Modulation of Catalytic Promiscuity during Hydrogen Sulfide Oxidation

Aaron P Landry; David P Ballou; Ruma Banerjee

doi:10.1021/acschembio.8b00258

. Author manuscript; available in PMC: 2019 Jun 15.

Published in final edited form as: ACS Chem Biol. 2018 May 10;13(6):1651–1658. doi: 10.1021/acschembio.8b00258

Modulation of Catalytic Promiscuity during Hydrogen Sulfide Oxidation

Aaron P Landry ¹, David P Ballou ¹, Ruma Banerjee ^1,^*

PMCID: PMC6449043 NIHMSID: NIHMS1011056 PMID: 29715001

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.

Graphical Abstract

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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 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 trans-sulfuration 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).

Figure 1. — Mitochondrial sulfide oxidation pathway. SQR is anchored in the inner membrane and catalyzes the first and 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.

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 reaction¹⁸ 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 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

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 (CoQ_ox) 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 may be in the case of MeSH (*path b*).

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 others^3,20 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 noncanonical 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 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 colon^27,28 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 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 reaction,^3,20 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, Supporting Information Figure 1). After 30 min of incubation with GSH, the absorbance change in ndSQR at 675 nm reached ~46% of that observed with sSQR (Figure 3C).

The k_obs for GSH-induced CT complex formation showed a linear dependence on GSH concentration (Figure 3D). With sSQR, a k_on value of 36.7 ± 2.2 M⁻¹ s⁻¹ was obtained from stopped-flow studies. A K_D of 36 ± 1 μM was obtained from spectral titration of the CT complex with GSH at 4 °C, which was used with the k_on value to calculate a k_off of 0.0013 ± 0.0001 s⁻¹ at 4 °C. In contrast, the k_on and k_off values for GSH-dependent CT complex formation with ndSQR were 0.38 ± 0.026 M⁻¹ s⁻¹ and 0.0018 ± 0.001 s⁻¹, respectively, at 4 °C, which were used to calculate an apparent K_D (k_off/k_on) of 4.7 ± 1.3 mM. The membrane environment thus impedes promiscuous addition of GSH to the SQR disulfide by decreasing k_on ~100-fold and increasing the K_D ~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.

Table 1.

Predicted Rates of CT Complex Formation in SQR

nucleophile	k_on,^a M⁻¹ _s⁻¹	estimated physiological concentration^b	predicted k_obs at physiological concentration,^c min⁻¹
sulfide	4.0 × 10^6d	15 nM (brain, liver)⁶	3.6
		1 μM (flatus)^27,39	240
		1.9−20 μM (oral cavity)³¹	456−4800
sulfite	1.03 × 10² (sSQR)^e	0.1−1 μM^47,48	0.00062−0.0062
	9.4 × 10² (ndSQR)		0.0056−0.056
GSH	36.7 (sSQR)	1−10 mM^25,26	2.2−22
	0.38 (ndSQR)		0.023−0.23
MeSH	6.3 × 10⁴	0.2 μM (flatus)^27,39	0.76
		0.2−4 μM (oral cavity)³¹	0.76−14.7

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Data were obtained with ndSQR at 4 °C, except where denoted.

Data are from the references denoted.

Predicted k_obs values were calculated as k_on for nucleophile × estimated physiological concentration.

Data are from Landry et al.¹⁸

Data are from Mishanina et al.⁴

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 k_obs for the GSH-induced CT decay was 0.0013 s⁻¹ ± 0.0003 s⁻¹ at 4 °C, similar to the k_off value determined for GSH dissociation from the CT complex on ndSQR (0.0018 s⁻¹). On the basis of these data, we propose that the mechanism for decay of the GSH-induced CT complex involves reversal to the 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 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 V_max 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 k_obs versus concentration of sulfite for the sulfite-induced CT complex with ndSQR (Supporting Information 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 k_on of 9.4 × 10² ± 46 M⁻¹ s⁻¹ at 4 °C was obtained (Supporting Information Figure 2C), which is ~9-fold higher than for sSQR (1.03 × 10² M⁻¹ s⁻¹ at 4 °C).⁴ A spectrally monitored titration of the CT complex with sulfite yielded a K_D of 30 ± 1 μM at 4 °C, which was used with the k_on value to calculate a k_off of 0.028 ± 0.002 s⁻¹ at 4 °C. For comparison, a ~5-fold greater K_D (165 μM) and a slightly smaller k_off (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 k_on 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.

