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Two-step reaction mechanism reveals new capability of against hydroxyl radical attack

Sarju Adhikaria, Ramon Crehuetb, Josep M. Angladab,1, Joseph S. Franciscoc,1, and Yu Xiaa,d,1

aDepartment of , Purdue University, West Lafayette, IN 47907; bDepartment of Biological Chemistry, Institute of Advanced Chemistry of Catalonia, IQAC-CSIC, E-08034 Barcelona, Spain; cDepartment of Chemistry, University of Pennsylvania, Philadelphia, PA 19104; and dDepartment of Chemistry, Tsinghua University, Beijing 100084, China

Contributed by Joseph S. Francisco, May 30, 2020 (sent for review April 9, 2020; reviewed by Richard A. O’ and Henry F. Schaefer III) Cysteine disulfides, which constitute an important component in group leads to H- transfer (13); thus-formed thiyl radicals are • biological buffer systems, are highly reactive toward the cycled in the –SS–/–SH/–S system via chemical reactions or en- • hydroxyl radical ( OH). The mechanistic details of this reaction, how- zymatic regulation (3). Although is not considered to be ever, remain unclear, largely due to the difficulty in characterizing an efficient antioxidant, due to its limited reactivity toward peroxyl • •− • unstable reaction products. Herein, we have developed a combined radicals (e.g., OOH and O2 ), the rate of its reaction with OH approach involving mass spectrometry (MS) and theoretical calcula- is under diffusion control (10); thus, its conversion to other • tions to investigate reactions of OH with cysteine disulfides (Cys– compounds directly impacts the steady state of –SS–/–SH redox S–S–R) in the gas phase. Four types of first-generation products system. It is believed that, in aqueous solutions, single-electron + were identified: protonated of the cysteine thiyl radical ( Cys– oxidation of the disulfide bond followed by bond cleavage into • + + • • • S ), cysteine ( Cys–SH), cysteine sulfinyl radical ( Cys–SO ), and cys- –S and –S+ is the dominant pathway by which OH reacts with + teine ( Cys–SOH). The relative reaction rates and prod- disulfide bonds (14, 15). The disulfide cleavage products can uct branching ratios responded sensitively to the electronic property • − further react with O2, OH, OH , or other ROS, forming a series of the R group, providing key evidence to deriving a two-step reac- – • of oxyacids of sulfur, including sulfenic acid ( SOH), sulfinic acid tion mechanism. The first step involved OH conducting a back-side – – ( SO H), and ( SO H) (3). For the reactions in CHEMISTRY – 2 3 attack on one of the sulfur , forming sulfenic acid ( SOH) and nonpolar environments, such as at the interface of lipid membrane – • thiyl radical ( S ) product pairs. A subsequent H transfer step within and plasma or in the hydrophobic regions of tertiary the product complex was favored for protonated systems, generat- structures, the role of cysteine disulfides is less well understood. – • – ing sulfinyl radical ( SO )andthiol( SH) products. Because sulfenic More relevant information here can be gleaned from reactions of • • acid is a potent scavenger of peroxyl radicals, our results implied OH with organic disulfides in the gas phase. In general, OH that cysteine disulfide can form two lines of defense against reac- substitution at the disulfide bond occurs more readily than any tive species, one using the cysteine disulfide itself and the possible H abstraction or C–S bond cleavage in gas-phase reac- other using the sulfenic acid product of the conversion of cysteine tions (16, 17). Due to disagreement on the identities of the first- disulfide. This aspect suggested that, in a nonpolar environment, generation reaction products, different elementary steps have cysteine disulfides might play a more active role in the antioxidant been proposed for the mechanism by which the disulfide bond is network than previously appreciated. cleaved. For instance, the earliest description of the mechanism disulfide bond | hydroxyl radical | reaction intermediate | mass spectrometry | antioxidant Significance

In this work, we harnessed mass spectrometry for online reac- he dynamic formation and cleavage of a disulfide linkage • between two cysteine residues has profound im- tion monitoring of hydroxyl radical ( OH) attack to cysteine T disulfide and acquired mechanistic details that had not been portance in cell biology, in particular for redox sensing, allosteric • regulation of protein functions, and enzyme catalysis (1, 2). achieved previously. Our findings suggest that OH substitution Equally important is the disulfide (–SS–)/ (–SH)/thiyl radical at the disulfide bond is a fast and predominant reaction channel • (–S ) redox system of low–molecular-weight sulfur compounds, in the gas phase, while subsequent transfer within the product pairs can be favorable in protonated systems. Notably, such as the redox pairs of and cysteine, the molar • ratios of which directly impact redox homeostasis in cells or by reacting with OH cysteine disulfide converts itself to a more potent antioxidant, sulfenic acid (–SOH), thus forming two lines plasma (3–5). Three types of reactions have been identified to be of defense against reactive oxygen species (ROS). These results responsible for chemical transformations between the –SS–, –SH, • provide insight into studying the antioxidant roles of cysteine and –S functional groups: nucleophilic substitution-eliminations, disulfide in nonpolar biological environment, such as at the in- specifically so-called thiol–disulfide interchange reactions (6), terface of lipid membrane and plasma. single-electron reactions of free or disulfide bonds (7), and

