A Pseudo-Hypervalent Sulfur Intermediate As An Oxidative Protective

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A Pseudo-Hypervalent Sulfur Intermediate As An Oxidative Protective Mechanism In The Archaea Peroxiredoxin Enzyme ApTPx Hisham M Dokainish, Daniel J Simard, and James W. Gauld J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04671 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 23, 2017

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The Journal of Physical Chemistry

A Pseudo-Hypervalent Sulfur Intermediate As An Oxidative Protective Mechanism In The Archaea Peroxiredoxin Enzyme ApTPx

Hisham M. Dokainish; Daniel J. Simard; James W. Gauld.*

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada

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Abstract Peroxiredoxins (Prxs) are a ubiquitous class of enzymes that have central roles in a number of important physiological processes. Using a multi-scale computational approach we have investigated the mechanism by which the active site cysteine (Cys50) in the typical 2-Cys Prx from Archaea (ApTPx) is oxidized by H2O2 to sulfenic acid. In addition, its further oxidation to give a sulfinic acid or its possible alternate intramolecular reaction to form an experimentally proposed hypervalent sulfurane was examined. Oxidation of Cys50 by H2O2 to give the sulfenic acid intermediate occurs in one step with a barrier of 82.1 kJ mol-1. A two-step pathway is proposed with a very low barrier of 16.5 kJ mol-1 by which it may subsequently react with an adjacent histidyl (His42) to form the pseudo-hypervalent sulfurane. This pathway also involves an adjacent aspartyl (Asp45) which helps alternate the protonation state of His42. The sulfurane's Cys50S…NdHis42 interaction was characterized using QTAIM, NCI, and NBO analyses and concluded to be a non-covalent interaction. Notably, this bond helps orient the

Cys50

SOH moiety such that it is less susceptible to oxidation by H2O2 to sulfinic acid. Significantly,

sulfurane formation is energetically favored to further H2O2 oxidation of

Cys50

SOH to a sulfinic acid,

providing a mechanism by which the active site Cys50 is protected against overoxidation.


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Introduction Peroxiredoxins (Prxs) are a class of ubiquitous enzymes with central roles in regulating signaling pathways as well as being potent antioxidants.1-3 Indeed, one of their key functions is the catalytic reduction of peroxide substrates such as hydrogen peroxide (H2O2) and alkyl-hydro-peroxide (ROOH).4 Notably, they are also very abundant in cells constituting up to 1% of the total soluble protein and can reach catalytic rates on the order of ~107 M-1 s-1.1 As a result, Prxs have been credited with reducing approximately 90% of the mitochondrial and approaching 100% of the cytoplasmic H2O2.3,5 Prxs are classified into 6 main groups based on sequence similarity: Prx1, Prx5, Prx6, Tpx, BCP and AhpE (in Mycobacterium tuberculosis).1,4 All groups contain a reducing thioredoxin (Trx) fold1,6,7 and possess similar active sites. In particular, they all contain a catalytic peroxidative cysteinyl (Cp), as well as proline (Pro), threonine (Thr)/serine (Ser), and arginine (Arg) residues.1,8,9 Prxs are further divided into three main groups: 1-Cys, typical 2-Cys and atypical 2-Cys.6 This classification is mainly dependent on the existence and location of a second mechanistic cysteinyl, known as the resolving cysteine (CR).1 Specifically, 1-Cys Prxs do not contain a CR1,6 while in typical 2-Cys Prxs (the largest subclass), the enzyme function is dependent on the presence of an intact homodimer where CP and CR are located on different monomers.4,10 In contrast, in atypical 2-Cys Prxs CP and CR are located on the same monomer.4,11 All classes of Prxs share a common catalytic cycle involving three main steps.1,4 These can be summarized as: (1) peroxidation; CP nucleophilically attacks the peroxide substrate to form a reactive Prx-sulfenic acid (-SPOH) intermediate, (2) resolution; the resolving CR or external thiol in atypical/typical 2-Cys Prx or 1-Cys Prx, respectively, reduces the sulfenic acid (–SPOH) to form an intra- or intermolecular disulfide bond, (3) recycling; regeneration of the active site via the reduction of the disulfide by an external thiol such as Trx.12 Several X-ray structures have been obtained of Prx family members, revealing similarities in binding of H2O2 (commonly denoted as HOA–OBH where OA is in close proximity to CP) within their active sites.9,13-15 In particular, the guanidinium side chain of the conserved Arginyl is situated within hydrogen bonding distance of (OA) of H2O2 and the side-chain of CP;6,9 this likely enables it to stabilize 3 ACS Paragon Plus Environment


