Redox and Nucleophilic Reactions of Naphthoquinones with Small Thiols and Their Effects on Oxidization of H2S to Inorganic and Organic Hydropolysulfides and Thiosulfate

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2–4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.


Introduction
1,4-Naphthoquinones and their derivatives (collectively termed NQs) are naturally occurring and synthetic compounds that are toxic at high levels, but at lower levels they have purported anti-inflammatory, anticancer, antibacterial, antiviral, antifungal, and antiparasitic actions as well as cytoprotective effects in essentially all organ systems [1][2][3][4][5][6][7][8][9][10][11][12]. These functions are derived in part from their ability to participate in redox cycling. As described by Kumagai et al. [13], oxidized NQs may undergo one-electron reduction to a semiquinone radical (NQ • ) and a second one-electron reduction to hydroquinone (NQH 2 ) in reactions catalyzed by cytochrome P450 reductase or other flavoprotein enzymes. Alternatively, the NQ may undergo a two-electron reduction to the fully reduced NQH 2 in a reaction catalyzed by DT diaphorase (NADPH quinone reductase). Both reactions use NADPH as the electron donor. The semiquinone is then reoxidized (autoxidized) by molecular oxygen to the NQ and

1,4-Naphthoquinone Oxidizes H 2 S to Inorganic and Organic Hydroper-and Hydropolysulfides in the Presence of Low-Molecular-Weight Thiols
We have previously shown that 1,4-NQ oxidizes H 2 S to S 2 -S 6 polysulfides [16]. Here we show that similar reactions occur in the presence of small thiols. LCMS identification of products produced by 1,4-NQ oxidation of H 2 S in the presence of Cys and GSH are shown in Figure 1. Incubation of H 2 S and 1,4-NQ with Cys produced both inorganic (H 2 S n , n = 2-5) and organic (CysS n H, n = 2-4) hydroper-and hydropolysufides; the extent of polysulfide production progressively decreased as the number of sulfur atoms increased ( Figure 1A). Most hydroper-and hydropolysulfides were produced in the first 10 min and declined thereafter, except for H 2 S 2 , which remained nearly constant, and CysS 2 H, which continued to increase. Incubation with GSH ( Figure 1B) produced an initial increase in H 2 S 2,3 and GSHS 2,3 at 10 min that declined thereafter. A small amount of GSHS 4 and GS 2 OH was observed at 2 min and GSOH was present at 2 and 10 min. No per-or polysulfides of Cys or GSH (RS n R; R = Cys, GSH; n ≥ 2) were observed. . GSH or Cys were added to 1,4-NQ followed by H 2 S. TME-IAM was added at 2, 10, 30, and 60 min and the reactions were stopped with 1% formic acid 30 min after the last (60 min) sample and subjected to mass spectrometry. A second aliquot of the 2 min sample was run after the 60 min sample ( ysulfide production progressively decreased as the numbe ure 1A). Most hydroper-and hydropolysulfides were pr declined thereafter, except for H2S2, which remained near continued to increase. Incubation with GSH ( Figure 1B) H2S2,3 and GSHS2,3 at 10 min that declined thereafter. A sm was observed at 2 min and GSOH was present at 2 and 10 Cys or GSH (RSnR; R = Cys, GSH; n ≥ 2) were observed. Values are expressed as relative area u were added to 1,4-NQ followed by H2S. TME-IAM was added a tions were stopped with 1% formic acid 30 min after the last (60 spectrometry. A second aliquot of the 2 min sample was run afte to evaluate the stability of the TME-IAM-sulfur adducts. The TME-IAM inorganic sulfur adducts appeared to under the curve (AUC) for a second t = 2 min sample tha the other 2-, 10-, 30-and 60-min samples ('✖' symbol in erably lower (50% or more in some samples) compared to in figures) to evaluate the stability of the TME-IAM-sulfur adducts.
The TME-IAM inorganic sulfur adducts appeared to deteriorate with time as the area under the curve (AUC) for a second t = 2 min sample that was saved and analyzed after the other 2-, 10-, 30-and 60-min samples (' of products produced by 1,4-NQ oxidation of H2S in the presence of Cys and GSH are shown in Figure 1. Incubation of H2S and 1,4-NQ with Cys produced both inorganic (H2Sn, n = 2-5) and organic (CysSnH, n = 2-4) hydroper-and hydropolysufides; the extent of polysulfide production progressively decreased as the number of sulfur atoms increased (Figure 1A). Most hydroper-and hydropolysulfides were produced in the first 10 min and declined thereafter, except for H2S2, which remained nearly constant, and CysS2H, which continued to increase. Incubation with GSH ( Figure 1B) produced an initial increase in H2S2, 3 and GSHS2,3 at 10 min that declined thereafter. A small amount of GSHS4 and GS2OH was observed at 2 min and GSOH was present at 2 and 10 min. No per-or polysulfides of Cys or GSH (RSnR; R = Cys, GSH; n ≥ 2) were observed. . GSH or Cys were added to 1,4-NQ followed by H2S. TME-IAM was added at 2, 10, 30, and 60 min and the reactions were stopped with 1% formic acid 30 min after the last (60 min) sample and subjected to mass spectrometry. A second aliquot of the 2 min sample was run after the 60 min sample (✖ in figures) to evaluate the stability of the TME-IAM-sulfur adducts.
The TME-IAM inorganic sulfur adducts appeared to deteriorate with time as the area under the curve (AUC) for a second t = 2 min sample that was saved and analyzed after the other 2-, 10-, 30-and 60-min samples ('✖' symbol in Figure 1A,B) was often considerably lower (50% or more in some samples) compared to the first 2 min sample. The TME-' symbol in Figure 1A,B) was often considerably lower (50% or more in some samples) compared to the first 2 min sample. The TME-IAM adducts of Cys compounds appeared fairly stable, while GSH adducts were at least 20% lower. These are points to consider when examining TME-IAM adducts over extended periods. They also suggest that the amount of hydroper-and hydropolysulfides, especially the inorganic compounds, produced in these experiments may be greater than the results indicate.
Superoxide dismutase (SOD) increases the autoxidation of NQs [17] and NQ oxidation of H 2 S [16]. The effects of superoxide dismutase (SOD) and catalase (Cat) on 1,4-NQ-mediated H 2 S oxidation in the presence of GSH were also examined. Neither enzyme, alone or together, appreciably affected H 2 S or GSH, whereas both, alone and in combination, decreased hydroper-and hydropolysulfide production (Supplemental Figure S1). These results contrast with our previous studies [16], where SOD increased polysulfide production from H 2 S and 1,4-NQ (measured by SSP4 fluorescence). This could be due to the limited variety of polysulfides detected with LCMS compared to SSP4, the direct effects of GSH and Cys on H 2 S oxidation by 1,4-NQ, or the fact that SSP4 reacts quickly with polysulfides before sulfoxides are produced.
We previously used the polysulfide-specific fluorophore SSP4 to demonstrate polysulfide production by the NQ oxidation of H 2 S [16]. In preliminary experiments in the present study, it appeared that GSH interfered with this method and, indeed, this was the case. To identify possible interference by GSH and Cys, we examined the effects of these compounds on the reactions between SSP4 and the mixed polysulfide K 2 S n , which was found by LCMS analysis of TME-IAM derivatized K 2 S n to contain 7.4 ± 1.2% H 2 S, 40.8 ± 5.9% H 2 S 2 , 41.9 ± 6.0% H 2 S 3 , 8.1 ± 1.3% H 2 S 4 , and 0.8 ± 0.2% H 2 S 5 (mean ± SE, n = 7).
As shown in Supplemental Figure S2A, when GSH was added concurrently to SSP4 and K 2 S n , SSP4 fluorescence was inhibited, whereas fluorescence of SSP4 and K 2 S n was unaffected when GSH was added 30 min later. Furthermore, both GSH and Cys concentrationdependently decreased fluorescence when they were added concurrently to either SSP4 and K 2 S n or SSP4, K 2 S n , and 1,4-NQ (Supplemental Figure S2B,C). Although 1,4-NQ appeared to decrease fluorescence in Supplemental Figure S2B,C, this did not appear to be the case with K 2 S n , as adding 1,4-NQ immediately or 10, 30, or 60 min after adding K 2 S n to SSP4 did not affect fluorescence (Supplemental Figure S2D). The effects of 1 mM propylamine (PA) on the SSP4 detection of 10 µM K 2 S n and 1 mM GSH were also examined (Supplemental Figure S2E). PA essentially halved SSP4 fluorescence, whereas GSH completely inhibited fluorescence unless it was added 2 h after PA and K 2 S n were mixed with SSP4. These experiments suggest that GSH and Cys directly and rapidly react with hydropolysulfides produced by the 1,4-NQ oxidation of H 2 S, whereas propylamine only partly affects the SSP4-K 2 S n interaction. They also suggest that 1,4-NQ does not catalyze further reactions with K 2 S n .

