−SH bond.

2. MATERIALS AND METHODS The iron porphyrin complexes (FePf and FeEs4 in Figure 1) are synthesized as previously reported.24,29,30 Sodium sulfide (Na2S) was purchased from Rankem, methanol-d (CH 3 OD), methanol-d 3 (CD3OH), methanol-d4 (CD3OD), and 5,5-dimethyl-1-pyrrilone-Noxide (DMPO) were purchased from Sigma-Aldrich (U.S.A.). Methanol (MeOH), dichloromethane (DCM), and carbontetrachloride (CCl4) were purchased from Rankem. All the electron paramagnetic resonance (EPR) spectra were recorded on a JEOL instrument. Resonance Raman (rR) data were collected using 413.1 nm excitation from a Kr+ ion source (Sabre Coherent Inc.) and a Trivista 555 triple spectrophotometer (gratings used in the three stages were 900, 900, and 1800/2400 grooves/mm) fitted with an electronically cooled Pixis CCD camera (Princeton Instruments). The irradiation power was limited to 10 mW at the sample to avoid degradation. Data were collected at room temperature or at 77 K (in a liquid N2 cooled finger dewar). UV−vis absorption spectra were 3917

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3. RESULTS 3.1. Absorption Spectroscopy. The absorption spectrum in the visible region of the α4-FeEs4 complex in DCM solution shows a Soret band at 422 nm and weaker Q bands at 517 nm, 577 nm, 635 nm, and 681 nm (Figure 2, black).35 This complex

population of this intermediate at RT. It could only be generated at high yields at cryogenic temperatures (section 4.2 and 4.3 below). 3.2. Resonance Raman (rR). The rR spectra of iron tetraphenylporphyrins have multiple modes which are sensitive to the oxidation and spin state of the iron center. For example, the marker bands ν4 (pyrrole half ring symmetric stretch involving N, C2 and C5 atoms of four pyrroles) and ν2 (pyrrole C3−C4 symmetric stretch involving all four pyrroles) of high spin (HS) FeII iron porphyrin complexes are observed at 1343−1345 cm−1 and 1540−1543 cm−1, respectively. Similarly, these vibrations occur at 1360−1363 and 1555 cm−1 for high spin FeIII, 1365−1366 cm−1 and 1563−1566 cm−1 for LS FeIII and 1369−1372 cm−1 and 1568−1570 cm−1 for FeIV=O species.38−40 In tetraphenylporphyrins both the ν4 and ν2 bands shift with oxidation and spin state with high fidelity and can be used to unambiguously assign the oxidation and spin state of the iron.24,38 Observation of an intense ν3 is generally associated with a 5C species.41,42 These vibrations are summarized in Table S2, Supporting Information. Resonance Raman(rR) data of the oxidized sample of FeEs4 shows that the ν4 and the ν2 bands are at 1361 and 1553 cm−1, respectively (Figure 4A, blue), which are characteristic of S = 5/2 FeIII porphyrins. When 1 equiv of Na2S (or NaSH) is added to the oxidized sample at room temperature, the oxidation and spin state marker ν4 and the ν2 bands shift to 1343 and 1543 cm−1, indicating reduction of FeIII porphyrin to FeII porphyrin (Figure 4A, green).38 However, when the sample is frozen rapidly ( 1 h). The ν4 and ν2 bands of this intermediate species are at 1369 and 1568 cm−1 (Figure 4B, orange), indicating that the intermediate is a 6C low-spin S = 1/2 FeIII species.38 Note that addition of 0.5 equiv of Na2S to the initial FeIII oxidized results in partial formation of the intermediate LS species at −80 °C, while the rest of the sample remains high spin FeIII (Figure S2, Supporting Information) suggesting that the formation of this intermediate requires a stoichiometric amount of sulfide.