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a Cp thiolate.6,9 The latter OA centre is also thought to hydrogen bond with the threonyl side chain hydroxyl. Meanwhile, the conserved prolyl is thought to protect CP from interacting with the solvent, helping minimize the possibility of Cys overoxidation.6,9 Due in part to the above described hydrogen bonds, the H2O2 moiety appears to be suitably positioned for CP nucleophilic attack, forming sulfenic acid.6,9 However, although existing X-ray structures highlight the roles of the active site residues, their specific roles in catalysis remain unclear.9 In addition to their central role in the mechanism of Prx's, sulfenic acids have been shown to be key intermediates of numerous redox processes in proteins.16 However, they are highly reactive and, due to their ability to act as a nucleophile or electrophile, can undergo a variety of reactions.16,17 In particular, they can be readily overoxidized to sulfinic and subsequently sulfonic acids in the presence of H2O2 or other oxidizing agents.16,17 These modifications are, in general, considered irreversible and lead to protein deactivation.18 Indeed, the reduction of Cp's sulfenic acid moiety (–SPOH) by CR, the second step of Prxs mechanism, is likely in competition with its overoxidation especially under conditions of oxidative stress.4,19 Furthermore, overoxidation of Cp in typical 2-Cys Prx occurs at a higher rate than in the atypical 2-Cys or 1-Cys Prx subclasses.20 This is thought to be due to the location of CR on the adjacent monomer in typical 2-Cys; larger structural rearrangements must occur before reduction of the sulfenic acid may proceed.4 Fortunately, a unique enzyme (sulfiredoxin) is able to reduce the sulfinic acid form of typical 2-Cys Prx to its precursor sulfenic acid form.20-22 However, this enzyme only occurs in eukaryotic organisms and is highly specific to typical 2-Cys Prx. This suggests the presence of alternative mechanisms in bacteria and Archaea for protecting typical 2-Cys and 1-Cys Prx from overoxidation and thus inactivation.20 Nakamura et al.23 performed an experimental and computational study on typical 2-Cys Prx (ApTPx) from Archaea. Based on X-ray crystallographic structures obtained they concluded that it is protected from overoxidation via the formation of a unique hypervalent sulfurane intermediate. More specifically, they concluded that the sulfur of CP50 forms a covalent bond of approximate length 2.2 Å with the imidazole Nd1 center of His42. It is noted that this His only occurs in some Prx proteins. They also performed calculations at the B3LYP/6-31G(d) level on several possible gas-phase model isolated 4 ACS Paragon Plus Environment

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sulfuranes. Two of these were suggested to be most probable; in both Cp50S is also bound to an hydroxyl and hydrogen (-SCp50(HO)(H)—Nd1His42), but they differ in the protonation state of His42, see scheme 1.23 Removal of the hydrogen ligand resulted in cleavage of the S—N bond,23 but this may reflect computational model choices (unpublished results). They also investigated the mechanism of sulfurane formation and suggested that it could not occur via direct reaction of sulfenic acid with imidazole due to an unfeasibly high reaction barrier of 215.9 kJ mol-1.23 Instead, they proposed that a neutral Cp thiol reduces the peroxide with concomitant formation of the sulfurane intermediate (Scheme 1).23 A similar S—N interaction involving a histidyl was previously reported in human 1-Cys Prx (hORF6), although the observed distance was suggested to instead reflect a hydrogen-bonding interaction.24,25 It is noted, however, that in sulfenamides a covalent single S—N bond length is typically in the vicinity of 1.7 Å.26,27

Scheme 1. The proposed mechanism23 for sulfurane formation in ApTPx. H S

Cys50

O

H2O2

S

H N

N His42

HN

Cys50

H His42 H2O

N (H+)

There is increasing interest in hypervalent sulfur-containing species due in part to a growing awareness of their potential key roles in biological systems.28 For example, they are thought to be involved in the catalytic mechanisms of methionine sulfoxide reductases (Msr's)29,30 and Sulfiredoxin (Srx).31 More recently, Iwaoka et al.28 characterized pseudo-hypervalent divalent sulfur species in phospholipase, ribonuclease A, insulin and lysozyme. Consequently, they suggested that such interactions may hold a broader importance for protein architecture and function. Indeed, such weak S…X (X = O, N or S) interactions are similar to halogen-bonds;32 the s hole bonding originates from a positive electrostatic potential on the sulfur that enables orbital interactions between the lone pair on X and an anti-bonding orbital involving the sulfur, nX®s*S.33 5 ACS Paragon Plus Environment


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In this study hybrid quantum mechanics/molecular mechanics (QM/MM)-based methods have been used to investigate formation of the initial sulfenic acid (i.e., hydrogen peroxide reduction) and subsequent proposed hypervalent sulfurane intermediate in Archaea typical 2-Cys Prx (ApTPx). QTAIM, NCIPLOT, and NBO analyses of the sulfurane have been performed to provide insights into the nature of its S…N interaction. Sulfurane formation in the related human peroxidase hORF6 has also been considered. Finally, for the first time, the mechanism for overoxidation of sulfenic to a sulfinic acid has also been examined.

