Modulation of catalytic promiscuity during hydrogen sulfide oxidation

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Modulation of catalytic promiscuity during hydrogen sulfide oxidation Aaron P Landry, David P Ballou, and Ruma Banerjee ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00258 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Modulation of catalytic promiscuity during hydrogen sulfide oxidation Aaron P. Landry, David P. Ballou and Ruma Banerjee*

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109

Running Title: SQR substrate promiscuity

*Address correspondence to: Ruma Banerjee, 4220C MSRB III, 1150 W. Medical Center Dr., University of Michigan, Ann Arbor, MI 48109–0600, Tel: (734) 615–5238; E-mail: [email protected]

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Abstract

The mitochondrial sulfide oxidation pathway prevents the toxic accumulation of hydrogen sulfide (H2S), a signaling molecule that is maintained at low steady-state concentrations. Sulfide quinone oxidoreductase (SQR), an inner mitochondrial membrane-anchored protein, catalyzes the first and committing step in this pathway, oxidizing H2S to persulfide. The catalytic cycle comprises sulfide addition to the active site cysteine disulfide in SQR followed by sulfur transfer to a small molecule acceptor, while a pair of electrons moves from sulfide, to FAD, to coenzyme Q. While its ability to oxidize H2S is well characterized, SQR exhibits a remarkable degree of substrate promiscuity in vitro that could undermine its canonical enzyme activity. To assess how its promiscuity might be contained in vivo, we have used spectroscopic and kinetic analyses to characterize the reactivity of alternate substrates with SQR embedded in nanodiscs (ndSQR) versus detergent-solubilized enzyme (sSQR). We find that the membrane environment of ndSQR suppresses the unwanted addition of GSH but enhances sulfite addition, which might become significant under pathological conditions characterized by elevated sulfite levels. We demonstrate that methanethiol, a toxic sulfur compound produced in significant quantities by colonic and oral microbiota, can add to the SQR cysteine disulfide and also serve as a sulfur acceptor, potentially interfering with sulfide oxidation when its concentrations are elevated. These studies demonstrate that the membrane environment and substrate availability combine to minimize promiscuous reactions that would otherwise disrupt sulfide homeostasis.

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Many enzymes can use alternative substrates that are structurally similar to their native ones, and yield different products.1 The prevalence of substrate promiscuity reflects its potential importance for the divergent evolution of enzymes as the same structural scaffold gains new functionality.2 Catalytic promiscuity is also a feature of several enzymes involved in hydrogen sulfide (H2S) production and clearance.3-5 Steady-state H2S levels are low in mammalian cells,6,7 but must increase transiently to enable signaling that occurs presumably via persulfidation of cysteine residues on target proteins.8-10 H2S is produced by cystathionine β-synthase and cystathionine γ-lyase in the transsulfuration pathway, via the promiscuous use of cysteine and homocysteine as substrates,11,12 as well as by 3-mercaptopyruvate sulfurtransferase involved in cysteine catabolism.13,14 While the concentrations needed to trigger H2S signaling remain to be established, it is a respiratory toxin at micromolar concentrations, inhibiting cytochrome c oxidase in the electron transport chain.15,16 Excessive H2S is efficiently detoxified by the mitochondrial sulfide oxidation pathway, which converts it to thiosulfate and sulfate via several enzyme-catalyzed reactions (Figure 1). The first and committing step in the sulfide oxidation pathway is catalyzed by sulfide quinone oxidoreductase (SQR), a flavoprotein disulfide reductase that resides in the inner mitochondrial membrane. The SQR reaction occurs in two half reactions consisting of: (i) sulfide oxidation via persulfide formation, and (ii) coenzyme Q10 (CoQ10) reduction (Figure 2). The first half reaction begins with the nucleophilic addition of sulfide to the active site cysteine disulfide, which is presumed to form a cysteine persulfide (Cys-SSH) on Cys379 thereby releasing the Cys201 thiolate. Interaction of the electron-rich Cys201 thiolate with the FAD cofactor produces a charge transfer (CT) complex, with an intense broad absorbance peak centered at ~675 nm.4,17 Resolution of the CT complex is predicted to be the rate-limiting step in the SQR reaction,18 and requires transfer of the sulfane sulfur from the Cys379 persufide to a small molecule acceptor. The sulfur transfer step regenerates the active site disulfide, while the electrons are relayed from the Cys201 thiolate to FAD. In the second half reaction, the electrons from FADH2 are 3 ACS Paragon Plus Environment

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transferred to CoQ10 in the mitochondrial inner membrane, regenerating the resting enzyme. The reduced CoQ10 provides electrons to complex III in the electron transport chain, and thus directly links sulfide oxidation to energy metabolism.19 Surprisingly, SQR exhibits considerable laxity in substrate specificity under in vitro conditions, utilizing several low molecular weight sulfur acceptors, including GSH, sulfite, sulfide, cyanide, cysteine and homocysteine.3,17,20 This relaxed substrate specificity has fueled controversy about the overall reaction catalyzed by SQR.3,17,18,20,21 Studies in our laboratory and others3,20 have led to the conclusion that GSH is the physiologically relevant acceptor. Indeed, kinetic simulations of the SQR reaction rate at physiologically relevant substrate concentrations support the role of GSH as the primary acceptor.3,18 In addition to exhibiting promiscuity for the sulfane sulfur acceptor, SQR also permits alternate substrates to add to its active site cysteine disulfide. Addition of sulfite leads to formation of a robust and stable CT complex that is resolved in the presence of sulfide, forming thiosulfate and FADH2.4 Although the relative slowness of this reaction makes it unlikely to be a significant contributor under physiological conditions, it could become more important under pathological conditions associated with elevated sulfite levels as seen in sulfite oxidase deficiency.22 Elevated sulfite promotes reactive oxygen species formation,23,24 which may further exacerbate non-canonical SQR activity by lowering GSH levels. In addition to sulfite, GSH also induces formation of a stable CT complex when added to solubilized SQR (sSQR).21 This relaxed substrate specificity in the first step of the reaction, combined with the relatively high intracellular GSH concentrations (1–10 mM),25,26 raises the obvious question as to how this activity is regulated in the cell so as to not adversely impact sulfide homeostasis. We had previously shown that incorporation of SQR into nanodiscs (ndSQR) yielded a soluble and monodisperse enzyme suitable for kinetic studies, which facilitated characterization of its catalytic mechanism in a membrane environment.18 In this study, we have compared the 4 ACS Paragon Plus Environment