Figure 2. UV−vis absorption spectra of the oxidized (in black) and reduced forms (in red) of FeEs4.

is reduced to its ferrous state in nonpolar dry deoxygenated solvent like DCM by the addition of a half equivalent of Na2S (S2− is a two electron reductant) dissolved in dry degassed methanol. This is indicated by the red shift of the Soret and the Q-band from 422 and 514 nm in the oxidized form (Figure 2, black) to 428 and 534 nm in the reduced form (Figure 2, red).8,29,36,37 The same reduction is observed when NaSH (0.5 eq) is used but not when H2S is used as a reducing agent (Figure S1, Supporting Information). The kinetics of reduction of the oxidized FeIII porphyrin to the reduced FeII porphyrin is monitored by following the absorption intensity at 549 nm in a DCM solution with 0.2% MeOH (required to dissolve Na2S). The reduced species is characterized by a significant enhancement of absorption intensity at 549 nm (used to monitor the kinetics in Figure 2). The reduction is found to involve at least two steps (Figure 3A), and the first step is too fast to quantify its kinetics at RT. The second step is independent of Na2S concentration (Table S1, Supporting Information) and proceeds with a first order rate of 0.026 ± 0.002 s−1 (Figure 3B blue). The reduction kinetics for this second step is slower in methanol-d4; i.e., the process has a H/D isotope effect with a kH/kD = 3. The kinetic trace clearly shows the formation of an initial intermediate species which then quickly decays to the final reduced species (t1/2 ≈ 20 s at RT), making it difficult to generate significant

Figure 3. (A) Kinetic trace of the reduction reaction at 421 nm. (B) Kinetic trace of the reduction reaction followed at 549 nm in MeOH (blue) and in MeOD (orange). The kinetic data have been fitted in the polynomial [A]0(1 − exp(−kt)) to calculate rate constant and isotope effect of the reduction reaction. 3918

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Figure 4. (A) rR spectra of the oxidized (FeEs4-oxidized), reduced (FeEs4-reduced), and the intermediate species (FeEs4-1Na2S-rapid freeze) generated by adding 1 equiv Na2S to FeEs4 at room temperature. (B) rR spectra of the oxidized (FeEs4-oxidized), reduced (FeEs4-reduced), and the intermediate species (FeEs4−SH) generated by adding 1 equiv Na2S at −80 °C to FeEs4. All spectra are recorded at 77K.

presence of a LS ferric species in solution under these conditions consistent with rR data (Figure 4B).44−46 As the temperature is gradually increased from −80 °C, the intensity of the 6C LS EPR signal slowly decreases to finally yield an EPR silent species at room temperature which is consistent with the formation of S = 2 high spin reduced FeII porphyrin species (Figure S4, Supporting Information).24 Upon addition of Na2S at −80 °C to the oxidized FePf complex a 6C FeIII LS species with g values of 2.38, 2.24, and 1.92 (Figure 6B, red) is formed. Similar to FeEs4, the FePf complex also gets reduced to FeII on warming the LS 6C Ferric intermediate to room temperature. The V/λ of the 6C LS FeIII species is determined from the g-values to be 5.33 and 5.28 for the intermediates obtained using FeEs4 and FePf, respectively (Figure S5, Supporting Information). The large V/λ is suggestive of the presence of a very covalent π anisotropic donor like HS−. The g-values and the V/λ are similar to that of resting P450 (g = 2.45, 2.26, 1.91, V/λ = 4.59) and related thiolate bound FeIIIporphyrin complexes.44,46,47 3.4. Identification of Intermediate. Since the addition of 1 equiv of NaSH or Na2S at −80 °C converts the initial FeIII from a high spin species to a LS species, the intermediate is likely to be a 6C hydrosulfide adduct of the ferric porphyrin complexes. Generally FeIII−SH vibrations should be observed in the same region as these of FeIII−SR (SR represents thiolate), i.e., between 380 and 420 cm−1 in iron porphyrins.48−50 Additionally, a FeIII−SH vibration may be affected by H/D isotope substitution of the bound −SH ligand and may be used to support the presence of a FeIII−SH bond.51 The synthesis of S34 labeled Na2S is complicated and beyond the scope of this group. The ν8 mode (Fe−Npyrrole symmetric stretching frequency) in rR data of the intermediate formed at −80 °C with the FePf complex shows significant distortion on deuterating the sulfide proton (Figure 7) by dissolving Na2S in CH3OD and CD3OD. Note that ν8 is a Fe−Npyrrole(porphyrin) symmetric stretching mode and should not have any involvement of the Fe−S bond. However, the Fe−S stretching mode often overlaps (not coupled) with the ν8 vibration as it is in the same energy region. This was also found to be the case for the high spin P450 active site as reported by Champion et al.38,52−54 Difference spectra shown in Figure 7a indicate that a vibration at 394 cm−1 downshifts to 387 and 378 cm−1 on deuteration with CD3OD and CH3OD, respectively. This is consistent with the presence of a bound SH ligand, and the difference between CD3OD and CH3OD suggests that the MeOH is bound as a trans axial ligand to the hydrosulfide. The coordination of the MeOH is

