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Enzyme Catalysis that Paves the Way for SSulfhydration via Sulfur Atom Transfer Gou-Tao Huang, and Jen-Shiang K. Yu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b03387 • Publication Date (Web): 05 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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The Journal of Physical Chemistry

Enzyme Catalysis that Paves the Way for S-Sulfhydration via Sulfur Atom Transfer Gou-Tao Huang† and Jen-Shiang K. Yu∗,†,‡ †

Department of Biological Science and Technology, and ‡ Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu City 300, Taiwan E-mail: [email protected] Phone: +886 (3)5729287. Fax: +886 (3)5729288

∗

To whom correspondence should be addressed National Chiao Tung University ‡ National Chiao Tung University †

1 ACS Paragon Plus Environment


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Abstract S-sulfhydration is generally anticipated to proceed through the transfer of the SH group (Nu−SH····− S−R → Nu– ····HS−S−R). The other route involves the sulfur atom (S0 ) transfer between two sulfhydryl anions (Nu−S– ····− S−R → Nu– ····− S−S−R) and is considered electrostatically unfavorable. Mercaptopyruvate sulfurtransferase (MST) catalyzes sulfur transfer from mercaptopyruvate to sulfur acceptors, and the first step of the reaction is the formation of cysteine (Cys248) persulfide via S-sulfhydration. Mechanistic studies on S-sulfhydration in MST using QM/MM methods show that the sulfur atom transfer initialized by the deprotonation of the Ser250/His74/Asp63 triad is kinetically preferred to the SH-promoted sulfur transfer. The calculated barrier of approximately 16 kcal mol−1 for the S0 transfer agrees well with experimental results. The electrostatic repulsion during the S0 transfer can be sophisticatedly reduced by the aid of the Cys248-Gly249-Ser250-Gly251-Val252-Thr253 (CGSGVT) loop. Electrostatic potentials and frontier orbitals are also analyzed for the persulfide anion surrounded by the loop. The sulfur atom transfer which is seldom regarded possible is therefore facilitated with the assistance of the triad and the loop in the enzyme.


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1

Introduction

In sulfur chemistry, persulfide compounds are reactive species so that it is usually difficult to synthesize and store them in laboratory. 1 Chemically unstable persulfides are intermediates in biological reactions, such as formation of the cysteine persulfide during catalysis of mercaptopyruvate sulfurtransferase (MST). Recently, it has been reported that persulfides and polysulfides (R−Sn −H) might be important signaling/effector molecules. 2,3 Accordingly, studies of persulfides have attracted attentions in chemistry and chemical biology. 4–8 Persulfide compounds have been prepared to study reactivity towards acid, base, electrophiles, and nucleophiles. 5,6,9–11 The synthetic persulfides are usually attached to bulky groups to prevent from decomposition. A persulfide group can also be protected by acylation; the addition of cysteine or glutathione (GSH) to the acyl-protected precursors yields persulfides, leading to the production of hydrogen sulfide (H2 S), 7,8 which was suggested to act as a cellular signaling molecule. 12,13 Sulfurtransferases catalyze sulfur transfer from a sulfur donor to a thiophilic acceptor. 3-Mercaptopyruvate and thiosulfate sulfurtransferases, abbreviated as MST and TST (also known as rhodanese), respectively, are members of the sulfurtransferase family, and their essential catalytic center is a cysteine residue. The sulfur-transfer reaction catalyzed by MST/TST involves the formation of the cysteine persulfide (or thiocysteine). 14–19 The MSTcatalyzed mechanism comprises two sulfur-transfer processes, shown in Scheme 1, in terms of S-sulfhydration and desulfhydration of the cysteine residue, i.e. a ping-pong mechanism. The sulfhydryl group of the cysteine residue is generally regarded as a deprotonated anion, which is more nucleophilic than its neutral form. The attack of the cysteine sulfhydryl anion on the sulfhydryl group of mercaptopyruvate produces thiocysteine and pyruvate. When pyruvate detaches from the catalytic site, other thiophilic compounds such as thiol molecules or cyanide can react with the terminal sulfur atom of the cysteine persulfide, and then the sulfhydryl form of the cysteine is regenerated. Hence, the catalytic cysteine in the enzyme cycles between a thiol and a perthiol. TST utilizes thiosulfate (S2 O2– 3 ) instead, as 3 ACS Paragon Plus Environment