kinetics of CT complex formation by GSH and sulfite in ndSQR versus sSQR to gain insights into how unwanted side reactions are averted in the cell, and how the membrane environment of SQR may play a role in this process. We also demonstrate that the substrate promiscuity of SQR extends to methanethiol (MeSH), which can serve both as a nucleophile and as a sulfur acceptor. MeSH, a toxic compound generated by microbiota in the colon27,28 and oral cavity,29,30 could potentially interfere with H2S metabolism when its concentrations are elevated. Our results demonstrate that the promiscuity of SQR is suppressed both by its membrane environment, and/or kinetically by the limited availability of alternate substrates in the intracellular milieu.

RESULTS AND DISCUSSION

Formation and decay of the GSH-induced CT complex in ndSQR. Formation of a CT complex has been reported when sSQR is exposed to GSH.21 GSH is a sulfur acceptor in the SQR reaction3,20 and formation of a CT complex with GSH would interfere with the canonical SQR reaction. To evaluate the contribution of this side reaction with GSH in a membranous environment, we compared the kinetics of GSH-mediated CT complex formation with sSQR versus ndSQR. Rapid mixing of sSQR with GSH generated within 74 s, a stable CT complex with a broad absorbance maximum at 675 nm and an isosbestic point at 505 nm (Figure 3A), similar to the CT bands previously seen with sulfide or sulfite.21 The GSH-induced CT complex also exhibits peaks at 370 and 425 nm and an isosbestic point at 435 nm. In contrast, the CT complexes induced by sulfide or sulfite, are associated with a single peak at 390 nm and an isosbestic point at 423 nm.4 As GSH is significantly larger than the other nucleophiles studied, we speculate that conformational changes induced by GSH influence the CT complex spectrum. A 425:675 nm ratio of ~1:0.7 was observed upon mixing sSQR with GSH. CT complex generation was significantly slower with ndSQR, and the absorbance change at 675 nm after 74 s was ~12% of that observed with sSQR under the same conditions (Figure 3B, Supplementary 5 ACS Paragon Plus Environment

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Figure 1). After 30 min incubation with GSH, the absorbance change in ndSQR at 675 nm reached ~46% of that observed with sSQR (Figure 3C). The kobs for GSH-induced CT complex formation showed a linear dependence on GSH concentration (Figure 3D). With sSQR, a kon value of 36.7 ± 2.2 M–1 s–1 was obtained from stopped-flow studies. A KD of 36 ± 1 µM was obtained from spectral titration of the CT complex with GSH at 4 °C, which was used with the kon value to calculate a koff of 0.0013 ± 0.0001 s–1 at 4 °C. In contrast, the kon and koff values for GSH-dependent CT complex formation with ndSQR were 0.38 ± 0.026 M–1s–1 and 0.0018 ± 0.001 s–1, respectively at 4 °C, which were used to calculate an apparent KD (koff/kon) of 4.7 ± 1.3 mM. The membrane environment thus impedes promiscuous addition of GSH to the SQR disulfide by decreasing kon ~100-fold and increasing the KD ~130-fold. The canonical sulfide-induced CT complex forming reaction is greatly favored on ndSQR at physiologically relevant concentrations for sulfide and GSH, and we estimate that addition of sulfide to ndSQR would be favored 15- to 150-fold over GSH at their respective cellular concentrations of 15 nM and 1–10 mM (Table 1). The kinetic advantage of sulfide over GSH addition is lost in sSQR, emphasizing the importance of the membrane environment in suppressing promiscuous reactivity. Next, we assessed the sulfide-mediated decay of the GSH-induced CT complex in ndSQR. Addition of sulfide to the partially-formed CT complex in the presence of GSH led to reduction of the remaining FAD within 1 min, owing to rapid sulfide-induced CT formation and subsequent sulfur transfer in the presence of excess GSH.18 The slow decay of the GSH-induced CT complex was monitored over the course of 60 min (Figure 3E). The kobs for the GSH-induced CT decay was 0.0013 s–1 ± 0.0003 s–1 at 4 °C, similar to the koff value determined for GSH dissociation from the CT complex on ndSQR (0.0018 s–1). Based on these data, we propose that the mechanism for decay of the GSH-induced CT complex involves reversal to free enzyme, followed by formation of the canonical sulfide-dependent CT complex. In this mechanism, dissociation of GSH from SQR is the rate-limiting step (Figure 2, path a), and 6 ACS Paragon Plus Environment


