Cytochrome c Reduction by H2S Potentiates Sulfide Signaling

Abstract

Hydrogen sulfide (H₂S) is an endogenously produced gas that is toxic at high concentrations. It is eliminated by a dedicated mitochondrial sulfide oxidation pathway, which connects to the electron transfer chain at the level of complex III. Direct reduction of cytochrome c (Cyt C) by H₂S has been reported previously but not characterized. In this study, we demonstrate that reduction of ferric Cyt C by H₂S exhibits hysteretic behavior, which suggests the involvement of reactive sulfur species in the reduction process and is consistent with a reaction stoichiometry of 1.5 mol of Cyt C reduced/mol of H₂S oxidized. H₂S increases O₂ consumption by human cells (HT29 and HepG2) treated with the complex III inhibitor antimycin A, which is consistent with the entry of sulfide-derived electrons at the level of complex IV. Cyt C-dependent H₂S oxidation stimulated protein persulfidation in vitro, while silencing of Cyt C expression decreased mitochondrial protein persulfidation in a cell culture. Cyt C released during apoptosis was correlated with persulfidation of procaspase 9 and with loss of its activity. These results reveal a potential role for the electron transfer chain in general, and Cyt C in particular, for potentiating sulfide-based signaling.

Figure 1.

Reduction of Cyt C by sulfide. (A) The initial spectrum of ferric Cyt C (10 μM) in 100 mM HEPES buffer [pH 7.4 (red)] shifts in the presence of 10 μM Na₂S (at 3, 5, 7, 10, 15, and 20 min, black lines) under aerobic conditions at 25 °C. (B) Kinetics of aerobic Cyt C reduction (10 μM) at 3, 5, and 10 μM Na₂S in 100 mM HEPES buffer (pH 7.4) at 25 °C. (C) Percent Cyt C [10 μM in 100 mM HEPES buffer (pH 7.4)] reduction as a function of sulfide concentration. The increase in absorbance at 550 nm was monitored after incubation with sulfide at 25 °C for 1 h under aerobic or anaerobic conditions. The red line is a sigmoidal fit to the experimental data with an S_0.5 of 2.9 ± 0.1 μM and an n of 2.4 ± 0.2. (D) pH dependence of Cyt C reduction by sulfide. The rate of Cyt C (2.5 μM) reduction by sulfide (500 μM) was determined by anaerobic stopped-flow spectroscopy as described in Methods. The data are the mean ± the standard deviation (SD) of four independent experiments as described in Methods. From the fit, estimates for pK_a1 and pK_a2 of 6.7 ± 0.1 and 8.5 ± 0.1, respectively, were obtained.

Figure 2.

Cyt C reduction kinetics and product analysis. (A) Kinetics of aerobic Cyt C reduction (10 μM) in the presence of 3, 5, and 10 μM Na₂S₂ in 100 mM HEPES buffer (pH 7.4) at 25 °C. Complete reduction with equimolar Na₂S₂ was seen within 5−10 min. (B) Kinetics of O₂ consumption in the presence of Cyt C and sulfide. Addition of sulfide to Cyt C [50 μM in 100 mM HEPES buffer (pH 7.4)] at 25 °C results in rapid initial consumption of O₂ followed by a slower phase. (C) Dependence of the concentration of O₂ consumed in the burst phase on sulfide concentration. A hyperbolic fit to the data (mean ± SD of three to five independent experiments) gives a K_act of 0.5 ± 0.1 mM and a maximal O₂ consumed of 35 ± 2 μM (red line), yielding a O₂ consumed:Cyt C stoichiometry of 0.7:1.0. (D) Consumption of sulfide and accumulation of thiosulfate and sulfite 2 h after addition of 800 μM Na₂S to 100 mM HEPES buffer (pH 7.4) without (black bars) or with (gray bars) 100 μM Cyt C at 25 °C under aerobic conditions. The initial levels of sulfide, thiosulfate, and sulfite were 756, 25, and 3.5 μM, respectively. (E) MS detection of sulfur products formed in the reaction of 10 μM Cyt C with 100 μM H₂S in ammonium carbonate buffer (pH 7.7) under aerobic conditions. The reaction was monitored for 15 min at room temperature. The observed spectrum is at the top, and the simulated spectra with an isotopic distribution are below.

Figure 3.

Cyt C stimulates H₂S-dependent persulfidation. (A) Mass spectrum of the reaction mixture (t = 6 min) containing 20 μM Cyt C, 100 μM H₂S, and 200 μM cysteine in ammonium carbonate buffer (pH 7.7) at 21 °C and simulated isotopic distributions for some of the identified species: 1, [CysSH + H]⁺; 2, [CysSSH + H]⁺; 3, [CysSSCys + H]⁺; 4, [CysSSCys + Na]⁺; 5, [2CysSSH + H]⁺. (B) Kinetics of [CysSSH + H]⁺ (m/z 151.9) and [CysSSCys + H]⁺ (m/z 241.0) formation. Data represent means ± SD of three independent experiments. (C) Persulfidation of HSA (10 μM) in phosphate-buffered saline (pH 7.4), incubated for 30 min at room temperature with 500 μM Na₂S (lane 1) and 2 μM (lane 2), 20 μM (lane 3), and 200 μM (lane 4) Cyt C. Data represent means ± SD (n = 3; *p < 0.001). (D) Persulfidation of purified (1 μM) EHTE-1 and (10 μM) ATR treated with either 100 μM H₂S (lane 1), 10 μM Cyt C (lane 2), or combination of both (lane 3). The results of two separate experiments are shown. Persulfidation (C and D) was detected by the CN-Cy3-based tag switch method (top panels), while equal loading was detected by Coomassie blue staining (bottom panels). The gel was artificially colorized using ImageJ to enhanced visualization of changes, and the fluorescence intensity scale is provided on the right. The persulfidation procedure is described in Supporting Materials and Methods. (E) Aerobic kinetics of Cyt C [20 μM in 100 mM HEPES buffer (pH 7.4) at 25 °C] reduction (monitored at 550 nm) by Na₂S (20 μM) with or without untreated or alkylated HSA (20 μM). Sulfide was added at time zero. (F) Kinetics of O₂ consumption after addition of 0.2 mM Na₂S to ferric Cyt C [50 μM in 100 mM HEPES buffer (pH 7.4)] with or without 50 μM HSA at 25 °C.