Therefore, to maximize the formation of the LS intermediate, 1 equiv of Na2S is added at −80 °C wherein almost quantitative yield of the intermediate is obtained. On warming up the samples to room temperature, the bands corresponding to this intermediate species disappear and new bands at 1343 and 1543 cm−1 are observed corresponding to FeII high spin state (Figure 4B, green). Thus, the conversion of initial FeIII high spin to the product FeII high spin takes place via a 6C LS FeIII intermediate. Encouraged by the stability of this intermediate at −80 °C, its presence is also tested in iron picket fence porphyrin (FePf) which has a hydrophobic distal pocket.43,28 The oxidized FePf shows the oxidation and spin state marker ν4 and the ν2 bands at 1362 and 1554 cm−1, respectively (Figure 5, blue), which are

Figure 5. rR spectra of the oxidized, reduced, and the intermediate species generated by adding 1 equiv of Na2S at −80 °C to FePf. All spectra are recorded at 77K.

typical of S = 5/2 FeIII porphyrins. On addition of 1 equiv of Na2S to the oxidized sample of FePf at −80 °C, the bands shift to 1368 and 1568 cm−1, indicating the formation of a 6C lowspin intermediate S = 1/2 FeIII species.38 When the sample is warmed to room temperature, the bands shift to 1345 and 1541 cm−1, indicating a reduced FeII high spin final product. 3.3. EPR. EPR spectra of the oxidized FeEs4 complex exhibit a characteristic g ≈ 6 axial EPR signal (Figure S3, Supporting Information) indicating that the Fe in the α4-FeEs4 complex is in a high-spin S = 5/2 FeIII state, consistent with rR data (Figure 5).35 Addition of 1 equiv of Na2S to the oxidized complex at −80 °C results in complete conversion of the high spin EPR signal to a 6C LS EPR signal (Figure 6A, red) with g values of 2.35, 2.19, 1.92. These values are consistent with the 3919

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Figure 6. EPR spectra of the intermediate Fe-SH species of (A) FeEs4 and (B) FeEs4. All spectra are recorded at 77 K.

Figure 7. rR spectra of Fe-SH intermediate generated by adding 1 equiv of Na2S dissolved in CH3OH (in blue) and CD3OH (in light green) at −80 °C to FePf. The isotope Fe-SD is generated by adding 1 equiv of Na2S in CD3OD (in red) and CH3OD (in brown) medium at −80 °C. All spectra are recorded at 77 K. Panels A and B represent the rR data of 6C Fe−SH intermediate in the region 200−550 cm−1, while panels C and D correspond to the rR data of Fe-SH intermediate in the region 600−1100 cm−1.

also indicated by a vibration at 539 cm−1 which shifts to 429 and 412 cm−1, respectively, upon deuteration with CH3OD and CD3OD. Moreover H/D isotope sensitive vibrations are observed at 344 and 300 cm−1 in protonated samples, which shift to 245 cm−1 on deuteration. Most importantly, these isotope shifts are not observed when CD3OH (no exchangeable deuterium) was used to dissolve Na2S (Figure 7b), which strongly suggests that a sulfide (HS−) ligand is bound to Fe and the modes with contributions from Fe−SH motion are shifting in energy upon deuteration. In higher frequency region, a large H/D isotope shift is observed from 896 to 792 cm−1 upon deuteration with CH3OD

or CD3OD, while this vibration is absent in the rR spectrum of the Fe−SH intermediate with CD3OH as a sixth ligand. Furthermore, a band at 1107 cm−1 in rR spectra of intermediate species is shifted to the 600−900 cm−1 region in CD3OH, CD3OD, and CH3OD. The H/D isotope shifts, in particular, the ones observed with CH3OH/CH3OD, clearly suggest the presence of a bound hydrosulfide in the intermediate isolated at −80 °C. A large number of isotope sensitive bands and the lack of detailed literature on the isotope shifts on such systems entail emulation of these vibrations (and the H/D shifts) computationally. Thus, these data are compared to density functional theory (DFT) calculated vibrations on a Me(H)O− 3920