occurs very slowly. For instance, in the presence of 5 mM GSH, the decay of the canonical sulfide-induced CT complex in ndSQR is ~1.5 s–1 at 4 °C,18 while the sulfide-mediated decay of the CT complex induced by GSH is ~1000-fold slower. Formation and decay of the sulfite-induced CT complex in ndSQR. Under Vmax conditions, sulfite is the most efficient sulfur acceptor for sSQR.3,17,18 Yet, sulfite can also add to the disulfide bond in the resting form of sSQR to generate a CT complex.4 The kinetics of sulfite addition to ndSQR, however, were not previously characterized. The absorption spectrum and the linear relationship of kobs versus concentration of sulfite for the sulfite-induced CT complex with ndSQR (Supplementary Figure 2A-B) are identical to that observed previously with sSQR.4 From the concentration dependence of the rate constant for CT complex formation, a kon of 9.4 102 ± 46 M–1s–1 at 4 °C was obtained (Supplementary Figure 2C), which is ~9-fold higher than for sSQR (1.03 102 M–1s–1 at 4 °C).4 A spectrally monitored titration of the CT complex with sulfite yielded a KD of 30 ± 1 µM at 4 °C, which was used with the kon value to calculate a koff of 0.028 ± 0.002 s–1 at 4 °C. For comparison, an ~5-fold greater KD (165 µM) and a slightly smaller koff (0.017 s–1) for sulfite were reported for sSQR.4 We had previously speculated that sulfite addition to sSQR4 might become an issue under pathological conditions e.g. sulfite oxidase deficiency that leads to high sulfite levels.22 We now report that in contrast to GSH, the kon for sulfite addition to ndSQR is enhanced ~9-fold compared to sSQR (Table 1). Therefore, the membrane environment could promote this SQR-impairing side reaction, if sulfite concentrations were elevated. Addition of sulfide to the sulfite-induced CT complex led to its decay with concomitant FAD reduction (Supplementary Figure 3), as seen with sSQR.4 Sulfide-mediated decay of the sulfiteinduced CT complex in ndSQR yielded a rate constant of 0.020 ± 0.005 s–1 at 4 °C, similar to the koff value determined for sulfite dissociation from the CT complex in ndSQR (0.028 ± 0.002 s–1). These data suggest that the dissociation of sulfite from SQR, as for GSH, is the rate-


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limiting step in the mechanism of the sulfite-induced CT complex decay (Figure 2, path a), and is comparatively slow versus the decay of canonical sulfide-induced CT complex. Formation and decay of the MeSH-induced CT complex in ndSQR. MeSH is a microbial product that is present at relatively high concentrations in the colon28 and in the oral cavity.31 The primary mechanism of MeSH toxicity is similar to that of H2S, which targets cytochrome c oxidase in the electron transport chain.32,33 MeSH detoxification activity is robust in colonic mucosa and the MeSH-derived H2S is ultimately oxidized to thiosulfate and sulfate.34 Elevated MeSH is observed in extraoral halitosis associated with mutations in SELENBP1, a selenoprotein recently designated as a MeSH oxidase converting MeSH to H2S, formaldehyde, and H2O2.35 Elevated MeSH levels are also associated with periodontitis,36 which is an independent risk factor for heart disease and diabetes37 and ischemic stroke.38 Given the promiscuity of SQR, we postulated that MeSH, like sulfide, might add to the active site disulfide to form a CT complex. Rapid mixing of ndSQR with MeSH generated a stable CT complex with kinetic (Supplementary Figure 4) and spectral features similar to the sulfide-induced CT complex, with absorbance maxima at 390 nm and 675 nm, isosbestic points at 423 nm and 505 nm, and a decrease in absorbance in the FAD-associated 450 nm feature (Figure 4A).4,17 The kobs for CT complex formation showed a linear dependence on MeSH concentration, yielding a kon of 6.3 104 ± 2.2 103 M–1s–1 at 4 °C (Figure 4B). A spectrally monitored titration of the CT complex with MeSH yielded a KD of 38 ± 1 µM 4 °C, which was used to calculate a koff of 2.4 ± 0.1 s–1 at 4 °C. Thus, MeSH can react with ndSQR to form a CT complex, albeit with a kon that is 63-fold lower than for sulfide (4 106 M–1s–1).18 The formation of a MeSH-induced CT complex raised the question of whether it can serve as an alternate sulfur source analogous to sulfide. Transfer of the outer sulfur from the putative Cys-SSMe mixed disulfide intermediate to a small molecule acceptor, such as a second equivalent of MeSH, GSH, sulfite, or sulfide, would regenerate the cysteine disulfide with concomitant reduction of FAD. While the sulfide-induced CT complex decays in the presence of 8 ACS Paragon Plus Environment


excess sulfide to produce persulfide and FADH2,4,17,18 the MeSH-induced CT complex is stable in ndSQR in the presence of excess MeSH (Figure 5A), indicating that it cannot utilize a second equivalent of MeSH as a sulfur acceptor. To test the possibility that MeSH can serve as a sulfur donor with GSH, sulfite, or sulfide as acceptors, the MeSH-induced CT complex was mixed with each of these to monitor CT complex decay and concomitant FAD reduction. While no change was elicited in the MeSH-induced CT absorption by GSH or sulfite, addition of sulfide led to CT complex decay and concomitant FAD reduction (Figure 5B). The sulfide-mediated decay of the MeSH-induced CT complex was then assessed by stopped-flow spectroscopy, and yielded a rate constant of 0.071 ± 0.002 s–1 at 4 °C (Figure 5C, Supplementary Figure 5). The decay of the MeSH-induced CT complex by sulfide is slower than the dissociation of MeSH (k = 2.4 ± 0.1 s–1), in contrast to the decay of the CT complexes induced by sulfite or GSH (Figure 2, path a). This suggests that in the presence of sulfide, dissociation of MeSH from the CT complex is slowed down. At present, we cannot distinguish between a sulfide attack on the mixed disulfide intermediate (Figure 2, path b) or MeSH dissociation followed by reaction with sulfide (Figure 2, path a). However, we note that resolution of the MeSH-induced CT complex is independent of sulfide concentration (0.1-1 mM). For comparison, the kcat for catalytic turnover under conditions in which sulfur transfer occurs from sulfide to GSH is 13 ± 2 s–1 at 4 ºC.18 Hence, the formation of CT complexes with MeSH, or any of the alternative nucleophiles, would ultimately impede SQR activity by trapping the enzyme in a slowly-decaying complex. Kinetic characterization of MeSH as a sulfur acceptor. We tested MeSH as a potential sulfur acceptor in the SQR reaction, using sulfide as the sulfur donor. Mixing ndSQR containing a sulfide-induced CT complex with MeSH led to its rapid decay and concomitant reduction of FAD (Figure 6A, Supplementary Figure 6). The kobs for CT complex decay was linearly dependent on the MeSH concentration, yielding a k of 5.4 ± 0.1 104 M–1s–1 at 4 °C (Figure 6B), which is similar to the kon value obtained for the nucleophilic addition of MeSH into the resting enzyme (6.3 104 M–1s–1 at 4 °C (Figure 4B)). 9 ACS Paragon Plus Environment