radical reactions (8). When the cell is under oxidative stress, Author contributions: J.M.A., J.S.F., and Y.X. designed research; S.A., R.C., J.M.A., J.S.F., low–molecular-weight sulfur compounds work synergistically with and Y.X. performed research; S.A., R.C., J.M.A., J.S.F., and Y.X. analyzed data; and S.A., other (e.g., ascorbic acid and tocopherol) in the R.C., J.M.A., J.S.F., and Y.X. wrote the paper. “antioxidant network” to combat elevated concentrations of re- Reviewers: R.A.O., University of Melbourne; and H.F.S., University of Georgia. • active oxygen species (ROS) (9, 10). The hydroxyl radical ( OH) is The authors declare no competing interest. among the most oxidative species produced in aerobic systems, Published under the PNAS license. •− typically generated from superoxide anion (O2 )andH2O2 by 1To whom correspondence may be addressed. Email: [email protected], frjoseph@sas. Fenton and/or Haber–Weiss reactions (7). Besides, peroxynitrous upenn.edu, or [email protected]. • • acid (ONOOH) readily decomposes to OH and NO2 at neutral This article contains supporting information online at https://www.pnas.org/lookup/suppl/ • pH in biological systems, thus serving as an in situ source of OH doi:10.1073/pnas.2006639117/-/DCSupplemental. • (11, 12). Direct interaction between OH and the thiol functional

www.pnas.org/cgi/doi/10.1073/pnas.2006639117 PNAS Latest Articles | 1of8 Downloaded by guest on September 30, 2021 • described a relatively simple picture, involving addition of OH onto the disulfide bond in R–S–S–R followed by rapid scission of • the disulfide bond into RS and RSOH (18). Butkovskaya and • Setser (19) later detected two product pairs: CH3SH/CH3SO and • CH3S /CH3SOH, with the former pair being found more favor- • able from reactions of dimethyl disulfide with OH. Bil et al. suggested H atom transfer in the product complex [CH3SOH– • • SCH3] to be a key step to account for the detection of CH3SO / • HSCH3 (20). However, the product pair CH3S /CH3SOH was not detected and thus ruled out in their mechanism. Obviously, the difficulties in identifying and quantifying the reactive inter- mediates have constituted a major hurdle for mechanistic studies of radical reactions. While electron paramagnetic resonance and other spectro- scopic methods are useful tools for detecting radical species (21), mass spectrometry (MS), which offers a distinct advantage in providing detailed molecular information, is increasingly being used to investigate the structure and reactivity of bio-radical ions in the gas phase (22–25). The recently developed electrospray ionization (ESI)–MS and direct MS for online reaction moni- toring have provided additional powerful tools for elucidating transient radical species in solution reactions (26–29). Given the • importance of –SS–/–SH/–S cycle in biology, we previously de- veloped an MS method to synthesize and characterize cysteine • • • thiyl (Cys–S ), perthiyl (Cys–SS ), and sulfinyl (Cys–SO )radicals in the gas phase (30–32). The method is based on performing radical reactions in the plume region of a nanoelectrospray ioni- zation (nano-ESI) source right before the sampling interface of a mass spectrometer. Such a setup provides a short sampling time Scheme 1. Online MS monitoring of reactions of OH• and cysteinyl disul- (submicrosecond to microsecond) so that bio-radicals can be de- fides (Cys–S–S–R) for various R groups with different electronic properties. tected and subsequently characterized using MS. For instance, two The Cys–S–S–R molecules became protonated at the group (+Cys–S– • pairs of reaction products, pep–S /pep–SOH and pep–SH/pep– S–R) when ionized using nano-ESI in positive mode. • SO resulting from disulfide bond cleavage in (pep–S– • S–pep), have been identified from reactions with OH (33). These and are supplied in SI Appendix. In brief, solutions containing results were consistent with the formation of two pairs of products equimolar amounts of the model compound and the internal upon disulfide