Computational Methods Molecular Dynamics Simulations: The Molecular Operating Environment (MOE) software package was used to prepare all starting structures for MD simulations and for subsequent analysis of the results.34 The NAMD program was used to perform all MD simulations.35 The default settings for both programs were used unless otherwise noted. For ApTPx, the X-ray structure of the C207S mutant with bound H2O2 (PDB ID: 3A2V)13 was chosen as a suitable initial structure for MD simulations on a monomer of the decameric form. This structure was chosen as it already contains active site-bound H2O2 and shows several key active site interactions. For MD simulations on human 1-Cys Prx (hORF6), the X-ray crystal structure (PDB ID: 1PRX)24,25 of the Prx-sulfenic acid derivative was used to provide a suitable starting structure for a monomer. Prior to MD simulations, MOE was used to prepare the structures by adding missing hydrogen atoms. In ApTPX, the position of His42 was modified manually allowing for His42…Cys50 interaction in accordance with their observed positioning in the experimental23 X-ray structure of the oxidized archaeal peroxiredoxin showing the hypervalent intermediate (PDB ID: 2ZCT). It is also noted that over the course of the MD simulation on the native enzyme, the histidyl was observed to be positionally flexible; a common conformation sampled putting it near Cys50 (Figure S3). The C207S mutation was left as is as it is remote (> 44 Å) from the current active site region of interest. The initial ionization state of appropriate groups was determined using the protonate 3D application in MOE and the PROPKA program.36,37 The generated structures were solvated up to 10 Å beyond every protein 6 ACS Paragon Plus Environment

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atom. Then, were minimized using the Amber12:EHT force field which was also used to parameterize H2O2, with all waters being described using the TIP3P forcefield.38,39 The minimized structures were then used as starting points for 500 ps equilibration MD simulations, done in NPT, to generate thermally relaxed structures; a protocol similar to that previously used successfully.40-42 In the MD simulations a time step of 2 fs was used while the PME method was used to calculate columbic interactions and the van der Waals interactions were truncated at 10 Å. This short equilibration time was chosen to maintain the original peroxide interactions from the X-ray complex structure as well as to avoid its diffusion during the simulation. In hORF6, the S–O bond in the sulfenic acid intermediate was broken to generate the reduced form of the enzyme while the peroxide substrate was manually docked in the active site prior to simulation. A third MD simulation was performed to simulate the overoxidation mechanism. More specifically, the QM/MM optimized structure of ApTPx sulfenic intermediate was used as starting structure and a H2O2 molecule was manually docked in the active site. The Cys50…H2O2 distance was constrained to 3 Å during the minimization, thus allowing for substrate binding adjustment in the active site. Later, similar solvation, minimization and MD simulation protocols as described above were used. QM/MM Models and Calculations: All calculations were performed within the ONIOM formalism using the Gaussian 09 suite of programs.43 The last structures of the previous MD simulations were used as starting structures for QM/MM calculations.44 The QM atoms were optimized at the M06-2X/631G(d,p)45 level of theory while the rest of the monomer was calculated using the AMBER96 force field as implemented in Gaussian 09.39 Frequency calculations were used to confirm the nature of the optimized stationary points using only the QM layer due to model size as has been previously successfully used.40,41 Relative energies were obtained using the ONIOM electrostatic embedding formalism via single points calculations at the ONIOM(M06-2X/6-311+G(2d,p):Amber96) on the above optimized structures. It is noted that we also assessed the use of other basis sets in conjunction with the M06-2X functional including 6-311+G(2df,p) and 6-311++G(3df,3pd), as well as ONIOM(MP2/6-31G(d,p):Amber96); and the above method used was found to be a suitable choice (Figure S1). It is noted that the calculated barriers are not corrected for entropic effects. 7 ACS Paragon Plus Environment


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Figure 1. Illustration of the ApTPx QM/MM model used in this study. The QM layer atoms are highlighted and shown in both wire and stick-ball representations.