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Under steady-state turnover conditions, the introduction of MeSH to a reaction mixture containing sulfide, CoQ1, and ndSQR yielded a kcat of 100 ± 5 s–1 at 25 °C (Figure 6C), which is ~20% higher than the value obtained under conditions where sulfide acts as both a sulfur donor and acceptor (84 ± 2 s–1 at 25 °C).18 With sulfide as the sulfur donor, the KM values for MeSH and sulfide as sulfur acceptors are similar (256 ± 33 µM and 230 ± 20 µM, respectively at 25 °C).18 When steady-state turnover was monitored at 4 °C, the kcat for sulfide oxidation in the presence of 100 µM MeSH was 6.6 ± 0.6 s–1, which is ~25% higher than the kobs of 5.2 ± 0.2 s–1 obtained under stopped-flow conditions at the same temperature for the pre-steady state sulfur transfer to MeSH. These data are consistent with our model that sulfur transfer from the active site Cys-SSH intermediate to an acceptor is the rate-determining step in the SQR reaction cycle.18 Thus, MeSH not only acts as a nucleophile to add to the ndSQR disulfide bond, but also appears to act as a sulfur acceptor to facilitate catalytic turnover (Figure 2). However, MeSH, unlike sulfide, does not act as both a sulfur donor and acceptor in the same catalytic turnover cycle. While accurate estimates of luminal H2S and MeSH concentrations are unavailable, their levels in flatus have been reported. The reported values, which are likely to be highly dependent on diet, microbiota and other variables, estimate that MeSH concentration is ~5-fold lower than H2S.27,39 The relative H2S versus MeSH levels also vary greatly in the oral cavity31 and correlate with the composition of the microbial community.30 MeSH levels are increased in periodontal disease, along with higher MeSH:H2S ratios reported in deep periodontal pockets.40 Our data suggest that when MeSH is elevated, it could potentially influence the sulfide oxidation activity of SQR, further impairing clearance of H2S generated during MeSH oxidation. As MeSH is far less abundant than GSH (Table 1), its contribution to sulfide oxidation is predicted to be negligible, despite its higher kcat/KM under steady-state turnover conditions (3.9 105 M–1s–1 at 25 °C) compared to GSH (kcat/KM = 1.6 104 M–1s–1 at 25 °C).18 However, in periodontal disease, which is marked by oxidative stress41 and decreased GSH,42,43 aberrant 10 ACS Paragon Plus Environment


utilization of MeSH in the SQR reaction might be promoted and would interfere with sulfide homeostasis. GSSH, generated by the SQR reaction, serves as a substrate for persulfide dioxygenase (PDO, also known as ETHE1), and is converted to sulfite and GSH in the presence of O2 (Figure 1).44,45 The analogous SQR reaction with MeSH as the sulfur acceptor would generate methane persulfide (MeSSH), whose fate is unknown. The conversion of sulfide to sulfite can be monitored in a coupled enzyme assay containing SQR and PDO. With GSH as the acceptor, full coupling between the ndSQR and PDO reactions was observed, with the experimentally determined ndSQR turnovers equaling the predicted number of turnovers (Table 2). However, significant O2 consumption by PDO was not observed when coupled to the ndSQR reaction containing sulfide and MeSH (Table 2). Thus, if MeSSH is generated in the SQR reaction, it is not subsequently processed by PDO. While MeSSH formation is suggested by the hyperbolic dependence of the SQR reaction on MeSH concentration, yielding a KM(MeSH) of 256 µM (Figure 6C), MeSSH could not be characterized directly due to its lability. The utilization of MeSSH as a sulfur donor by the broad specificity sulfurtransferases rhodanese3 or TSTD146 would regenerate MeSH, without net metabolism of MeSH occurring via this set of reactions. In summary, we have demonstrated that substrate promiscuity in the initial catalytic step of the SQR reaction is minimized by its membrane environment and by the more favorable kinetics associated with the canonical sulfide oxidation reaction. Under normal cellular conditions where GSH levels are high, the membrane environment of SQR suppresses nucleophilic addition of GSH to the active site cysteine disulfide, while the low concentrations and lower kon for MeSH and sulfite render them less competitive with respect to sulfide. Under conditions of high sulfite as observed in sulfite oxidase deficiency,22 or high MeSH concentrations as observed in periodontal disease,40 nucleophilic addition of these molecules into the active site cysteine disulfide in SQR might become more significant, impairing sulfide homeostasis. Finally, we


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demonstrate the potential for forming MeSSH via sulfur transfer from SQR-bound sulfide to MeSH.

METHODS

Materials. The following reagents were purchased from Millipore Sigma: CoQ1, GSH, sodium methanethiol, sodium sulfide nonahydrate, and sodium sulfite. 1,2-diheptanoyl-sn-glycero-3phophocholine (DHPC) was purchased from Avanti Polar Lipids. Purification of sSQR and ndSQR. Human SQR was purified as detergent-solubilized enzyme as described previously.3 The sSQR was incorporated into nanodiscs containing 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine using the MSP1E3D1 membrane scaffold protein as described previously.18 Spectral analysis of ndSQR. Electronic absorption spectra of ndSQR were recorded in 100 mM potassium phosphate buffer, pH 7.4. The same buffer containing 0.03% (v/v) DHPC was used for the detergent-solubilized enzyme. The concentrations of sSQR and ndSQR were estimated using an extinction coefficient at 450 nm of 11,500 M–1cm–1 for the enzyme-bound FAD cofactor.17 To determine KD values for nucleophiles, CT complex formation was monitored in sSQR or ndSQR (10 µM) in 100 mM potassium phosphate buffer, pH 7.4 at 4 °C. Spectra were recorded after incubation with small aliquots of GSH (0–200 µM), sulfite (0–120 µM), or MeSH (0–120 µM). The CT complex absorbance at 675 nm was plotted as a function of nucleophile concentration and fitted to a 3-parameter sigmoidal equation using the SigmaPlot software. SQR activity assays. Steady-state assays were conducted in 100 mM potassium phosphate buffer, pH 7.4 at 25 °C by monitoring the reduction of CoQ1 at 278 nm (∆εox-red = 12,000 M–1cm– 1