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the sulfur source, and the mechanism of S-sulfhydration is similar to that in MST. TST is supposed to involve itself in the detoxification of cyanide that converts toxic CN– to SCN– (Scheme 1b). 20–22 MST and TST possess high similarity in amino acid sequences, which may imply their structural akins. 23 Their active-site pockets feature a cradle-like structure composed of a hexapeptide sequence, namely Cys-Gly-Ser-Gly-Val-Thr in MST and CysArg-Lys-Gly-Val-Thr in TST; these hexapeptide sequences form loops connecting the betastrand and the helix in the C-terminal domain in MST/TST. The terminal sulfur atom of the cysteine persulfide is surrounded by four amide NH groups of the loop, as observed in the crystal structures. 14–19 It has been reported that the hexapeptide loop can stabilize the persulfide group by electrostatic interactions. 14 Site-directed mutagenesis studies of rat a) S-sulfhydration

Gly MST Cys S Thr

Asp His Ser Gly

O +

O

MST Cys SS Gly O

Val

Gly

SH

Thr

3-mercaptopyruvate

MST

Asp His Ser

O +

O O

Val

pyruvate

MST

b) desulfhydration

Gly

Asp His Ser

MST Cys SS Gly Thr MST

Val

Gly +

CN

MST Cys S

RSH

Thr

sulfur acceptor

Asp His Ser Gly Val

+

SCN RSSH

MST

Scheme 1: Sulfur transfer catalyzed by MST, consisting of a) S-sulfhydration and b) desulfhydration. The loop of the active site, Cys-Gly-Ser-Gly-Val-Thr, is shown in blue while the triad, Ser/His/Asp, is shown in green. liver rhodanese showed that the mutations of Arg and Lys to Gly and Ser increase the activity of MST and decrease native rhodanese activity, which indicates that the loop affects specific recognition of their substrates of mercaptopyruvate and thiosulfate. 24 In a recent Xray crystallographic study of human MST 19 (PDB code: 4JGT), it was demonstrated that 4 ACS Paragon Plus Environment

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Arg188 and Arg197 are two significant residues for substrate-binding in MST, anchoring the carboxylate and ketone groups of mercaptopyruvate into the active site pocket; however, only one Arg residue is observed near the active site of TST. In addition, the Ser/His/Asp triad, where Ser of the loop is in the C-terminal domain while His and Asp belong to the N-terminal domain, is significant in the catalysis of MST. 18 Experiments demonstrated that the addition of benzoyl-Arg-p-nitroaniline, a serine protease inhibitor, could lead to decrease of MST activity. It was hence suggested that the role of the triad is to help the sulfur transfer by the hydrogen bonding between the hydroxyl group of Ser and the carbonyl oxygen atom of mercaptopyruvate. 18 Nevertheless, such a Ser/His/Asp triad is absent in TST. The substrate-bound MST enzyme was first crystallized by Yadav et al in 2013. 19 According to electron density analysis in X-ray crystallography, two possible structures were assigned in the active site: 1) the cysteine persulfide bound to a pyruvate, and 2) the structure with an S−S bond linked covalently between the sulfur atoms of Cys and mercaptopyruvate. The formation of the latter was explained by a side reaction, where the cysteine persulfide attacks another mercaptopyruvate substrate. Several sulfur acceptors, for example, 2-mercaptoethanol, GSH, and thioredoxin, were used to react with the cysteine persulfide, and the kinetics of the production of H2 S was further analyzed in the reaction. 19 The mechanism of the MST/TST-catalyzed sulfur transfer has been proposed based on crystallographic structures and mutation tests; however, the mechanistic detail of the sulfur transfer is still not well known. In this study, computational approaches are employed to investigate the mechanism of S-sulfhydration of the cysteine (Scheme 1a) and to analyze the roles of the triad and the loop. Human mercaptopyruvate sulfurtransferase was chosen as the enzyme studied. Classical molecular dynamics simulation was first performed to obtain water-solvated enzyme structures. Combining quantum mechanical (QM) and molecular mechanics (MM) methods, the QM/MM methodology was used to study the sulfur-transfer reaction, where the chemical reaction to be investigated is treated at the QM level while the environmental effect of the enzyme is considered at the MM level.