bond cleavage from gas-phase studies and also standard (O-ethylated , I–S–S–I, 10 μM each in water) were suggested the feasibility of using MS to monitor the presence of ionized using nano-ESI in positive ion mode and subjected to sulfur radicals during reactions. • online reactions with OH radicals produced by performing di- Leveraging the detailed molecular information that MS can SI Appendix provide for radical reactions, in the current work we aimed to electric barrier discharge of helium in ambient air ( , • delineate the mechanism of the reaction of OH with the cysteine Fig. S1) (31). In this study, we chose to perform reactions in positive ion mode because higher ionization efficiency for Cys– disulfide bond in the gas phase. Gaining this type of knowledge is – – important for understanding the intrinsic reactivity of the disulfide S S R was obtained than that from negative ion mode and the internal standard could not be ionized in negative ion mode. It bond, and it is relevant to reactions occurring in nonpolar mi- • croenvironments of biological systems. A series of cysteine disul- should be noted that similar reaction phenomena of OH attack to fide derivatives was investigated (Cys–S–S–R, structure shown in cysteinyl disulfide bond were observed regardless of the ion po- Scheme 1) using a combined experimental and theoretical ap- larity (33). Although minor amounts of reactive species such as H proach. The electronic character of the disulfide bond was mod- atoms, O atoms, singlet oxygen, and ozone were also expected to ulated by changing the inductive effect of the connecting R group. have formed under the discharge conditions (34), their contribu- For MS experiments, we focused on optimizing the formation of tions to the observed reaction phenomena were expected to be the first-generation products and performing a cross-group com- insignificant. This expectation was due to a couple of factors: H and O atoms having short (nanosecond-scale) lifetimes and being parison of the reaction reactivities resulting from the R substitu- • ents. We were interested in describing the effect of the electronic quickly converted to OH upon collisions with water molecules character of the disulfide bond on its reactivity and the product (35), and ozone (36) or singlet oxygen (37) reacting very sluggishly branching ratio. High-level theoretical calculations were carried with cystine disulfide. According to the ion evaporation model, out to provide detailed descriptions of the thermodynamics and developed to explain the ionization of small molecules by ESI kinetics of the reactions. A two-step reaction mechanism was (38), cysteine disulfides should most likely be in the form of • • derived, with this mechanism involving rapid OH substitution on protonated ions when encountering OH. Note that because of either sulfur atom, leading to cleavage of the disulfide bond and the lack of basic functional groups for protonation in the tested R – – • – • – groups (except for R = cysteine), the disulfide cleavage products formation of Cys SOH/R S and Cys S /R SOH. For protonated • • reaction systems, subsequent transfer of H within the product involving the R side chain, viz. R–S ,R–SH, R–SO ,andR–SOH, complex was indicated to be a significant process, and to be assisted could not be detected using MS. For this reason, the same types of by a stabilizing effect of hydrogen bonding in the transition states. ionic products were expected to be observed from the disulfide cleavages of all of the tested Cys–S–S–R compounds, albeit with Results and Discussion perhaps different relative abundances of these ionic products due Reaction Phenomena of •OH and Cys–S–S–R. Experimental details to the impact of the R substituent. Fig. 1A summarizes the • • for conducting the reactions between OH and Cys–S–S–Rinthe expected main reaction products from OH attack on different • plume region of a nano-ESI source have been reported previously sulfur atoms in the disulfide bond. The attack of OH on the