QM/MM models were generated from the last conformer of the equilibrated MD simulations of the ApTPx/H2O2, ApTPx(sulfenic acid)/H2O2 and hORF6/H2O2 complexes, respectively. All MM atoms 15 Å away from the Cys50 sulfur were kept fixed in the optimization. For ApTPx, the QM layer included H2O2, Cys50, Pro48, Thr47, the backbone of Val40 and the R groups of His42, Asp45, Glu53, Arg126 and Arg149 (Figure 1). For studies on overoxidation of the sulfenic acid, in addition to these residues and H2O2, Pro43, Ala44 and a water molecule were also included in the QM layer. In the investigations on hORF6, the QM-layer consisted of H2O2, Cys47, backbone of Val46, Pro45, Thr44, His39, Pro40, part of Ser38 backbone, and the side chains of Arg132, Arg155, and Glu50, as well as three H2O. The nature of the S…N interaction was examined using the Quantum Theory Atoms-in-Molecules (QTAIM) AIM2000 and the NCIPLOT (Figure S2) programs.46,47 Natural Bond Orbital (NBO) analyses were also used to examine various interactions and to determine atomic partial charges in the optimized structures.


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Results and discussion The H2O2 active site-bound reactant complex. The optimized structure of the substrate-bound active site of ApTPx (RC), with selected bond lengths, is shown in Figure 2. It is noted that upon optimization the protonated His42 imidazole transferred a proton to the side-chain carboxylate of Asp45 with which it remains hydrogen bonded. As a result, in RC Cys50 has an anionic thiolate (Cys50S–), while the side-chains of His42 and Asp45 are both neutral. However, more importantly the optimized structure of RC in general exhibits similar interactions to those observed in an X-ray structure13 of a mutated ApTPx with bound H2O2. In RC the anionic charge on the sulfur of Cys50 is stabilized via four moderate hydrogen bond interactions with the side chains of Arg126 (2.36 and 2.39 Å), His42 (2.24 Å) and Thr49 (2.54 Å), Figure 2. The H2O2 moiety also forms multiple hydrogen bonding interactions with several residues upon binding within the active site. In particular, its OB oxygen and OAH group forms moderately strong hydrogen bonds with the backbone amide -NH- of Val49, r(OB…HNVal49)=1.91 Å, and sidechain hydroxyl oxygen of Thr47, r(OBH…OThr47)=1.94 Å, respectively. In addition, its OA center forms two weaker hydrogen bonds, both with lengths of 2.33 Å, with the backbone amide -NH- of Cys50 and side-chain guanidinium ion of Arg126 (Figure 2). Notably, and as has been previously suggested,48,49 these hydrogen bonds appear to help polarize the H2O2 within the active site. For instance, NBO analyses were used to compare the charges on the oxygen's of H2O2 when bound within the active site, with isolated H2O2 both in the gas-phase (dielectric constant (e)=1) and a homogeneously polar proteinlike environment (e=4). In the latter two scenarios, the negative charge on the H2O2 oxygen’s is symmetric with values of -0.47 and -0.48, respectively. However, when H2O2 is bound in the ApTPx active site, the HOA–OBH bond becomes slightly polarized with charges of -0.52 and -0.49 for OA and OB, respectively. Meanwhile, the ÐH-OB-OA-H dihedral angle decreases significantly from 179.9° to 112.0°, further highlighting the protein's influence on the H2O2 moiety. It is noted that the Cys50S center has a charge of -0.63 while the Cys50S...OA distance is approximately 3.2 Å.



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Figure 2. Illustration of the QM/MM optimized active site-bound substrate, reactive complex (RC), of ApTPx. For clarity only the QM layer is shown.

Sulfurane intermediate formation in ApTPx. The first step in the overall mechanism is reduction of the peroxide moiety. More specifically, the Cys50 thiolate nucleophilically attacks the nearest oxygen (OA) of the H2O2 moiety. This step occurs via TS1 at a cost of 82.1 kJ mol-1 relative to RC (Figure 3). The resulting Cys50-derived sulfenic acid intermediate (I1) lies considerably lower in energy than RC by 88.9 kJ mol-1. It is noted that similar to that previously described for peroxidredoxins,48,49 the Arg126 residue may facilitate this step. In particular, it helps stabilize the Cys50S- thiolate via a hydrogen bonding interaction and also forms a strong hydrogen bond with OA of the hydrogen peroxide. Sulfenic acid (I1) formation occurs with concomitant transfer of two protons. Specifically, the proton from the hydrogen peroxide's OAH group transfers onto its leaving OB oxygen to give a water molecule (H2OB), while the guanidinium ion of Arg126 transfers a proton onto OA which is now the sulfenic acid's oxygen (Cys50SOAH; Figure 4). The side chain hydroxyl of Thr47 also forms a strong hydrogen bond with the sulfenic acid oxygen with a Thr47

OH…(H)OASCys50 distance of 1.71 Å. 10 ACS Paragon Plus Environment

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The sulfenic acid intermediate I1 can readily undergo proton loss from its

Cys50

SOH group to the

guanidinium of Arg126 with which it is hydrogen bonded. This step proceeds via TS2 with a barrier of just 4.8 kJ mol-1 to give the sulfenate-type intermediate I2 which lies 15.0 kJ mol-1 lower in energy than I1 (Figure 3). In I2, the

Cys50

S–O bond has shortened slightly by 0.02 Å to 1.64 Å while the negative

charge on the oxygen has increased from -0.93 to -1.09. Similar to that observed in I1, the

Cys50

SO–

oxyanion is stabilized by markedly strong hydrogen bonds with both the side chain Thr47 hydroxyl and guanidinium ion of Arg126 with distances of 1.56 and 1.43 Å, respectively (Figure 4). In addition, it also forms a moderately strong hydrogen bond with the newly formed water with a Cys50S–…H2O length of 1.93 Å.

