Concerted ElectronâProton Transfer (EPT) in the Oxidation of Cysteine

Article pubs.acs.org/JPCC

Concerted Electron−Proton Transfer (EPT) in the Oxidation of Cysteine Christopher J. Gagliardi,† Christine F. Murphy,‡ Robert A. Binstead,§ H. Holden Thorp,∥ and Thomas J. Meyer*,§ †

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States Office of the Dean of the Graduate School, Princeton University, Princeton, New Jersey 08540, United States § Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599−3290, United States ∥ Office of the Provost, Washington University in St. Louis, St. Louis, Missouri 63130, United States ‡

S Supporting Information *

ABSTRACT: Cysteine is the most acidic of the three common redox active amino acids with pKa = 8.2 for the thiol compared to pKa = 10.1 for the phenol in tyrosine and pKa ≈ 16 for the indole proton in tryptophan. Stopped-flow and electrochemical measurements have been used to explore the role of proton-coupled electron transfer (PCET) and concerted electron−proton transfer (EPT) in the oxidations of Lcysteine and N-acetyl-cysteine by the polypyridyl oxidants M(bpy)33+ (M = Fe, Ru, Os) and Ru(dmb)33+ (bpy is 2,2′-bipyridine, and dmb is 4,4′-dimethyl-2,2′bipyridine). Oxidation is rate limited by initial 1e− electron transfer to M(bpy)33+, with added proton acceptor bases, by multiple pathways whose relative importance depends on reaction conditions. The results of these studies document important roles for acetate (AcO−) and phosphate (HPO42−) as proton acceptor bases in concerted electron−proton transfer (EPT) pathways in the oxidation of L-cysteine and N-acetyl-cysteine with good agreement between rate constant data obtained by electrochemical and stopped-flow methods.

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INTRODUCTION The amino acids tyrosine, cysteine, and tryptophan play important roles as electron transfer carriers and mediators in biology with important examples appearing in photosystem II, class I ribonucleotide reductase, and DNA photolyase.1−8 In tyrosine and cysteine oxidation, proton coupled electron transfer (PCET) reactions and half reactions in which protons and electrons are gained or lost, play an important role in avoiding charge build up. Concerted electron−proton transfer (EPT) pathways, with electrons and protons transferred simultaneously, are used to avoid high-energy protonated or deprotonated intermediates that arise in 1e− pathways involving electron transfer (ET) alone or proton transfer (PT) alone. As an example, E°′ ≈ 1.5 V vs NHE9−11 for tyrosine oxidation to TyrOH•+ while E°′ ≈ 1.0 V for oxidation of the TyrOH--histidine base adduct to TyrO•---+H-histidine, a couple important in photosystem II.3,4 In proteins, pendant bases or water, with pKa (H3O+) = −1.74 have been suggested to act as EPT proton acceptors as a way of avoiding high-energy intermediates such as TyrOH•+.12 Protein structures with redox-active cysteine residues typically include an associated carboxylate base such as aspartate or histidine for tyrosine oxidation, which enable EPT to occur.3,4,13,14 Tryptophan lacks a readily dissociable proton and, in peptides, is often found in solvent-exposed sites without an associated base. Mechanistically, its oxidation chemistry is dominated by sequential electron transfer followed © 2015 American Chemical Society

by proton transfer (ET−PT). In a recent study on N-acetyltryptophan (NAceTrpNH) oxidation by Os(bpy)33+, there was no evidence for EPT pathways with added phosphate or Tris buffers over an extended range of buffer concentrations.15 Evidence for buffer base effects and noninteger pH dependences in metal complex assemblies has been reported by Hammarström and co-workers in a series of photochemical studies. This observation is proposed to arise from acid−base equilibria involving an amine group on a ligand, although related phenomena have been observed in assemblies without acidic amines.16−18 Tyrosine oxidation by the oxidants M(bpy)33+ (M = Fe, Ru, Os) with added bases is dominated by multiple site-electron proton transfer (MS-EPT). In this pathway, concerted electron−proton transfer occurs to separate e− and H+ acceptors (Scheme 2).19−22 Related observations have been made at tin-doped indium oxide (ITO) electrodes derivatized by surface binding of the electron transfer mediator [RuII(bpy)(4,4′−(HO)2P(O)CH2)2bpy)2]2+ (bpy = 2,2′-bipyridine; 4,4′(HO)2P(O)CH2)2bpy = 4,4′-bis-methylenephosphonato-2,2′bipyridine)19,20 and in the solution oxidation of cis− OsIII(bpy)2(py)(OH)2+ to OsIV(bpy)2(py)(O)2+.23 PCET and EPT have been the subject of recent reviews.3−6,24 Received: January 13, 2015 Revised: March 3, 2015 Published: March 4, 2015 7028

DOI: 10.1021/acs.jpcc.5b00368 J. Phys. Chem. C 2015, 119, 7028−7038

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The Journal of Physical Chemistry C