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.2006639117 Adhikari et al. Downloaded by guest on September 30, 2021 sulfur atom connected to R should produce protonated cysteine subsequent reaction products, i.e., I–SO2H(m/z 182.1, Fig. 1B), • thiyl radical (+Cys–S , m/z 121) and cysteine (+Cys–SH, m/z contributed relatively little to the spectrum. Therefore, we kept • • 122), while protonated cysteine sulfinyl radical (+Cys–SO , m/z the relative ion abundance of +I–SO at a constant 10% for all 137) and cysteine sulfenic acid (+Cys–SOH, m/z 138) should reactions to ensure that the same reaction conditions were reproduced for each individual Cys–S–S–R compound even result from an attack on the Cys side of sulfur atom. Possible • reaction products involving C–S bond cleavage or H abstraction though the actual number density of OH and the reaction time were not detected above noise level; thus, these reaction chan- could not be precisely controlled using our current experimental nels were not investigated in this study. methods. It is worth noting that doubly protonated ions were In order to perform relative reaction rate comparisons, we first only observed from I–S–S–I at relatively low ion abundances but • – – – investigated the reaction of OH with the internal standard (I– not for the rest of Cys S S R compounds. Their presence S–S–I, O-ethylated cystine). Fig. 1B shows the reaction spectrum; should not cause interferences to the measurements of reactions involving Cys–S–S–R because the degree of reaction was moni- as predicted, four types of disulfide cleavage product ions were • • • + – + – – – m/z m/z detected: +I–S (m/z 149), +I–SH (m/z 150), +I–SO (m/z 165), tored using %( I SO / I S S I) and their value ( 149) + – m/z was different from reaction products involving Cys–S–S–R. and I SOH ( 166). Due to an overlap of the signal of the – – – – – – – – – doubly protonated form of I–S–S–I(m/z 149.1) with the signal of Reactions of Cys S S Cys, Cys S S CH3, and Cys S S + • C(O)–OCH were used as examples to demonstrate the char- singly protonated I–S form (also m/z 149.1), the actual amount 3 • acteristics of reactions of a symmetric disulfide bond vs. asym- of +I–S produced was likely lower than that indicated by the metric disulfide bond connected to an electron-donating group spectrum. By changing the rate of the helium gas flow in the (EDG) and electron-withdrawing group (EWG), respectively. discharge area and changing the position of the nano-ESI tip For Cys–S–S–Cys, the four expected reaction products were relative to the location of the discharge, different degrees of • clearly detected (Fig. 1C), showing patterns similar to that of the reaction of I–S–S–I with OH were tested (SI Appendix, Fig. S2). + • • internal standard; while for Cys–S–S–CH3 (Fig. 1D), the Cys–S + – • We found that by keeping the amount of I SO formed at and +Cys–SH signals were stronger than the +Cys–SO and ∼ – – – m/z B 10% of that of the remaining I S S I ions ( 297, Fig. 1 ), +Cys–SOH signals. The spectrum resulting from the reaction of the first-generation reaction products predominated while the Cys–S–S–C(O)OCH3 (Fig. 1D), however, presented a drastically • different product partitioning: +Cys–SO was detected as the predominant reaction product, while the signals corresponding A to the other three ionic products were much weaker. Overall, our results were consistent with the product branching ratios being CHEMISTRY sensitive to the electronic properties of the R group. • Although the actual number density of OH and the reaction time could not be precisely measured, the use of the internal B standard allowed us to compare the reactivities of all seven tested Cys–S–S–R model compounds. The %reaction of each Cys–S–S–R was defined as the sum of the peak areas of all first- generation reaction products divided by the peak area of the remaining +Cys–S–S–R ions. This value was further normalized to the %reaction of I–S–S–I to provide %rel. reactivity of each Cys–S–S–R compound. All experiments were repeated three times; the average %rel. reactivity values and associated SDs were C reported. As shown in Fig. 2A,Cy–S–S–R showed higher reac- tivities toward OH• with R being an EDG, i.e., –CH3 (100 ± 2%), –C3H7 (110 ± 17%), –Ph (120 ± 6%), than with it being an EWG, i.e., –CH2CF3 (23 ± 10%), –C(O)CH3 (64 ± 40%), –C(O) OCH3 (39 ± 7%). Interestingly, Cys–S–S–Cys showed a lower reactivity (66 ± 16%) than did the internal standard (100%), D despite the two compounds only differing in the modification of the ethyl at the carboxylic group. These results indicated that the –OH group in the might play a role in affecting the energetics of disulfide bond cleavage. • The preference of OH to attack the cysteinyl sulfur (Cys–S) (%selectivity)in+Cys–S–S–R was evaluated by calculating the + • + E summed ion abundances of Cys–SO and Cys–SOH from all first-generation products. A value of 50% selectivity would sug- • gest that the OH radical has an equal probability of attacking either sulfur atom of the disulfide bond. Approximately such a value (specifically 53 ± 12%) was in fact found for Cys–S–S–Cys, while 45 ± 4% was found for that of I–S–S–I(SI Appendix, Fig. S2B). A selectivity value greater than 50% would indicate that • the OH preferentially reacts with the cysteinyl sulfur (Cys–S), • Fig. 1. (A) Expected products of the reactions of OH with protonated • while a value less than 50% would indicate a preference for the Cys–S–S–R. The attack of OH on the sulfur atom linked to Cys- or R-side leads S–R moiety. As shown in Fig. 2B, the %selectivity values were to different products, while only protonated products can be detected by • clearly clustered into two groups based on whether an EWG or MS. Nano-ESI mass spectra of the reactions of OH with (B)I–S–S–I (O-ethy- • lated cystine), (C)Cys–S–S–Cys, (D)Cys–S–S–CH , and (E)Cys–S–S–C(O)OCH . EDG R group was used. Specifically, OH showed a high pref- 3 3 erence to attack the cysteinyl sulfur when R was an EWG, with Insets show magnified regions of the spectra of the first-generation reaction %selectivity ± – ± products. The same molar amount of I–S–S–I was added to each of the the values here at 70 9% ( CH2CF3), 68 13% Cys–S–S–R solutions in order to provide similar reaction conditions for all [–C(O)CH3], and 91 ± 1% [–C(O)OCH3]. For all of the cases in model compounds. which R was an EDG, the %selectivity values were all less than

