Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in

Apr 30, 2016 - Sulfidic off-odors arising during wine production are frequently removed by Cu(II) fining. In part 1 of this study (10.1021/acs.jafc.6b...
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Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron- and Copper-Catalyzed Oxidation Gal Y. Kreitman,† John C. Danilewicz,‡ David. W. Jeffery,§ and Ryan J. Elias*,† †

Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania 16802, United States 44 Sandwich Road, Ash, Canterbury, Kent CT3 2AF, United Kingdom § School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia ‡

S Supporting Information *

ABSTRACT: Sulfidic off-odors arising during wine production are frequently removed by Cu(II) fining. In part 1 of this study (10.1021/acs.jafc.6b00641), the reaction of H2S and thiols with Cu(II) was examined; however, the interaction of iron and copper is also known to play an important synergistic role in mediating non-enzymatic wine oxidation. The interaction of these two metals in the oxidation of H2S and thiols (cysteine, 3-sulfanylhexan-1-ol, and 6-sulfanylhexan-1-ol) was therefore examined under wine-like conditions. H2S and thiols (300 μM) were reacted with Fe(III) (100 or 200 μM) alone and in combination with Cu(II) (25 or 50 μM), and concentrations of H2S and thiols, oxygen, and acetaldehyde were monitored over time. H2S and thiols were shown to be slowly oxidized in the presence of Fe(III) alone and were not bound to Fe(III) under model wine conditions. However, Cu(II) added to model wine containing Fe(III) was quickly reduced by H2S and thiols to form Cu(I) complexes, which then rapidly reduced Fe(III) to Fe(II). Oxidation of Fe(II) in the presence of oxygen regenerated Fe(III) and completed the iron redox cycle. In addition, sulfur-derived oxidation products were observed, and the formation of organic polysulfanes was demonstrated. KEYWORDS: H2S, thiols, copper, iron, oxidation, wine aroma, reaction mechanism

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INTRODUCTION Non-enzymatic wine oxidation, in which polyphenols interact with oxygen, is now known to be catalyzed by trace concentrations of transition metals in wine, particularly iron (Fe) and copper (Cu).1,2 During this oxidation process, O2 can be reduced to water in four discrete one-electron steps,1 resulting in the formation of reactive intermediate oxygen species3 that can oxidize wine constituents.4−6 However, recently, it was proposed that, under wine-like conditions, Fe(II) reduces an intermediate Fe(III)−oxygen complex in a concerted two-electron reduction to produce H2O2 from O2 without the formation of an intermediate hydroperoxyl radical (Figure 1).7 Similar results were obtained for the Cu(I)-

which results in ethanol oxidation by forming the intermediate 1-hydroxyethyl radical (1-HER).9 In low O2 concentrations, 1HER will be oxidized by Fe(III) to yield acetaldehyde (AC); however, at higher O2 concentrations, O2 is known to add to 1HER to yield the 1-hydroxyethylperoxyl radical (1-HEPR) (Figure 2). Recent work suggests that, rather than 1-HEPR releasing AC and hydroperoxyl radicals, 1-HEPR is reduced to peroxide by the presence of reduced metal complexes.8 Peroxide can then undergo a Fenton-like reaction to form the alkoxyl radical that will subsequently be reduced to 1,1dihydroxyethane that dehydrates to AC. Fe(III) catalyzes the oxidation of wine polyphenols containing catechol or pyrogallol moieties to form intermediate semiquinone radicals, which are further oxidized to o-quinones. The reaction is accelerated by nucleophiles, such as bisulfite and thiols.10,11 In this latter process, quinones are reduced back to catechols by reaction with sulfite10 or undergo Michael-type addition reactions with sulfite or thiols,12,13 effectively driving the reaction forward by consuming the product of phenolic oxidation. Fe(III) may also interact with thiols directly, which could either have deleterious effects by causing the oxidative loss of important aroma compounds, such as 3-sulfanylhexan-1ol (3SH), or a beneficial effect by reacting with hydrogen sulfide (H2S).10,14 The presence of thiols in wine may,

Figure 1. Reduction of oxygen by Fe(II) to yield hydrogen peroxide without the release of hydroperoxyl radicals.

mediated reduction of oxygen, where no evidence of an intermediate hydroperoxyl radical was observed.8 In combination, these metals act synergistically, with copper playing an important role in the overall wine oxidation process by accelerating the reaction of Fe(II) with oxygen to regenerate Fe(III);2 presumably, copper facilitates Fe(III)/Fe(II) redox cycling. Once H2O2 is formed, it is reduced by Fe(II) through the Fenton reaction to yield the highly reactive hydroxyl radical, © XXXX American Chemical Society