For stopped-flow mixing experiments, buffered aqueous solutions were prepared in 0.8 M NaCl to maintain constant ionic strength. For mixing studies, Os(bpy)33+ was generated from solutions of Os(bpy)32+ by bubbling with Cl2 gas, followed by purging with argon. The concentration of Os(bpy) 3 3+ was determined spectrophotometrically in CH3CN, ε = 39300 cm−1 M−1 at 306 nm.34 To ensure constant pH during the experiments, pH was checked and adjusted before each measurement and evaluated again at the end of the measurement. Deuteration. For electrochemical experiments, deuterated L-cysteine was prepared by dissolving protonated L-cysteine in D2O and deprotonating by adding NaOD. The resulting product was isolated by distillation that left deuterated Lcysteine in the bottom of the reaction flask. Proton content was monitored by 1H NMR. Deuterated buffers were prepared similarly and isolated by distillation. The deuterated products were subsequently redissolved in D2O and the pD measured. In mixing experiments, deuterated N-acetyl-cysteine was prepared by dissolving N-acetyl-cysteine in D2O, where the H/ D exchange rate is instantaneous. Buffers were prepared by dissolving either acetate and deuterated acetic acid or sodium phosphate and deuterated phosphoric acid to the appropriate concentration and pH/pD value, with pD = pH + 0.4 on a H2O-calibrated pH meter. Solution pH values were adjusted by adding DCl or NaOD to the solutions and measuring the pH/ pD. Stopped−Flow Kinetics. Kinetics experiments were performed on a HI-TECH SF-61DX2 double-mixing stoppedflow spectrophotometer fitted with either a single-beam photodiode array detector and xenon light source for multiwavelength analysis or dual-beam photomultiplier tubes (Hamamatsu R928) and a tungsten light source for single wavelength measurements. Initial studies were performed with a MG-6050 diode array (1.5 ms integration time) with the KinetAsyst software, while later work utilized an MG-6560S diode array detector (3 ms integration time) operated by the Kinetic Studio software (TgK Scientific Ltd.). In most cases, measurements were obtained with a 1 cm optical path. Sample temperatures were controlled by a Thermo Haake A28 water bath and monitored with the internal Pt-100 sensor of the mixing unit. Solutions containing buffer, N-acetyl-cysteine, and dipicolinic acid at known pH values and separate solutions having the same composition with added Os(bpy)33+ were deaerated with argon for 30 min prior to kinetic measurements. Timescales from 1 to 100 s were suitable for diode array detection. The latter mode was used with an automatic shutter system (Uniblitz LS3) for timescales longer than 3 s as a precaution against photolysis of Os(bpy)33+ by the intense xenon light source. Higher-quality kinetic traces were obtained with photomultiplier detection and a tungsten lamp, which provided better time resolution and lower noise. Typically, the kinetics were monitored with a pseudo-first-order excess of Nacetyl-cysteine (200 μM) with Os(bpy)33+ at 20 μM with 500− 5000 μM added dipicolinic acid (Figure 2). In this region, the observed kinetics were uncomplicated and reproducible allowing for kinetic analysis with HI-TECH KinetAsyst or Kinetic Studio software. Electrochemistry. Cyclic voltammetry experiments were performed with a BAS100B/W or CH Instruments CHI-601D series potentiostat in conjunction with a three-electrode cell described previously.33,36 For experiments testing the effect of dipicolinic acid on rates, a two compartment cell was used with

Cysteine is a thiol and, with pKa = 8.2, is a stronger acid than either tyrosine (pKa = 10.1) or tryptophan (pKa ≈ 16). From electrochemical measurements, the reduction potential for the cysteine radical cation couple, CysSH•+/CysSH, is >1.5 V vs NHE, and the radical cation is highly acidic with pKa (CysSH•+) < −2.0.25 Although expected to play an important role, EPT in cysteine oxidation has not been systematically addressed. Stanbury and co-workers have reported on the results of a detailed study on electron transfer oxidation of cysteine by Mo(CN)83− with E°′(Mo3−/4−) = 0.77 V vs NHE and in a follow up report using Fe(bpy)2(CN)2+ and Fe(bpy)(CN)4−.26,27 In each case, it was found that trace Cu(II) dominates the kinetics of cysteine oxidation and of other thiols such as glutathione.26−29 They also showed that complications arising from Cu(II) can be circumvented by adding dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid), which coordinates to Cu(II). Cysteine oxidation by chlorine dioxide has also been investigated by Margerum and coworkers.30 More recently, Alvarez and co-workers reported on a role for PCET in the oxidation of glutathione by IrCl62− with electrochemical simulations used to elucidate buffer effects and reveal mechanistic details.31 This report has been the subject of considerable discussion surrounding the origin of the reported base dependent increase in kobs as well as the participation of Cu(II) in thiol oxidation catalysis.32,33 The goal of the current study was to investigate a possible role for EPT pathways in cysteine oxidation as part of a larger study on the role of PCET and EPT in biological redox cofactors.

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EXPERIMENTAL SECTION Reagents and Materials. Buffered aqueous solutions were prepared from water purified with a Milli-Q purification system (Synthesis A10) with added NaCl at 0.8 M to maintain constant ionic strength. The buffers (acetate, histidine, phosphate, and Tris), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO) and were used as received. The concentration of buffer components was calculated based on the Henderson− Hasselbalch equation. The pKa values used in the calculations are standard values for each buffer in aqueous solution. Nacetyl-L-cysteine (NAceCysSH) and dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid) were obtained from Alfa Aesar and L-cysteine (CysSH) from Sigma-Aldrich (Figure 1). These

Figure 1. Structures of the dominant forms of cysteine (CysSH) and N-acetyl-cysteine (NAceCysSH) at neutral pH and of dipicolinic acid, pyridine-2,6-dicarboxylic acid.

reagents were used as received. Buffered solutions were adjusted to the correct pH using HCl or NaOH with a Fisher Scientific Accumet AB15 pH meter. Buffer and solution conditions used to establish the rate law for cysteine oxidation are discussed below. N- acetyl- L-cysteine was used in place of L-cysteine in mixing studies in order to mitigate the effect of trace copper catalysis (see the Supporting Information). 7029


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Figure 2. Oxidation of N-acetyl-cysteine (200 μM) by Os(bpy)33+ (20 μM) at pH 5.76, (I = 0.8 M, NaCl; T = 20 °C) in acetate buffer, AcOH/AcO− (500 mM) with DPA (500 μM). (A) Stopped-flow kinetic trace for Os3+ → Os2+ monitored at 480 nm. (B) Plot of ln(A∞−A) vs time (kobs = 5.56 s−1).35

