Báo cáo khoa học: Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria

Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria

Tác giả: Tatjana M. Hildebrandt and Manfred K. Grieshaber

Lĩnh vực: Zoophysiology

Nội dung tài liệu: Nghiên cứu này khám phá con đường trao đổi chất phức tạp của quá trình oxy hóa sulfide thành thiosulfate trong ty thể của động vật có vú và động vật không xương sống. Nó xác định ba enzyme chính tham gia: sulfide: quinone oxidoreductase (SQR), sulfur dioxygenase và sulfur transferase. Quá trình này bắt đầu bằng việc SQR oxy hóa sulfide thành lưu huỳnh dạng persulfide, sau đó sulfur dioxygenase oxy hóa persulfide thành sulfite, và cuối cùng sulfur transferase chuyển đổi sulfite và một persulfide khác thành thiosulfate. Nghiên cứu nhấn mạnh vai trò sinh lý quan trọng của con đường này trong việc điều hòa sulfide nội sinh và giải độc, đặc biệt là ở các sinh vật thích nghi với môi trường giàu sulfide.

Mục lục chi tiết:

• Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria
• Keywords
• Correspondence
• (Received 27 February 2008, revised 15 April 2008, accepted 29 April 2008)
• doi:10.1111/j.1742-4658.2008.06482.x
• Hydrogen sulfide is a potent toxin of aerobic respiration, but also has physiological functions as a signalling molecule and as a substrate for ATP production. A mitochondrial pathway catalyzing sulfide oxidation to thiosulfate in three consecutive reactions has been identified in rat liver as well as in the body-wall tissue of the lugworm, Arenicola marina. A membrane-bound sulfide : quinone oxidoreductase converts sulfide to persulfides and transfers the electrons to the ubiquinone pool. Subsequently, a putative sulfur dioxygenase in the mitochondrial matrix oxidizes one persulfide molecule to sulfite, consuming molecular oxygen. The final reaction is catalyzed by a sulfur transferase, which adds a second persulfide from the sulfide: quinone oxidoreductase to sulfite, resulting in the final product thiosulfate. This role in sulfide oxidation is an additional physiological function of the mitochondrial sulfur transferase, rhodanese.
• Hydrogen sulfide was known as a toxic pollutant long before its physiological functions became apparent. The main effects of sulfide poisoning are the loss of central respiratory drive due to lesions in the brain stem, and inhibition of cytochrome oxidase, leading to impaired aerobic energy metabolism [1]. Sulfide-rich environments occur naturally in the sediments and grass marshes of the intertidal zone, in deep-sea hydrothermal vents, and in the hypolimneon of eutrophic lakes [2]. Moreover, mammalian as well as invertebrate tissues, such as the body wall of the lugworm Arenicola marina, enzymatically produce sulfide [3,4].
• In mammals, H2S acts as a gaseous transmitter and regulates several physiological processes [5]. Altered sulfide metabolism is associated with a number of disorders, such as Alzheimer’s disease, Down’s syndrome and ulcerative colitis [6-8]. Within a narrow concentration range, the effects of sulfide change from physiological to highly toxic, and therefore regulatory mechanisms are necessary to control endogenous sulfide levels within the physiological range.
• Sulfide is catabolized mainly by oxidation to non-toxic sulfur compounds. Mammals rapidly oxidize sublethal concentrations of sulfide to sulfate and excrete it in the urine [1,9]. The main sites of sulfide oxidation are liver and colon tissues, and the enzyme activity is present in the mitochondria [10,11]. Thiosulfate is produced as an obligate intermediate by a putative sulfide oxidase, which has not yet been identified [12]. A mitochondrial pathway also catalyzes sulfide oxidation in many sulfide-adapted invertebrates including polychaetes, crustaceans and bivalves [2]. The lugworm A. marina is often used to study strategies of sulfide tolerance, because it is highly abundant in sandy to muddy intertidal flats, where high micromolar concentrations of sulfide regularly occur [2].
• Abbreviations
• Mitochondrial sulfide oxidation in A. marina is linked to the electron transport chain at the level of ubiquinone via a sulfide: quinone oxidoreductase (SQR) [13]. As in most of the invertebrates studied so far, thiosulfate is the main excretion product of sulfide oxidation in A. marina [14], but the complete enzyme system catalyzing mitochondrial thiosulfate production is still unknown. In this paper, we provide further insights into the biochemical pathway of sulfide oxidation to thiosulfate in both mammalian and invertebrate mitochondria.