) as described previously.3,17 Stock solutions of CoQ1 were prepared aerobically in 10 mM



potassium phosphate, pH 6.8, containing 10% (v/v) DHPC. The kinetic parameters KM and Vmax were obtained by fitting the data sets to the Michaelis-Menten equation. Stopped-flow spectroscopy. All rapid-mixing spectroscopic experiments were carried out in 100 mM potassium phosphate buffer, pH 7.4, at 4 °C on an SF-DX2 double mixing stopped-flow system from Hi-Tech Scientific, equipped with a photodiode array detector (300-700 nm range). The spectra and kobs for CT complex formation in ndSQR in the presence of acceptors were obtained in single mixing experiments as described previously.4 The spectra and kobs for the disappearance of the sulfide-induced CT complex in the presence of MeSH were obtained in double mixing experiments in which the final sulfide concentration was 20 µM as described previously,18 with MeSH concentrations kept ≤100 µM to minimize competition from MeSHinduced CT complex formation. The reported concentrations are those before mixing 1:1 (v/v). All kinetic traces at selected wavelengths were monophasic, and were fitted to a singleexponential equation using the KinetAsyst software. ndSQR activity assay in a coupled enzymatic reaction. Human PDO was purified as described previously.45 Oxygen consumption by PDO was measured using a Clark oxygen electrode housed in a 1.5 mL Gilson type chamber at 25 °C with a magnetic stirrer. The assays were carried out with PDO (0.5 µM) in 100 mM potassium phosphate, pH 7.4, coupled to the SQR reaction containing Na2S (300 µM), GSH (35 mM) or methanethiol (1 mM), and CoQ1 (300 µM). The reactions were initiated by injection of Na2S followed immediately by injection of ndSQR (10 nM). Oxygen consumption was recorded on a Kipp and Zonen BD single channel chart recorder and was expressed as µmol O2 consumed per min. Coupling of ndSQR and PDO activity was estimated as the ratio of observed turnovers (µmol O2 consumed / µmol ndSQR) to the predicted ndSQR turnovers (duration of reaction kcat for acceptor). A ratio of 1:1 represents complete coupling. For predicted ndSQR turnovers, the kcat values for acceptors


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used were 128 s-1 for GSH as determined previously,18 and 100 s–1 for MeSH as determined in this study.

ASSOCIATED CONTENT Supporting Information: Supporting information (Figures S1-S6) is available free of charge via the ACS Publications website at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

ORCID Aaron P. Landry: 0000-0003-1161-7092 Ruma Banerjee: 0000-0001-8332-3275

Notes No competing financial interest exists.

ACKNOWLEDGEMENTS This work was supported in part by the National Institutes of Health (GM112455 to R.B. and F32GM122357 to A.P.L). The authors thank P. Yadav for discussions on experiments with ndSQR and MeSH, which were based on preliminary experiments with sSQR. 14 ACS Paragon Plus Environment


REFERENCES 1. Khersonsky, O., and Tawfik, D. S. (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective, Annu. Rev. Biochem. 79, 471-505. 2. Jensen, R. A. (1976) Enzyme recruitment in evolution of new function, Annu. Rev. Microbiol. 30, 409–425. 3. Libiad, M., Yadav, P. K., Vitvitsky, V., Martinov, M., and Banerjee, R. (2014) Organization of the human mitochondrial hydrogen sulfide oxidation pathway, J. Biol. Chem. 289, 30901–30910. 4. Mishanina, T. V., Yadav, P. K., Ballou, D. P., and Banerjee, R. (2015) Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase, J. Biol. Chem. 290, 25072–25080. 5. Banerjee, R. (2017) Catalytic promiscuity and heme-dependent redox regulation of H2S synthesis, Curr. Opin. Chem. Biol. 37, 115–121. 6. Furne, J., Saeed, A., and Levitt, M. D. (2008) Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values, Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R1479–1485. 7. Vitvitsky, V., Kabil, O., and Banerjee, R. (2012) High turnover rates for hydrogen sulfide allow for rapid regulation of its tissue concentrations, Antioxid. Redox Signal. 17, 22–31. 8. Mustafa, A. K., Gadalla, M. M., Sen, N., Kim, S., Mu, W., Gazi, S. K., Barrow, R. K., Yang, G., Wang, R., and Snyder, S. H. (2009) H2S signals through protein S-sulfhydration, Sci. Signal. 2, ra72. 9. Mishanina, T. V., Libiad, M., and Banerjee, R. (2015) Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways, Nat. Chem. Biol. 11, 457–464. 10. Filipovic, M. R., Zivanovic, J., Alvarez, B., and Banerjee, R. (2018) Chemical Biology of H2S Signaling through Persulfidation, Chem. Rev. 118, 1253–1337. 11. Chiku, T., Padovani, D., Zhu, W., Singh, S., Vitvitsky, V., and Banerjee, R. (2009) H2S biogenesis by human cystathionine gamma-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia, J. Biol. Chem. 284, 11601–11612. 12. Singh, S., Padovani, D., Leslie, R. A., Chiku, T., and Banerjee, R. (2009) Relative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions, J. Biol. Chem. 284, 22457–22466. 13. Shibuya, N., Tanaka, M., Yoshida, M., Ogasawara, Y., Togawa, T., Ishii, K., and Kimura, H. (2009) 3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide and bound sulfane sulfur in the brain, Antioxid. Redox Signal. 11, 703–714. 14. Yadav, P. K., Yamada, K., Chiku, T., Koutmos, M., and Banerjee, R. (2013) Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase, J. Biol. Chem. 288, 20002–20013. 15. Nicholls, P., and Kim, J. K. (1982) Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system, Can. J. Biochem. 60, 613–623. 16. Nicholls, P., Marshall, D. C., Cooper, C. E., and Wilson, M. T. (2013) Sulfide inhibition of and metabolism by cytochrome c oxidase, Biochem. Soc. Trans. 41, 1312–1316. 17. Jackson, M. R., Melideo, S. L., and Jorns, M. S. (2012) Human sulfide:quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite, Biochemistry. 51, 6804–6815. 18. Landry, A. P., Ballou, D. P., and Banerjee, R. (2017) H2S oxidation by nanodisc-embedded human sulfide quinone oxidoreductase, J. Biol. Chem. 292, 11641–11649. 19. Goubern, M., Andriamihaja, M., Nubel, T., Blachier, F., and Bouillaud, F. (2007) Sulfide, the first inorganic substrate for human cells, FASEB J. 21, 1699–1706.