Effects of GSH and Cys on Thiosulfate Production by H 2 S and 1,4-NQ
Thiosulfate is also a product of H 2 S oxidation by quinones and naphthoquinones [16,18,19]. To determine if Cys or GSH affected thiosulfate (TS) production, H 2 S was incubated with 1,4-NQ in the presence of these thiols, and thiosulfate production was measured with Ag nanoparticles (Supplemental Figure S3A). No TS production was detected when GSH was added and only minimal production in the presence of Cys. SOD did not increase TS production in either GSH or Cys supplemented conditions. Additional experiments were then conducted to determine if the apparent inhibitory effects of GSH and Cys were due to these thiols reacting with some intermediate that then decreased the opportunity for thiosulfate production or if they directly interfered with the thiosulfate assay. As shown in Supplemental Figure S3B, when the compounds were sequentially added in the order AgNP, thiosulfate, GSH or Cys, relatively little thiosulfate was detected with GSH, whereas Cys did not appear to substantially affect thiosulfate detection. However, little thiosulfate was detected with either GSH or Cys when the AgNPs were added last (Supplemental Figure S3C). The inhibitory effects of GSH were also evident when different concentrations of GSH were added simultaneously to thiosulfate and AgNP, whereas they were less apparent when GSH was added ten minutes later (Supplemental Figure S3D). These studies show that both GSH and Cys interfere with the AgNP-thiosulfate assay, most likely by reacting directly with the AgNP.
The HPLC-MBB assay was then used to further examine the effects of GSH and Cys on thiosulfate production. As shown in Supplemental Figure S3E, both GSH and Cys decreased thiosulfate production by approximately 30%. This is far less of an effect than that observed with the AgNPs, although it confirms that much of the apparent inhibition seen with the thiols and AgNPs is not a direct effect on thiosulfate production, nevertheless, it does appear that both GSH and Cys have an inhibitory effect.

Effects of GSH and Cys on Absorbance Spectra of H 2 S Reactions with 1,4-NQ
We [16] have previously shown that oxidized 1,4-NQ has absorbance peaks at 203 nm, 246 nm, and 252 nm, a shoulder at 261 nm, and a broad peak at~342 nm. Reduction of 1,4-NQ to 1,4-NQH 2 with nitrogen (N 2 ) produced a new peak at 240 nm, decreased the 252 peak, and blue-shifted the 342 nm peak to 333 nm ( Figure 2A). Na 2 S has a peak at 228 nm and a peak at 203 nm which is a nonvolatile impurity ( Figure 2B). Reduction of 1,4-NQ with H 2 S ( Figure 2C) produced similar effects as reduction with N 2 , although 246 nm and 252 nm peaks were smaller, and the 342 nm peak was blue-shifted to~305 nm. Mixed polysulfides, produced by the incubation of elemental sulfur (S 8 ) with Na 2 S, have shoulders at approximately 295 nm and 376 nm ( Figure 2B). GSH has a single stable peak at 203 nm ( Figure 2D), and Cys has a sharp peak at 203 nm that increased with time and a shoulder at~230 nm that initially increased then decreased over the 1576 s observation period ( Figure 2E).  over the 1576 s sampling period. Although GSH decreased the 246 nm and 252 nm peaks, characteristic of oxidized 1,4-NQ, the lack of a peak at 240 nm (characteristic of reduced 1,4-NQH 2 ) suggests that the 1,4-NQH 2 is rapidly reoxidized to give a spectrum of the oxidized NQ-GS adduct. Addition of H 2 S to 1,4-NQ with GSH ( Figure 2H,I) produced a faster decrease in the 246 nm and 252 nm peaks, a faster increase in the 307 nm peak, and a pronounced nadir at 240 nm that was not apparent with 1,4-NQ and GSH alone. The peaks at 307 nm and 417 nm observed after the addition of GSH to 1,4-NQ were not affected by addition of H 2 S.
The addition of Cys to 1,4-NQ ( Figure 2J,K) produced a transient shift in the 203 nm peak to 209 and then back to 203 nm. The 246 nm and 252 nm peaks decreased within the first 15-20 s, a shoulder appeared around 260 nm, and new peaks appeared at 304 nm and 447 nm. Over time, the 246 nm and 252 nm peaks decreased even further, the 260 nm peak became pronounced, and the 304 nm and 447 nm peaks increased. In addition to the spectrum observed with 1,4-NQ and Cys alone, adding H 2 S to 1,4-NQ and Cys produced a new peak at 226 nm and a nadir at 242 nm, and the loss of the 246 nm and 252 nm peaks occurred within the first few seconds after adding H 2 S ( Figure 2L,M).
In general, the effects of GSH and Cys on the 1,4-NQ spectrum were similar. Both produced new peaks at 256 nm, 307 nm, and 417 nm (GSH) and 260 nm, 304 nm, and 447 nm (Cys), although Cys appeared to be more effective than GSH in reducing 1,4-NQ based on faster and more pronounced spectral shifts with Cys than with GSH. H 2 S also appeared to produce similar effects when added to GSH/1,4-NQ and Cys/1,4-NQ, especially at 226/7 nm and 242 nm. The pronounced peaks produced by GSH and Cys at 417 nm and 457 nm, respectively, which were not seen with either oxidized 1,4-NQ or reduced 1,4-NQH 2 , are suggestive of one or more new molecules, most likely sulfur adducts and their semiquinones. This was confirmed with EPR and LCMS analysis (cf. Sections 2.5.1 and 2.5.2).