The addition of sulfide to the sulfite-induced CT complex led to its decay with concomitant FAD reduction (Supporting Information Figure 3), as seen with sSQR.⁴ Sulfide-mediated decay of the sulfite-induced CT complex in ndSQR yielded a rate constant of 0.020 ± 0.005 s⁻¹ at 4 °C, similar to the k_off 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-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 (Supporting Information Figure 4) and spectral features similar to the sulfide-induced CT complex, with absorbance maxima at 390 and 675 nm, isosbestic points at 423 and 505 nm, and a decrease in absorbance in the FAD-associated 450 nm feature (Figure 4A).^4,17 The k_obs for CT complex formation showed a linear dependence on MeSH concentration, yielding a k_on of 6.3 × 10⁴ ± 2.2 × 10³ M⁻¹ s⁻¹ at 4 °C (Figure 4B). A spectrally monitored titration of the CT complex with MeSH yielded a K_D of 38 ± 1 μM 4 °C, which was used to calculate a k_off of 2.4 ± 0.1 s⁻¹ at 4 °C. Thus, MeSH can react with ndSQR to form a CT complex, albeit with a k_on that is 63-fold lower than for sulfide (4 × 106 M–1 s–1).¹⁸

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 k_obs for CT complex formation on MeSH concentration. The data represent the mean ± SD for three independent experiments.

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 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, the 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, Supporting Information 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 k_cat for catalytic turnover under conditions in which sulfur transfer occurs from sulfide to GSH is 13 ± 2 s⁻¹ at 4 °C.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.

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 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 its decay is observed. (B) ndSQR (10 μM) was preincubated 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 preincubated 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.

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, Supporting Information Figure 6). The k_obs for CT complex decay was linearly dependent on the MeSH concentration, yielding a k of 5.4 ± 0.1 × 10⁴ M⁻¹ s⁻¹ at 4 °C (Figure 6B), which is similar to the k_on value obtained for the nucleophilic addition of MeSH into the resting enzyme (6.3 × 10⁴ M⁻¹ s⁻¹ at 4 °C (Figure 4B)).

Figure 6. — Presteady state sulfur transfer kinetics to MeSH and steadystate CoQ₁ reduction in ndSQR. (A) ndSQR (40 μM) was rapidly mixed 1:1 (v/v) with Na₂S (80 μM) and aged for ~35 ms 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 k_obs for the disappearance of the CT complex on the MeSH concentration. The data represent the mean ± SD 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 the addition of ndSQR (1 nM). The data represent the mean ± SD for three independent experiments, each run in duplicate.

Under steady-state turnover conditions, the introduction of MeSH to a reaction mixture containing sulfide, CoQ₁, and ndSQR yielded a k_cat 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 K_M 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 k_cat for sulfide oxidation in the presence of 100 μM MeSH was 6.6 ± 0.6 s⁻¹, which is ~25% higher than the k_obs of 5.2 ± 0.2 s⁻¹ obtained under stopped-flow conditions at the same temperature for the presteady 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 k_cat/K_M under steady-state turnover conditions (3.9 × 10⁵ M⁻¹ s⁻¹ at 25 °C) compared to GSH (k_cat/K_M = 1.6 × 10⁴ M⁻¹ s⁻¹ at 25 °C).¹⁸ However, in periodontal disease, which is marked by oxidative stress⁴¹ and decreased GSH,^42,43 aberrant 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 K_M(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 sulfur-transferases rhodanese³ or TSTD1⁴⁶ would regenerate MeSH, without net metabolism of MeSH occurring via this set of reactions.

Table 2.

Rate of SQR-Dependent Sulfide Oxidation Detected by O₂ Consumption in a Coupled Assay with PDO

reaction^a	O₂ consumption rate, μmol O₂/min	SQR turnover ratio^b
ndSQR, Na₂S, GSH, CoQ₁	0.0041 ± 0.001
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

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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.