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2

Computational Details

Model reactions where the sulfur transfer occurs between mercaptopyruvate and MeS– were first analyzed (see Section 1 of ESI) before the sulfur transfer in enzyme is studied. Preliminary calculations showed that the M06-2X density functional 25 predicts the energetic trend well in accordance with the high level CCSD(T) method. 26 M06-2X was hence employed at the QM level in the following QM/MM calculations. The crystal structures of the human mercaptopyruvate sulfurtransferases (PDB code: 4JGT and 3OLH) were obtained from the Protein Database website. The crystal structure of 4JGT includes the pyruvate substrate, which is the product of the sulfur transfer reaction. The protonation states of the enzymatic residues were predicted with the PropKa 3.1 package. 27–30 It should be noted that the sulfhydryl group of the catalytic Cys248 was considered deprotonated according to the previous study that the pKa of the essentially catalytic Cys is about 6.5 in rhodanese while the normal value of a sulfhydryl group of cysteine is 8.3. 31 This unusually low pKa value is attributed to the specific active-site environment, where the amide groups of the loop are supposed to stabilize the sulfhydryl anion. Both Arg188 and Arg197, which bind mercaptopyruvate into the active site by hydrogen bonding, were considered cationic to effectively seize the carboxylate and carbonyl groups of mercaptopyruvate. 24 The Ser250/His74/Asp63 triad forms a hydrogen-bond network shown in Figure 1. Detailed setup and results of molecular dynamics (MD) were described in Section 2 of ESI. MD simulation was run using the Gromacs 5.0.2 package. 32 The snapshot with the lowest potential energy during MD simulation was taken to set up the next QM/MM calculation using Our own N -layered Integrated molecular Orbital and molecular Mechanics (ONIOM). 33–36 In the snapshot structure, the water molecules either within 3 ˚ A of protein or within 22 ˚ A of active-site residues were retained. The final geometry, consisting of 12117 atoms, was utilized as the real system in the ONIOM calculation. The model system, namely the QM region, includes the loop (Cys248-Gly249-Ser250-Gly251Val252-Thr253, CGSGVT), the residues of Leu38, Asp63, His74, Arg188, and Arg197, the 6 ACS Paragon Plus Environment

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Arg197 NH NH HN H H

O

Leu38

O Asp63 O

H Arg188 NH HN

NH

O O

SH

NH H

O Cys248 H H OH H O N W2 H O O W1 N H H

N

Ser250 OH O

NH

His74 O

N H

HN

S

HN

O

H N

H O HN

O O SH

O the loop: Cys248-Gly249-Ser250-Gly251-Val252-Thr253

Figure 1: Active-site region of the 4JGT enzyme. The QM atoms are shown in black and red, while the MM atoms are colored in gray. The backbone of the hexapeptide loop is decorated with magenta outer glow.



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substrate, and two water molecules (W1 and W2), shown in Figure 1. The N- and Cterminal sides of the loop were capped by the carbonyl group of Thr247 and the amino group of Ala254, respectively. The two water molecules in the QM layer formed hydrogen bonds with the carboxylic group of the substrate observed in the crystal structure of 4JGT. Significant interactions involved in mercaptopyruvate and Cys248, in terms of the effects of the loop and the triad, were covered in the QM layer. Geometry optimizations were carried out with the two-layer ONIOM(QM:MM) method, where the QM layer was calculated at the M06-2X level combined with the basis sets of 6-31G* for H, C, N and O atoms and 6-31+G* for S, while the MM region was computed using the AMBER ff99SB force field 37 for protein and the TIP3P model 38 for waters. The ONIOM calculations were done using the electrostatic embedding (EE) scheme. Detailed description for the ONIOM setup can be found in Section 3.1 of ESI. During the course of geometry optimizations, residues and waters within 15 ˚ A of the QM region were set flexible while other outer atoms were kept frozen. Minima and transition states were verified by frequency calculations. Furthermore, single-point energies based on the optimized structures were evaluated using the larger basis set of 6-311++G**, and zero-point corrections in enthalpy (∆H) and free energy (∆G) are still based on the frequency calculations at the M06-2X/6-31G*:Amber level with diffuse functions adding to sulfur atoms in the QM layer (Tables S4 and S6). All of the ONIOM computations were carried out with the Gaussian 09 software. 39 Electrostatic potential (ESP) and electron localization function (ELF) were used to analyze electron distribution of lone pairs in thiolate and persulfide anion. 40,41