H 2 S Reaction with Specific Naphthoquinones Consumes O 2 and Is Variously Affected by GSH and Cys
We have previously shown that para-quinones and NQs catalytically oxidize H 2 S to polysulfides and consume oxygen in the process [16,18,19]. The measurement of oxygen consumption also provides an alternative approach to fluorophores for examining the effects of GSH and Cys on the H 2 S/NQ reactions.
As shown in Figure 3A, O 2 consumption increased when 1,4-NQ was added to H 2 S and this was further increased by the addition of either Cys or GSH, with Cys being more efficacious. This effect was not observed with either cystine or methionine, suggesting that a free SH or S − is required. GSH and Cys also concentration-dependently increased O 2 consumption by 1,4-NQ and H 2 S ( Figure 3B-D). The maximal rate of O 2 consumption by either 10 or 30 µM 1,4-NQ and GSH or with 30 µM 1,4-NQ and Cys appeared to occur with GSH/Cys concentrations between 30 and 100 µM. The effects of GSH and Cys on O 2 consumption by H 2 S and juglone were essentially similar to 1,4-NQ ( Figure 3E).
The effects of GSH and Cys on O 2 consumption by H 2 S and other NQs were more variable ( Figure 3F-J). O 2 consumption by lawsone and H 2 S was minimal and only slightly affected by either GSH or Cys ( Figure 3F). GSH decreased O 2 consumption by 2-MNQ ( Figure 3G), increased it with menadione ( Figure 3H), and, after a delay, increased it with plumbagin ( Figure 3I,J). Cys greatly increased O 2 consumption by H 2 S and 2-MNQ together and had no effect with menadione or plumbagin under the same conditions. Collectively, these results indicate that substitutions on the parent 1,4-NQ profoundly and specifically affect the effects of GSH and Cys on O 2 consumption. Given the fact that the concentration of these thiols was at least 30-fold greater than the NQs, it is likely that the effects are mediated through S-adducts on the NQ rather than thiol sequestration or other reactions with polysulfides.

Formation of 1,4-NQ Thiol S-Adducts and Their Effects on H 2 S Oxidation
Naphthoquinones with an open position on the 2 or 3 carbon, adjacent to the carbonyls, may react with thiol or amine nucleophiles and form adducts via a 1,4 reductive Michael addition reaction [14,17]. Importantly, S-adducts with low-molecular-weight (LMW) thiols, such as GSH and Cys, as well as with protein thiolates, have been suggested to affect intracellular antioxidant mechanisms [14,15], but the expected low intracellular concentration of exogenously administered NQs relative to GSH argue against a direct effect. S-adducts may also directly affect protein function, e.g., Keap-1 [6]. The effects of these adducts on H 2 S oxidation are not known and given the relatively high intracellular concentrations of GSH and protein thiolates, it is important to determine if these adducts affect H 2 S oxidation and thereby offer dual functionality. These questions are examined in the following sections.

EPR Examination of GSH, Cys and H 2 S Reactions with 1,4-NQ
EPR spectra of 1,4-NQ semiquinone and reactions between oxidized 1,4-NQ and increasing concentrations of GSH, Cys, propylamine, and H 2 S are shown in Figure 4A-E. There is no noticeable spectrum of oxidized 1,4-NQ dissolved in either 21% oxygen or 0% oxygen, but if reduced to the hydroquinone (1,4-NQH 2 ) with NaBH 4 (0.15:1. NaBH 4 :1,4-NQ), a 1,4-NQ semiquinone becomes evident ( Figure 4A). This likely is the result of a comproportionation reaction between the newly reduced 1,4-NQH 2 and the remaining oxidized 1,4-NQ. Addition of GSH to 1,4-NQ produced a somewhat similar semiquinone spectrum at a low GSH:NQ ratio; however, this disappeared as the GSH:NQ ratio approached 0.5:1 ( Figure 4B). Similar EPR spectra were observed for the reactions between Cys and 1,4-NQ, again with the loss of signal as the Cys:NQ ratio approached 0.5:1 ( Figure 4C). Although these reactions were carried out in buffer equilibrated with room air, it is likely that the solution quickly became hypoxic as even 600 µM thiol (0.15:1 thiol:1,4-NQ ratio) reacting with the NQ will quickly consume all the oxygen (~200 µM, see Section 2.5.4 below). The EPR spectrum of 0.25:1 propylamine was identical to that of reduced 1,4-NQ. The spectrum was first observed at a propylamine:1,4-NQ ratio of 0.5:1 and persisted to the highest ratio (10:1) examined ( Figure 4D).
An EPR spectrum first appeared at an H 2 S:1,4-NQ ratio of 0.15:1 ( Figure 4E), and it was identical to that produced by the 1,4-NQ semiquinone ( Figure 4A), including the hyperfine splitting from the two equivalent protons 2 and 3 and the four equivalent protons 5, 6, 7, and 8. This spectrum quickly changed to a different pattern at a H 2 S:1,4-NQ ratio of 0.25:1, with a quintet overlapped with other unresolved EPR features. The single quintet suggests a radical species with hyperfine splitting from only four protons. This radical species was likely due to the product from the Michael addition reaction of HS − to 1,4-NQ, substituting the protons on carbons 2 and 3 with SH groups. The oxidized 1,4-NQ and reduced 1,4-NQ-2SH adduct (2,3-SH-1,4NQ) then comproportionated to generate the semiquinone radicals of 1,4-NQ and 2,3-SH-1,4-NQ. At higher H 2 S:1,4-NQ ratios (0.25:1 and above), the yield of radical species increased significantly, showing more hyperfine splittings that are distinct from that of the 1,4-NQ semiquinone radical ( Figure 4E). The complicated EPR signatures are likely due to multiple radical species, probably resulting from the further substitution of protons at carbons 5, 6, 7 or 8 by excess HS − . Repeating the above experiment with 1,4-NQ in buffer that was not sparged with N 2 produced essentially similar results, albeit with an improved signal suggesting the contribution of autoxidation. Collectively, these results indicate the H 2 S produces semiquinone radicals unique to this thiol.
The EPR spectrum with 1,4-NQ and Cys (Supplemental Figure S4B) was identical to that of the 1,4-NQ semiquinone. Interestingly, the semiquinone signal could be turned off or on depending on the Cys:1,4-NQ ratio. The spectrum was silent with a 1:1 Cys:1,4-NQ ratio (Supplemental Figure S4B(a)); the spectrum appeared if the reaction solution was mixed with 1,4-NQ, dropping the Cys:1,4-NQ ratio to 0.3:1 (Supplemental Figure S4B(b)). On the other hand, if the Cys:1,4-NQ ratio was decreased only to 0.6:1 by mixing 1:1 Cys:1,4-NQ reaction solution with less 1,4-NQ, only small EPR signals were observed (Supplemental Figure S4B(c)). When this solution was further diluted with 1,4-NQ to a 0.3:1 Cys:1,4-NQ ratio, the strong signal of the 1,4-NQ semiquinone reappeared (Supplemental Figure  S4B(d)). This supports observations that the 1,4-NQ-Cys semiquinone is sensitive to the 1,4-NQ:Cys ratio and that a 0.5:1 ratio is the transition point. These results are suggestive of a reversible comproportionation reaction being the principal pathway for the formation of the semiquinones. This also implies that the comproportionation reaction is slower than the reduction of NQ by the thiols.