Figure 3. (A) Reaction of Os(bpy)33+ (20 μM) with N-acetyl-cysteine (1500 μM) and DPA (2000 μM) at pH 6.0 (I = 0.8 M, NaCl; T = 20.0 ± 0.1 °C). (B) Reaction of Os(bpy)33+ (20 μM) with N-acetyl-cysteine (1500 μM) and DPA (2000 μM) at pH 4.5 (I = 0.8 M, NaCl; T = 20.0 ± 0.1 °C). All traces fit to single exponential kinetics. If added to control pH, buffer concentrations were sufficiently low, < 1 mM, that base-assisted pathways were unimportant (see text). In a series of 10 replicates, kobs = 55.0 ± 0.1 s−1 for (A) and 2.54 ± 0.07 s−1 for (B).

separate solution volumes of 3 mL. Solutions were deaerated for 15 min with argon prior to measurement and with headspace deaeration maintained throughout the experiment. The working electrode was tin-doped indium oxide coated glass (ITO) purchased from Delta Technologies (Stillwater, MN). In kinetics, experimental working electrode areas of 0.32 cm2 or 1 cm2 were used, with the area defined by Kapton tape or an Oring. The reference electrode was a Teflon-coated Ag/AgCl microelectrode purchased from Cypress Systems, Inc. (Lawrence, KS) or a saturated calomel reference electrode (SCE) purchased from BioAnalytical Systems (BASi, Lafayette, IN). The auxiliary electrode was a platinum wire purchased from Sigma-Aldrich (St. Louis, MO). ITO electrodes were treated before use by sonication in Milli-Q water for 15 min, isopropanol for 15 min, and two washes with Milli-Q water for 15 min each. ITO electrodes were laid flat and allowed to

dry overnight. Experimental volumes for most kinetic experiments were typically 50−100 μL. In voltammetric measurements, the potential was scanned positively from 0.2 to 1.2 V vs NHE for 6 consecutive scans in the buffer solution before a final background scan was taken. In sequence, a scan of buffer plus metal complex was recorded and then a scan of buffer with metal complex and L-cysteine. After the scan was complete, the ITO electrode was discarded and a new electrode used for the next set of experiments. Cyclic voltammograms (CV) were background-corrected by subtracting scans of solutions containing only buffer from those with complex and cysteine added.

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RESULTS AND DISCUSSION Stopped-Flow Measurements. Stopped-flow measurements at a variety of pHs and thiol concentrations were used to establish the rate law for N-acetyl-cysteine (NAceCysSH)

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Figure 4. Plots of kobs/[NAceCysSH] vs 1/[H+] for oxidation of N-acetyl-cysteine by Os(bpy)33+(20 μM), pH 4−6 (I = 0.8 M, NaCl; T = 20 °C) with 2000 μM added DPA. Each data point is the average of 10 replicates with most error bars smaller than the data point symbols. (A) 500 and (B) 3000 μM N-acetyl-cysteine. The linear fits to eq 2 yielded k′ = (3.49 ± 0.03) × 10−2 s−1 and kH2O = (4.8 ± 1.6) × 102 M−1 s−1.40 Additional plots for 1000, 1500, 2000, and 2500 μM N-acetyl-cysteine are in shown in Figure SI.1 of the Supporting Information, with all data summarized in Table SI.1 of the Supporting Information.

oxidation with Os(bpy)33+ as the oxidant. Absorbance−time traces in solutions containing NAceCysSH in pseudo-first-order excess were typically close to exponential, as shown in Figure 3. A series of experiments were conducted with N-acetylcysteine from 500 to 3000 μM with 20−100 μM Os(bpy)33+ over the pH range from 4.0 to 6.0, with low concentrations or no added buffer in order to avoid participation by base-assisted pathways (see below). As noted above, DPA (2000 μM) was added to avoid complications from trace Cu(II). At each pH, the kinetics were first-order in [Os3+] and first-order in Nacetyl-cysteine. These observations are consistent with the rate law in eq 1 with [CysSH]T, the total concentration of N-acetylcysteine. −d[Os3 +]/dt = kobs[Os3 +][CysSH]T

Scheme 1. PT−ET (k′) and EPT to Water (kH2O) Pathways for NAceCysSH Oxidation with RSH Either NAceCysSH or CysSH

(1)

From the results in Figure 4, kobs is pH-dependent. The dependence is consistent with eq 2 under conditions where the pH was significantly below pKa = 8.2 for cysteine with [CysSH] ∼ [CysSH]T. +

kobs = k H2O + k′/[H ]

CysSH, H 2O + Os(bpy)33 + ⇌ CysSH, H 2O, Os(bpy)33 + : KA ,M3+

(2)

(6)

+

The appearance of the term inverse in [H ] is consistent with initial deprotonation of Cys−SH followed by oxidation of the N-acetyl-cysteine anion, CysS− (PT−ET, eqs 3 and 4), with k′ = kCys− Ka,Cys−SH followed by radical coupling to give the disulfide (Scheme 1). Interpretation of the acid-independent term (kH2O) is ambiguous. Oxidation by this pathway could occur by initial outer sphere oxidation to give Cys−SH•+, by oxidation of the thiolate tautomer, HOOC(H)(NH3+)CH2S−, as proposed for tyrosine oxidation by Savéant and coworkers,11,37,38 by MS-EPT oxidation of Cys−SH with water as the proton acceptor base (eqs 5−7) or by a combination of the three. −