Adhikari et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 30, 2021 A reaction mechanism to be the predominant pathway for both neutral and protonated Cys–S–S–R compounds (SI Appendix, Figs. S3–S7). The distance between the approaching oxygen atom and the attacked sulfur atom was indicated from our cal- culations to, in general, range between 1.82 and 2.17 Å, and the OSS angle generally ranged between 132° and 155°, similar to the • reaction of OH with hydropersulfides (41). In addition, intra- molecular hydrogen bonds were indicated to play a role in sta- B bilizing the transition states. Shorter hydrogen bonds, indicative of stronger bonds, were calculated for the protonated species than ... for the corresponding neutral species. For example, the N–H O bond in the low-lying transition state of reaction 2b of +Cys–S–S– ... CH3 showed a length of 1.794 Å (Fig. 3A), while O–H O showed a length of 1.867 Å in that of 2a of Cys–S–S–CH3 (Fig. 3B). A new reaction channel, involving proton-coupled electron transfer (PCET), was found to be competitive for the protonated • C species, and to lead to the formation of [Cys–S–S–R] +:

+ • •+ Cys –S–S–R + OH → [Cys–S–S–R] + H2O (PCET).

The calculations indicated a PCET branching ratio between 22% and 32%, but also indicated further dissociation at the S–S bond or S–R bond to not be energetically favorable. In addition, ions • corresponding to [Cys–S–S–R] + were not detected experien- tially. Therefore, this process was concluded to not contribute

• significantly to disulfide bond cleavage. Fig. 2. (A) %Rel. reactivity values of Cys–S–S–R toward OH, i.e., normalized – – – – – – – Table 1 summarizes kinetics data together with branching ra- to I S S I. The Cys S S R compound exhibited higher reactivities with R tios for reactions 1 and 2 from the calculations. When the neutral being an EDG. (B) %Selectivity values of OH attack on the Cys–S bond rel- ative to that on the S–R bond within Cys–S–S–R. (C) Values of %H transfer cysteine disulfides were considered, the summed rate constants for • 10 12 leading to the formation of +Cys–SO and +Cys–SH, respectively. disulfide cleavage (ktotal) ranged between 5.9 × 10 and 4.2 × 10 −1 −1 M ·s , with R groups of –C(O)OCH3 and –CH3 yielding the slowest and fastest reactions, respectively. For reactions involving 50%, viz. 26 ± 5% for –CH3,38± 5% for –C3H7, and 40 ± 3% for –Ph.

Theoretical Calculations. In order to rationalize these MS results, AB we carried out a series of theoretical calculations. These com- putational methods consisted of a combination of the molecular mechanics (MM) methods, ab initio density functional theory with the BH&HLYP functional, and domain-based local pair- natural orbital coupled cluster including perturbative triplet ex- citation methods [DLPNO–CCSD(T)]. The kinetics calculations were carried out in the framework of multiconformer transition state theory (MC-TST). The following elemental reactions were envisaged as the first steps of the disulfide bond cleavage: CD Cys–S–S–R +•OH → Cys–SOH +•S–R, [1a]

• Cys–S–S–R +•OH → Cys–S + HOS–R. [2a]

We also considered protonated species (+Cys–S–S–R) in the reactions, with the proton bound to the primary amine of the cysteine moiety:

+ + Cys–S–S–R +•OH → Cys–SOH +•S–R, [1b] EF

+ + • Cys–S–S–R +•OH → Cys–S + HOS–R. [2b]

Regarding R substituents, –CH3 and –phenyl were chosen to represent EDG groups, while –CH2CF3 and –C(O)OCH3 were used as examples of weak and strong EWG groups, respectively. Previous reports showed that a carbon-centered radical or H atom can attack either of the sulfur atoms in a disulfide bond, Fig. 3. Relevant geometrical parameters for the low-lying transition states following either a back-side mechanism (with an attacking angle = – = – ∼ of (A) reaction 2b for R CH3,(B) reaction 2a for R CH3,(C) reaction 3b between 140° and 180°) or a front-side mechanism ( 90° attack- for R = –CH3,(D) reaction 3b for R = –C(O)OCH3,(E) reaction 4b for R = –CH3, ing angle), but with the back-side attack being more favorable and (F) reaction 4b for R = –C(O)OCH3. Distances are in angstroms and angles (39–42). The results of our calculations indicated the back-side in degrees.

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.2006639117 Adhikari et al. Downloaded by guest on September 30, 2021 Table 1. Calculated rate constant (k) values and branching ratio (Γ) values for two different types of each of the • Cys–S–S–R+ OH (reactions 1a and 1b) and Cys+–S–S–R+•OH (reactions 2a and 2b) reactions, and the rate

constant (ktotal) values for the whole reactions and the corresponding branching ratios −1 −1 −1 −1 −1 −1 R kS1,M ·s kS2,M ·s ktotal,M ·s ΓS1,% ΓS2,%

• Cys–S1–S2–R+ OH → Products 11 12 12 –CH3 4.4 × 10 4.1 × 10 4.6 × 10 9.6 90.4 –Phenyl 5.5 × 1011 1.1 × 1012 1.6 × 1012 34.0 66.0 12 12 12 –CH2CF3 1.1 × 10 1.6 × 10 2.7 × 10 40.2 59.8 10 9 10 –C(O)OCH3 5.7 × 10 2.2 × 10 5.9 × 10 96.3 3.7 + • Cys–S1–S2–R+ OH → Products 9 12 12 –CH3 2.7 × 10 1.6 × 10 1.6 × 10 0.2 99.8 –Phenyl 1.8 × 1012 3.5 × 1012 4.8 × 1012 34.0 66.0 12 12 12 –CH2CF3 2.9 × 10 2.9 × 10 5.8 × 10 50.0 50.0 12 12 12 –C(O)OCH3 6.9 × 10 1.3 × 10 8.1 × 10 84.7 15.3

Computed using transition state theory with DLPNP–CCSD(T) energies and BH&HLYP free energy corrections. kS1 stands for the • • attack of the OH on the S atom linked to Cys (reactions 1a and 1b); kS2 stands for the attack of the OH on the S atom linked to the R group (reactions 2a and 2b). A temperature of 298 K was used for all reactions.