SQR turnover ratios were calculated as described under Methods.

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 k_on 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 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-3-phophocholine (DHPC) was purchased from Avanti Polar Lipids.

Purification of sSQR and ndSQR.

Human SQR was purified as a detergent-solubilized enzyme as described previously.³ The sSQR was incorporated into nanodiscs containing 1-palmitoyl-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 at 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 K_D values for nucleophiles, CT complex formation was monitored in sSQR or ndSQR (10 μM) in 100 mM potassium phosphate buffer, at pH 7.4 and 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 three-parameter sigmoidal equation using the SigmaPlot software.

SQR Activity Assays.

Steady-state assays were conducted in 100 mM potassium phosphate buffer, at pH 7.4 and 25 °C, by monitoring the reduction of CoQ₁ at 278 nm (Δε_ox‑red = 12 000 M⁻¹ cm⁻¹) as described previously.^3,17 Stock solutions of CoQ₁ were prepared aerobically in 10 mM potassium phosphate, at pH 6.8, containing 10% (v/v) DHPC. The kinetic parameters K_M and V_max 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, at pH 7.4 and 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 k_obs for CT complex formation in ndSQR in the presence of acceptors were obtained in single mixing experiments as described previously.⁴ The spectra and k_obs 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 MeSH-induced 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 single-exponential 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, at 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 micromoles of O₂ consumed per minute. 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 × k_cat for acceptor). A ratio of 1:1 represents complete coupling. For predicted ndSQR turnovers, the k_cat values for acceptors used were 128 s⁻¹ for GSH as determined previously¹⁸ and 100 s⁻¹ for MeSH as determined in this study.

Supplementary Material

Supporting Information

NIHMS1011056-supplement-Supporting_Information.docx^{(924.5KB, docx)}

ACKNOWLEDGMENTS

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.

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00258.

Figures S1–S6 (PDF)