3 3.1

Results and Discussion S-sulfhydration

The S-sulfhydration can proceed through the sulfur transfer in the form of either the sulfur atom (S0 ) or the SH group. Figures 2 and 3 show the mechanisms of the sulfur atom transfer 8 ACS Paragon Plus Environment

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and the SH-promoted sulfur transfer, respectively, and both reaction pathways start from the same reactant, PyrSH. For clarity only the significant residues of Cys248 and Ser250 of the triad as well as the substrate are depicted in Figures 2 and 3 while clearer visualization for the active site region is shown in Figures S12-S14 of ESI. The pathway of the sulfur atom transfer is composed of three steps, the deprotonation, the S0 transfer, and the proton abstraction (Figure 2). In contrast, the SH-promoted transfer is a single-step process (Figure 3). Computed enthalpies and free energies for the two pathways are given in Figure 4, and PyrS is set as the energetic reference in the energy profiles. Detailed energy data are given in Table S4-S7 of ESI.

3.1.1

S0 transfer

The sulfur atom transfer is initialized by the deprotonation of the sulfhydryl group of the substrate (Figure 2). In this deprotonation step, Ser250 in the triad acts as the base that takes the proton of the SH group off through two simultaneous proton shuttles among Ser250, His74, and the sulfhydryl group of mercaptopyruvate. The barrier of the deprotonation (PyrSH → PyrSH-tsH) is 8.1 kcal mol−1 in ∆G, and the formation of PyrS is exergonic by 3.3 kcal mol−1 (Figure 4). In PyrS the hydroxyl group of Ser250 interacts with the sulfhydryl anion of the substrate at the S– ···H distance of 2.36 ˚ A (Figure 2). However, when the sulfur atom transfer happens, Ser250 and the carbonyl group of mercaptopyruvate form a hydrogen bond, indicated by the O···H distance of 1.63 ˚ A in PyrS-tsS. Thus it can be anticipated that the role of the hydrogen bond is to stabilize the negatively-charged oxyanion of the pyruvate enolate in PyrS-tsS and Pyr, which supports the proposition for the key role of the hydrogen-bond interaction between Ser250 and mercaptopyruvate. 18,24 In addition to Arg188 and Arg197, Ser250 is also one significant residue for stabilizing the substrate. In order to convert to the initial protonation state in the triad, the pyruvate enolate in Pyr has to abstract the hydroxyl proton of Ser250. Because the hydrogen bond of 1.46 ˚ A between the oxyanion and Ser250 is present (Figure 2), the formation of the enol pyruvate occurs



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deprotonation Asp63 O Asp63 O 1.84 Å

O

NH

O

1.72 Å 2.10 Å

O

S

1.67 Å

His74

S His74

S

H

3.41 Å

N

3.32 Å

N H 1.14 Å O 1.41 Å

1.43 Å 1.62 Å

O

OH

SH

Asp63 O

NH

O O

O

1.80 Å

O

O

2.83 Å

O

Ser250

Cys248

O

NH

O HN

HO

S

1.80 Å

His74

2.36 Å

3.35 Å

Ser250

S

Ser250 PyrSH-tsH

Cys248

Cys248 PyrSH

PyrS S0 transfer

Asp63 O O

O proton abstraction

NH

O

1.63 Å

HN

HO

O

1.65 Å

1.72 Å

His74

2.27 Å

S Asp63 O

2.46 Å

Cys248

O Asp63 O

O

O

H

O 1.80 Å

OH

O

1.69 Å

OH

H

N

2.12 Å

1.56 Å

Asp63 O

1.21 Å 1.29 Å

O

Ser250

S

N

Ser250

O

PyrS-tsS

1.77 Å

His74 S

His74 S

1.48 Å

O

NH O

NH 1.04 Å

O

O

Cys248 O

NH 1.46 Å

1.68 Å

HN

HO

1.70 Å

His74

Pyr-tsH

2.12 Å

S

S Cys248

Ser250

S

2.12 Å

Ser250

S Pyr-enol

Cys248 Pyr

Figure 2: Mechanism of the sulfur atom transfer. Selected bond lengths are given. The other QM regions (Leu38, Arg188, Arg197, two water molecules, and the loop) are not depicted for clarity.


