Figure 1.

Stimulation of O₂ consumption by sulfide. Representative oxygen consumption traces (blue) and corresponding O₂ consumption rate (OCR, red traces) acquired with normoxic (a) or hypoxia-treated SW480 cells (b), following the addition of cells (4 × 10⁶), rotenone (Rot., 5 μM) either alone (top traces) or plus antimycin A (Ant. A, 5 μM, bottom traces), and subsequent injection of a sulfide solution (3-5 mM) at increasing rates (10 nL·s⁻¹, 20 nL·s⁻¹, 40 nL·s⁻¹, 80 nL·s⁻¹, and 160 nL·s⁻¹, corresponding, respectively, to injections #1 to #5). (c, d) OCR values obtained from the oxygraphic traces, respectively shown in (a) and (b), measured at basal condition and upon sulfide injection at increasing rates after addition of rotenone alone (full symbols) or rotenone plus antimycin A (hollow symbols). Mitochondrial H₂S consumption in normoxic cells was calculated by determining the OCR measured at the highest non-inhibitory H₂S injection rate (highlighted with grey bar in (c)) and subtracting the OCR measured after the addition of rotenone (horizontal dashed line in (c)), yielding ΔOCR_(-Ant). Then, the ΔOCR at the corresponding sulfide injection in the antimycin A-containing measurement was calculated in the same manner, yielding ΔOCR_(+Ant). By calculating ΔOCR_(‐Ant) − ΔOCR_(+Ant), the genuine mitochondrial H₂S-dependent OCR (OCR_mitH2S) was determined. Finally, OCR_mitH2S was multiplied by 1.33 to account for the number of H₂S molecules consumed per O₂ molecule, yielding an estimated sulfide oxidizing activity of 12.7 nM H₂S·s⁻¹·10⁶ cells⁻¹. Employing the same procedure for cells exposed to hypoxia (b, d), an activity of 9.5 nM H₂S·s⁻¹·10⁶ cells⁻¹ was estimated.