+

CysSH ⇌ CysS + H : K a,Cys − SH

CysSH, H 2O, Os(bpy)33 + → CysS• + H3O+ + Os(bpy)32 + : kEPT,H2O

Outer-sphere oxidation (eqs 8 and 9) may not be important given the 0.7 V difference between E°′ for the Os(bpy)33+/2+ (E°′ = 0.80 V vs NHE) and estimated CysSH/CysSH•+ (E°′ > 1.50 V vs NHE) couples. Os(bpy)33 + + CysSH → Os(bpy)32 + + CysSH•+

(8)

CysSH•+ → 1/2CysSSCys + H+(rapid)

(9)

The products of L-cysteine oxidation have been characterized by Stanbury and co-workers with the 1e − oxidant octacyanomalybdate(V) and shown to be largely cystine (RSSR) and cysteinesulfinate (RSO2−) as determined by 1H NMR measurements.26 The formation of the cysteine disulfide (eq 10) from the deprotonated cysteine radical (2RS• → RSSR) occurs rapidly with a reported rate constant of ∼1010

(3)

Os(bpy)33 + + CysS− → Os(bpy)32 + + CysS•: k Cys− (4)

CysSH + H 2O ⇌ CysSH, H 2O: KA ,H2O

(7)

(5) 7031


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The Journal of Physical Chemistry C M−1 s−1, making it the dominant oxidation product under our conditions.26−28 The thiyl radical can also react with thiolate anion to give the cystine radical anion (eq 11), with k ∼ 109 M−1 s−1, followed by further oxidation to RSSR.26,39 2CysS• → CysSSCys (rapid)

(10)

CysS• + CysS− → CysSSCys•− (rapid)

(11)

Stopped-flow mixing was used to investigate the oxidation of N-acetyl-cysteine by Os(bpy)33+ in acetate (AcO−/HOAc) buffer. The experiments were carried out at low [Os(bpy)33+] with [HOAc]/[OAc−] = 0.1, conditions under which the expression for kobs in eq 12c simplifies to eq 13. ⎡ KA[B−] ⎤⎛ k1k 2 ⎞ kobs k′ = k H 2O + ⎥⎜ ⎟ + + ⎢ [CysSH]T [H ] ⎣ 1 + KA[B−] ⎦⎝ k −1[HB] ⎠

From eq 2 and the slopes and intercepts of the plot of kobs/ [NAceCysSH] versus [H+]−1 in Figure 4, k′ = Ka,Cys−SHk2 = (3.49 ± 0.03) × 10−2 s−1 and kH2O = (4.8 ± 1.6) × 102 M−1 s−1. Similar values for k′ and kH2O were found by digital simulations of cyclic voltammograms under comparable conditions for Lcysteine oxidation. From stopped-flow measurements and Ka,Cys−SH = 6.30 × 10−9 M−1, the outer-sphere rate constant for oxidation of L-cysteine anion is k2 = (5.5 ± 0.5) × 106 M−1 s−1. The two pathways are illustrated in Scheme 1. Stopped-Flow Measurements with Added Bases. A proposed mechanism for oxidation of N-acetyl-cysteine with added bases is shown in Scheme 2. It is based on the known

(13)

Over an extended range of buffer concentrations, the observed kinetic behavior was consistent with eq 13 (note Figure 5). Under these conditions, KA[AcO−]/(1 + KA[AcO−])

Scheme 2. Base-Assisted Pathways for Cysteine Oxidation by M3+ = Os(bpy)33+ with Added Buffer Base B− with RSH Either NAceCysSH or CysSH

Figure 5. Plot of kobs/[NAceCysSH] vs [AcO−] (0−0.7 M, pH = 5.76) for oxidation of N-acetyl-cysteine by Os(bpy)33+ as a function of added base in 0.8 M NaCl; T = 20 °C, DPA (1 mM), Os(bpy)33+ (20 μM), N-acetyl-cysteine (100 μM), y-int = 2.6 × 104 M−1 s−1.

− −d[Os3 +] ⎛ KA[CysSH]T [B ] ⎞ 3 + =⎜ ⎟[Os ] − dt ⎝ 1 + KA[B ] ⎠

≈ KA[AcO−] and, from the slope of the line in Figure 5, kobs ∼ Ka{k1k2/k−1[AcOH]}. The y-intercept of 2.6 × 104 M−1 s−1 from the plot at pH 5.76 is in good agreement with kH2O + k′/ [H+] = 2.0 × 104 M−1 s−1, calculated from kH2O = (4.8 ± 1.6) × 102 M−1 s−1 and k′ = (3.5 ± 0.03) × 10−2 s−1 measured independently. The stopped-flow experiments were extended to phosphate buffers (H2PO4−/HPO42−) at pH 6.2 under conditions: k2[Os3+] ≪ k−1[HB], [H2PO4−]/[HPO42−] = 10, where the MS-EPT pathway in Scheme 2 is dominant and the rate constant expression in eq 12c simplifies to eq 14.

⎞ ⎛ k1k 2 ⎟ × ⎜kEPTKA′ + 3+ k 2[Os ] + k −1[HB] ⎠ ⎝

⎡ KA[B−] ⎤ kobs k′ = k H 2O + + ⎥(kEPTKA′ ) ⎢ [CysSH]T [H+] ⎣ 1 + KA[B−] ⎦

mechanism for tyrosine oxidation by M(bpy)33+.21,22 The corresponding rate law is given in eq 12a and, including the buffer base independent k′ and kH2O terms, by eq 12b with [CysSH]T the total concentration of cysteine.