Figure 4.

Cyt C silencing decreases protein persulfidation. (A) Under normoxic conditions, silencing of Cyt C in HeLa cells (confirmed by Western blot analysis) led to a small but measurable decrease in basal persulfidation levels, which was increased by the mitochondrially targeted H₂S donor, AP39 (200 nM). The scale bar is 5 μm. (B) Exposure of HeLa cells to hypoxia for 1 h results in increased endogenous H₂S levels as detected by MeRho-Az. The scale bar is 10 μm. (C) Under hypoxic conditions, the levels of protein persulfidation are high but significantly reduced by Cyt C silencing. The scale bar is 10 μm. Representative microscopy images are given on the left, and quantitative image analysis is given on the right. The results represent the average fluorescence intensity (F. I.) detected from ≥40 cells ± the standard error of the mean of five independent images (*p < 0.001).

Figure 5.

Sulfide stimulates O₂ consumption by mammalian cells and controls persulfidation of mitochondrial proteins. (A) Cartoon showing electrons from H₂S oxidation enter the ETC at the level of complex III via the reduced quinone pool. Entry into complex IV via Cyt C can be monitored in the presence of antimycin A, a complex III inhibitor. (B and C) O₂ consumption rates of HT29 and HepG2 cells, respectively, before (control) and after treatment with 2 μg mL⁻¹ antimycin A (AA) followed by 20 μM Na₂S. Finally, antimycin A-treated cells were exposed to 5 mM KCN. The data are means ± SD of 12 (B) or 6 (C) independent experiments; the p value shows the statistical significance for the difference between antimycin A with and without sulfide-treated cells. (D) Protein persulfidation in purified mitochondria is affected by the ETC. Functional mitochondria were isolated from S. cerevisiae and preincubated with 2.5 μg mL⁻¹ antimycin A or 10 mM KCN for 10 min at 37 °C or treated with 1 μM H₂S for 20 min at 37 °C. Persulfidation was monitored by the improved tag swtich method. The first lane represents the basal persulfidation level in untreated functional mitochondria. The total protein load is shown in the gel on the right.

Figure 6.

Cyt C-mediates procaspase 9 persulfdation and activity. (A) Murine recombinant procaspase 9 (1 μM) was incubated with 5 μM H₂S for 30 min at 37 °C in the absence (lane 1) or presence of 0.5 (lane 2) and 2 μM (lane 3) Cyt C. Persulfidation was detected by the CN-Cy3-based tag switch method (top panel), while equal loading was confirmed by Coomassie blue staining (bottom panel). (B) H₂S inhibits Cyt C-induced caspase 9 activation in cell lysates. Cyt C (500 nM) was added to freshly prepared HeLa cell lysates, and caspase 9 activity was monitored using a fluorescence caspase 9 kit. Simultaneous addition of Cyt C and H₂S (10 μM) inhibited the Cyt C-induced increase in fluorescence. (C) Cyt C-induced caspase 9 activation in Jurkat cells is inhibited by H₂S donors. Apoptosis was induced by staurosporine (ST, 2.5 μM), and caspase 9 activity was monitored by flow cytometry using the caspase 9 FITC staining kit 4 and 6 h after induction. Treatment of cells with GYY4137 (100 μM) or ammonium tetrathiomolybdate (ATTM, 200 μM) reduced fluorescence. (D) Inhibition of caspase 3 activation in cells treated with antimycin A and the slow-releasing H₂S donor, GYY4137 (100 μM). HeLa cells were incubated with antimycin A (300 μM) overnight to induce the release of Cyt C from mitochondria. GYY4137, when used, was added 2 h prior to antimycin A. Caspase 3 activity was monitored in cell lysates as described for panel B. (E) Persulfidation of procaspase 9 in HeLa cells treated with 2.5 μM ST (1) that were either pretreated for 2 h (2) or treated for the last 2 h (3) with GYY4137 (100 μM): (left) total procaspase 9 levels and (right) persulfidation (PSSH) levels of procaspase 9. PSSH levels were quantified by measuring the difference in fluorescence between DTT-treated and untreated samples and normalizing this to the total procaspase 9 levels. The strategy used for sample processing to detect total versus persulfidated procaspase 9 is explained in the Supporting Information and Supporting Figure 5.

Figure 7.

Protein persulfidation catalyzed by the Cyt C/H₂S couple.