Page 16 of 26

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20. Hildebrandt, T. M., and Grieshaber, M. K. (2008) Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria, FEBS J. 275, 3352–3361. 21. Augustyn, K. D., Jackson, M. R., and Jorns, M. S. (2017) Use of Tissue Metabolite Analysis and Enzyme Kinetics To Discriminate between Alternate Pathways for Hydrogen Sulfide Metabolism, Biochemistry. 56, 986–996. 22. Shih, V. E., Abroms, I. F., Johnson, J. L., Carney, M., Mandell, R., Robb, R. M., Cloherty, J. P., and Rajagopalan, K. V. (1977) Sulfite oxidase deficiency. Biochemical and clinical investigations of a hereditary metabolic disorder in sulfur metabolism, N. Engl. J. Med. 297, 1022–1028. 23. Grings, M., Moura, A. P., Parmeggiani, B., Marcowich, G. F., Amaral, A. U., de Souza Wyse, A. T., Wajner, M., and Leipnitz, G. (2013) Disturbance of brain energy and redox homeostasis provoked by sulfite and thiosulfate: potential pathomechanisms involved in the neuropathology of sulfite oxidase deficiency, Gene. 531, 191–198. 24. Vincent, A. S., Lim, B. G., Tan, J., Whiteman, M., Cheung, N. S., Halliwell, B., and Wong, K. P. (2004) Sulfite-mediated oxidative stress in kidney cells, Kidney Int. 65, 393–402. 25. Kosower, N. S., and Kosower, E. M. (1978) The glutathione status of cells, Int. Rev. Cytol. 54, 109–160. 26. Meister, A. (1988) Glutathione metabolism and its selective modification, J. Biol. Chem. 263, 17205–17208. 27. Suarez, F., Furne, J., Springfield, J., and Levitt, M. (1997) Insights into human colonic physiology obtained from the study of flatus composition, Am. J. Physiol. 272, G1028– 1033. 28. Suarez, F., Furne, J., Springfield, J., and Levitt, M. (1998) Production and elimination of sulfur-containing gases in the rat colon, Am. J. Physiol. 274, G727–733. 29. Persson, S., Edlund, M. B., Claesson, R., and Carlsson, J. (1990) The formation of hydrogen sulfide and methyl mercaptan by oral bacteria, Oral Microbiol. Immunol. 5, 195–201. 30. Takeshita, T., Suzuki, N., Nakano, Y., Yasui, M., Yoneda, M., Shimazaki, Y., Hirofuji, T., and Yamashita, Y. (2012) Discrimination of the oral microbiota associated with high hydrogen sulfide and methyl mercaptan production, Sci. Rep. 2, 215. 31. Blanchette, A. R., and Cooper, A. D. (1976) Determination of hydrogen sulfide and methyl mercaptan in mouth air at the parts-per-billion level by gas chromatography, Anal. Chem. 48, 729–731. 32. Waller, R. L. (1977) Methanethiol inhibition of mitochondrial respiration, Toxicol. Appl. Pharmacol. 42, 111–117. 33. Finkelstein, A., and Benevenga, N. J. (1986) The effect of methanethiol and methionine toxicity on the activities of cytochrome c oxidase and enzymes involved in protection from peroxidative damage, J. Nutr. 116, 204–215. 34. Furne, J., Springfield, J., Koenig, T., DeMaster, E., and Levitt, M. D. (2001) Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa, Biochem. Pharmacol. 62, 255–259. 35. Pol, A., Renkema, G. H., Tangerman, A., Winkel, E. G., Engelke, U. F., de Brouwer, A. P. M., Lloyd, K. C., Araiza, R. S., van den Heuvel, L., Omran, H., Olbrich, H., Oude Elberink, M., Gilissen, C., Rodenburg, R. J., Sass, J. O., Schwab, K. O., Schafer, H., Venselaar, H., Sequeira, J. S., Op den Camp, H. J. M., and Wevers, R. A. (2018) Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis, Nat. Genet. 50, 120–129. 36. Iatropoulos, A., Panis, V., Mela, E., Stefaniotis, T., Madianos, P. N., and Papaioannou, W. (2016) Changes of volatile sulphur compounds during therapy of a case series of patients with chronic periodontitis and halitosis, J. Clin. Periodontol. 43, 359–365. 16 ACS Paragon Plus Environment