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most directly in the active site. As expected, the barrier of the proton abstraction without zero-point and thermal corrections included is low (1.3 kcal mol−1 , listed in Table S4), while the process is barrierless in terms of ∆G and ∆H. Finally, the formation of the enol form of pyruvate (enol-pyruvate) is exergonic by 1.2 kcal mol−1 . When the enol-pyruvate is released from the active site to water, it can tautomerize to the more stable ketone form in water. 42 Furthermore, different enzyme configurations were used to evaluate the conformational effect on the sulfur atom transfer (Figures S15 and S16 in ESI). It is found that the influence of the enzyme configuration on the barrier of the S0 -transfer step is small. Note that water molecules appear not to be involved during the reaction. The water W1 which is closest to the active site forms a hydrogen bond with the carboxylic group of the substrate and the hydroxyl group of Thr253 (Figure 1), and is hence unable to approach the transferred sulfur atom, as observed in the crystal structure. Actually, the transferred sulfur atom is surrounded by the loop so that there is not enough space to accommodate water molecules, and therefore the effect of water molecules on the S-sulfhydration is not considered in the study.

3.1.2

SH-promoted sulfur transfer

Compared to the sulfur atom transfer, the SH-promoted sulfur transfer does not require the deprotonation of the sulfhydryl group in mercaptopyruvate. Mechanistically, the transfer of the SH group is followed by the proton transfer, yielding the persulfide anion and the ketopyruvate, as shown by the dotted arrows in Figure 3. Attempts to find the transition state involving the transfer of the SH group failed, and the hydropersulfide intermediate could not be located in the ONIOM calculations. For the located transition state (PyrSH-tsSH), the vibrational mode of the imaginary frequency involves mainly the proton transfer, mixed with minor contribution from the sulfur transfer. Model reactions also demonstrate that in the transition states of the SH transfer, the sulfur transfer can be coupled with the proton transfer (Figure S1) depending on a dielectric field of environment. The intrinsic



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reaction coordinate 43 (IRC) calculation confirms that PyrSH-tsSH is linked to PyrSH along the same reaction coordinate (Figure S10). The pKa values of a hydropersulfide 44 (RSS−H) and the α-proton of ketone (RC(O)CH2 −H) are about 6.2 and 20, respectively, which rationalizes that a hydropersulfide is chemically labile for an enolate. This might also indicate that the hydropersulfide intermediate could not exist as a stable intermediate in the active site. In regard with structural change, the hydrogen bond between the hydroxyl oxygen atom of Ser250 and the hydrogen atom of the transferred SH group elongates to 2.5 ˚ A in PyrSH-tsSH compared with that of 2.1 ˚ A in PyrSH (Figure 3). The distance between the two sulfur atoms of 2.63 ˚ A in PyrSH-tsSH suggests that the persulfide bond is not yet formed. The SH-promoted transfer (PyrSH → PyrSH-tsSH) requires a high activation energy of about 40 kcal mol−1 (Figure 4), and hence the pathway disfavors in kinetics whereas the product Pyr-keto is more stable than Pyr-enol obtained from the S0 transfer. This large barrier is likely due to the absence of the hydrogen bond between Ser250 and the carbonyl oxygen atom of the substrate. SH-promoted sulfur transfer O O O O

1.53 Å

2.27 Å

S 1.46HÅ

OH

2.50 Å

2.63 Å

O

S

O O

SH

2.10 Å

OH

Ser250

Cys248

O

S

PyrSH-tsSH S

OH

O

3.32 Å

2.11 Å

S

Ser250

Cys248

Ser250

Cys248 Pyr-keto

PyrSH O O O S

H

S

OH

Ser250

Cys248

Figure 3: Mechanism of the SH-promoted sulfur transfer. Selected bond lengths are given. The other QM regions (Leu38, Asp63, His74, Arg188, Arg197, two water molecules, and the loop) are not depicted for clarity.