Figure 3. Potential energy surface obtained (see Computational Methods) for the formation of a pseudo-sulfurane intermediate in ApTPx. In order to form the proposed23 sulfurane intermediate with its

Cys50

S...NHis42 interaction, the

protonated imidazole of His42 must lose a proton from its NπH moiety, i.e., that not hydrogen bonded 11 ACS Paragon Plus Environment


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to Asp45 (Nτ). Unfortunately, there appears to be no suitable base positioned near the imidazole of His42. However, in I2 the His42

His42

NπH moiety weakly interacts with the sulfenate sulfur of Cys50 at an

NH...SCys50 distance of 2.70 Å (Figure 4). Thus, the possibility of a proton transfer from

His42

NπH to

the sulfenate sulfur of Cys50 was considered and proceeds via TS3 with a quite low barrier of just 16.5 kJ mol-1. Importantly, the resulting sulfenic acid tautomer (Cys50SHO) intermediate I3 formed lies 11.5 kJ mol-1 lower in energy than I2 (Figure 3).

Figure 4. Optimized structures (see Computational Methods) of the intermediates and transition structures obtained for the mechanism of pseudo-sulfurane formation. The atoms shown represent the QM layer only, with the highlighted residues being those directly involved in the reaction. 12 ACS Paragon Plus Environment

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It is noted that TS3 involves a double proton transfer; the above mentioned transfer from onto

Cys50

His42

Nπ H

S and the other from the neutral carboxylic side-chain of Asp45 onto His42 imidazole's Nτ

nitrogen. The latter transfer is indicated by the fact that the relevant

His42

Nτ…H+ and

Asp45

COO–…H+

distances are now 1.14 and 1.47 Å, respectively (Figure 4). This transfer ensures the His42 imidazole remains neutral within the present computational model. A considerable shortening of the S–O bond by 0.08 Å to 1.56 Å is observed upon forming I3. The oxygen of the sulfenic acid tautomer maintains its three hydrogen bond interactions observed in I2 with the side chains of Thr47 and Arg126, and the previously formed active site water. Now, however, there is also a non-covalent nN®s*S interaction between the imidazole's His42

the

His42

Nπ nitrogen and sulfur of the Cys50-derived sulfenic acid tautomer with a

Nπ...SCys50 distance of 2.76 Å. This interaction was analyzed using QTAIM and a bond path between His42

Nπ and

Cys50

S centers was obtained with a critical point electron density (r) of 0.020 and

Laplacian (Ñr2) of 0.017, also suggesting a non-covalent interaction.47,50-53 NBO analysis of I3 indicates that there is a positive charge on Cys50S of 1.05 and a negative charge on His42Nπ of -0.63. It is also noted that the optimized ÐNπ–S–O angle of 164.1° in I3 is in close agreement with the available experimental X-ray structure.13 The calculated

His42

Nπ–SCys50 distance in the pseudo-hypervalent intermediate I3 is longer than that

observed experimentally.23 This may reflect at least in part well-known challenges in computational modelling of in particular, weak non-covalent interactions. The impact of key active site interactions or further reactions on the strength of the

Cys50

occurrence of a hydrogen bond between transfer from

His42

NτH to

Asp45

S–NHis42 bond was also examined. In particular, given the

His42

NτH and the carboxylate of Asp45, the impact of proton

COO– was considered; i.e., the His42 imidazole now has greater negative

charge. This transfer can proceed via TS4 to give the deprotonated sulfurane-type intermediate I4. It is noted that the lower energy of TS4 relative to I4 is a common artefact due to the use of single-point energy corrections for TS's on comparatively flat PESs. It simply indicates that the forward or reverse reaction essentially occurs without a barrier. While the latter is moderately higher in energy that I3 by 43.7 kJ mol-1, it is still significantly lower in energy than RC by 71.7 kJ mol-1. In I4 the His42 imidazole is now formally anionic while the Asp45 side chain is now neutral. The sulfenic S–O bond 13 ACS Paragon Plus Environment


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has slightly increased to 1.58 Å while the

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Nπ–SCys50 bond has shortened significantly by 0.18 Å to