REFERENCES

(1).Khersonsky O, and Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem 79, 471–505. [DOI] [PubMed] [Google Scholar]
(2).Jensen RA (1976) Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol 30, 409–425. [DOI] [PubMed] [Google Scholar]
(3).Libiad M, Yadav PK, Vitvitsky V, Martinov M, and Banerjee R (2014) Organization of the human mitochondrial hydrogen sulfide oxidation pathway. J. Biol. Chem 289, 30901–30910. [DOI] [PMC free article] [PubMed] [Google Scholar]
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(20).Hildebrandt TM, and Grieshaber MK (2008) Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria. FEBS J. 275, 3352–3361. [DOI] [PubMed] [Google Scholar]
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(22).Shih VE, Abroms IF, Johnson JL, Carney M, Mandell R, Robb RM, Cloherty JP, and Rajagopalan KV (1977) Sulfite oxidase deficiency. Biochemical and clinical investigations of a hereditary metabolic disorder in sulfur metabolism. N. Engl. J. Med 297, 1022–1028. [DOI] [PubMed] [Google Scholar]
(23).Grings M, Moura AP, Parmeggiani B, Marcowich GF, Amaral AU, de Souza Wyse AT, 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. [DOI] [PubMed] [Google Scholar]
(24).Vincent AS, Lim BG, Tan J, Whiteman M, Cheung NS, Halliwell B, and Wong KP (2004) Sulfite-mediated oxidative stress in kidney cells. Kidney Int. 65, 393–402. [DOI] [PubMed] [Google Scholar]
(25).Kosower NS, and Kosower EM (1978) The glutathione status of cells. Int. Rev. Cytol 54, 109–160. [DOI] [PubMed] [Google Scholar]
(26).Meister A (1988) Glutathione metabolism and its selective modification. J. Biol. Chem 263, 17205–17208. [PubMed] [Google Scholar]
(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. [DOI] [PubMed] [Google Scholar]
(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. [DOI] [PubMed] [Google Scholar]
(29).Persson S, Edlund MB, Claesson R, and Carlsson J (1990) The formation of hydrogen sulfide and methyl mercaptan by oral bacteria. Oral Microbiol. Immunol 5, 195–201. [DOI] [PubMed] [Google Scholar]
(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. [DOI] [PMC free article] [PubMed] [Google Scholar]
(31).Blanchette AR, and Cooper AD (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. [DOI] [PubMed] [Google Scholar]
(32).Waller RL (1977) Methanethiol inhibition of mitochondrial respiration. Toxicol. Appl. Pharmacol 42, 111–117. [DOI] [PubMed] [Google Scholar]
(33).Finkelstein A, and Benevenga NJ (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. [DOI] [PubMed] [Google Scholar]
(34).Furne J, Springfield J, Koenig T, DeMaster E, and Levitt MD (2001) Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa. Biochem. Pharmacol 62, 255–259. [DOI] [PubMed] [Google Scholar]
(35).Pol A, Renkema GH, Tangerman A, Winkel EG, Engelke UF, de Brouwer APM, Lloyd KC, Araiza RS, van den Heuvel L, Omran H, Olbrich H, Oude Elberink M, Gilissen C, Rodenburg RJ, Sass JO, Schwab KO, Schafer H, Venselaar H, Sequeira JS, Op den Camp HJM, and Wevers RA (2018) Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis. Nat. Genet 50, 120–129. [DOI] [PMC free article] [PubMed] [Google Scholar]
(36).Iatropoulos A, Panis V, Mela E, Stefaniotis T, Madianos PN, 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. [DOI] [PubMed] [Google Scholar]
(37).Southerland JH, Taylor GW, Moss K, Beck JD, and Offenbacher S (2006) Commonality in chronic inflammatory diseases: periodontitis, diabetes, and coronary artery disease. Periodontol. 40, 130–143. [DOI] [PubMed] [Google Scholar]
(38).Dorfer CE, Becher H, Ziegler CM, Kaiser C, Lutz R, Jorss D, Lichy C, Buggle F, Bultmann S, Preusch M, and Grau AJ (2004) The association of gingivitis and periodontitis with ischemic stroke. J. Clin. Periodontol 31, 396–401. [DOI] [PubMed] [Google Scholar]
(39).Suarez FL, Springfield J, and Levitt MD (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. [DOI] [PMC free article] [PubMed] [Google Scholar]
(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. [DOI] [PubMed] [Google Scholar]
(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. [DOI] [PMC free article] [PubMed] [Google Scholar]
(42).Chapple IL, Brock G, Eftimiadi C, and Matthews JB (2002) Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease. Mol. Pathol 55, 367–373. [DOI] [PMC free article] [PubMed] [Google Scholar]
(43).Sculley DV, and Langley-Evans SC (2003) Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation. Clin. Sci 105, 167–172. [DOI] [PubMed] [Google Scholar]
(44).Tiranti V, Viscomi C, Hildebrandt T, Di Meo I, Mineri R, Tiveron C, Levitt MD, 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. [DOI] [PubMed] [Google Scholar]
(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. [DOI] [PMC free article] [PubMed] [Google Scholar]
(46).Libiad M, Motl N, Akey DL, Sakamoto N, Fearon ER, Smith JL, and Banerjee R (2018) Thiosulfate sulfurtransferase-like domain-containing 1 protein interacts with thioredoxin. J. Biol. Chem 293, 2675–2686. [DOI] [PMC free article] [PubMed] [Google Scholar]
(47).Ji AJ, Savon SR, and Jacobsen DW (1995) Determination of total serum sulfite by HPLC with fluorescence detection. Clin. Chem 41, 897–903. [PubMed] [Google Scholar]
(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 40, 3000–3004. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

NIHMS1011056-supplement-Supporting_Information.docx^{(924.5KB, docx)}

[R1] (1).Khersonsky O, and Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem 79, 471–505. [DOI] [PubMed] [Google Scholar]

[R2] (2).Jensen RA (1976) Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol 30, 409–425. [DOI] [PubMed] [Google Scholar]

[R3] (3).Libiad M, Yadav PK, Vitvitsky V, Martinov M, and Banerjee R (2014) Organization of the human mitochondrial hydrogen sulfide oxidation pathway. J. Biol. Chem 289, 30901–30910. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] (4).Mishanina TV, Yadav PK, Ballou DP, and Banerjee R (2015) Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase. J. Biol. Chem 290, 25072–25080. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] (5).Banerjee R (2017) Catalytic promiscuity and heme-dependent redox regulation of H2S synthesis. Curr. Opin. Chem. Biol 37, 115–121. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] (6).Furne J, Saeed A, and Levitt MD (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. [DOI] [PubMed] [Google Scholar]