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Figure 8. Calculated IR spectra of DFT optimized structure of sulfide bound FeTPP adduct with CH3OH (in blue), CD3OH (in light green), CH3OD (in brown), and CD3OD (in red) as the sixth ligand. Please note that the intensities of the transitions may not be compared to experimental data as these calculations reflect IR absorbance. Panels A and B represent the rR data of 6C Fe−SH intermediate in the region 200−550 cm−1, while panels C and D correspond to the rR data of Fe−SH intermediate in the region 600−1100 cm−1.

Figure 9. (A) DFT optimized structure of sulfide bound FeTPP adduct with MeOH as the trans axial ligand. (B) Displacement vectors of the DFT predicted Fe-SH stretch at 393 cm−1. The mode shows the Fe−SH vibration mixed with bending modes of the bound methanol.

FeIII−SH tetraphenylporphyrin (TPP) species where the protons are systematically changed to deuterium. The distal architectures are not included in the computational model to reduce the computational time involved. Theoretically, using a simple harmonic oscillator approximation, a Fe−SH vibration at 394 cm−1 should shift only by 4−6 cm−1 on deuteration.51 Thus, these large H/D isotope effects (11 cm−1) likely suggest the mixing of Fe−S−H vibrations with vibrations of the bound methanol (Figure 9B). This proposal is supported by difference in the rR data obtained in CH3OD and CD3OD. In the past the Fe−S vibrations in the 5C high spin active site of P450 could be obtained by exciting at 350−365 nm.53,54 However, recent reports have shown that exciting the Soret can also excite the Fe−S vibrations in 6C LS

thiolate bound ferric porphyrin complexes.44,48 This could be due to the mixing of the sulfide (HS−) lone pair with porphyrin π* as indicated by the DFT calculated ground state wave function (Figure S9, Supporting Information). The sulfide (HS−) bound FeEs4 also shows vibrations at 388 and 352 cm−1 (Figure S6, Supporting Information, blue to red) which shift to 378 and 338 cm−1, respectively, on deuteration with CD3OD. Thus, the rR data on the intermediate strongly suggest that it is a MeOH bound 6C LS FeIII−SH species. Theoretical vibrational calculations are used to further support these assignments (Figure 8A−D). Geometry optimized DFT calculations on FeTPP based 6C LS MeOH-FeIII−SH species for these species were done using both B3LYP and BP86 functionals. The calculated frequencies 3921

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Figure 10. EPR of DMPO adduct of (A) FeEs4 and (B) FePf. All spectra are recorded at 77 K.

strongly support the assignment of the intermediate species as a 6C-LS methanol bound FeIII−SH species. Thus, the H/D isotope effect on the kinetics, the EPR data, and rR data (and H/D isotope shift) on the cryogenically trapped intermediate all indicate the formation of a LS, 6C, and MeOH (solvent) bound MeOH−FeIII−SH species during the reduction of ferric porphyrin by sulfide in an organic solvent. This implies that the electron transfer from sulfide to FeIII follows an inner sphere mechanism. 3.5. Fate of Hydrosulfide (HS−). It is understood that the III Fe −SH adduct (trapped at −80 °C) is the intermediate which decays to FeII at room temperature. To determine the fate of hydrosulfide (HS−), the reductant, the FeIII−SH was prepared at −80 °C and incubated with 5,5-dimethyl-1-pyrrilone-N-oxide (DMPO) at room temperature. EPR of the so-formed adduct revealed a dominant anisotropic radical signal with g values at 2.078, 2.062, and 2.037 characteristic of the thiyl adduct of DMPO (Figure 10).58 This suggests that a hydrosulfide radical (HS•) is formed as the FeIII−SH adduct gets reduced to FeII; i.e., the reaction proceeds via a homolytic cleavage of the Fe-SH bond. The products of the reaction, i.e., reduction of FeIII to FeII, were analyzed using GC-MS to understand the fate of NaSH. The results indicate formation of elemental sulfur, when compared with that of GC-MS data of pure sulfur powder, as the end product (Figure S7, Supporting Information).