His42

2.58 Å. Concomitantly, the ÐNπ–S–O angle has increased slightly to 166.1°. A QTAIM analysis of I4 suggests that the

Nπ–SCys50 non-covalent interaction has strengthened as indicated by an increase in

His42

values of the density (r) and Laplacian (Ñr2) at the critical point to 0.030 and 0.023, respectively. It is noted that the nature of this non-covalent interaction in I3 and I4 was also confirmed using the NCIPLOT program (Figure S2). NBO analysis indicates that there is also a slight increase in negative charge on the sulfurane oxygen by 0.01 to -1.10 and nitrogen by 0.05 to -0.68. This suggests that Asp45 may help enhance or moderate the strength of the non-covalent

His

Nπ…SCys interaction in the

sulfurane-like intermediate through its hydrogen bond with His42. We also considered protonation of I4 by an active site residue other than the now neutral Asp45

COOH. Given the residues with which the sulfurane's oxygen hydrogen bonds, one possible

candidate is Arg126. Indeed, proton transfer from the guanidinum of Arg126 onto the sulfurane oxygen leads via TS5 to formation of intermediate I5 (Figure 3). Importantly, however, the latter lies significantly higher in energy than I3 by 130.6 kJ mol-1, and is 15.2 kJ mol-1 higher in energy than RC. Thus, this reaction would seem unlikely to occur within the enzyme. The last step in the native mechanism of ApTPx is formation of a disulfide bond. It is noted that in order for this to occur sulfurane formation must be reversible so as to enable sulfenic acid reduction and subsequent disulfide formation. The present potential energy surface obtained (Figure 3) suggests that the sulfurane I4 could be readily reduced to the sulfenate (I2) or sulfenic acid (I1) with relatively low barriers of just 28.0 and 31.3 kJ mol-1, respectively.

Oxidation of the sulfenic acid in ApTPx. Presumably, sulfurane formation occurs more readily than oxidation of the sulfenic acid intermediate to its corresponding sulfinic (Cys50SOOH) acid derivative. However, in order to gain further insights into the protective role of sulfurane formation, the mechanism for oxidation of the sulfenic acid to sulfinic acid by H2O2 was examined.


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As noted in the Computational Methods, the QM/MM optimized structure of the sulfenic acid intermediate (I1) was used as the starting point for the MD simulations. Two complexes were considered which differed only in whether the imidazole of His42 was neutral or protonated. However, only the results for the case of neutral His42 are discussed herein as the barrier obtained for protonated His42 was unfeasibly high at 183.3 kJ mol-1 (not shown). Optimized structures of the resulting reactant, transition structure and product complexes, with selected bond lengths, are shown in Figure 5.

Figure 5. Optimized structures (only QM-region shown) of the reactant, transition structure, and product complexes obtained for the mechanism of sulfenic acid oxidation by H2O2. For clarity, the QMregion groups directly involved in the reaction are highlighted.

During the MD simulation, and as suggested experimentally,54 consistent binding of H2O2 within the active site only occurred upon rotation about the Cys50 Cb–S bond such that the sulfenic acid (Cys50SOH) moiety was directed away from the H2O2 binding site (see, for example, Figure 5). This may at least in part explain the role of sulfurane formation in overoxidation protection; presence of a noncovalent

S...NHis42 interaction may help hinder such a rotation. The QM/MM optimized structure of

Cys50

I1 with H2O2 also bound in the active site (RCOx) shows similar hydrogen bonding interactions between the H2O2 moiety and active site residues to those observed in RC (cf. Figure 4). Specifically, its OA center hydrogen bonds to the backbone amide -NH- of Cys50, and the side chain Thr47 hydroxyl and 15 ACS Paragon Plus Environment


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Arg126 guanidinium with distances of 2.06, 2.57, and 1.68 Å, respectively. Meanwhile, its OB oxygen hydrogen bonds with the backbone amide -NH- of Val49 with r(OB…HNVal49)=1.99 Å. Importantly, the sulfenic acid –SOH group is hydrogen bonded to the imidazole of His42. As a result, its sulfur center is directly exposed to the H2O2 with an

Cys50

S…OA distance similar to that observed in RC of 3.09 Å (cf.

Figure 4). Oxidation of the sulfenic acid by reaction with H2O2 to give a sulfinic acid occurs in one step via TSOx at a cost of 121.6 kJ mol-1. The sulfinic acid product complex (PCOx) lies markedly lower in energy than the initial reactant complex RCOx by 179.4 kJ mol-1. Thus, while further oxidation is highly exergonic, the barrier for reaction is considerably higher than that obtained for sulfurane formation (16.5 kJ mol-1; Figure 3). It is noted that experimentally16,17 it has been suggested that overoxidation is irreversible.