• protonated Cys–S–S–R species, the charged species was calculated abundances of +Cys–SH and +Cys–S (for +Cys–SH%) or to those • • to react about two times slower than the neutral species containing of +Cys–SO and +Cys–SOH (for +Cys–SO %).Fig.2C summa- – – – – the CH3 group, whereas for the CH2CF3, phenyl, and C(O) rizes %H transfer values for all seven Cys–S–S–R compounds. The + OCH3 groups, the reaction of the charged species was predicted to %H transfer values for the formation of Cys–SH were determined be, respectively, about three times, four times, and two orders of to be in the range of 44 to 83%, suggesting a substantial transfer of • magnitude faster than the corresponding neutral species. These H. Because no ion signals corresponding to +Cys–SH and +Cys–S differences can be attributed to the presence of stabilizing hy- were detected above the noise level for R = –C(O)OCH3,its drogen bond interactions, which are in general stronger in reac- + – Cys SH% could not be obtained accurately. Regarding the for- CHEMISTRY + – – – • tions involving protonated species. Experimentally, Cys S S Cys mation of +Cys–SO , only for R groups of –C(O)CH and –C(O) was found slightly less reactive than +Cys–S–S–R with R being an 3 OCH were the %H transfer values determined to be relatively high EDG (R = –CH ,CH , –Ph, Fig. 2A). This difference might be 3 3 3 7 (>70%), while the rest of the compounds showed similarly low due to both the electron-donating character and small steric hin- drance of these R groups as compared to Cys. It is worth pointing degrees of H transfer, between 30 and 43%. The reaction rate constants as well as transition states relating out that because reactions happened in the plume region of nano- 3 4 SI Appendix ESI, a fraction of ions of Cys–S–S–R might be microsolvated when reactions and are summarized in Table 2, and , reacting with •OH. They could adopt different ionic structures Figs. S9 and S10: such as zwitterionic form, and thus contribute to differences in – +• – → – • + – [3a] reactivities observed from experiments to that of calculations. Cys SOH S R Cys SO HS R, Analysis of the branching ratios for reactions 1 and 2 (Table 1) + • + • suggested a general preference of •OH to attack the more Cys–SOH + S–R → Cys–SO + HS–R, [3b] electron-rich S atom for both neutral and protonated systems. For instance, for +Cys–S–S–Ph, the branching ratio for the at- – • + – → – +• – [4a] • Cys S HOS R Cys SH OS R, tack of OH on the sulfur atom bonded to R (S2, ΓS2) was cal- – + • + culated to be 66%. The calculation using C(O)OCH3 as the R Cys–S + HOS–R → Cys–SH +•OS–R. [4b] group, in contrast, indicated a preference for attack on the S + Γ atom bonded to the Cys moiety (S1), with an S1 of 85%; and In general, for the various R groups, the reactions involving – • that using CH3 as the R indicated a high preference for S2, transfer of H from HO–S–Rto+Cys–S to form +Cys–SH (re- which may have been due to both the electron-donating char- action 4b) were computed to be the fastest reactions, with cal- acter and small steric hindrance of this R group considering that 11 12 −1 −1 • culated rate constants between 8.0 × 10 and 2.3 × 10 M ·s OH prefers back-side attack to the disulfide bond. These data except for R = –CH2CF3 where the computed rate constant was − − showed a good linear correlation with experimental results, with 2.4 × 108 M 1·s 1. Notably, these values are between 4 and 10 R2 coefficients of correlation ( ) of 0.923 and 0.969 for neutral and orders of magnitude faster than those of the corresponding protonated systems, respectively (plots shown in SI Appendix, Fig. S8). Our experimental data showed about equal selectivity + to each sulfur atom in a symmetric disulfide bond as in Cys– Table 2. Calculated rate constant (k) values at 298 K for + S–S–Cys and I–S–S–I. However, for a disulfide bond embedded hydrogen transfer in reactions 3 and 4 in the hydrophobic pocket of a protein, the local structure as well − − k,M 1·s 1 as hydrogen bonding will likely be the main factors determining • the selectivity of OH attack. • R Reaction 3a Reaction 4a Reaction 3b Reaction 4b Besides disulfide cleavage resulting from OH substitution, + + • – × 4 × 6 × 4 × 11 detection of Cys–SH and Cys–SO products in the MS data CH3 1.9 10 4.5 10 4.9 10 8.0 10 4 7 4 11 suggested the occurrence of a transfer of H between the products –Phenyl 1.7 × 10 1.9 × 10 6.9 × 10 2.4 × 10 – × 4 × 4 × 8 × 8 produced in reactions 1 and 2. These reactions are listed as re- CH2CF3 6.5 10 3.2 10 1.2 10 2.4 10 – × 4 × 2 × 11 × 12 actions 3 and 4, with a and b corresponding to the neutral and C(O)OCH3 3.9 10 9.0 10 3.6 10 2.3 10 %H transfer charged reactions, respectively. values were calcu- Computed using transition state theory with DLPNP–CCSD(T) energies + + • lated from the experimental data as Cys–SH% and Cys–SO and BH&HLYP free energy corrections. The tunneling corrections were com- % by normalizing their relative ion abundances to the summed puted using the Wigner approach.