(12a)

(14)

⎛ KA[CysSH]T [B−] ⎞ 3 + k′ −d[Os3 +] = k H 2O + + ⎟[Os ] ⎜ − dt [H +] ⎝ 1 + KA[B ] ⎠

A best fit of the data to eq 14 is shown in Figure 6 with the parameters KA = 16 M−1 and KA′kEPT = 2.2 × 105 M−2 s−1 and y intercept = 7.7 × 104 M−1 s−1. In the analysis, KA′kEPT and KA were varied until the lowest residual values were obtained at the y-intercept with [HPO42−] = 0 M. Fits were sensitive to changes in KA of ±1 and in KA′kEPT of ±5%. The intercepts obtained by this procedure are in good agreement with kH2O + k′/[H+] = 5.6 × 104 M−1 s−1 obtained from the independent study described above. The value KA′kEPT = 2.2 × 105 M−2 s−1 for the oxidation of N-acetyl-cysteine by the MS-EPT pathway in Scheme 2 is in reasonable agreement with 7.2 × 105 M−2 s−1 obtained for oxidation of L-cysteine in the electrochemical study described

⎞ ⎛ k1k 2 ⎟ × ⎜kEPTKA′ + 3+ k 2[Os ] + k −1[HB] ⎠ ⎝ (12b)

⎡ KA[B ] ⎤ kobs k′ = k H 2O + ⎥ + + ⎢ [CysSH]T [H ] ⎣ 1 + KA[B−] ⎦ −

⎞ ⎛ k1k 2 ⎟ × ⎜kEPTK′A + 3+ k 2[Os ] + k −1[HB] ⎠ ⎝ (12c) 7032


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Systems, West Lafayette, IN).46 CV simulations were carried out over a wide range of buffer concentrations and buffer ratios under conditions that isolated the individual pathways in Scheme 2 in order to explore the limiting forms of the rate law in eq 12. Cysteine shares with tyrosine and tryptophan a kinetically slow response at ITO (tin-doped indium oxide) electrodes, with only small currents in excess of the background observed in oxidative scans to the oxidative limit of the solvent (Figure 7). The CVs in Figures 7 and 8 show the effect of added L-

Figure 6. Plot of kobs/[NAceCysSH] vs [HPO42−] (0−0.1 M, pH = 6.2) for oxidation of N-acetyl-cysteine by Os(bpy)33+ as a function of added base. I was held constant at 0.8 M with NaCl; T = 20 °C, DPA (1000 μM), Os(bpy)33+ (20 μM), N-acetyl-cysteine (100 μM), y-int = 7.7 × 104 M−1 s−1. Experimental data (green) and a least-squares residual fit (black) to eq 14 are shown.

below. Similarly, from the stopped-flow measurements, KA = 16 M−1 for the association complex between NAceCysSH and HPO 4 2− and K A = 50 M −1 for L-cysteine from the electrochemical study. There is a kinetically equivalent interpretation of the dependence on HPO42− shown in Figure 6, where Os(bpy)33+, HPO42− ion pair formation is followed by formation of an association complex with NAceCysSH to give the outer-sphere assembly Os(bpy)33+, HPO42−, NAceCysSH, followed by EPT oxidation. Kinetic Isotope Effects (KIE). Stopped-Flow. Stoppedflow kinetic measurements on the oxidation of N-acetylcysteine by Os(bpy)33+ were repeated in D2O with the AcO−/ AcOD buffer concentration varied from 10 to 300 mM at pH = pD = 5.76. Under these conditions in H2O, the mechanism is dominated by the PT−ET pathway in Scheme 1 with kobs/ [CysSH]T given by eq 13. Analysis of the data under these conditions in D2O compared to H2O gave a kinetic isotope effect of kH/kD = 2.2 ± 0.5, independent of the buffer concentration. This experimental value includes the equilibrium isotope effect for the RSH/RS− acid−base pre-equilibrium (Scheme 1). This value is high for a solvent kinetic isotope effect for outer-sphere electron transfer but corresponds well to previously reported isotope effects on the ionization of thiol groups in water and deuterium oxide for which KRSH/KRSD = 2.0−2.5.41,42 Similarly, kinetic measurements in D2O with [D2PO4−/ DPO42−] = 10, 200, and 600 mM at pH = pD = 6.2 with 10:1 [D2PO4−]/[DPO42−], gave kH/kD = 3.76 ± 0.4, 3.2 ± 0.1, and 2.9 ± 0.1, respectively. This observation is consistent with an important role for EPT with HPO42− as the proton acceptor base with pKa = 7.2 for H2PO4− with a lesser contribution from H2PO4− with pKa = 2.1 for H3PO4.31 In water, under these conditions, the MS-EPT pathway in Scheme 2 dominates the reaction. Electrochemistry. The oxidation of L-cysteine by M(bpy)33+ (M = Fe, Ru, Os) and Ru(dmb)33+ (bpy is 2,2′bipyridine and dmb is 4,4′-dimethyl−2,2′−bipyridine) was investigated in parallel by an electrocatalytic procedure applied earlier to oxidation of tyrosine and tryptophan.15,21,22,43−45 It is based on the numerical simulation of cyclic voltammetric (CV) waveforms by the simulation program DigiSim (BioAnalytical

Figure 7. Cyclic voltammograms (300 mV/s) in 50 mM [H2PO4−/ HPO42−] (1:1 acid/base) at pH 7.2 (I = 0.8 M, NaCl; T = 23 ± 2 °C). ITO background (blue); CysSH (100 μM, green); Os(bpy)32+ (20 μM, black); CysSH (100 μM) + Os(bpy)32+ (20 μM, red). Note: CVs are not background subtracted in order to illustrate the ITO and ITO + CysSH backgrounds.