Sulfurane formation in human 1-Cys hORF6. As noted in the introduction, 1-Cys Prxs lack the presence of a second cysteine.1 This suggests that they may also have a mechanism for protection of the sulfenic acid intermediate. Indeed, in an X-ray crystal structure25 of human 1-Cys hORF6 an imidazole nitrogen of His39 is 3.0 Å from the sulfur of Cys47 suggesting a hydrogen bonding interaction. Furthermore, the carboxylate side chain of Glu50 is situated near the imidazole of His39. In the present MD simulations on the solvated reactant complex (see Computational Methods) a water molecule forms a consistent hydrogen bonding bridge between the side chains of His39 and Glu50. This suggests a path by which His39 may become deprotonated as appears needed if a sulfurane-type intermediate similar to that of ApTPx is to be formed. Thus, the potential for formation of a similar pseudo-hypervalent sulfurane intermediate in hORF6 was examined. Optimized structures of the initial reactant and key intermediate complexes obtained are shown in Figure 6.


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Figure 6. Optimized structures (only QM-region shown) of the reactant and intermediate structures obtained for sulfenic acid formation and oxidation in hORF6. Those groups within the QM-region directly involved in the reaction are highlighted for clarity.

The QM/MM optimized structure of the H2O2-bound reactive complex (RChORF6) exhibits similar active site residue…H2O2 interactions to those observed in RC for ApTPx. In particular, the hydrogen peroxide's OB oxygen hydrogen bonds with the backbone amide -NH- of Val46 with a distance of 2.08 17 ACS Paragon Plus Environment


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Å. Concomitantly, its OA oxygen hydrogen bonds with the backbone amide -NH- of Cys47, and the side chain Thr44 hydroxyl and Arg132 guanidinium with distances of 2.02, 2.29, and 1.59 Å, respectively (cf. Figure 2). Similarly, the thiolate of Cys47 is stabilized by four hydrogen bonds; two with Arg132, and one each with His39 and a water with distances of 2.39, 2.27, 2.02, and 2.02 Å respectively. It should also be noted that the protonated imidazole of His39 is strongly hydrogen bonded to a water, r(His39NH…Ow)=1.52 Å, which in turn is strongly hydrogen bonded to the carboxylate of Glu50, r(OwH…-OOCGlu50)=1.48 Å. Similar to that observed for ApTPx, the corresponding sulfenic acid intermediate (I1hORF6) is lower in energy than RChORF6 though now by -111.3 kJ mol-1. Furthermore, the previously protonated imidazole of His39 has transferred its NτH proton to the bridging water which has itself donated a proton to the Glu50 carboxylate. After these transfers the strong His39…H2Ow…Glu50 hydrogen bond bridge is retained with His39Nτ…HOw and Ow…HOOCGlu50 distances of 1.79 and 1.50 Å, respectively. In addition, and again analogous to that observed for ApTPx, the guanidinium of Arg132 has transferred a proton onto the sulfenic acid's oxygen while the

His39

NπH…SCys47 hydrogen bond has lengthened from

2.02 to 2.36 Å (Figure 6). The analogous subsequent pseudo-sulfurane like intermediate I2hORF6 is also calculated to lie decidedly lower in energy than the sulfenic acid intermediate I1hORF6, but now by 55.1 kJ mol-1. Thus, at least thermodynamically, formation of a pseudo-sulfurane intermediate may be possible in human 1Cys hORF6. It is noted that in ApTPx the corresponding sulfurane-like intermediate I3 is 26.5 kJ mol-1 lower in energy than the corresponding sulfenic acid intermediate I1 (cf. Figure 3). The newly formed Nπ...SCys47 interaction in I2hORF6 has a length of 2.87 Å which is 0.11 Å longer than observed for the

His39

corresponding intermediate I3 in ApTPx. However, QTAIM analysis of I2hORF6 gave r and Ñr2 values at its critical bond point of 0.017 and 0.015. respectively. Again, such values are usually indicative of a weak non-covalent His39Nπ...SCys47 interaction.47,50-53 It is also noted that with formation of I2hORF6 the side chain Glu50COOH group has transferred a proton back via the bridging water onto the His39Nτ center while the sulfenic acid

Cys47

SOH moiety in I1hORF6 has transferred its proton back to the guanidinium of

Arg132. Thus, I2hORF6 can be thought as containing a sulfenic acid tautomer and neutral imidazole. 18 ACS Paragon Plus Environment

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We also examined the impact of deprotonation of the

His39

NτH group in I2hORF6, though now

indirectly by Glu50 via the hydrogen bonding water bridge. This proton transfer, is thermodynamically unfavorable with the resulting I3hORF6 complex lying significantly higher in energy than I2hORF6 by 67.5 kJ mol-1 (Figure 6). Notably, and as observed in ApTPx, the

Cys

S...NHis interaction has shortened

markedly to 2.78 Å while the electron density and Laplacian at its critical point has concomitantly increased slightly to r=0.022 and Ñr2=0.017, respectively. This appears to further support the suggestion that in addition to a mechanistic role an Asp/Glu…His interaction may also influence the stability of the CysS...NHis interaction.

