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 O2·mg−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.
Figure 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) (μm). (A) Oxidation of 50 μm 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 μm 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 μm GSSH had been consumed.
Download figure to PowerPoint
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).
|Compartment||Activity of the pathway|
|Mitochondria||14.39 ± 2.37||25.43 ± 2.22|
|Membranes||56.63 ± 8.21||226.32 ± 11.41|
|Matrix||138.11 ± 27.95||133.16 ± 14.19|
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-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 . 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 μm 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 1D 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 U·mg−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 . 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 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 . 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 184.108.40.206) . 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).
Figure 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).
Download figure to PowerPoint
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 (μm)||Km SO32− (μm)||Activity (U·mg−1)||Km S2O32− (mm)||Km KCN (mm)|
|Rat||4.15 ± 0.52||57.3 ± 7.1||7.93 ± 0.39||615.2 ± 106.9||12.4 ± 1.6||0.31 ± 0.02|
|Lugworm||8.52 ± 1.00||70.0 ± 20.2||46.3 ± 5.0||3.29 ± 0.46||4.32 ± 0.26||7.51 ± 0.52|
|Bovine||0.40 ± 0.11||61.3 ± 17.8||21.8 ± 3.6||94.5 ± 5.6||16.2 ± 2.0||0.087 ± 0.009|