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SH-promoted sulfur transfer PyrSH-tsSH 43.2 [47.3]

S0 transfer deprotonation

PyrS-tsS 15.9 [15.1]

PyrSH-tsH 11.4 [15.3]

proton abstraction Pyr-tsH

PyrSH G 3.3 H [8.2] in kcal mol

Pyr 2.7 [2.6]

PyrS 1

0.0 [0.0]

1.0 [1.1]

Pyr-enol 1.2 [ 0.1] Pyr-keto 7.7 [ 3.0]

ONOIM level: M06-2X/6-311++G**//M06-2X/6-31G* for H, C, N, O:Amber99sb 6-31+G* for S

Figure 4: Energy profiles for the sulfur atom transfer and the SH-promoted sulfur transfer. The enthalpies (∆H) and free energies (∆G) are relative to PyrS.



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The calculations of the S-sulfhydration show that the sulfur atom transfer (∆G‡ : 15.9 kcal mol−1 ) is kinetically preferred to the SH-promoted sulfur transfer (∆G‡ : 43.2 kcal mol−1 ). Ser250 of the triad plays two roles in the overall reaction of the sulfur atom transfer: 1) the deprotonation triggering the sulfur atom transfer; 2) the stabilization for the pyruvate enolate by the hydrogen-bond interaction. The effect of the loop on the sulfur atom transfer is extensively analyzed in the following section.

3.2

Effect of the CGSGVT loop on S0 transfer

Figure 5 shows the active site structures in PyrS, Pyr-tsS, and Pyr, where the sulfur atom to be transferred is labeled by S1 and the sulfur atom of Cys248 by S2. Based on the computed NH/OH···S distances, the amide groups of Gly249, Ser250, Val252, and Thr253 as well as the hydroxyl group of Thr253 interact with the S1 atom in PyrS-tsS. These five sets of H···S electrostatic interactions also assist to stabilize the persulfide anion in Pyr. In contrast, the other two H3 and H7 atoms of Gly251 and Ala254 stay close to the S2 atom. The structural information shows that the hydrogen atoms of the amide groups in the CGSGVT loop provide a positively-charged environment to stabilize the sulfhydryl/persulfide anion of Cys248. Analyses on the model anions of MeS– and MeSS– (Figures S2 and S3) show that the electrons of the three lone-pairs of the terminal sulfur atom distribute around the lateral side along the C−S/S−S bond, which implies that the donut-like electron domain of the lone pairs interacts with the NH/OH groups of the loop in the active site. The electrostaticinteraction model illustrated in Figure 6a is therefore proposed: the sulfhydryl group of Cys248 leans, in a side-on manner, against the electron-deficient region of the sulfur atom being transferred so as to reduce the repulsion between them, while the NH/OH groups of the loop stabilize the lateral lone-pair electrons of the S0 atom. In this model, there is no direct contact between the lone-pair electrons of the two sulfhydryl anions, and the Coulomb repulsion between the anions (Cys248−S– ····− S−Nu) could be reduced by the support of the 14 ACS Paragon Plus Environment

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loop. His74 His74 Arg197 Arg197 C1 C1 H2 S1

S1

H1

H6 H1

H4

H4

H2

H3

H5 H6

H3 S2

H5 S2

H7 H7

PyrS

PyrS-tsS

His74

loop residue

S1 H6

H1

H3

H5

S2

Pyr

C1···S1

1.86

2.27

3.12

S2···S1

3.35

2.46

2.12

Gly249

H1···S1

2.70

2.28

2.22

Ser250

H2···S1

2.81

2.39

2.40

Gly251

H3···S1

3.79

3.13

2.95

Val252

H4···S1

2.78

2.49

2.47

Thr253

H5···S1

2.65

2.32

2.28

Thr253

H6···S1

2.52

2.25

2.23

Ala254

H7···S1

4.70

3.99

3.71

Gly251

H3···S2

2.20

2.32

2.43

Thr253

H6···S2

2.84

2.90

2.93

Ala254

H9···S2

2.40

2.46

2.50

C1

H2

PyrS-tsS

Cys248

Arg197

H4

PyrS

H7

Pyr

distance in Å

Figure 5: Geometries of the active sites in PyrS, PyrS-tsS, and Pyr. The other QM regions, Leu38, Asp63, Arg188, and the two water molecules, are not depicted for clarity. Selected distances are listed. Experimental studies reported that persulfides can react with either nucleophiles or electrophiles. 5,6,9–11 An electrophilic attack occurs at the terminal sulfur atom while a nucleophile can attack either a terminal or an inner sulfur atom; however, only the terminal sulfur atom of the cysteine persulfide is reactive in the MST owing to the specific geometry of the active site. To address whether the cysteine persulfide anion in the active site of MST is nucleophilic 15 ACS Paragon Plus Environment