Figure 8. Cyclic voltammograms (300 mV/s) in 300 mM [H2PO4−/ HPO42−], (I = 0.8 M NaCl), T = 23 ± 2 °C. Os(bpy)32+ (20 μM) on ITO (black), Os(bpy)32+ (20 μM) + CysSH (100 μM), 10:1 acid/base buffer ratio, pH 6.2 (red), 1:1 acid/base, pH 7.2 (blue), 10:1 acid/ base, pH 8.2 (green).

cysteine (CysSH) and buffer on the wave form for −e −

Os(bpy)3 2+ ⎯⎯⎯→Os(bpy)33+. The Os(bpy)33+/2+ couple is electrochemically reversible under these conditions with E°′ = 0.80 V versus NHE. As shown by the data in Figure 8 with H2PO4−/HPO42− as −e −

the buffer, the Os(bpy)32+ ⎯⎯⎯→Os(bpy)33+ waveform with added L-cysteine varies with both buffer base concentration and pH. This behavior is qualitatively similar to tyrosine oxidation with 7033


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The Journal of Physical Chemistry C significant rate enhancements observed under the same conditions.21,22 Digital Simulation of CV Waveforms. DigiSim uses the Butler−Volmer equation as a starting point to simulate digitized voltammetric waveforms (eq 15). i = FAks[C0(0, t )e−αF(E − E°)/ RT − CR(0, t )e(1 − α)F(E − E°)/ RT ] (15)

In eq 15, i is the current, A is electrode area, (E − E°) = η is the overpotential, the difference between the applied potential and E1/2 for the M(bpy)33+/2+ couple, α is the transfer coefficient, and ks is the interfacial electron transfer rate constant. CO and CR are the concentrations of oxidized and reduced forms of the substrate as a function of distance from the electrode (x) and time (t).22,46,47 In the simulations, Fick’s laws of diffusion are used to define boundary conditions with the concentrations dependent on the diffusion coefficient, the slope of the concentration gradient between the bulk solution and the electrode interface, and the scan rate, which defines the diffusion layer thickness.48 As shown in Figures 7 and 8, with added L-cysteine, current enhancements are observed on oxidative CV scans through the Os(bpy)33+/2+ couple. They arise from reoxidation of Os(bpy)32+ formed by L-cysteine reduction of Os(bpy)33+ in the double layer near the electrode. Simulation of voltammograms without added L-cysteine gave ks = 0.01 cm s−1 and DOs = 6.0 × 10−6 cm2 s−1 for the Os(bpy)33+/2+ couple.46,47 In analyzing the data with added L-cysteine, a literature value was used for the diffusion coefficient for cysteine (3.0 × 10−6 cm2 s−1).49−51 Reduction potentials for the metal complex couples used in the study ranged from E°′ = 0.80 V versus NHE for the Os(bpy)33+/2+ couple to 1.25 V for the Ru(bpy)33+/2+ couple versus NHE in 0.05 M phosphate; 0.8 M NaCl at 23 ± 2 °C. Cyclic voltammetric data for oxidation of L-cysteine without added bases were fit in DigiSim by assuming the electrochemical mechanism shown in eqs 16−18 and the reaction stoichiometry in eq 9.29,30 Os(bpy)32 + → Os(bpy)33 + + e−

Figure 9. Cyclic voltammograms (300 mV/s) illustrating the dependence of the current−potential profile on total buffer concentration, [H2PO4−] + [HPO42−] at pH 7.2 (I = 0.8 M, NaCl; T = 23 ± 2 °C). Os(bpy)32+ (20 μM, black), Os(bpy)32+ (20 μM) + LCys (100 μM) in 300 (blue) and 50 (red) mM [H2PO4−] + [HPO42−]. ⎡ K [HPO2 −] ⎤ kobs k′ A 4 ⎢ ⎥(kEPTK′A) = k H2O + + 2− [CysSH]T [H +] ⎣ 1 + KA[HPO4 ] ⎦ (19)

From the stopped-flow results on N-acetyl-cysteine at relatively high concentrations of added buffer, the term (kH2O + k′/[H+]) in eq 12c under these conditions is negligible, giving eq 20. From this equation and the plot of kobs−1 versus [HPO42−]−1 in Figure 10B, KAKA′kEPT = 4.2 × 108 M−3 s−1. From the y-intercept, KA′kEPT = 1.0 × 107 M−2 s−1 and from the intercept/slope ratio, KA ∼ 50 M−1. ⎡ K [HPO2 −] ⎤ kobs A 4 ⎥ = (kEPTKA′ )⎢ 2− [CysSH]T 1 K [HPO + ⎣ A 4 ]⎦

At high buffer ratios with [H2PO4−]/[HPO42−] > 10, the MS-EPT pathway dominates and, in the limit KA[HPO42−] ≪1, eq 20 becomes eq 21. Under these conditions, the mechanism in Scheme 2 simplifies to Scheme 3 with a simulation of CV data shown in Figure 11.

(16)

kobs

Os(bpy)33 + + CysSH ⎯→ ⎯ Os(bpy)32 + + CysSH ox

(17)

CysSH ox → 1/2CysSSCys + 1/2H+ (rapid)

(18)

(20)

kobs = kEPTKAKA′ [HPO24 −] [CysSH]T

In an initial series of experiments with no added buffer at pH = 5.76 (I = 0.8 M, NaCl; T = 23 ± 2 °C), simulation of CVs with [Os(bpy)33+] = 20 μM, [CysSH] = 100 μM, and DOs(bpy)33+ = 6.0 × 10−6 cm2 s−1 gave kobs = (2.0 ± 0.9) × 105 M−1 s−1 compared to kobs = kH2O + k′/[H+] = 2.05 × 104 M−1 s−1 for Os(bpy)33+ oxidation of N-acetyl-cysteine under the same conditions. Limiting Forms: Saturation in Buffer Anion. Figure 9 illustrates the response of the CV waveform to an increase in total buffer concentration with [H2PO4−] = [HPO42−] at pH 7.2. A saturation onset appears at [HPO42−] > 80 mM (Figure 10A) consistent with formation of the H-bonded adduct, CysS−H---OP(OH)O22− (Scheme 2). Saturation kinetics appeared over the entire pH range studied in this series of experiments. In the limit that proton transfer within the adduct is rapid, the general rate law in eq 12c simplifies to eq 19, which is eq 14 written specifically for HPO42− as the proton acceptor base.