EPR Examination of GSH, Cys and H 2 S Reactions with Other NQs
The EPR spectra of reactions of juglone, plumbagin, and 2-MNQ with GSH, Cys, and H 2 S are shown in Supplemental Figures S5-S7, respectively.
Reduction of juglone with NaBH 4 (0.25:1 NaBH 4 :juglone ratio) produced a spectrum of a juglone semiquinone radical (Supplemental Figure S5A). Similar EPR signatures were observed in reactions of juglone with low levels of GSH or Cys (Supplemental Figure S5A-C). The EPR intensity decreased in the reactions with increasing GSH and disappeared when the GSH:juglone ratio reached 1:1 when the experiment was performed in room-air equilibrated buffer, whereas it disappeared when the ratio reached 0.5:1 when the experiment was performed in anoxic conditions. The EPR signatures were also significantly different from that of the juglone semiquinone radical, likely due to multiple radical species. In the reaction with Cys, the EPR signal disappeared when the Cys:juglone ratio was~0.25:1 and above. Addition of H 2 S to juglone produced an EPR spectrum that was essentially identical to that of the juglone semiquinone radical at a H 2 S:juglone ratio of~0.15:1; at higher H 2 S:juglone ratios this EPR lineshape changed significantly (Supplemental Figure S5D). The strongest signal was observed at a 2:1 H 2 S:juglone ratio and decreased thereafter.
The EPR spectrum of plumbagin reduced with NaBH 4 (0.25:1 NaBH 4 : plumbagin ratio) showed a linewidth significantly broader than those observed for the reduction of juglone with NaBH 4 . The reason is unclear but may be related to the 2-methyl group on plumbagin (Supplemental Figure S6A). The EPR spectra of the reactions of plumbagin with different ratios of GSH in either room air or anoxia were overall similar and broader compared to those observed in the reactions of GSH with juglone (Supplemental Figure S6A,B). Reactions of plumbagin with low ratios of H 2 S (H 2 S:plumbagin~0.15:1-0.25:1) produced spectra that were identical to that of the NaBH 4 -plumbagin spectrum but became more complicated with higher intensities and a narrower linewidth as the ratio of H 2 S:plumbagin was increased, the signal size reaching a maximum at a 1:1 H 2 S:plumbagin ratio and declining thereafter.
Reduction of 2-MNQ with NaBH 4 produced a distinct spectrum; however, unlike either 1,4-NQ, plumbagin, or juglone, no EPR signal was observed in the reaction of 2-MNQ with either GSH or Cys (Supplemental Figure S7A,B). Conversely, the addition of H 2 S to 2-MNQ produced a spectrum that was essentially identical to that produced by NaBH 4 , and the intensity appeared to increase as the H 2 S:2-MNQ concentration ratio increased from 0.05:1 to 5.0:1 (Supplemental Figure S7C).
Collectively, these results suggest that at low concentrations H 2 S reduced juglone to the NQH 2 , but as the H 2 S concentration was increased a different, or several different NQ radicals, possibly due to SH-substituted quinones, were formed. Low concentrations of H 2 S also reduced plumbagin to the NQH 2 , while at high concentrations H 2 S generated plumbagin semiquinone radicals as well as other NQ radicals, which were likely due to SH-substituted plumbagin quinones. However, it should be noted that all the g values were typical of an oxygen-centered radical, not a sulfur-centered radical.

Oxygen Consumption during Formation of GSH and Cys Adducts
We next examined oxygen consumption during reactions between 1,4-NQ and GSH or Cys to determine if the 1,4-NQ semiquinone, detected by EPR (Figure 4), was the result of O 2 -dependent autoxidation, comproportionation, or both. Oxygen was rapidly consumed when either 200 µM GSH or 280 µM Cys was added to 4 mM 1,4-NQ ( Figure 5A). This ratio, 0.05:1 for GSH:1,4-NQ and 0.07:1 for Cys:1,4-NQ, is the same thiol:NQ ratio that produced a strong EPR signal (Figure 4). Progressively higher thiol:1,4-NQ ratios consistently and rapidly consumed oxygen, which suggests that while oxygen may be involved in the initial reaction(s) between the thiols and 1,4-NQ, subsequent reactions occurred in anoxia or extreme hypoxia. It is also likely that oxygen becomes progressively less relevant in the EPR spectrum as the thiol:NQ ratio increases. The rapid decline in oxygen tension that was consistently observed also suggests that adduct formation is a rapid reaction followed by rapid autoxidation of either the reduced naphthoquinone-S adduct (NQH 2 -S) or a reduced NQ that was produced by a reaction between NQH 2 -S and oxidized NQ.   To further examine oxygen consumption by thiol-NQ reactions, we used lower concentrations of thiols and 1,4-NQ that we predicted would not consume all of the oxygen. As shown in Figure 5B-E, incremental additions of thiols to 100 µM 1,4-NQ or incremental additions of 1,4-NQ to 100 µM GSH or Cys produced essentially identical concentrationdependent increases in oxygen consumption, albeit slightly more oxygen was consumed with Cys-NQ than with GSH-NQ. Furthermore, the amount of oxygen consumed was essentially equivalent to the amount of thiol or 1,4-NQ that was sequentially added. This suggests that the amount of oxygen consumed in these reactions is stoichiometrically related to and limited by the net amount of 1,4-NQ-thiol produced and not a catalytic process.
Somewhat unexpectedly, the amount of oxygen consumed by consecutive additions of 25 µM thiol to 100 µM 1,4-NQ progressively decreased such that there was little oxygen consumed after the second addition of GSH or the third addition of Cys ( Figure 5F-I). However, the cumulative amount of oxygen consumed by serial additions of 25 µM GSH was similar to that produced by a single 125 µM GSH bolus when corrected for the total elapsed time. This suggests that reactions with these low concentrations of compounds are somewhat offset by continuous oxygen diffusion into the reaction chamber, which is evident from the increase in oxygen tension within 10-15 min after the compounds are mixed. Regardless, it is evident that addition of either GSH or Cys reduces 1,4-NQ and is rapidly reoxidized by molecular oxygen.
The observation that the formation of 1,4-NQ-thiol adducts consumed oxygen prompted further investigation into other NQ-thiol adducts (Supplemental Figure S8). Oxygen consumption by the addition of 300 µM GSH to 30 µM NQs progressively decreased in the order 1,4-NQ~juglone > plumbagin > menadione > 2-MNQ with essentially no oxygen consumed when GSH was added to 2-MNQ. Superoxide dismutase did not appreciably affect oxygen consumption in any of these reactions. Oxygen consumption by the addition of Cys under similar conditions was greater than that for the reaction of GSH with the respective NQ and decreased from 1,4-NQ to 2-MNQ, again with little oxygen being consumed by the latter. Superoxide dismutase decreased oxygen consumption in the reaction of 1,4-NQ with Cys, but it did not appreciably affect oxygen consumption in any of the other reactions. These results show that (1) the effects of the formation of thiol adducts on oxygen consumption decrease with an increase in the complexity of the side groups on the quinoid ring, (2) more oxygen is consumed, and at a faster rate, in the formation of Cys adducts compared to GSH adducts as there is likely less steric hindrance, and (3) with the exception of the reaction of 1,4-NQ with Cys, SOD has little effect on the formation of these adducts.