• Table 1. Rates of sulfide and persulfide oxidation. Rates of H2S oxidation in isolated rat and lugworm mitochondria (nmol O2-mg-1. min¯¹), sulfide: quinone oxidoreductase activities of mitochondrial membranes (nmol reduced decyl ubiquinone mg¯¹ min¯¹), and rates of GSSH oxidation in mitochondrial matrix fractions (nmol O2-mg-1. min¯¹).
• Results
• Mitochondrial sulfide oxidation
• Mitochondria isolated from both rat liver and lugworm body-wall tissue quantitatively converted sulfide to thiosulfate, consuming corresponding amounts of molecular oxygen (Fig. 1A). The respiratory rates of isolated lugworm mitochondria were similar with added succinate (27.3 ± 3.5 nmol O2mg¯¹·min¯¹) or sulfide (Table 1) as a substrate, but the respiratory control ratio was slightly higher with the carbon substrate (1.96 ± 0.41 versus 1.65 ± 0.17). In rat liver mitochondria, sulfide oxidation was significantly less coupled than succinate respiration (respiratory control ratios 1.41 ± 0.23 and 3.56 ± 0.98, respectively), with oxygen consumption rates for succinate respiration (58.0 ± 7.7 nmol O2·mg¯¹·min¯¹) fourfold higher than those for sulfide oxidation (Table 1). Myxothiazole and cyanide, which are inhibitors of complex III and complex IV of the respiratory chain, respectively, completely blocked sulfide oxidation.
• Sulfide: quinone oxidoreductase
• Membrane preparations from mitochondria of rat liver as well as lugworm body-wall tissue were able to reduce externally added decyl ubiquinone in the presence of sulfide (100 µm) as a substrate. When 30 μμ decyl ubiquinone was reduced, approximately the same concentration of sulfide was consumed, and stoichiometric amounts of cyanolysable sulfane sulfur (detected as SCN¯) were produced (Fig. 1B). Neither thiosulfate,
• Fig. 1. Stoichiometries of mitochondrial sulfide oxidation and partial reactions in rat liver and in the body-wall musculature of Arenicola marina: decrease in substrate concentrations (white bars) and increase of product concentrations (grey bars) (μμ). (A) Oxidation of 50 μΜ H2S by isolated mitochondria required oxygen, and thiosulfate was produced. (B) Mitochondrial membranes oxidized H2S to persulfides, which were detected as SCN after cyanolysis. Samples were taken after exactly 30 μμ of the artificial electron acceptor decyl ubiquinone had been reduced. (C) Mitochondrial matrix fractions produced S2O32 from GSSH and O2. (D) Isolated sulfur dioxygenases oxidized GSSH to SO32, consuming O2. (E) Purified sulfur transferases produced S2O32 from SO32 and GSSH. (F,G) Isolated sulfur dioxygenases mixed with purified persulfide transferase (F) or bovine rhodanese (G) converted GSSH and O2 to S2O32. Samples were taken after 79.3 ± 4.5 μΜ GSSH had been consumed.
• SQR activity was four times higher in lugworm than in rat mitochondrial membranes (Table 1). Substrate saturation was achieved at low micromolar sulfide concentrations (Km = 9.94 ± 1.36 μμ for lugworm and 2.87 ± 0.29 µm for rat membranes). The SQR activity of rat mitochondrial membranes decreased when the ambient sulfide concentration exceeded 300 μμ, whereas SQR activity of lugworm mitochondrial membranes was not inhibited by up to 2 mm H2S. Hardly any SQR activity was detectable if KCN was omitted from the assay. Thus, the persulfide acceptor is essential for the SQR reaction in vitro.