(Table 1) indicate that the BP86 functional reproduces both the intraligand and metal ligand vibrations well (B3LYP results in Table S1 show substantially weaker metal ligand vibrations). A theoretically calculated FTIR spectrum can be used to gauge the isotope shifts in different normal modes to compare it with the experimental data, albeit the intensities cannot be compared. A vibration at 394 cm−1 was calculated to have significant Fe−SH motion (Figure 7A−C). This mode also show significant mixing with bending modes of the bound MeOH. On replacing the hydrosulfide (HS−) ligand with deuterated sulfide (DS−) and CH3OH with CD3OD or CH3OD (as the sixth ligand), this vibration shifts to 389 cm−1. The calculated H/D shift of 394 cm−1 to 389 cm−1 (Figure 7A, ν8 calculated at 383 cm−1 and does not shift on dueteration) agrees quite well with the experimentally observed shift from 394 to 387 cm−1 and 378 cm−1 in CD3OD and CH3OD, respectively, in the FePf complex. The energy of the Fe−S vibration is consistent with those reported for LS ferric thiolate systems in the literature.48,55 The calculations also predict a Fe−O(Me)H bending mode of the bound MeOH at 543 cm−1, which should shift to 428 and 432 cm−1 on deuteration with CD 3 OD and CH 3 OD (Figure 8A), respectively. This agrees reasonably well with the experimental vibration at 539 cm−1 which shifts to 429 and 412 cm−1 on deuteration with CH3OD and CD3OD, respectively. An earlier report by Kitagawa showed the Fe−O(Me)H vibration of a MeOH bound Fe(OEP), with a 2-methyl-imidazole axial ligand, to be at 524 cm−1, which is consistent with our observation.56 The weaker Fe−O(Me)H vibration, when compared to Fe(OEP) bound MeOH, in these complexes is because the greater push effect of the negatively charged thiolate axial ligand (as compared to the neutral 2-methyl-imidazole), which weakens the trans axial Fe−O(Me)H bond.57 Finally the C− O stretching frequency of the bound MeOH, experimentally observed at 896 cm−1 which shifts to 792 cm−1 upon deuteration, is calculated to be at 1007 cm−1, which shifts to 953 cm−1 in CD3OD and 960 cm−1 in CD3OH (Figure 8C). The H−O−CH3 bending vibration of Fe bound methanol at 1065 cm−1 in Fe-SH−CH3OH species shifts to 858 cm−1 in CD3OH and CH3OD and 772 cm−1 in CD3OD. The band at 1107 cm−1 in the experimental spectrum obtained in CH3OH, which shifted into the 900−600 cm−1 region on using CD3OD and CH3OD, can tentatively be assigned as the CH3−O−H bending mode of the Fe bound MeOH. No shifts are observed for the ν8, ν4, and ν2 modes either in the experimental data or in the computed vibrations. Thus, the calculated vibrations are in reasonably good agreement with the experimental data and

4. DISCUSSION The mechanism of reduction of ferric porphyrin complexes by hydrosulfide in an organic solvent is investigated. The reduction from an initial FeIII high spin to a final FeII high spin follows an inner-sphere mechanism and proceeds via the formation of MeOH−FeIII−SH intermediate which decays with a first order rate constant of 0.026 ± 0.002 s−1 at room temperature to the final FeII. The rate of reduction has an H/D isotope effect of 3, suggesting that the reduction step (rate determining) involves protons (Figure 3B).59,60 In biological systems like hemoglobin and cytochrome c, histidine (imidazole in synthetic mimics) is present as an axial headgroup coordinated to iron-porphyrin as the sixth ligand. Since methanol and imidazole are both sigma donor ligands, it is expected that replacing a methanol with imidazole should not substantially affect the reactivity of Fe− SH. However, it has been shown that H-bonding to the bound histidine strengthens the Fe−S bond.48 The reducing agent NaSH/Na2S (in solution) gets oxidized to elemental sulfur after reducing FeIII to FeII.8 Since oxidation of hydrosulfide (−SH) to elemental sulfur is a two-electron process, it is only expected 3922