Received: February 5, 2016 Revised: April 29, 2016 Accepted: April 30, 2016

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DOI: 10.1021/acs.jafc.6b00642 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Reduction of hydrogen peroxide to produce hydroxyl radicals by the Fenton reaction and subsequent formation of the 1-hydroxyethyl radical. The 1-hydroxyethyl radical is further oxidized by oxygen or Fe(III) to eventually yield AC.

removal of undesirable sulfidic off-odors in comparison to copper alone. Recent work has examined the reaction of H2S with Cu(II)19 but did not take into account the presence of iron, which could be present in ∼10-fold excess in wine compared to copper.20 The aim of this present study was to elucidate the mechanism underlying Fe-mediated thiol oxidation under wine-like conditions, which builds on the findings of the first part of this larger study involving copper alone (10.1021/ acs.jafc.6b00641). Because the interaction of iron and copper plays an important role in polyphenol oxidation, it was of interest to understand whether these metals also interacted synergistically in the oxidation of H2S and thiols. As noted previously,8 the concentration of thiols, such as glutathione and cysteine analogues, far exceeds that of H2S that are likely to occur in wine. The oxidation of H2S in the presence of greater concentrations of Cys, as a representative thiol, was therefore investigated as a result of its relevance to the copper fining operation in winemaking.

therefore, play an important role in mediating wine oxidation, although the mechanism by which sulfhydryl compounds (i.e., species containing an −SH moiety) directly interact with iron and copper in wine remains poorly understood. Such information is important to winemakers for them to make informed decisions about managing oxidation to improve wine quality. Studies performed with glutathione (GSH) in a pH range (3−7) have shown that Fe(II) is spontaneously produced when GSH is added to Fe(III) (Figure 3).15,16 The same has been

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Figure 3. Proposed mechanism for initial Fe(III) reduction by thiols showing that resulting Fe(II) is not coordinated to sulfur after disulfide is formed.

MATERIALS AND METHODS

Chemicals. L-Cysteine (Cys), monobromobimane (MBB), 6sulfanylhexan-1-ol (6SH), and diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma-Aldrich (St. Louis, MO). 2,4Dinitrophenylhydrazine (DNPH) was purchased from MCB Laboratory Chemicals (Norwood, OH) and L-tartaric acid, 3SH, and 5,5′dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Copper(II) sulfate pentahydrate was purchased from EMD Chemicals (Gibbstown, NJ), Tris hydrochloride was purchased from J.T. Baker (Center Valley, PA), and sodium hydrosulfide hydrate (as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Iron(III) chloride hexahydrate was purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or highperformance liquid chromatography (HPLC) grade, and solutions were prepared volumetrically, with the balance made up with Milli-Q water, unless specified otherwise. Model Wine Experiments. Model wine was prepared by dissolving tartaric acid (5 g/L) in water, followed by the addition of ethanol to yield a final concentration of 12% (v/v). The solution was adjusted to pH 3.6 with sodium hydroxide (10 M) and brought to volume with water. For H2S and Cys, an aqueous stock solution of each (approximately 0.5 M) was freshly prepared, whereas 6SH and 3SH were added directly by syringe during experimentation. Aqueous stock solutions of Cu(II) sulfate and Fe(III) chloride (0.1 and 0.4 M, respectively) were

shown with Cys at low pH, because the Fe(III)−Cys complex is unstable and quickly reacts to yield Fe(II) and cystine.17 Previous work has failed to provide evidence of free thiyl radical generation under those conditions,15 and disulfide is seemingly formed in situ before being released from the metal complex. Resulting Fe(II) remains bound to GSSG and is only released when excess GSH is present; however, unlike Cu(I), which coordinates strongly with thiols, Mössbauer spectroscopy showed that Fe(II) is not bound to sulfur. It was concluded that coordination to GSSG, GSH, and also Cys occurred by interaction with carboxylate groups under acidic conditions (pH < 4).15,16 As discussed above, Fe(II) produced can be reoxidized to Fe(III) by reacting with O2, with the reaction markedly accelerated by copper. Recent work in model systems has demonstrated that tartaric acid determines the reduction potential of the Fe(III)/Fe(II) couple in wine,18 but it may be possible that thiols also affect that potential. This is of particular interest to copper-containing systems, because H2S and thiols keep copper in its reduced Cu(I) state under wine-like conditions.8 In view of the known interaction of iron and copper in relation to wine oxidation, it is of interest to examine the effect of the metal combination in the B

DOI: 10.1021/acs.jafc.6b00642 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

voltage was 25 V; the source temperature was 120 °C; and the desolvation gas flow was 650 L/h.