Adhikari et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 30, 2021 neutral compounds (reaction 4a, Table 2). Analysis of these data suggested that protonated ions were likely responsible for the reaction products detected experimentally; moreover, the analy- ses indicated that formation of +Cys–SH should be detectable on the same timescale as disulfide bond cleavage (reactions 1 and 2, Table 1). In addition, these high values of the rate constants may be attributed to the combination of two factors, namely the strength of the interaction, between the proton- ated amine and the oxygen atom of the when R = –C(O)OCH3 (1.745 Å, Fig. 3F), and the early transition state according the Hammond postulate, as in the case of reaction • 4b = – – Scheme 2. Two-step reaction mechanism for the OH attack on the disul- for R CH3, where the S H distance of the H atom being – – – E fide bond in Cys S S R. H transfer was found to be a competitive process for transferred is quite large (1.736 Å, Fig. 3 ). The differences in +Cys–S–S–R. the reactivity between compounds with different substitute in this reaction are mainly due to different contribution of these two effects. However, the reason for relatively low k4b associated indicated to be rapid for most neutral cysteine disulfides, with rate 12 −1 −1 with R = –CH2CF3 was not fully understood. The above- constants on the order of 10 M ·s , but about two orders of described predictions made were in good accordance with the magnitude slower for R being an EWG. For protonated com- • substantial %H transfer observed for +Cys–SH formation accord- pounds, however, the rate constant of OH substitution on a ing to the MS data (Fig. 2C). For reaction 3b, analysis of the disulfide bond connected to an EWG was indicated to be increased results of calculations predicted quite scattered values of the rate to the same level as for R being an EDG, due to the stabilizing k × 4 −1· −1 = – constants, with a value of 4.9 10 M s for R CH3 and effect of hydrogen bonding for the low-lying transition states. Hy- × 11 −1· −1 = – 3.6 10 M s for R C(O)OCH3. These data were con- drogen bonding was also modeled to help stabilize the product sistent with the experimental observations, in particular the high- complex and further facilitate rapid H transfer before product %H transfer = – C est observed for R C(O)OCH3 (88%, Fig. 2 ). separation in protonated systems (reaction 4b), explaining the %H + • + However, explaining such a large difference between the observation of Cys–SO and Cys–SH in the MS experiments. transfer values of the different tested compounds by solely refer- Analysis of our calculation results suggested that H transfer would ring to the different inductive effects of their R substituents not predominate for neutral species on the detection timescale would be at odds with a previous study indicating a lack of any used in this study. strong effect of the identity of R of alkylthiols (RS–H) on the S–H bond dissociation energy (RS–H BDE: ∼360 kJ/mol) (43). Summary 3b Analysis of the low-lying transitions states of reaction sug- We have developed a combined experimental and theoretical • gested an important role played by hydrogen bond interactions approach to study reactions of the OH radical with a series of in stabilizing the transition state, thus favoring the formation of cysteine disulfides (Cys–S–S–R) in the gas phase. The ability to + – • Cys SO . For instance, the hydrogen bond between the proton- use MS to perform online identification and relative quantitation ated amine and the carbonyl group was calculated to be 1.6 Å for – of first-generation reaction products provided mechanistic de- the compound containing an R of C(O)OCH3 (an early transi- tails that had not been achieved for this reaction previously. A tion state, Fig. 3C), much shorter than the hydrogen bond in – ··· – – D two-step reaction mechanism has been proposed. In contrast to H2N H S CH3 (2.2 Å), with its R being CH3 (Fig. 3 ). We solution reactions where single-electron transfer is prevalent, noticed that for Cys–S–S–Cys and I–S–S–I, which contained a • OH substitution at the disulfide bond is a fast and predominant symmetric disulfide bond, the detected %H transfer values were + reaction channel in nonpolar environment, forming product not 50%. In fact, H transfer leading to the formation of Cys–SH • pairs containing sulfenic acid (–SOH) and thiyl radical (–S )at (70%) or +I–SH (71%, SI Appendix, Fig. S2B) was higher than • • the cleavage site (Scheme 2). For protonated cysteine disulfides, that of +Cys–SO (43%) or +I–S (34%, SI Appendix, Fig. S2B). subsequent H transfer within each of the product pairs could be This is because the disulfide bond is no longer symmetrical in these two compounds given that the proton can only reside on competitive due to the stabilizing effect of hydrogen bonding in + • the transition states. Both experimental and theoretical results one side of the molecule. H transfer from Cys–SOH to Cys–S • • leads to the formation of +Cys–SO (reaction 3b), while H trans- supported the idea that reactions of OH and cysteine disulfide + • + compounds produce four types of sulfur species, namely –SOH, fer from Cys–SOH to Cys–S leads to the formation of Cys– • • 4b = – –SO , –SH, and –S , which are important to the overall sulfur SH (reaction ). Using the calculation results from R CH3 in – Cys–S–S–R (Table 2) as a simplified estimation for Cys–S–S– redox cycle. Of these species, SOH is a potent scavenger for k = × 7 −1· −1 Cys, it is clear that reaction 4b is significantly enhanced relative peroxyl radicals via H atom transfer ( 3 10 M s ) (44, to reaction 3b when compared to their corresponding neutral 45), presenting a contrast to its disulfide bond precursor and the reactions (reactions 4a and 3a). This difference might lead to reduced thiol both of which show limited reactivity toward per- the higher %H transfer values for forming +Cys–SH or +I–SH oxyl radicals (46). This implies that after the cysteine disulfide • • • than +Cys–SO or +I–SO (SI Appendix, Scheme S2). Hydrogen reacts directly with OH, the in situ formed reaction product, bond interactions discovered in the cysteinyl disulfide system –SOH, can join the antioxidant network of other peroxyl scav- also presented a distinct difference from the calculated results engers (such as vitamin E), thus forming two lines of defense. • Also of importance is the formation of a sulfinyl radical from H of OH reactions with small organic disulfides (20, 41). • Combining the accumulated pieces of evidence from the ex- transfer within [–SOH... S–] product complex. Because the perimental and computational studies, we derived a two-step re- sulfinyl radical is much less reactive than the thiyl radical (32), • action mechanism to account for OH attack on cysteine disulfides this process basically detoxifies of thiyl radical. However, in a (Scheme 2). According to this proposed mechanism, in the first nonpolar biological system, such as at the interface of lipid • step, OH performs a back-side attack on one of the sulfur atoms, membrane or in the hydrophobic regions of protein tertiary with a preference for the electron-richer one—with this attack structures, the local environment will largely modulate the re- • • leading to OH substitution and subsequent disulfide bond cleav- activity of the cysteine disulfide toward OH as well as the rate of age and hence forming the sulfenic acid (–SOH) and thiyl radical subsequent H transfer. In summary, while cysteine disulfide has • (–S ) pair of products. Note that hydroxyl radical substitution was been considered to be as an inefficient antioxidant, our study