(21)

Kinetic Isotope Effects. CV. CV measurements on the oxidation of L-cysteine by Os(bpy)33+ were repeated at pH 5.5 in a 0.05 M deuterated phosphate buffer at a buffer ratio of [D2PO4−]/[DPO42−] = 15:1. In this limit, oxidation is dominated by MS-EPT with kobs/[CysSH]T given by eq 20. Analysis of CV waveforms under these conditions gave kD = 2.3 × 105 M−2 s−1 with kH/kD = 3.2. This KIE is comparable to the value obtained for oxidation of N-acetyl-cysteine by stopped flow measurements and consistent with a significant contribution from MS-EPT. Summary: Oxidation of L-Cysteine and N-AcetylCysteine by Os(bpy)33+. A summary of rate and equilibrium constants derived from stopped-flow and electrochemical measurements on oxidation of L-cysteine and N-acetyl-cysteine by Os(bpy)33+ with added H2PO4−/HPO42− buffers is given in Table 1. The agreement between kinetic parameters derived from the two independent techniques is satisfactory with small 7034


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Figure 10. (A) Plot of kobs (M−1 s−1) vs [HPO42−] in [H2PO4−] = [HPO42−] buffers at pH 7.2 in 0.8 M NaCl at T = 23 ± 2 °C, for the oxidation of L-Cys (100 μM) by Os(bpy)33+ (20 μM). (B) As in (A), plot of kobs−1 vs [HPO42−]−1. Slope = (KAKA′kEPT)−1 = 2.4 × 10−9 M3 s; intercept = (KA′kEPT)−1 = 1.0 × 10−7 M2 s, KA = 50 ± 10 M−1.

differences attributable, in part, to the difference in substrates between the two studies. Rate Comparisons. Oxidation of the Redox Active Amino Acids CysSH, TyrOH, and NAceTrpNH by M(bpy)33+. The results of a series of studies based on oxidation of L-tyrosine, L-tryptophan, N-acetyl-tryptophan, N-acetylcysteine, and L-cysteine by M(bpy)33+ (M = Os, Fe, Ru) derivatives are now available.15,19,21,22 In comparing the results of these studies, there are features of relevance to their roles as redox cofactors (see below). With buffer and pH conditions of relevance to biological reactions, electron transfer oxidation of tyrosine (TyrOH) and cysteine (CysSH) is presumably dominated by MS-EPT given their relatively acidic protons and the presence of high concentrations of phosphate buffers. For tryptophan in NAceTrpNH, without an acidic proton with pKa ≈ 16 for the indole N−H, initial electron transfer followed by proton transfer (ET−PT) is the favored pathway.15 Kinetic parameters for oxidation of the three redox active amino acids are presented in Table 2 for the MS-EPT pathways that dominate under conditions (pH 7.5, 0.05 M phosphate buffer) of direct relevance to their roles as redox cofactors in redox active enzymes such as the oxidoreductases. Notable in the data is the increase in rate for MS-EPT oxidation of CysSH (KAKA′kEPT) and TyrOH and for ET−PT oxidation of NAceTrpNH as E°′ for the M(bpy)33+/2+ couple increases. In an earlier study, a detailed analysis of the role of the driving force in the MS-EPT oxidation of TyrOH was presented.22 Oxidation of L-Cysteine by M(bpy)33+ (M = Fe, Ru, Os) and Ru(dmb)33+ with the Buffer Bases OAc−, Suc−, His, and HPO42−. The results of a more extensive study on Lcysteine oxidation by M(bpy)33+ (M = Fe, Ru, Os) and Ru(dmb)33+ has also been investigated by CV simulation with the results reported elsewhere.52 For purposes of comparison, data for these oxidants with HPO42− (pKa = 7.2) or Tris (pKa = 8.1) as the common proton acceptor base are listed in Table 2. Notable in the data is the increase in KAKA′kEPT for MS-EPT oxidation of CysSH (KAKA′kEPT) as E°′ (M(bpy)33+/2+) and the driving force of oxidation increase. Similarly, in the oxidation of L-cysteine by Os(bpy)33+, the driving force dependence of KAKA′kEPT arising from variations in the base strength of proton acceptor base over a pKa range from 4.7 to 7.2 is shown in Table 3. A similar variation was observed earlier in the MS-EPT

Scheme 3

Figure 11. Simulation (red dots) of a CV (300 mV/s) in 300 mM 10:1 H2PO4−:HPO42− (I = 0.8 M, NaCl; T = 23 ± 2 °C), Os(bpy)32+ (20 μM) + CysSH (100 μM), pH 6.2, based on the mechanism in Scheme 3.