[R7] (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 Signaling 17, 22–31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] (8).Mustafa AK, Gadalla MM, Sen N, Kim S, Mu W, Gazi SK, Barrow RK, Yang G, Wang R, and Snyder SH (2009) H2S signals through protein S-sulfhydration. Sci. Signaling 2, ra72. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R9] (9).Mishanina TV, Libiad M, and Banerjee R (2015) Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways. Nat. Chem. Biol 11, 457–464. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] (10).Filipovic MR, Zivanovic J, Alvarez B, and Banerjee R (2018) Chemical Biology of H2S Signaling through Persulfidation. Chem. Rev 118, 1253–1337. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] (11).Chiku T, Padovani D, Zhu W, Singh S, Vitvitsky V, and Banerjee R (2009) H2S biogenesis by human cystathionine gammalyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia. J. Biol. Chem 284, 11601–11612. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] (12).Singh S, Padovani D, Leslie RA, 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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] (13).Shibuya N, Tanaka M, Yoshida M, Ogasawara Y, Togawa T, Ishii K, and Kimura H (2009) 3-Mercaptopyruvate sulfur-transferase produces hydrogen sulfide and bound sulfane sulfur in the brain. Antioxid. Redox Signaling 11, 703–714. [DOI] [PubMed] [Google Scholar]

[R14] (14).Yadav PK, 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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] (15).Nicholls P, and Kim JK (1982) Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system. Can. J. Biochem 60, 613–623. [DOI] [PubMed] [Google Scholar]

[R16] (16).Nicholls P, Marshall DC, Cooper CE, and Wilson MT (2013) Sulfide inhibition of and metabolism by cytochrome c oxidase. Biochem. Soc. Trans 41, 1312–1316. [DOI] [PubMed] [Google Scholar]

[R17] (17).Jackson MR, Melideo SL, and Jorns MS (2012) Human sulfide:quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite. Biochemistry 51, 6804–6815. [DOI] [PubMed] [Google Scholar]

[R18] (18).Landry AP, Ballou DP, and Banerjee R (2017) H2S oxidation by nanodisc-embedded human sulfide quinone oxidoreductase. J. Biol. Chem 292, 11641–11649. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] (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. [DOI] [PubMed] [Google Scholar]

[R20] (20).Hildebrandt TM, and Grieshaber MK (2008) Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria. FEBS J. 275, 3352–3361. [DOI] [PubMed] [Google Scholar]

[R21] (21).Augustyn KD, Jackson MR, and Jorns MS (2017) Use of Tissue Metabolite Analysis and Enzyme Kinetics To Discriminate between Alternate Pathways for Hydrogen Sulfide Metabolism. Biochemistry 56, 986–996. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] (22).Shih VE, Abroms IF, Johnson JL, Carney M, Mandell R, Robb RM, Cloherty JP, and Rajagopalan KV (1977) Sulfite oxidase deficiency. Biochemical and clinical investigations of a hereditary metabolic disorder in sulfur metabolism. N. Engl. J. Med 297, 1022–1028. [DOI] [PubMed] [Google Scholar]

[R23] (23).Grings M, Moura AP, Parmeggiani B, Marcowich GF, Amaral AU, de Souza Wyse AT, 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. [DOI] [PubMed] [Google Scholar]

[R24] (24).Vincent AS, Lim BG, Tan J, Whiteman M, Cheung NS, Halliwell B, and Wong KP (2004) Sulfite-mediated oxidative stress in kidney cells. Kidney Int. 65, 393–402. [DOI] [PubMed] [Google Scholar]

[R25] (25).Kosower NS, and Kosower EM (1978) The glutathione status of cells. Int. Rev. Cytol 54, 109–160. [DOI] [PubMed] [Google Scholar]

[R26] (26).Meister A (1988) Glutathione metabolism and its selective modification. J. Biol. Chem 263, 17205–17208. [PubMed] [Google Scholar]