Oxygen Consumption during Formation of NQ-Propylamine Adducts
N-propylamine (PA) reportedly reacts in 5 min with 1,4-NQ in water at room temperature and physiological pH to form 1,4-NQ-PA adducts with 95% yield [21]. Several experiments were performed to determine if PA reactions with 1,4-NQ also consumed oxygen and if NQ-PA adducts affected H 2 S oxidation. As shown in Supplemental Figure S9A, adding 1 mM 1,4-NQ to 1 mM PA did not affect oxygen consumption, whereas oxygen consumption progressively increased when 2.5 mM and 5 mM PA were added to 1 mM 1,4-NQ. Adding 150 µM 1,4-NQ to 5 mM PA also increased oxygen consumption suggesting that both the 1,4-NQ and PA concentrations are important. This was confirmed by consecutive additions of 1,4-NQ to 5 mM PA that produced a concentration-dependent increase in oxygen consumption (Supplemental Figure S9B). These results also show that oxygen is consumed when a 1,4-NQ-PA adduct is formed. However, compared to other reductants, i.e., GSH or Cys ( Figure 5), or dithiothreitol or ascorbic acid [16], higher PA concentrations are needed to react with 1,4-NQ and consume oxygen, even 1 mM 1,4-NQ and 5 mM PA only consumed approximately 160 µM of oxygen, which is far less than that consumed by 1,4-NQ with either GSH or Cys ( Figure 5).
The effects of propylamine on H 2 S oxidation by 1,4-NQ are shown in Supplemental Figure S9E,F. Propylamine did not affect oxygen consumption when added to H 2 S, 1,4-NQ plus H 2 S, or H 2 S with 1,4-NQ and GSH. These results show that once a 1,4-NQ-PA adduct is formed, it is relatively impervious to subsequent attack by GSH. However, PA did not affect the GSH-stimulated increase in H 2 S oxidation. This suggests that either the PA was displaced by GSH or that GSH increased oxygen consumption by removing inorganic persulfide products from the 1,4-NQ-catalyzed oxidation of H 2 S, and/or by forming GSH sulfoxides. It also suggests that if PA tightly occupies both the 2 and 3 carbons on 1,4-NQ, oxidation of H 2 S occurs at the carbonyl groups.

H 2 S Oxidation and Polysulfide Production by NQ-Thiol, NQ-Propylamine and NQ-H 2 S Adducts
It was clear from our preliminary studies that elevated concentrations of GSH and Cys inhibited the SSP4 detection of polysulfides (Supplemental Figure S2). To overcome this, we incubated high concentrations (1 mM) of NQs with similar concentrations of GSH, Cys, PA, and H 2 S for one hour to allow the adducts to form. The adducts were then diluted to a final NQ concentration of 10 µM or 30 µM. By incubating equimolar concentrations of NQ with thiols or PA, we assumed that much of the free thiol and PA would be consumed in forming the adducts and the concentration of the remaining unreacted compounds would be further reduced by the 1/100 or 1/30 dilution.
The effects of NQ-sulfur and NQ-amine adducts on H 2 S oxidation as measured by polysulfide production are shown in Figure 6 and Supplemental Figures S10-S13. Polysulfide production (SSP4 fluorescence) by 1,4-NQ-GSH and 1,4-NQ-Cys adducts was significantly greater, nearly twice as much in most instances, than that produced by 1,4-NQ alone. Overall, more polysulfides were produced by the GSH adducts. 1,4-NQ-PA or 1,4-NQ-H 2 S adducts only marginally and inconsistently affected polysulfide production ( Figure 6).   Polysulfide production was also doubled by juglone-GSH adducts; however, it was not affected by juglone-Cys or juglone-PA adducts, with the exception of a potent inhibitory effect when 30 µM juglone-PA adduct was incubated with 300 µM H 2 S (Supplemental Figure S10).
Menadione-GSH adducts produced nearly ten-fold increases in polysulfide production; Cys adducts doubled polysulfide production at 10 µM menadione-Cys but did not affect it at 30 µM (Supplemental Figure S12). Menadione-H 2 S adducts increased polysulfide production by two-to three-fold, whereas PA did not have any noticeable effect.
2-MNQ adducts were noticeably different, the GSH adducts did not affect polysulfide production, whereas polysulfide production was tripled by the Cys adducts (Supplemental Figure S13). Propylamine adducts did not affect polysulfide production and only the 30 µM 2-MNQ-H 2 S increased polysulfides.
Since we were not sure that we had completely removed GSH or Cys from the above reactions, we then incubated 10 µM NQs with 300 µM H 2 S and then added 10 µM of either GSH or Cys to determine if these low residual concentrations of thiol affected the SSP4 detection of polysulfides. As shown in Supplemental Figure S14A, the effects of 10 µM GSH on polysulfide production were relatively small compared to the effects observed when the NQs were pre-incubated with GSH ( Figure 6 and Supplemental Figures S10-S13). Adding 10 µM Cys directly to 1,4-NQ (Supplemental Figure S14B) had a more pronounced effect on polysulfide production than adding 10 µM GSH to 1,4-NQ (Supplemental Figure S14A), but other than that, adding 10 µM Cys directly to other NQs had only a minimal effect on polysulfide production. It is also clear from these figures that the onset of polysulfide production was the most rapid for juglone followed by 1,4-NQ and then plumbagin and even later for menadione and 2-MNQ. These observations correlate with our previous observations [16] on the propensity for juglone and 1,4-NQ to rapidly reoxidize after an initial comproportionation reaction, while the others with more substitutions on the quinoid ring depend more on an initial oxidation by oxygen, which is a slower and less favorable process. The slow but sustained H 2 S oxidation by menadione and 2-MNQ was confirmed by following this reaction for 4 h (Supplemental Figure S14C,D).