Table 1. Comparison of Rate and Equilibrium Constants for the Oxidation of L-Cysteine and N-Acetyl-Cysteine by Os(bpy)3 3+ Obtained by DigiSim CV Analysis and StoppedFlow Measurements with Added H2PO4−/HPO42− Buffer in 0.8 M NaCl at 23 ± 2°C KA (M−1)

substrate a

NAceCysSH LCysSHb

c

16.0 50.0 ± 1.5

KA′kEPT (M−2 s−1)

k1k2/k−1 (s−1)

2.2 × 10 7.2 ± 0.5 × 105

5.5 ± 0.5 × 105 8.3 ± 1.7 × 105

5c

Stopped-flow. bCV simulation. cValues from least-squares residual fit to data in Figure 6 discussed in the text above. a

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Table 2. Kinetic Parameters for the MS-EPT Oxidation of L−Tyrosine (TyrOH)22,52 and L-Cysteine (CysSH) and for the ET− PT Pathway for NAceTrpNH15 by Analysis of CV Wave Formsa amino acid

metal mediator 3+

TyrOH TyrOH TyrOH TyrOH CysSH CysSH CysSH CysSH NAceTrpNH NAceTrpNH NAceTrpNH NAceTrpNH

Ru(bpy)3 Ru(dmb)33+ Fe(bpy)33+ Os(bpy)33+ Ru(bpy)33+ Ru(dmb)33+ Fe(bpy)33+ Os(bpy)33+ Ru(bpy)(dmb)23+ Ru(dmb)33+ Fe(bpy)33+ Fe(dmb)33+

buffer base

E°′ (V vs NHE)

KAKA′kEPT (M−2 s−1)

1.25 1.06 1.03 0.80 1.25 1.06 1.03 0.80 1.13 1.06 1.03 0.86

× × × × × × × ×

phosphate phosphate phosphate phosphate phosphate phosphate phosphate phosphate Tris Tris Tris Tris

7.0 3.9 3.2 7.3 4.7 4.0 4.5 2.5 − − − −

5

10 105 105 103 106 105 105 105

kET (M−1 s−1) − − − − − − − − 2.3 1.7 1.1 4.4

× × × ×

107 107 107 104

Data for CysSH and TyrOH oxidations in 50 mM H2PO4−/HPO42− at pH 6.2 and for NAceTrpNH in 50 mM Tris buffer at pH 7.1. In all cases I = 0.8 M, NaCl; T = 23 ± 2 °C. a

microscopically more complex concerted pathways is due to a high barrier to electron transfer imposed by initial oxidation to the RSH•+ radical cation. The appearance of multiple pathways in tyrosine and cysteine oxidation is in contrast to tryptophan which has no acidic protons and, in most cases, is constrained mechanistically to outer-sphere electron transfer.15 The presence of relatively acidic protons in cysteine (pKa = 8.2) and tyrosine (pKa = 10.1) provides access to EPT and PT−ET pathways that circumvent the high-energy 1e− intermediates TyrOH•+ and CysSH•+. Detailed structural analysis of redox enzymes is limited,53,54 but in vivo studies of active enzymes are consistent with preferential oxidation of TyrOH and CysSH by MS-EPT with long-range electron transfer occurring to an electron acceptor and short-range proton transfer to an associated base.1,5−7 The available evidence for tryptophan oxidation points to longrange electron transfer followed by proton transfer to an available acceptor base.6,55

Table 3. Buffer Base Dependence of the Kinetic Parameter KAKA′kEPT Derived from CV Simulations for the Oxidation of CysSH by Os(bpy3)3+ in 50 mM Buffers with I = 0.8 M, NaCl; T = 23 ± 2°C metal mediator

buffer base

pKa

pH

Os(bpy)33+ Os(bpy)33+ Os(bpy)33+ Os(bpy)33+

phosphate histidine succinate acetate

7.2 6.1 5.6 4.8

6.2 5.1 4.6 3.8

KAKA′kEPT (M−2 s−1) 2.5 1.8 3.2 1.5

× × × ×

105 105 104 104

oxidation of TyrOH.21,22 The driving force contribution to MSEPT from proton transfer from CysSH•+ to the acceptor base (B) is given by ΔG°′ = 0.059{pKa(CysSH•+) − pKa(HB+)}. As noted above, a detailed analysis of the free-energy dependence for MS-EPT oxidation of TyrOH with variations in ΔG°′ from both the buffer base and E°′ for the M(bpy)33+/2+ couple was published earlier.22 It was not possible to apply the same analysis to L-cysteine oxidation because values for pKa(CysSH•+) and E°′ for the CysSH•+/CysSH couple are not available.

■

ASSOCIATED CONTENT

■

AUTHOR INFORMATION

S Supporting Information *

■

Rates of outer-sphere electron transfer for CysSH and CysS−, additional plots for the stopped-flow analysis of rates for outersphere electron transfer of N-acetyl-cysteine by Os(bpy)33+, observed rate constants, cyclic voltammograms of Os(bpy)33+, and further DPA analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

CONCLUSIONS The results of kinetic studies on the oxidation of N-acetylcysteine and L-cysteine by the oxidants M(bpy)33+ (M = Fe, Ru, Os), by either spectroscopic monitoring with stopped-flow mixing or simulation of cyclic voltammetry waveforms, give comparable results. Small differences in kinetic parameters between the two are due, in part, to the different cysteine derivatives used in the two studies. Closely related patterns of oxidative reactivity exist for oxidation of tyrosine and cysteine. In the oxidation of both by M(bpy)33+, initial 1e− oxidation is rate-limiting with electron transfer occurring by multiple competing pathways: (i) direct oxidation by outer-sphere electron transfer, MS-EPT oxidation with water the acceptor base, oxidation of the tautomer, HOOC(H)(NH3+)CH2S−, or by a combination of the three; (ii) proton transfer followed by electron transfer oxidation of CysS− (PT-ET); (iii) with added proton acceptor bases, MSEPT with electron transfer occurring in concert with proton transfer to the H-bonded base; or (iv) by rate-limiting proton transfer within the H-bonded adduct CysSH---B. The relative importance of the competing pathways varies with pH and buffer ratio. The appearance of buffer base effects and the

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation under Grant CHE−1362481. REFERENCES

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Concerted ElectronâProton Transfer (EPT) in the Oxidation of Cysteine

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Concerted ElectronâProton Transfer (EPT) in the Oxidation of Cysteine