37. Southerland, J. H., Taylor, G. W., Moss, K., Beck, J. D., and Offenbacher, S. (2006) Commonality in chronic inflammatory diseases: periodontitis, diabetes, and coronary artery disease, Periodontol. 2000. 40, 130–143. 38. Dorfer, C. E., Becher, H., Ziegler, C. M., Kaiser, C., Lutz, R., Jorss, D., Lichy, C., Buggle, F., Bultmann, S., Preusch, M., and Grau, A. J. (2004) The association of gingivitis and periodontitis with ischemic stroke, J. Clin. Periodontol. 31, 396–401. 39. Suarez, F. L., Springfield, J., and Levitt, M. D. (1998) Identification of gases responsible for the odour of human flatus and evaluation of a device purported to reduce this odour, Gut. 43, 100–104. 40. Yaegaki, K., and Sanada, K. (1992) Volatile sulfur compounds in mouth air from clinically healthy subjects and patients with periodontal disease, J. Periodontal Res. 27, 233–238. 41. D'Aiuto, F., Nibali, L., Parkar, M., Patel, K., Suvan, J., and Donos, N. (2010) Oxidative stress, systemic inflammation, and severe periodontitis, J. Dent. Res. 89, 1241–1246. 42. Chapple, I. L., Brock, G., Eftimiadi, C., and Matthews, J. B. (2002) Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease, Mol. Pathol. 55, 367–373. 43. Sculley, D. V., and Langley-Evans, S. C. (2003) Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation, Clin. Sci. (Lond.). 105, 167–172. 44. Tiranti, V., Viscomi, C., Hildebrandt, T., Di Meo, I., Mineri, R., Tiveron, C., Levitt, M. D., Prelle, A., Fagiolari, G., Rimoldi, M., and Zeviani, M. (2009) Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy, Nat. Med. 15, 200–205. 45. Kabil, O., and Banerjee, R. (2012) Characterization of patient mutations in human persulfide dioxygenase (ETHE1) involved in H2S catabolism, J. Biol. Chem. 287, 44561–44567. 46. Libiad, M., Motl, N., Akey, D. L., Sakamoto, N., Fearon, E. R., Smith, J. L., and Banerjee, R. (2018) Thiosulfate sulfurtransferase-like domain-containing 1 protein interacts with thioredoxin, J. Biol. Chem. 293, 2675–2686. 47. Ji, A. J., Savon, S. R., and Jacobsen, D. W. (1995) Determination of total serum sulfite by HPLC with fluorescence detection, Clin. Chem. 41, 897–903. 48. Togawa, T., Ogawa, M., Nawata, M., Ogasawara, Y., Kawanabe, K., and Tanabe, S. (1992) High performance liquid chromatographic determination of bound sulfide and sulfite and thiosulfate at their low levels in human serum by pre-column fluorescence derivatization with monobromobimane, Chem. Pharm. Bull. (Tokyo). 40, 3000–3004.


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TABLE 1 Predicted rates of CT complex formation in SQR

Nucleophile

kon a

Estimated physiological concentration b

M-1s-1 sulfide

sulfite

4.0 106 d

1.03 102 (sSQR) e

15 nM (brain, liver)6

3.6

1 µM (flatus)27,39

240

1.9–20 µM (oral cavity)31

456–4800

0.1–1 µM47,48

0.00062–0.0062

9.4 102 (ndSQR) GSH

36.7 (sSQR)

0.0056–0.056 1–10 mM25,26

0.38 (ndSQR) MeSH

4

6.3 10

Predicted kobs at physiological concentration c min-1

2.2–22 0.023–0.23

0.2 µM (flatus)27,39

0.76

0.2–4 µM (oral cavity)31

0.76–14.7

a

Data were obtained with ndSQR, except where denoted. Data are from the references denoted. c Predicted kobs values were calculated as kon for nucleophile estimated physiological concentration. d Data are from Landry et al.18 e Data are from Mishanina et al.4 b



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TABLE 2 Rate of SQR-dependent sulfide oxidation detected by O2 consumption in a coupled assay with PDO

Reaction a ndSQR, Na2S, GSH, CoQ1

O2 consumption rate µmol O2/min 0.0041 ± 0.001

SQR turnover ratio b

PDO, Na2S, GSH, CoQ1

0.032 ± 0.01

ndSQR, PDO, Na2S, CoQ1

0.125 ± 0.007

0.13:1

ndSQR, PDO, Na2S, GSH, CoQ1

1.1 ± 0.17

1:1

ndSQR, PDO, Na2S, MeSH, CoQ1

0.007 ± 0.001

0.008:1

a

Reactions were run at 25 °C in 100 mM potassium phosphate, pH 7.4. The concentrations of the reaction components where denoted are: ndSQR (10 nM), PDO (0.5 µM), Na2S (300 µM), GSH (35 mM), MeSH (1 mM), and CoQ1 (300 µM). The data are the average ± SD of three independent experiments. b SQR turnover ratios were calculated as described under Methods.


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intermembrane space

SO

SO42matrix

PDO GSH H2S

2O2 SO3 GSSH

Rhd

SSO32-

SQR Qox Qred

III

IV

inner membrane Figure 1. The mitochondrial sulfide oxidation pathway. SQR is anchored in the inner membrane and catalyzes the first committing step of sulfide oxidation. PDO, Rhd, SO, III and IV denote the persulfide dioxygenase (or ETHE1), rhodanese, sulfite oxidase, and complexes III and IV of the electron transport chain, respectively. The oxidized sulfur derived from H2S is shown in red.



CoQred 379

Cys S HS

1 Cys S

Nuc

201

path a

CoQox

379

379

Cys S SH Ac

Cys S

2

3 Ac SH

Cys S

201

Cys S

201

FADox

FADox GSH, SO3

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2- 379

Cys S Nuc

2'

MeS SH

FADred

HS path b

Cys S

201

FADox Ac: GSH, sulfite, MeSH, sulfide, cysteine, homocysteine Nuc: GSH, sulfite, MeSH Figure 2. Reaction mechanism for formation of the canonical or alternative CT complexes in SQR. Addition of sulfide to the disulfide bond in SQR (1) leads to formation of the canonical CT complex (2). Sulfur transfer from the Cys379 persulfide (Cys-S-SH) to an acceptor (Ac: GSH, sulfite, MeSH, sulfide, cysteine, or homocysteine) regenerates the disulfide bond in SQR with concomitant reduction of FAD (3). The transfer of electrons from reduced FAD to oxidized coenzyme Q10 (CoQox) regenerates the resting enzyme. Conversely, addition of a nucleophile (Nuc: GSH, sulfite, or MeSH) to the disulfide bond of the resting enzyme forms an alternate CT complex (2´). In the presence of sulfide, resolution of the alternative CT occurs either via dissociation of the nucleophile followed by formation of a sulfide-dependent CT complex, as in the case of GSH or sulfite (path a), or via sulfur transfer to the sulfide acceptor, as in the case of MeSH (path b).