Effects of Thiol:NQ Ratio on Polysulfide Production from H 2 S
As NQ-thiol adducts clearly affect H 2 S oxidation, we examined the effects of the ratio of thiol to NQ on polysulfide production. One mM of NQ was incubated with various concentrations of thiols for 1 h in an open container to autoxidize the NQ-thiol adduct, then it was diluted to 10 µM NQ and incubated with 300 µM H 2 S and SSP4 in a tape-covered well-plate for 90 min. As shown in Figure 7A, GSH concentration-dependently increased polysulfide production for all NQs except 2-MNQ. Plumbagin-GSH adducts appeared to be the most efficacious; 0.063 mM GSH per mM PB doubled polysulfide production and 1 mM GSH:1 mM PB produced more polysulfides than any other NQ-thiol combination. The general order of sensitivity was PB > Mdn > 1,4-NQ~Jug. Polysulfide production by 2-MNQ was slightly increased when incubated with 0.25 mM GSH but decreased at progressively higher GSH concentrations; however, these reactions were only monitored for 90 min and the effects of menadione and 2-MNQ could have been underestimated. Except for 2-MNQ (and to a lesser extent, menadione), the effects of Cys were less concentration dependent ( Figure 7B). Polysulfide production was increased at a 0.063:1 mM Cys:NQ for both 2-MNQ-and Mdn-Cys adducts and these effects at 90 min were also likely to be underestimated.
Addition of BSA to 1,4-NQ decreased absorbance of the 252 nm peak, increased absorbance of the 260 nm and 265 nm peaks, and produced a broad peak at~407 nm. The decrease in absorbance at 252 nm and increases at 260 nm and 407 nm were consistent with the reduction of 1,4-NQ by Cys ( Figure 2F,G) and suggest that the 1,4-NQ-BSA adduct is on the reactive β93 Cys of BSA. Addition of BSA to 1,4-NQ produced an EPR spectrum that was identical to reduction of 1,4-NQ with NaBH 4 and reaction with Cys at low Cys:1,4-NQ concentration ratio ( Figure 4A,C). An identical spectrum was observed when 1,4-NQ was reacted with hemoglobin. These experiments suggest that 1,4-NQ-protein thiol adducts retain their ability to redox cycle.
We initially tried to determine if the 1,4-NQ bovine serum albumin (BSA) adduct (1,4-NQ-BSA) could oxidize H 2 S to polysulfides using SSP4; however, BSA concentrationdependently inhibited the SSP4 reaction with polysulfides (Supplemental Figure S15E). Parenthetically, it is also noteworthy that the SSP4/polysulfide (K 2 S n ) reaction is approximately 75% inhibited over the range of plasma BSA (55-75 µM), which may call into question the use of SSP4 to measure polysulfides in the presence of proteins. We then examined the effects of the 1,4-NQ-BSA adduct on H 2 S consumption as BSA did not appear to substantially interfere with the H 2 S/AzMC reaction (Supplemental Figure S15F). As shown in Supplemental Figure S15F, 1,4-NQ consumed approximately 40% of the H 2 S in 30 min and the 1,4-NQ-BSA adduct consumed 30% of the H 2 S. These results show that the 1,4-NQ-BSA adduct retains the ability to oxidize H 2 S, albeit with a slight reduction in efficiency. Interestingly, and for reasons unknown, BSA also appeared to slightly increase fluorescence from the AzMC/H 2 S reaction (Supplemental Figure S15F,G), but neither BSA alone nor BSA added to 1,4-NQ affected oxygen consumption (Supplemental Figure S15H).
Collectively, these results show that NQ-thiol adducts can affect the catalytic efficiency of NQs to oxidize H 2 S. This appears to be specific for different thiols, for the type and location of substitutions on the NQ benzene and quinoid rings, and for the relative abundance of GSH, Cys, and reactive protein Cys.

Discussion
Naphthoquinones are well-known for their ability to participate in redox cycling and to function as electrophiles that form adducts with thiols and amines. We have previously shown that 1,4-naphthoquinone (1,4-NQ) and its derivatives (NQs) catalytically oxidize H 2 S to inorganic hydropolysulfides, thiosulfate, and related sulfoxides [16]. Here we show that these inorganic products of NQ-catalyzed H 2 S oxidation may then react with other small thiols to form biologically relevant organic hydropolysulfides and sulfoxides. We also show that NQ-thiol adducts specifically affect the catalytic efficacy of NQs and that these reactions depend on both the specific structure of the NQ and the nature of the thiol involved.

Production of Thiol Hydropersulfides, Hydropolysulfides and Sulfoxides
Inorganic hydroper-and hydropolysulfides could be initially produced by several mechanisms: (1) two one-electron oxidation reactions with the carbonyl oxygen that form two hydrosulfide radical intermediates that then combine to produce hydropersulfide (H 2 S 2 ), (2) a single two-electron reaction that forms elemental sulfur S 0 which then combines with H 2 S to form H 2 S 2 and (3) or by consecutive Michael addition reactions of two H 2 S on a quinoid carbon that releases H 2 S 2 . These possible mechanisms are under current investigation. Regardless, the present studies show that organic hydroper-and hydropolysulfides can be derived from sulfur exchange between the organic thiol and H 2 S 2 (Equation (1)) or direct addition of elemental sulfur to the organic thiol (Equation (2)). Note: while it is recognized that the pKas become progressively lower with increasing sulfur atoms, for simplicity, the sulfur reactants and products are shown in the fully protonated form.
H 2 S 2 + GSH/Cys -> GSH/CysS 2 H + H 2 S (1) Continuation of these reactions can produce organic hydropolysulfides with as many as four sulfur atoms, as shown in Figure 1. We did not observe any GSH-S n -GSH or Cys-S n -Cys (n = 2-6) polysulfides with LCMS, most likely because these will react with excess H 2 S to give the thiol and the hydroper-and hydropolysulfides, where R=GSH or Cys (Equation (3)), A small amount of GSHS 4 and GS 2 OH was also observed at 2 min and GSOH was present at 2 and 10 min. These oxygenated products appear to be due to reactions shown in Equations (4)-(7), as described by Winterbourn [23].
and this becomes part of a chain reaction, followed by, to regenerate superoxide and continue the chain. SOD would be expected to inhibit these reactions, which is what we observed (Supplemental Figure S1).

1,4-NQ-GSH/Cys Adducts
Reduction of 1,4-NQ with NaBH 4 results in a semiquinone EPR spectrum that could be the result of comproportionation between the residual oxidized 1,4-NQ and the newly formed, reduced 1,4-NQH 2 , or a one-electron oxidation of the reduced 1,4-NQH 2 with oxygen as the electron acceptor. The fact that these semiquinones could be formed in the absence, or near absence of oxygen (Supplemental Figures S5 and S6) indicates that comproportionation is probably a major factor in the formation of the semiquinones. This finding, coupled with the fact that SOD does not have much effect on the rate or extent of the oxygen consumption of NQ with GSH, one can infer that at large molar excess of NQ over GSH (4:1), oxygen consumption (reaction of the semiquinone with oxygen to make superoxide), and, therefore, superoxide formation is slow and that GSH reduction of NQ is fast. Thus, there is little GSH to react with superoxide and make a chain reaction. In either case, the fully oxidized NQ will be produced by a second oxygen-dependent reaction.
Similarities in EPR spectra of 1,4-NQ reactions with low concentrations of either GSH or Cys (Figure 4) suggest that both GSH and Cys also reduce 1,4-NQ while forming the thiol adduct (Equation (8) Although the slightly altered spectrum may reflect a small population of the NQ-thiol semiquinone, it appears that most of the semiquinone is not one of a thiol adduct. This suggests that the reduced quinone-thiol adduct readily reduces the unreacted, oxidized quinone (Equation (9)): 1,4-NQ + 1,4-NQH 2 -GS(Cys) -> 1,4-NQ-GS(Cys) + 1,4-NQH 2 (9) There is an interesting loss of EPR signal when the thiol:1,4-NQ ratio is 0.5:1. We propose that at this ratio, half of the initial 1,4-NQ will be reduced to the 1,4-NQH 2 -GS/Cys adduct, and half will remain as the oxidized 1,4-NQ. Since all the oxygen will have been consumed (the oxygen concentration in buffer is <265 µM, only 10% of the thiol concentration), comproportionation will have to account for most of the semiquinone formation. If 1,4-NQ and 1,4-NQH 2 -GS(Cys) adducts cannot comproportionate, or do so very slowly, then there will be no semiquinone formation and no EPR signal. This is consistent with what we observed. It is also possible that the reduced 1,4-NQH 2 -GS(Cys) completely reduces the oxidized 1,4-NQ producing equal amounts of 1,4-NQH 2 and 1,4-NQ-GS(Cys), again hindering comproportionation. However, this scenario is less likely as this reaction appears to require enzymatic catalysis [24].
We also found that with considerably lower concentrations of 1,4-NQ (10-30 µM), some, but not all, of the oxygen was readily consumed upon addition of either GSH or Cys (Figures 3 and 5). Furthermore, the amount of oxygen consumed was essentially equivalent to the amount of 1,4-NQ-thiol adduct expected to be produced, regardless of whether 1,4-NQ or thiol was in excess ( Figure 5). This suggests that this is a stoichiometric reaction and not a catalytic process. We also observed that the formation of the 1,4-NQH 2 -Cys adduct consistently consumed slightly more oxygen than formation of the 1,4-NQH 2 -GSH adduct. We interpret this as a two-step process where a small fraction of the 1,4-NQH 2 -Cys is autoxidized to 1,4-NQ-Cys but then reacts with a second Cys to again reduce the adduct, this time to 1,4-NQH 2 -2Cys which is also autoxidized. This is consistent with our LCMS observations of only one GSH forming an adduct with 1,4-NQ, whereas both one and to a lesser extent, two Cys adducts were observed ( Figure 4F,G).