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Inorganic Chemistry Scheme 1. Proposed Mechanism of Reduction of FeIII to FeIIa

The hydrosulfide (HS−) forms a six-coordinate FeIII−SH−MeOH adduct which then decays to yield the reduced FeII. The hydrosulfide radical (SH·) so formed precipitates as elemental sulfur. a

that 1 equiv of hydrosulfide (−SH) reduces two equivalents of FeIII to FeII. However, the reaction proceeds via homolytic cleavage of the FeIII−SH bond and in the process releases hydrosulfide radical (HS·) (trapped with DMPO). This hydrosulfide radical (SH·) released then reduces another FeIII porphyrin to FeII porphyrin and forms elemental sulfur in the process. The 6C MeOH−Fe−SH intermediate decays to the FeII porphyrin at a slower rate than it is formed, which allows trapping and characterization of this intermediate. Thus, the homolytic dissociation of the FeIII−SH bond to form FeII and hydrosulfide radical (SH•) (trapped with DMPO) is the ratedetermining step (rds) of the reaction which shows a H/D isotope effect of 3. This is also consistent with the fact that the rds is independent of sulfide concentration. The ΔG# of this homolytic dissociation can be estimated to be 19.6 kcal/mol (using the experimental k = 0.0265 s−1). Further ΔS# for this homolytic bond dissociation step can be expected to be positive as the number of particles increase in the homolytic dissociation rds. Three spin states are possible for the FeIII− SH intermediate species (S = 1/2, 3/2, 5/2). Calculated thermochemical parameters of homolytic dissociation of FeIII− SH adduct (Figure 11) from the three possible spin states are

Table 2. Thermochemical Parameters of Homolytic Cleavage of Fe−SH of Figure 11 to its Final FeII State spin of FeIII

ΔH (kcal/mol)

ΔS (kcal mol−1 K−1)

TΔS (kcal/ mol)

ΔG (kcal/mol)

S = 1/2 S = 3/2 S = 5/2

53.75 43.00 31.80

0.041 0.030 0.031

12.41 9.02 9.48

41.34 33.98 22.33

values in Table 2 clearly imply that dissociation from the S = 1/ 2 surface is associated with a very high energy barrier. However, dissociation from the S = 5/2 surface has a ΔG of 22.3 kcal/ mol, which is comparable to the experimental result. This computed value of ΔG (dissociation from S = 5/2) is very close to the experimentally estimated ΔG# 19.0 kcal/mol. Thus, dissociation most likely occurs from the S = 5/2 spin state, which would entail transition from the S = 1/2 to S = 5/2 surface along the reaction coordinate via two consecutive spin flips. In summary, a 6C solvent bound LS FeIII−SH species is identified as an intermediate formed in the reduction of FeIII porphyrin with sufide (HS−) in organic solvents. This implies that the reduction follows an inner-sphere mechanism. The homolytic dissociation of the Fe−S bond of this intermediate is the rate-determining step of the reaction with a H/D kinetic isotope effect (KIE) of 3.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02878.

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Figure 11. DFT optimized structure of sulfide bound Fe porphyrin adduct with MeOH as the sixth ligand used for the energy calculations.

Kinetic, EPR data, GC-MS data, displacement vector representations and the optimized coordinates (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

shown in Table 2. To avoid computational expenses, energy calculations were done on an unsubstituted porphyrin (Figure 11). Potential energy scan of the homolytic dissociation of the Fe−SH bond did not reveal the presence of a transition state, rather showing a smooth dissociation curve (Figure S8, Supporting Information). Previous reports suggest that similar homolytic dissociations may not have a distinct transition state and may possess an endothermic reaction barrier.61,62 The ΔG

ORCID

Abhishek Dey: 0000-0002-9166-3349 Author Contributions #

K.M. and A.S. contributed equally.

Notes

The authors declare no competing financial interest. 3923

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ACKNOWLEDGMENTS This work is funded by the department of science and technology Grant SB/S1/IC-25/2013. K.M. and A.S. acknowledge CSIR fellowships.

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