freshly prepared. H2S, Cys, 6SH, or 3SH were added to air-saturated model wine (1 L, 300 μM), followed by thorough mixing. For Fe experiments, Fe(III) (200 μM) was added to all H2S and thiol treatments and thoroughly mixed. For Fe and Cu combination experiments, Fe(III) (200 μM) and Cu(II) (50 μM) were consecutively added to H2S, 6SH, or 3SH solutions. For Cys experiments, Fe(III) (100 μM) and Cu(II) (25 μM) were consecutively added and mixed thoroughly. For thiol experiments in combination with H2S and Fe/Cu, H2S was added to the thiol treatment and mixed prior to the addition of metal stock solutions. H2S (50 or 100 μM), Fe(III) (200 μM), and Cu(II) (50 μM) were added to Cys, 6SH, and 3SH. For Cys experiments with low metal concentrations, H2S (50 μM), Fe(III) (100 μM), and Cu(II) (25 μM) were added and thoroughly mixed. The resulting treatment solutions were immediately transferred to 60 mL glass biological oxygen demand (BOD) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and bottles were capped immediately with ground glass stoppers, eliminating headspace. The glass reservoir of the BOD bottles was topped off with water daily. The bottles were stored in the dark at ambient temperature. One BOD bottle was sacrificed per time point per replicate and used for further analyses. All experiments were conducted in triplicate and contained their own series of sacrificial bottles. Determination of Oxygen Consumption. Glass BOD bottles were fitted with PSt3 oxidots, and oxygen readings were taken per time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Further details were reported in part 1 (10.1021/ acs.jafc.6b00641).8 Spectrophotometric Measurements. Ultraviolet−visible (UV− vis) spectra of the treatments were recorded at each time point using 10 mm quartz cuvettes (model wine blank) and measured using an Agilent 8453 UV−vis spectrophotometer (Agilent, Santa Clara, CA). Determination of the Fe(III) concentration was achieved by measurement of absorbance at 336 nm associated with the Fe(III)− tartrate complex.7 For H2S, Cys, 6SH, and 3SH, the total concentration was analyzed using Ellman’s assay. Further details were reported in part 1 (10.1021/ acs.jafc.6b00641).8 HPLC Analyses. For the mixed H2S and thiol treatments, MBB derivatization and analysis of the thiol concentration were performed using negative electrospray ionization (ESI−) HPLC−tandem mass spectrometry (MS/MS) as described in part 1 (10.1021/ acs.jafc.6b00641).8 The mass transition of sulfide-dibimane was monitored at m/z 413 → 191; the mass transition of Cys-bimane was monitored at m/z 310 → 223; the mass transition of 3SH-bimane was monitored at m/z 323 → 222; and the mass transition of the internal standard 6SH-bimane was monitored at m/z 323 → 222. External standard curves prepared for sulfide-dibimane, Cys-bimane, and 3SH-bimane were normalized to the 6SH-bimane internal standard. In the case of the 6SH/H2S combination experiment, external calibration curves were made the same day prior to analysis and used without the addition of the 6SH-bimane internal standard. AC was measured in model wine treatment solutions as its 2,4dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10−220 μM) by HPLC as described in part 1 (10.1021/ acs.jafc.6b00641).8 Polysulfides were formed by the reaction of H2S (300 μM) with Cu(II) (50 μM) and Fe(III) (200 μM). A sample was derivatized using MBB as described above with the same HPLC separation parameters. Mass spectra were obtained using ESI− and full scan between m/z 100 and 1000. 6SH and 3SH polysulfanes were obtained by adding H2S (100 μM), Fe(III) (200 μM), and Cu(II) (50 μM) to 6SH or 3SH (300 μM). The organic polysulfanes were detected by UV absorbance at 210 nm and verified using MS detection with ESI+ and full scan between m/z 100 and 1000. Mobile phases consisted of 0.1% (v/v) formic acid (A) and 0.1% (v/v) formic acid in acetonitrile (B) with a linear gradient according to the following program: 0 min, 5% B; 20 min, 95% B; 28 min, 95% B; 28.1 min, 5% B; and 38 min, 5% B. The ESI capillary spray voltage was set to 4 kV; the sample cone

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RESULTS AND DISCUSSION Reaction of Fe(III) with H2S and Thiols in Model Wine. The reactivity of Fe(III) with the following treatments was investigated in model wine: (1) Cys, which also represents homo-Cys and Cys derivatives; (2) 6SH, which represents primary thiols; (3) 3SH, which represents secondary thiols; and (4) H2S, because it is one of the primary targets associated with sulfidic off-odors. Unlike the Cu(II) experiments described in part 1 (10.1021/acs.jafc.6b00641), in which 2 mol equiv of thiols and 1.4 equiv of H2S were immediately consumed (i.e., within 5 min),8 there was no initial uptake of these substances when Fe(III) was added (Figure 4A). In the case of H2S, although there was no appreciable consumption observed within the first few hours of the experiment, it reacted faster than the other thiol compounds, with its concentration