1,4-NQ-Propylamine Adducts
Our oxygen consumption experiments suggested that 1,4-NQ-propylamine adducts were slower to form compared to GSH or Cys and required elevated ratios of propylamine to 1,4-NQ. This was confirmed by our EPR studies that showed that while the 1,4-NQpropylamine semiquinone spectrum was identical to the 1,4-NQ semiquinone, it did not appear until the propylamine:1,4-NQ ratio was 0.5:1. Furthermore, it did not become appreciably reduced until the ratio was 5:1 and was still evident at a 10:1 ratio ( Figure 4D). These results are similar to what we observed with 1,4-NQ and either GSH or Cys, albeit at a considerably higher propylamine:1,4-NQ concentration ratio, and we attribute the difference to the amine being less reactive than the thiols. Grant et al. [25] observed the EPR signals of 1,4-NQ adducts with GSH, N-acetylcysteine and glycine. They also needed a considerably higher ratio of glycine to 1,4-NQ than N-acetylcysteine to 1,4-NQ (20:1 versus 0.25:1, respectively) to achieve comparable results and they also concluded that thiols were considerably more reactive than amines.
Our studies also suggested that once formed, the 1,4-NQ-propylamine adducts are relatively impervious to attack from either GSH or Cys. We do not know if propylamine can be displaced from 1,4-NQ by H 2 S; however, the observations that, in most cases, propylamine neither increased nor decreased polysulfide production when H 2 S was added to a variety of NQs suggest that thiol adducts (especially GSH adducts), but not propylamine adducts, specifically affect NQ catalyzed H 2 S oxidation.

Adducts of Other NQs with GSH/Cys
The presence of side groups clearly and specifically affect adduct formation. Reduction of either juglone or plumbagin with a low concentration of NaBH 4 produced semiquinones that were considerably more complex than the 1,4-NQ semiquinone (cf. Figure 4 and Supplemental Figures S5 and S6). The radicals formed at the low (0.05:1) GSH or Cys:juglone ratio is dominated by the juglone semiquinone radical, although they also exhibit some features of another radical. If the experiment is performed in anoxia, the juglone semiquinone radical disappears when the GSH:juglone ratio exceeds 0.5:1, whereas in 21% oxygen the previously faint radical becomes the dominant species, possibly a 2GS-juglone species, and it persists, albeit progressively fainter until the ratio exceeds 1:1. This radical was not as obvious with Cys even in 21% oxygen.
The radical observed with plumbagin and GSH does not appear to be the plumbagin semiquinone radical but probably that of a plumbagin-GS semiquinone. Also, GSH appeared to react with greater difficulty with plumbagin than it did with juglone, as suggested by the persistent observation of significant radical formation at a GSH:plumbagin ratio of 1:1. This is reminiscent of the persistence of an EPR signal we observed at high propylamine:1,4-NQ ratios ( Figure 4D). Inbaraj and Chignell [26] incubated HaCaT Keratinocytes with plumbagin or juglone and observed that while plumbagin stoichiometrically converted GSH to GSSG, suggesting a redox cycling pathway, juglone appeared to preferentially form a juglone-GSH adduct.
Although a semiquinone radical is formed when 2-MNQ is reduced with NaBH 4 , no radicals were evident with either GSH or Cys (Supplemental Figure S7). The decrease in propensity for semiquinone radical formation with GSH or Cys as the side chain complexity of the NQ increased is also observed as a decrease in oxygen consumption during adduct formation with more complex NQs (Supplemental Figure S8).

Effects of GSH, Cys and Propylamine Adducts on H 2 S Oxidation
Incubating relatively high concentrations of NQs with equal concentrations of GSH, Cys, propylamine, and H 2 S for an extended period in air-equilibrated conditions before diluting them to low-micromolar concentrations allowed us to evaluate the effects of oxidized NQ adducts on H 2 S oxidation, we assume with relatively little interference from unreacted compounds (Figures 6 and 7 and Supplemental Figures S10-S13). Consistent with other experiments, 10 µM NQs were generally more efficacious than 30 µM NQs and only 10 µM adducts will be considered.
Although propylamine forms adducts with NQs and consumes oxygen in the process (Supplemental Figure S9), it had minimal effect on H 2 S oxidation to polysulfides. Formation of a 1,4-NQ-propylamine adduct was slower than either the 1,4-NQ-GSH or 1,4-NQ-Cys adduct, and either thiol would out-compete propylamine if all were added within 15 min of each other. However, once a propylamine adduct was formed it appeared to prevent GSH from forming an adduct with NQ.

NQ-H 2 S Adducts
H 2 S-NQ reactions are unique in that H 2 S may not only form an adduct with the NQ, but some of the products of H 2 S oxidation may also form adducts. This is supported by our observations that the EPR spectra from H 2 S-NQ reactions were unique to this thiol (cf. Figure 4 and Supplemental Figures S5 and S6). While addition of 600 µM H 2 S to 4 mM 1,4-NQ, juglone, or plumbagin (0.15:1 H 2 S:NQ) produced a spectrum that was essentially identical to the semiquinone produced by reduction of the NQ with NaBH 4 , and similar to those observed with GSH and Cys, the NQ-H 2 S spectrum did not disappear when the H 2 S:NQ ratio increased above 0.5:1, and now it no longer resembled the original semiquinone. These adducts could be due to reactions of NQ with a variety of inorganic per-and polysulfides or even sulfoxides. Ogata et al. [27] demonstrated that thiosulfate forms an adduct with benzoquinone in aqueous solutions. How, or if these other adducts affect H 2 S oxidation remains to be determined.

Autoxidation of NQ Adducts and Their Parent Compounds
Redox cycling is implicit in the NQ-catalyzed oxidation of H 2 S. While autoxidation reactions may affect the rate and nature of sulfur metabolism, they were not the primary goal of this study. Buffington et al. [20] compared autoxidation rate (H 2 O 2 production) of parent NQs and their GSH conjugates after reduction by DT-diaphorase and NADPH and they reported that the glutathionyl conjugates "autoxidize at rates higher than those for the unsubstituted parent compounds". Indeed, they showed that H 2 O 2 production by 3-GS-1,4-NQ was twenty times greater than that produced by 1,4-NQ and 3-GS-menadione produced ten times more H 2 O 2 than menadione. However, they also found that H 2 O 2 production by juglone was ten times greater than that produced by 3-GS-juglone, and plumbagin produced five times as much H 2 O 2 as 3-GS-plumbagin [20]. Moreover, they observed that autoxidation of either juglone or plumbagin consumed fifty times as much H 2 O 2 as autoxidation of 1,4-NQ. Clearly these values are not reconcilable with our studies, and they support our hypotheses that NQ oxidation of H 2 S is a unique process. Furthermore, relevant autoxidation processes and mechanisms need to be considered in future work in this field.