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a)

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b) Arg197 H

O

Nu

O Arg188 N H

S0

Ser250

O O

H

Nu

Nu

S

N

S0

S S

loop

N H

H N

H

S

S

O electron-deficient region

Cys248

S

Nu = pyruvate enolate

1

S lone-pair electrons

Figure 6: Electrostatic-interaction model for the S0 transfer. or electrophilic, the loop moiety in Pyr-enol is analyzed by the single-point calculation at the M06-2X/6-311++G** level. The ESP map on the isosurface of ρ = 0.0025 gives a maximal electrostatic potential of −0.05 a.u. in the electron-deficient region of the terminal sulfur atom (Figure 7a), while the electrostatic interactions of H···S can be observed with the isosurface of ρ = 0.02 (Figure 7b). The Vmax value of −0.05 a.u. in the loop is less negative than the value of −0.18 a.u. in the terminal sulfur atom of the MeSS– model (Figure S2b and Table S1), which indicates that the electron-deficiency effect is stronger in the persulfide anion surrounded by the loop. The enhancement of the electron-deficient phenomenon can be explained by the inductive effect that the lone-pair electrons of the terminal sulfur are pulled outward by the NH/OH groups of the loop to result in a decreased electron distribution along the S−S axial direction. Frontier orbitals of the loop moiety are plotted in Figures 7c and 7d. The HOMO and LUMO+3 are π ∗ and σ ∗ orbitals of the two sulfur atoms, respectively. The nucleophilic attack of the persulfide anion might be sterically unfavorable because the lobes of HOMO is surrounded by the loop. The alternative electrophilic attack along the direction of the S1−S2 axis orients directly towards the substrate in the active site pocket. The observations based on the electrostatic and orbital analyses support the conclusion that the persulfide anion tends to be more electrophilic under the effect of the loop.


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(a)

(b)

ρ = 0.0025 −0.08

ρ = 0.02 −0.06

0.00 a.u.

0.06 a.u.

electron-deficient region on S1 Vmax on S1 = −0.05 a.u.

Vmax on S1 = 0.06 a.u.

(c)

(d) HOMO π*S−S

LUMO+3 σ*S−S

Figure 7: Electrostatic potential and frontier orbitals in the CGSGVT loop of Pyr-enol. Electrostatic potential is mapped on the total electron density with isosurfaces of a) 0.0025 and b) 0.02. c) HOMO and d) LUMO+3 are plotted according to the isosurface of electron density of 0.02.



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The S-sulfhydration of Cys248 is illustrated in Figure 6b, in which the sulfur atom transfer is represented in the form of Cys−S– −−−S0 −−−Nu– , where Nu– is the pyruvate enolate. The two nucleophiles in terms of Cys−S– and Nu– compete with each other attacking the sulfur atom S0 in the sulfur transfer reaction. The two moieties in the reactants (Cys−S– and NuS– ) as well as in the products (Cys−SS– and Nu– ) are anions, and the repulsive interactions between the anions can be sophisticatedly diminished by the support of the CGSGVT loop. In analogy, the desulfhydration shown in Scheme 1b can be regarded as the reverse reaction of the S-sulfhydration, where Nu– is either cyanide or thiolate. Based on the assumption that both S-sulfhydration and desulfhydration proceed via the same mechanism, the barriers for the S0 transfer are expected to be comparable. The calculated activation free energy of approximately 16 kcal mol−1 is in agreement with the experimentally derived values of 16 ∼ 18 kcal mol−1 estimated from kcat of 0.3 ∼ 6.1 s−1 with sulfur acceptors of glutathione, L-homocysteine, L-cysteine, thioredoxin, dihydrolipoic acid, cyanide, and dithiothreitol, 19