Figure 4. Reaction of H2S or thiols upon the addition of Fe(III) (200 μM) to 6SH, H2S, Cys, or 3SH (300 μM) in air-saturated model wine. (A) Consumption of H2S or thiols, (B) percentage of Fe(III)−tartrate based on absorbance at 336 nm, and (C) O2 consumption. Error bars indicate the standard deviation of triplicate treatments. C

DOI: 10.1021/acs.jafc.6b00642 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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relative rate of Fe(II) reoxidation by O2. Because tartaric acid determines the reduction potential of the Fe(III)/Fe(II) redox couple in the model system described here, it is likely that the reoxidation of Fe(II) will proceed as described previously (Figure 1).7 Fe(II) is expected to reduce O2 by a concerted two-electron mechanism, yielding an Fe(III)−dioxygen complex that directly hydrolyzes to H2O2 without release of hydroperoxyl radicals. H2O2 should then undergo reduction via the Fenton reaction in the presence of Fe(II) to yield hydroxyl radicals that will subsequently oxidize ethanol (Figures 1 and 2). Fe behaves as a redox catalyst, cycling electrons from thiols and H2S to O2. On the basis of the overall sequence of reactions, it would be expected that three electrons would come from thiols or H2S and one electron would come from ethanol to reduce O2 to water. Consequently, it would be expected that the O2/thiol molar reaction ratio would be 1:3 and the O2/H2S ratio would be 1:1.5 because H2S is capable of reducing 2 equiv of Fe(III) as it is oxidized to ground-state sulfur.25 The treatment containing H2S resulted in the greatest uptake of O2 in the presence of Fe(III). Of the 262 μM H2S that reacted (Figure 4A), 135 μM O2 was consumed (Figure 4C), giving a 1:1.9 O2/H2S molar reaction ratio. However, roughly 66% Fe(III) had also been reduced to Fe(II) (∼132 μM) (Figure 4B), which would have required ∼66 μM H2S. Subtracting that amount from total reacted H2S would give ∼196 μM uptake corresponding to the 135 μM O2 uptake, thus lowering the O2/H2S molar reaction ratio to ∼1:1.5, as anticipated from the proposed mechanism (Figures 1 and 2). Fe(III) is reduced to some extent by Cys, likely in the same manner proposed in Figure 3, and 192 μM Cys (Figure 4A) reacted to reduce Fe(III) with subsequent consumption of 49 μM O2 (Figure 4C). However, roughly 17.5% (35 μM) Fe(II) remained at the end of the reaction, which corresponded to 35 μM Cys uptake. Subtracting this amount results in 157 μM Cys oxidized with the corresponding 49 μM O2 uptake, giving a O2/ thiol molar ratio of ∼1:3.2, which is in agreement with the proposed mechanism. (Figures 1 and 2). As a result of the inability of 6SH and 3SH to outcompete tartaric acid to form an Fe(III) complex, the oxidation of 6SH and 3SH was extremely slow and the O2/thiol molar reaction ratios could not be calculated (panels A and C of Figure 4). Low concentrations of AC (15−30 μM) were formed in the Cys and H2S systems (data not shown), demonstrating that the Fenton reaction does proceed in the system described. The formation of AC is thought to proceed as described in Figure 2. It was expected that a higher concentration of AC would be formed in the H2S system. In a previous study in which the Fenton reaction was investigated in model wine with iron only, up to 90% 1-HER radical was intercepted by thiol-containing compounds, with the resulting thiyl radical likely then quickly dimerizing to yield a disulfide.26 Fe(III) and Cu(II) Reduction by Thiols and H2S. The interaction of iron and copper plays an important synergistic role in wine oxidation, and it was important to investigate whether these metals impacted H2S and thiol oxidation. The treatments described above were employed again, this time using a combination of Cu(II) (50 μM) and Fe(III) (200 μM). The Cu(II) concentration was chosen to remain consistent with part 1 of this investigation (10.1021/acs.jafc.6b00641), and the concentration ratios chosen because wines typically have 5−10-fold higher relative concentrations of iron to copper.20 In this experiment, Cys reacted rapidly and was completely consumed within 5 min (data not shown);

declining as Fe(III) was reduced and O2 was consumed (panels B and C of Figure 4). A total of 262 μM H2S was consumed after 144 h elapsed, and a total of 192 μM Cys was consumed after 193 h. Both 6SH and 3SH reacted extremely slowly, with negligible losses (