Conclusion In this study a detailed computational investigation on possible oxidation protection mechanisms in an Archaeal typical 2-Cys Prx (ApTPx) enzyme has been performed. Molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) methods have been used to examine oxidation of the active site cysteinyl thiolate (Cys50S–) by H2O2 to give a sulfenic acid (Cys50SOH), and its subsequent intramolecular reaction to form a pseudo-hypervalent sulfurane. Natural Bond Order (NBO) and Quantum Theory of Atoms in Molecules (QTAIM) analyses were performed to help elucidate the nature of key interactions. The overall rate-limiting step for formation of a pseudo-hypervalent sulfurane (I3) from the initial Cys50

S– thiolate is the initial oxidation of the active site Cys50S–with H2O2 (RC) to give Cys50SOH and H2O

(I1). This process occurs in one step with a barrier of 82.1 kJ mol-1. Subsequent subsequent formation of a sulfrane-like intermediate (I3) then occur in two-steps with much lower barriers. More specifically, I1 first undergoes deprotonation of its

Cys50

SOH group to give a sulfenate (Cys50SO–) intermediate (I2)

which is stabilized by hydrogen bonds with both active site residues (Thr47 and Arg126) and water. The

Cys50

SO– moiety then reacts with the imidazole of His42 to give the sulfurane-type intermediate

(I3). This latter step, with a barrier of just 16.5 kJ mol-1, is the rate-limiting step for conversion of I1 to the sulfurane-type intermediate I3. QTAIM and NBO analyses characterize the His42Nπ…SCys50 bond as a non-covalent nNπ®s*S type interaction. 19 ACS Paragon Plus Environment


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The present findings indicate that the reverse reaction, reformation of I1 from I3 thus allowing the formers reduction by a resolving Cys, should be facile with an overall barrier of 31.3 kJ mol-1. In contrast, oxidation of Cys50SOH by H2O2 to give the sulfinic acid derivative occurs with a notably higher barrier of 121.6 kJ mol-1. Furthermore, an aspartate (Asp45) hydrogen bonded to the imidazole of His42 may play an important role in the formation and stability of the pseudo-hypervalent sulfurane. In particular, it helps mediate the ionization state of His42 and thus formation of the non-covalent His42

Nπ…SCys50 interaction. The potential for the broader applicability of the present findings for formation of analogous species

in related Prx enzymes was also explored. Specifically, the related Prx enzyme 1-Cys human hORF6 also contains an active site cysteinyl (Cys47) thiol positioned near a histidyl (His39) imidazole that is itself near to a side chain glutamyl (Glu50) carboxylate. The potential reactant and intermediate complexes were optimized for hORF6 and appear to follow similar trends and interactions to those observed in ApTPx, e.g., the sulfurane was the lowest energy intermediate obtained.

Supporting Information. Cartesian coordinates and energies of the optimized structures reported here. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Telephone: (519) 253-3000, ext.3992. Fax: (519) 973-7098

Present Addresses † Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada

ACKNOWLEDGMENT 20 ACS Paragon Plus Environment

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We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding and Compute Canada and SHARCNET for additional computational resources. D.J.S. also thanks the Ontario Graduate Scholarship (OGS) program and the NSERC CGS-Master’s program for financial support.

ABBREVIATIONS ApTPx, Archaeal thioredoxin peroxidase; ONIOM, own n-layered integrated molecular orbital and molecular mechanics; QTAIM, Quantum Theory Atoms-in-Molecules; NBO natural bond orbital; Prxs, peroxiredoxins; Tpx thioredoxins; BCP, bacterioferritin comigratory protein; AhpE, alkyl hydroxyperoxide reductase E; CR, resolving cysteine; Cp, catalytic/peroxidative cysteine; MOE, Molecular Operating Environment; MD, molecular dynamics; PME, Particle Mesh Ewald; QM/MM, quantum mechanics/molecular mechanics; QM, quantum mechanics; RC, reactant complex; TS, transition state; IC, intermediate complex; SUMO protease, small ubiquitin-like modifier protease; hORF6, human open reading frame 6;

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TOC Graphic

? Cysteine

Sulfinic acid

Sulfenic acid

Pseudo-hypervalent Sulfurane


A Pseudo-Hypervalent Sulfur Intermediate As An Oxidative Protective

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