[R27] (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. [DOI] [PubMed] [Google Scholar]

[R28] (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. [DOI] [PubMed] [Google Scholar]

[R29] (29).Persson S, Edlund MB, Claesson R, and Carlsson J (1990) The formation of hydrogen sulfide and methyl mercaptan by oral bacteria. Oral Microbiol. Immunol 5, 195–201. [DOI] [PubMed] [Google Scholar]

[R30] (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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] (31).Blanchette AR, and Cooper AD (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. [DOI] [PubMed] [Google Scholar]

[R32] (32).Waller RL (1977) Methanethiol inhibition of mitochondrial respiration. Toxicol. Appl. Pharmacol 42, 111–117. [DOI] [PubMed] [Google Scholar]

[R33] (33).Finkelstein A, and Benevenga NJ (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. [DOI] [PubMed] [Google Scholar]

[R34] (34).Furne J, Springfield J, Koenig T, DeMaster E, and Levitt MD (2001) Oxidation of hydrogen sulfide and methanethiol to thiosulfate by rat tissues: a specialized function of the colonic mucosa. Biochem. Pharmacol 62, 255–259. [DOI] [PubMed] [Google Scholar]

[R35] (35).Pol A, Renkema GH, Tangerman A, Winkel EG, Engelke UF, de Brouwer APM, Lloyd KC, Araiza RS, van den Heuvel L, Omran H, Olbrich H, Oude Elberink M, Gilissen C, Rodenburg RJ, Sass JO, Schwab KO, Schafer H, Venselaar H, Sequeira JS, Op den Camp HJM, and Wevers RA (2018) Mutations in SELENBP1, encoding a novel human methanethiol oxidase, cause extraoral halitosis. Nat. Genet 50, 120–129. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] (36).Iatropoulos A, Panis V, Mela E, Stefaniotis T, Madianos PN, 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. [DOI] [PubMed] [Google Scholar]

[R37] (37).Southerland JH, Taylor GW, Moss K, Beck JD, and Offenbacher S (2006) Commonality in chronic inflammatory diseases: periodontitis, diabetes, and coronary artery disease. Periodontol. 40, 130–143. [DOI] [PubMed] [Google Scholar]

[R38] (38).Dorfer CE, Becher H, Ziegler CM, Kaiser C, Lutz R, Jorss D, Lichy C, Buggle F, Bultmann S, Preusch M, and Grau AJ (2004) The association of gingivitis and periodontitis with ischemic stroke. J. Clin. Periodontol 31, 396–401. [DOI] [PubMed] [Google Scholar]

[R39] (39).Suarez FL, Springfield J, and Levitt MD (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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R40] (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. [DOI] [PubMed] [Google Scholar]

[R41] (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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R42] (42).Chapple IL, Brock G, Eftimiadi C, and Matthews JB (2002) Glutathione in gingival crevicular fluid and its relation to local antioxidant capacity in periodontal health and disease. Mol. Pathol 55, 367–373. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R43] (43).Sculley DV, and Langley-Evans SC (2003) Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation. Clin. Sci 105, 167–172. [DOI] [PubMed] [Google Scholar]

[R44] (44).Tiranti V, Viscomi C, Hildebrandt T, Di Meo I, Mineri R, Tiveron C, Levitt MD, 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. [DOI] [PubMed] [Google Scholar]

[R45] (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. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R46] (46).Libiad M, Motl N, Akey DL, Sakamoto N, Fearon ER, Smith JL, and Banerjee R (2018) Thiosulfate sulfurtransferase-like domain-containing 1 protein interacts with thioredoxin. J. Biol. Chem 293, 2675–2686. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R47] (47).Ji AJ, Savon SR, and Jacobsen DW (1995) Determination of total serum sulfite by HPLC with fluorescence detection. Clin. Chem 41, 897–903. [PubMed] [Google Scholar]

[R48] (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 40, 3000–3004. [DOI] [PubMed] [Google Scholar]

PERMALINK

Modulation of Catalytic Promiscuity during Hydrogen Sulfide Oxidation

Aaron P Landry

David P Ballou

Ruma Banerjee

Abstract

Graphical Abstract

Figure 1.

Figure 2.

RESULTS AND DISCUSSION