• Persulfide oxidation in the mitochondrial matrix
• SQR produces sulfane sulfur, so the complete reaction of sulfide to thiosulfate requires at least one further oxidative step. Therefore, we looked for an enzyme activity capable of elemental sulfur oxidation. In combination with 1 mm glutathione (GSH), elemental sulfur was oxidized to thiosulfate in an oxygen-dependent manner by isolated matrix fractions of rat and lugworm mitochondria (Fig. 2A,B). The sulfane sulfur-
• Oxygen concentration (μm)
• Concentration of sulfur compound (µм)
• Fig. 2. Original traces of persulfide oxidation and partial reactions in the mitochondrial matrix of rat liver and the body-wall musculature of Arenicola marina. Time courses of complete reactions of 80 μμ GSSH (●) were recorded, together with the corresponding concentrations of O2 (-), S2O32- (□) and SO32- (▲). (A) 65 µg-mL-¹ rat mitochondrial matrix; (B) 48 µg-mL¯¹ lugworm mitochondrial matrix; (C) 20 µg-mL-¹ partially purified rat sulfur dioxygenase; (D) 9 µg-mL-¹ partially purified lugworm sulfur dioxygenase; (E) 3 µg·mL-¹ purified rat sulfur transferase; (F) 2 µg-mL-¹ partially purified lugworm sulfur transferase.
• oxidizing enzyme activity is tentatively referred to as sulfur dioxygenase, as it corresponds to an enzyme catalyzing the glutathione-dependent oxidation of elemental sulfur in acidophilic thiobacilli, in which glutathione persulfide (GSSH) is the actual substrate [15]. GSSH was generated by injection of a saturated acetonic sulfur solution into the GSH-containing assay mixture. In the rat and lugworm mitochondrial matrix, half maximal sulfur dioxygenase activities were achieved in the presence of 0.31 ± 0.03 and 0.22 ± 0.03 mm GSH, respectively. Neither elemental sulfur nor GSH were metabolized when added separately. The acetone used as a solvent for elemental sulfur did not inhibit sulfur dioxygenase activity.
• Persulfide oxidation rates were identical in the rat and lugworm mitochondrial matrix (Table 1). Complete oxidation of the 79.3 ± 4.5 μμ GSSH that was used as a substrate required about 40-50 µm molecular oxygen, with thiosulfate being the main product (Fig. 1C). Only small amounts of sulfite and sulfate (≤ 5 µM) accumulated. No oxygen consumption or thiosulfate production was detectable with sulfide as a substrate.
• Two enzymes were necessary to convert sulfane sulfur to thiosulfate in the mitochondrial matrix. The sulfur dioxygenase produced sulfite and only minor amounts of thiosulfate from GSSH and molecular oxygen (Figs ID and 2C,D). No further cofactors were necessary, but the sulfur dioxygenase activity increased by about 25% in the presence of ascorbate (2.5 mm). The sulfur dioxygenase could be purified 44-fold from rat liver and 72-fold from lugworm body-wall tissue, resulting in specific activities of 0.87 ± 0.04 and 0.85 ± 0.24 Umg¯¹, respectively. Based on the results of size-exclusion chromatography, both enzymes had a molecular weight of about 46 kDa. Unfortunately the yields were rather low (3-7%), and homogeneity was not achieved as the sulfur dioxygenase activity rapidly disappeared after ion chromatography.
• The final reaction step was catalyzed by a sulfur transferase, which is defined as an enzyme that transfers sulfane atoms from a donor molecule to a thiophilic acceptor substrate [16]. The sulfur transferases isolated from both rat and lugworm mitochondria stoichiometrically transferred persulfide groups from GSSH to sulfite, producing thiosulfate (Figs 1E and 2E,F), and this activity is referred to as persulfide transferase activity. The functional role of the sulfur transferase for thiosulfate production during persulfide oxidation was demonstrated by size-exclusion chromatography of the mitochondrial matrix constituents (Fig. 3). Only if the sulfur dioxygenase-containing fractions (peak fractions from 55.5-57.5 mL) were
• Fig. 3. Size-exclusion chromatography (Su- perdex 75) of mitochondrial matrix fractions from (A) rat liver and (B) the body-wall musculature of Arenicola marina. Conditions: 0.1 м phosphate buffer (pH 7.4), flow rate=0.5 mL min¯¹, fraction volume = 2 mL. Volume activities (percentage of maximal activity) were determined for sulfur dioxygenase () and rhodanese (▲). In addition, the concentrations (µM) of SO32- (0) and S2O32 (A) (the products of persulfide oxidation) in each fraction mixed with the same volume of fraction 28 (55.5-57.5 mL elution volume) are shown. Gray lines indicate the absorbance at 280 nm.