according to the Eyring-Polanyi equation. In addition, it was recently reported that upon deprotonation, tritylhydropersulfide (TrtSSH) undergoes a disproportionation, leading to the formation of S8 . 6 The disproportionation was discovered to correlate with the strength of bases, and thus the deprotonated persulfide is likely to be the dominant reactive species, leading to the sulfane sulfur transfer (TrtS– −−−S0 −−− − SSn Trt). This experimental evidence could further support the possibility of the sulfane sulfur transfer in the form of the S0 atom that is initialized by the deprotonation. For TST (rhodanese) with similar activity to MST, the triad is absent in the active site. The substrates of TST, thiosulfate (S2 O2– 3 ) and cyanide, usually exist in deprotonated forms at physiological pH so that the deprotonation step is not essential for the sulfur atom transfer. The existence of the triad might be one of the critical factors for the specific selection of the major target substrates between MST and TST.


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4

Conclusions

The mechanism of the S-sulfhydration in the two forms of the sulfur atom and the SH group is investigated in the MST enzyme. The sulfur atom transfer is a multi-step process consisting of the deprotonation, the S0 transfer, and the proton abstraction, whereas the SH-promoted sulfur transfer is a single-step reaction. The calculations show that the sulfur atom transfer (∆G‡ : 15.9 kcal mol−1 ) is more kinetically favorable than the SH-promoted sulfur transfer (∆G‡ : 43.2 kcal mol−1 ). The calculated barrier of approximately 16 kcal mol−1 for the sulfur atom transfer is in accordance with experimental results for the MST-catalyzed sulfur transfer reaction. The Ser250/His74/Asp63 triad assists the deprotonation of the sulfhydryl group of mercaptopyruvate, which promotes the subsequent sulfur atom transfer. Furthermore, the hydrogen bond between the hydroxyl group of Ser250 and the carbonyl oxygen atom of mercaptopyruvate stabilizes the pyruvate enolate. The electrostatic-interaction model based on the ESP and ELF analyses demonstrates that the CGSGVT loop of MST supports the sulfur atom transfer by the electrostatic interactions of NH/OH···S0 . The steric and inductive effects caused by the loop render the persulfide anion more electrophilic, and facilitate an attack by thiophilic nucleophiles.



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Supporting Information Available • Model reactions of MeS– with mercaptopyruvate. • Classical molecular dynamics simulation. • Detailed description of ONIOM calculations. The authors declare no competing financial interest. This material is available free of charge via the Internet at http://pubs.acs.org/.

Acknowledgement The authors are indebted to the Ministry of Science and Technology, Taiwan, for financial support under Grants NSC 100-2627-B-009-001, MOST 102-2113-M-009-013, and MOST 103-2113-M-009-014-MY3, and the ”Center for Bioinformatics Research of Aiming for the Top University Program” of NCTU and MoE, Taiwan. G.T.H. acknowledges the postdoctoral fellowship from MOST 104-2811-M-009-001 and 105-2811-M-009-001.

References (1) Park, C.-M.; Weerasinghe, L.; Day, J. J.; Fukuto, J. M.; Xian, M. Persulfides: Current Knowledge and Challenges in Chemistry and Chemical Biology. Mol. BioSyst. 2015, 11, 1775–1785. (2) Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T. et al. Reactive Cysteine Persulfides and SPolythiolation Regulate Oxidative Stress and Redox Signaling. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 7606–7611. (3) Kimura, H. Hydrogen Sulfide and Polysulfides as Signaling Molecules. Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 2015, 91, 131–159.


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Perthiol: Reversible Hydrogen Transfer and Perthiyl Radical Formation. Free. Radic. Res. 1994, 20, 387–400.


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Graphical TOC Entry sulfur atom (S0) transfer electrostatic potential

HO Ser250

O

ρ = 0.0025

O

−0.08

N H O N H

O O O

S

H N H N loop

S0

Cys248

0.00 a.u.

O O

−1

S

O S

Cys248

S

electron-deficient region

S

Cys248

S electron-deficient region

lone-pair electrons


Enzyme Catalysis that Paves the Way for S ... - ACS Publications

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