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A

B 425 nm

0.12 370 nm

0.12

450 nm

450 nm

675 nm

0.08

0.08 435 nm

0.04

505 nm

0.04

0

675 nm

0 350

C

450 550 650 Wavelength (nm) 450 nm

0.12

350

D 0.4

0.01 0

0

10 20 30 Time (mins)

675 nm 0.04

sSQR ndSQR

0.03 0.02

0.08

450 550 650 Wavelength (nm)

0.2

505 nm

0 350

E

0


0

10 20 GSH (mM)

30

0.04

0.12 425 nm 0.08

0.02 0 0

20 40 60 Time (min)

675 nm

0.04

0 350


Figure 3. Kinetics of the GSH-induced CT complex formation in ndSQR and its sulfide-mediated decay. (A) and (B), sSQR (20 µM) (A) or ndSQR (20 µM) (B) with and without 0.03% (v/v) DHPC, respectively, rapidly mixed 1:1 (v/v) with GSH (10 mM) with CT complex formation (thick black line) monitored over 74 s. (C) ndSQR (10 µM) incubated with GSH (5 mM), with CT complex formation monitored over 30 min (thick black line). The inset shows the increase in absorbance at 675 nm versus time. (D) Dependence of the kobs for CT complex formation in sSQR (open circles) versus ndSQR (closed circles) on the GSH concentration. The data represent the mean ± S.D. of two independent experiments. (E) ndSQR (10 µM) pre-incubated with GSH (5 mM) for 1 h, followed by addition of Na2S (500 µM), with the spectra recorded over 1 h. The dashed line denotes the initial spectrum before addition of Na2S. Decay of the GSHinduced CT complex leading to FAD reduction (thick black line) was observed. The inset shows the change in absorbance of the CT complex at 675 nm versus incubation time with Na2S. The data are representative of three independent experiments. 22 ACS Paragon Plus Environment


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A 390 nm 450 nm

0.12

675 nm

0.08 423 nm

0.04

505 nm

0 350

B


100 80 60 40 20 0

0

0.4 0.8 1.2 Methanethiol (mM)

Figure 4. Formation of the MeSH-induced CT complex in ndSQR. (A) ndSQR (20 µM) rapidly mixed 1:1 (v/v) with MeSH (400 µM), with CT complex formation (thick black line) monitored over 3 s. (B) Dependence of the kobs for CT complex formation on MeSH concentration. The data represent the mean ± S.D. for three independent experiments.


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A 0.1

MeSH CT

0.08 0.06 sulfide CT

0.04 0.02 0

0.1

1

B

10 Time (s)

100

0.16 390 nm

ndSQR

0.12 675 nm CT

0.08

+GSH, +SO32-

0.04 +Na2S 0 300


C

700

390 nm 0.12 675 nm 0.08 0.04 0 350


Figure 5. Sulfide-mediated decay of the MeSH-induced CT complex in ndSQR. (A) ndSQR (20 µM) rapidly mixed 1:1 (v/v) with MeSH (1 mM, black trace) or Na2S (1 mM, gray trace) and monitored at 675 nm over a period of 285 s or 72 s for decay of the MeSH and sulfide-induced CT complexes, respectively. The sulfide-induced CT complex was formed in the dead time of the instrument and only it decay is observed. (B) ndSQR (10 µM) was pre-incubated with MeSH (1 mM) for 30 s to generate the CT complex, followed by the addition of GSH (1 mM), sulfite (1 mM), or Na2S (1 mM), and incubation for 1 min. The data are representative of two independent experiments. (C) ndSQR (20 µM) was pre-incubated with MeSH (400 µM) for 2 min and followed by rapid 1:1 (v/v) mixing with Na2S (200 µM). The spectra were recorded over 74 s, with decay of the MeSH-induced CT complex leading to FAD reduction (thick black line). The data are representative of three independent experiments. 24 ACS Paragon Plus Environment


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A 0.12 390 nm 450 nm

0.08

675 nm 565 nm

0.04 495 nm 0 350

B


6

4

2

0 0

20

C

40 60 80 100 120 Methanethiol (µM)

120 80 80

60 40

40 20 0

0

0.2 0.4 0.6 0.8 1.0 Methanethiol (mM)

0

Figure 6. Pre-steady state sulfur transfer kinetics to MeSH and steady-state CoQ1 reduction in ndSQR. (A) ndSQR (40 µM) was rapidly mixed 1:1 (v/v) with Na2S (80 µM) and aged for ~35 msec to form the CT complex, followed by rapid mixing with 40 µM MeSH (1:1 (v/v)). The reaction was monitored for 3 s, during which time the CT complex decayed and FAD was reduced (thick black line). (B) Dependence of the kobs for the disappearance of the CT complex on the MeSH concentration. The data represent the mean ± S.D. for two independent experiments. (C) Modulation of sulfide-driven CoQ1 reduction by MeSH (0‒0.8 mM) in 100 mM potassium phosphate buffer, pH 7.4, with 60 µM CoQ1, 0.06 mg ml-1 BSA, and 150 µM Na2S. The reactions were initiated by addition of ndSQR (1 nM). The data represent the mean ± S.D. for three independent experiments, each run in duplicate.


Modulation of catalytic promiscuity during hydrogen sulfide oxidation

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