H 2 S and Polysulfide Measurements in Buffer
Fluorophore experiments were performed in 96-well plates and fluorescence was measured with a SpectraMax M5e plate reader (Molecular Devices, Sunnyvale, CA, USA). Compounds were pipetted into 96-well plates and the plates were covered with tape to minimize H 2 S loss due to volatilization. Excitation/emission (Ex/Em) wavelengths were per the manufacturer's recommendations. 7-azido-4-methylcoumarin (AzMC, 365/450 nm) and 3',6'-Di(O-thiosalicyl) fluorescein (SSP4, 482/515 nm) have been shown to have sufficient specificity relative to other sulfur compounds and reactive oxygen and nitrogen species (ROS and RNS, respectively) to effectively identify H 2 S (AzMC) and per-and hydropolysulfides (H 2 S 2 and H 2 S n where n = 3-7 or RS n H where n > 1 or RS n R = where n > 2) [28][29][30]. As both AzMC and SSP4 are irreversible, they provide a cumulative record of H 2 S and polysulfide production, but they do not reflect cellular concentrations at any specific time.

Mass Spectrometry
Inorganic polysulfides were identified using liquid chromatography electrospray ionization high-resolution mass spectrometry (LCMS) using a micrOTOF-Q II Mass Spectrometer (Bruker Daltronics, Billerica, MA, USA) coupled to an UltiMate 3000 (Thermo HAuCl 4 ·4H 2 O. A total of 1 mL of 20 mM AgNO 3 was mixed with 200 µL of 0.5 mM HAuCl 4 in 98 mL of Milli-Q water at room temperature. A total of 1 mL of 5.0 mM TA was added, and the mixture was vigorously stirred, turning yellow within 30 min. The AgNPs were stored at 4 • C until use. Thiosulfate standards were made fresh daily, and a standard curve was prepared by plotting the concentration against (A 0 − A)/A 0 , where A 0 and A are absorbances of AgNPs without and with thiosulfate, respectively. Dong et al. [32] reported a linear standard curve; however, we note that the standard curve was non-linear but reproducible.
In a typical experiment, H 2 S, 1,4-NQ, and either GSH or Cys were placed in 96-well plates and the plates were covered with tape for 60 min to minimize H 2 S volatilization during the reaction, then the tape was removed to allow excess H 2 S to dissipate for an additional 60 min as we noted some H 2 S interference with this assay. A total of 30 µL of the buffer was then added to 200 µL of the AgNP in 96-well plates and the absorbance was measured after 60 min at 419 nm.
Thiosulfate was also measured using a variation of the high-performance liquid chromatography-monobromobimane (HPLC-MBB) assay reported previously [33,34]. HPLC-fluorescence analysis was performed with an UltiMate 3000 HPLC (Thermo Fisher Scientific, Waltham, MA, USA) with fluorescence detection (Ultimate FLD-3100 fluorescence detector, Thermo Fisher Scientific). A Shim-pack VP-ODS column (5 µm, 4.6 × 150 mm, Shimadzu Scientific, Kyoto, Japan) was used with mobile phases A (MilliQ water containing 0.25% formic acid) and B (methanol containing 0.25% formic acid). Samples (10 µL) were injected onto the column and run with a linear gradient (0-75% B) at a flow rate of 0.8 mL/min and a column temperature of 30 • C. The detector excitation/emission was set to 370/485 nm. Thiosulfate concentration was derived from the standard curve prepared on the same day. In these experiments, 50 µL of Na 2 S (60 µM), 1,4-NQ (6 µM), and either GSH (200 µM) or Cys (200 µM), all final concentrations, were added to Eppendorf tubes; the tubes were then closed for 70 min to allow the reaction to proceed. The tubes were then opened for an additional 60 min to allow H 2 S volatilization and 10 µL of 25 mM MBB in acetonitrile was added and the samples incubated in the dark at room temperature for 30 min to convert the thiosulfate into thiosulfatebimane.

Absorbance Spectra
Absorbance spectra were measured with an Agilent HP 8453 spectrometer (Agilent Technologies, Santa Clara, CA, USA). Experiments were performed at room temperature with PBS equilibrated with room air (21% O 2 ) or PBS sparged for 30 min with nitrogen (0% O 2 ) and spectra from 190 to 1100 nm were obtained at 4-6 s intervals for at least 25 min. In a typical experiment, the naphthoquinone was added, and three consecutive spectra obtained before addition of other reagents. Figures are displayed over 190-500 nm for clarity as most of the effects on absorbance were observed over this range.

Electron Paramagnetic Resonance (EPR) Spectrometry
X-band EPR spectra were recorded at 295 K on a Bruker EMX spectrometer (Billerica, MA, USA). The instrument parameters were frequency, 9.30 GHz; MW power, 1 or 4 mW; range, 20 G; modulation frequency, 100 kHz; modulation amplitude, 0.1 or 0.2 G; and time constant, 0.17 s. EPR samples were prepared by diluting 16 mM 1,4-NQ DMSO stocks in PBS buffer (pH 7.4, final 1,4-NQ concentration 4 mM) and reacted with GSH, Cys, or H 2 S prepared in PBS buffer. H 2 S stock was prepared by dissolving Na 2 S in PBS. For hypoxia reactions, 1,4-NQs were diluted into N 2 -sparged PBS buffer and the solutions were further sparged with N 2 while H 2 S was prepared by dissolving Na 2 S into N 2 -sparged PBS. Reaction solutions were then transferred into capillary tubes for EPR measurements. To prepare 1,4-NQ semiquinone radical, 4 mM 1,4-NQs were reacted with 0.15-0.25× sodium borohydride (NaBH 4 ).
The simulation of EPR spectra was conducted using Simfonia (Bruker). The parameters used in the simulation were g = 2.0044 and hyperfine splitting of 3.21 G for protons 2 and 3 and 0.63 G for protons 5, 6, 7, and 8. The parameters for simulating the spectrum of the SH-substituted 1,4-NQ were g = 2.0044 and hyperfine splitting of 0.69 G for protons 5, 6, 7, and 8.

H 2 S Oxidation and Polysulfide Production by 1,4-NQ-Thiol and 1,4-NQ-Amine Adducts
Since thiols interfere with the SSP4 detection of polysulfides from H 2 S/NQ reactions (see Results, Section 2.1), we prepared concentrated adducts and then diluted them prior to adding H 2 S and SSP4. One mM of GSH, Cys, or propylamine (PA) was added to 1 mM 1,4-NQ and allowed to react for 1-2 h in an open vessel to form the adduct and then allow it to autoxidize. We also assumed that most if not all the thiol or PA would be consumed in this reaction. The aliquots were then diluted in buffer in 96-well plates to a final concentration of 10 µM or 30 µM, leaving little or no free thiol or PA. H 2 S-NQ adducts were initially reacted for 30 min in a closed container to minimize H 2 S volatilization; the containers were then opened for another hour to allow all the remaining free H 2 S to dissipate and to oxidize the NQ-H 2 S adduct. SSP4 and H 2 S were then added, and fluorescence was measured over 90 min and compared to control.