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Hydrogen sulfide (H2S) is a potent inhibitor of cytochrome c oxidase (COX) and, thus, of mitochondrial respiration . Since H2S was reported to induce a suspended animation-like status characterized by reduced energy expenditure and hypothermia , we sought to determine the effect of hypothermia on mitochondrial respiratory capacity and H2S-related COX inhibition. We further studied the influence of variations in pH on both variables.
All measurements were conducted in digitonin-permeabilised cultured peritoneal macrophages using high-resolution respirometry  (Oxygraph-2 k, Oroboros, Austria). Maximum mitochondrial respiration (1 to 2 Mio cells/ml respiration medium) was achieved in the uncoupled state by adding pyruvate, malate, glutamate and succinate as respiratory substrates. Then, in one of the two chambers of the oxygraph, mitochondrial respiration was inhibited stepwise by incremental concentrations of the H2S donor Na2S (1 to 64 μM). In the parallel chamber, the identical inhibitor titration sequence was preceded by the inhibition of the respiratory chain by rotenone and antimycin A followed by the selective stimulation of the COX after addition of ascorbate and TMPD. COX excess capacity (% of OXPHOS) was calculated based on the ratio of inhibition of mitochondrial respiration with full operating respiratory chain versus the COX-stimulated condition. This experimental sequence was repeated at 37°C and 25°C with a medium pH of 7.1 and then at 37°C with a pH of 6.8 and 7.7.
COX excess capacity (median (quartiles)) was significantly higher at 25°C than at 37°C (134 (113; 140) vs 61 (47; 79)), most likely due to the almost halved mitochondrial respiratory capacity at hypothermia (50 (37; 63) vs 95 (81; 103) pmolO2/s × Mio cells). Changing the medium pH from 6.8 to 7.7 significantly increased the COX excess capacity (91 (79; 103) vs 71 (64; 82) pmolO2/s × Mio cells), which again was related to the significantly lower mitochondrial respiratory capacity with more acidic conditions (80 (70; 89) vs 94 (85; 98)).
Our results suggest that COX excess capacity is temperature as well as pH dependent in peritoneal macrophages. This effect may protect cells from H2S toxicity at low temperatures and high pH values.
Supported by the Deutsche Forschungsgemeinschaft (KFO 200).