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.2006639117 Adhikari et al. Downloaded by guest on September 30, 2021 • suggested that by reacting it with OH to form a more potent ion selection in Q1 and ion acceleration into a Q2 collision cell followed by antioxidant in nonpolar environment, disulfide may be more product analysis in a Q3 linear ion trap. Ion-trap CID consisted of precursor actively involved in the antioxidant network to combat elevated ion selection in Q1 and ion transfer through Q2 to Q3 with minimum acti- levels of ROS than previously appreciated. vation energy followed by reisolation, accumulation, and application of di- polar excitation to effect CID in Q3. To induce radical reactions, the nano-ESI • Methods plume of disulfide was allowed to interact with OH in the afterglow region Materials. All reagents and solvents were purchased from commercial sources of an atmospheric-pressure helium low-temperature plasma enabled in a and were used without further purification. S-methyl methanethiosulfonate T-shaped glass tube placed in front of the entrance of the mass spectrometer (MMTS), L-cysteine, DL-cysteine, thioacetic acid, methoxycarbonyl chloride, (SI Appendix, Fig. S1) (16). benzyl mercaptan, 1-propantethiol, 2,2,2-trifluoroethanethiol, acetyl chlo- ride, ethanol, anhydrous methanol, and trimethylamine were purchased Computational Details. The computational methods employed in this work – – – from Sigma-Aldrich. Syntheses of cysteine cysteinyl disulfides (Cys S S R, includes a combination of MM methods (50), ab initio density functional Scheme 1) were achieved according to procedures described in the literature theory with the BH&HLYP functional (51), domain-based local pair natural (details provided in SI Appendix)(47–49). The ethyl ester of cystine was orbital coupled cluster including perturbative triplet excitation [DLPNO– synthesized and used as an internal standard to compare the reaction rates CCSD(T)] methods (52), and kinetics calculations in the framework of MC-TST using different cysteine disulfide derivatives. Working solutions for positive (53). A full detail of theoretical approaches used is discussed in SI Appendix, mode nano-ESI were prepared in deionized H2O (ultrapure purification system at 0.03 μS·cm). The extents of reaction were online monitored by which also includes a detailed description of the electronic features of the MS analysis. processes investigated and the most relevant transition states of the different reactions investigated. MS and Online Radical Reactions. All MS data were collected on a 4000 QTRAP triple quadrupole/linear ion trap (LIT) mass spectrometer equipped with a Data Availability. All data are included in the manuscript and SI Appendix. nano-ESI source made in the laboratory. Analyst software 1.6.2 was used for data acquisition, processing, and instrument control. Typical MS parameters ACKNOWLEDGMENTS. Y.X. acknowledges financial support from National used during the study were a spray voltage of ±1,500 to 1,800 V, curtain gas Natural Science Foundation of China (Grant 21722506). R.C. and J.M.A. pressure of 10 psi, declustering potential of ±20 V, and scan rate of acknowledge funding from the Ministry of Economic Affairs and Digital 1,000 Da/s. MS1 mass analysis was performed in LIT mode in Q3. Beam-type Transformation (Grant CTQ2016-78636-P) and from the Generalitat de collision-induced dissociation (CID) was performed by performing precursor Catalunya (Grant 2017SGR348).

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