![]() Therefore, to observe SQR activity with a minimal interference of the inhibiting effects of sulfide, it is advisable to maintain the sulfide concentration in the 10 μ M range or below. ![]() Because of the positive feedback loop, this recovery phase shows an acceleration of oxygen consumption that culminates short before returning to the initial rate. However, even in conditions of severe inhibition, sulfide-consuming reactions continue at a low rate and moreover nonbiological processes (evaporation auto-oxidation) participate to an unavoidable sulfide concentration decline. 5–7), and this is heavily influenced by the maximal activity of SOU that is present. Concentrations in the 10–100 μ M range are usually required to observe a net decrease in cellular/mitochondrial oxygen consumption ( Figs. It exists therefore a range of concentrations of sulfide with which no decrease or even an increase in oxygen consumption is observed although a net decrease in electron transfer in mitochondrial respiratory chain (and deterioration of the ATP production) is likely to occur. Schematically, if SOU is not limiting, more than a 67% inhibition of the electron flux in the mitochondrial respiratory chain would be necessary to result a net decrease in oxygen consumption. However, the presence of the dioxygenase in SOU complicates the situation because when sulfide is oxidized, two-thirds of this oxygen consumption is explained by the dioxygenase of SOU and one-third by complex IV ( Fig. ![]() When other mitochondrial oxidations (normal carbon metabolism) take place at the same time than sulfide oxidation a net decrease in oxygen consumption rate after sulfide injection directly evidences the inhibition by sulfide ( Figs. Accordingly, if SOU activity is modest with regard to that of complex IV, a considerable inhibition of the latter (hence high concentration of sulfide) might be tolerated before the sulfide oxidation rate declines ( Fig. In fact sulfide inhibits the complex IV in a non-competitive manner ( Cooper & Brown, 2008) and therefore decreases the maximal activity without affecting the affinity for oxygen. The mechanism for this inhibition lies in the dependence of SOU activity from the activity of complex IV. A net inhibition of sulfide oxidation by a too high sulfide concentration is detected when a further increase in sulfide concentration in fact decreases the SOU reaction rate (above 8 μ M in Fig. With this approach, the estimation of the apparent K m for sulfide oxidation by isolated mitochondria led to a value around 2 μ M ( Szabo et al., 2014), thus lower but close enough to the concentration causing a detectable inhibition of complex IV (4–5 μ M and more, see above). When SOU is present, the addition of sulfide in the high nanomolar and low micromolar range causes a sharp increase in the oxygen consumption rate to be distinguished from the injection artifact ( Fig. ![]() This situation is closer to physiology as sulfide oxidation is supposed to occur simultaneously with all the other “normal” organic substrates available, but the interpretation is more complicate. Another possibility is to administrate sulfide in the presence of a preexisting mitochondrial/cellular respiratory rate supported by endogenous or exogenous carbon-containing substrate(s). This occurs probably because carbon metabolism stalls and pathways other than complex I able to yield hydrogen to coenzyme Q run out of substrates. This is the case when the mitochondrial complex I is inhibited (usually with rotenone), and this blockade of the reoxidation of NADH slows down cellular respiration to a value close to that obtained with complete inhibition of mitochondrial respiratory chain. SOU activity could be best evidenced in cells/mitochondria in conditions where no other substrate than sulfide is usable. Frédéric Bouillaud, in Methods in Enzymology, 2015 4.3 Concentration dependence of SOU activity
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