• combined with specific later-eluting fractions (63- 69 mL for rat and 69-75 mL for lugworm samples) could GSSH be converted to thiosulfate (Fig. 3, open triangles). The main mitochondrial sulfur transferase, rhodanese, is normally detected by its in vitro activity as a thiosulfate : cyanide sulfur transferase on the basis of thiocyanate production [17]. For both animals studied, rhodanese activity (Fig. 3, filled triangles) co-eluted with the thiosulfate-producing enzyme, which was purified to homogeneity from rat liver. It comprised a single polypeptide of about 35 kDa (Fig. 4A), and the sequence of tryptic peptides was identical with the published rhodanese sequence (EC 2.8.1.1) [18]. The final purification product of the lugworm sulfur transferase was enriched 406-fold, but still contained myoglobin and two proteins of approximately 16.5 kDa that could not be identified (Fig. 4B).
• Fig. 4. Silver-stained SDS-PAGE gels of purified sulfur transferases. m, molecular mass standard (size indicated). (A) Sulfur transferase from rat liver mitochondrial matrix after size-exclusion and cation-exchange chromatography (4 µg). (B) Sulfur transferase from the matrix fraction of mitochondria isolated from the body-wall musculature of A. marina after size-exclusion chromatography (5.5 µg).
• When mixed with the isolated sulfur transferases, the sulfur dioxygenases from rat and lugworm mitochondria oxidized persulfides to thiosulfate, showing a stoichiometry similar to that for the matrix fractions (Fig. 1F). The sulfur transferase could be functionally replaced by bovine rhodanese purchased from Sigma- Aldrich (Taufkirchen, Germany) (Fig. 1G).
• The affinities of the sulfur transferases from rat and bovine liver, as well as the lugworm enzyme, for the substrates of the persulfide transferase reaction, persulfides and sulfite, were significantly higher than for the substrates of the rhodanese reaction, thiosulfate and cyanide (Table 2). The Km values for GSSH and sulfite were in the low micromolar range, whereas millimolar
• concentrations of thiosulfate were required for half- maximal rhodanese activity. Differences were seen with respect to the enzyme activities at substrate saturation (Table 2). The rat liver sulfur transferase produced thiocyanate about 150 times faster than thiosulfate, and for bovine rhodanese the ratio was 230. In contrast, the lugworm sulfur transferase had more than twice as much persulfide transferase activity as rhodanese activity.
• Table 2. Kinetic properties of the persulfide transferase and rhodanese activities of sulfur transferases. Persulfide transferase activities (1 unit = 1 µmol S2O32 min¯¹) and rhodanese activities (1 unit = 1 µmol SCN-min¯¹) of sulfur transferases purified from rat liver and the body-wall musculature of Arenicola marina and of bovine liver rhodanese purchased from Sigma-Aldrich. The Km values were determined by fitting the data to the Michaelis-Menten equation using SIGMAPLOT version 9.01 (Systat Software) and the enzyme kinetic module 2.0.
• Persulfide sulfur transferase
• Thiosulfate sulfur transferase (rhodanese)
• Activity (U-mg-1)
• Km GSSH (μΜ)
• Km SO32- (μμ)
• Activity (U-mg-1)
• Km S2O32- (мм)
• Km KCN (тм)
• Rat
• Lugworm
• Bovine
• Sulfur dioxygenase
• H2S2O3
• O2+ H₂O
• H2SO3
• 2 H2S 2 SQR-SSH
• S
• SQR
• Sulfur transferase
• O2 2 H₂O
• IIII
• IV
• Matrix
• Cytosol
• Fig. 5. Proposed model of mitochondrial sulfide oxidation. A membrane-bound sulfide: quinone oxidoreductase (SQR) oxidizes sulfide (H2S) to the level of elemental sulfur, simultaneously reducing a cysteine disulfide such that a persulfide group is formed at one of the cysteines (SQR-SSH) [13]. The electrons are fed into the respiratory chain via the quinone pool (Qox/Qred), and finally transferred to oxygen by cytochrome oxidase (complex IV). A sulfur dioxygenase in the mitochondrial matrix oxidizes persulfides to sulfite (H2SO3), consuming