1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15-20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100-400 μg/ml) or 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2-3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicyl-hydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 μg/mg protein, a finding consistent with the lack of antimycin binding sites. 5. The Km O2 was less than 2 μM for the cyanide-sensitive respiration and 4-5 μM for the cyanide-insensitive respiration. 6. ADP stimulated the oxidation of pyruvate and succinate by mitochondria of the wild type and the mutant Cl1 by 2-4-fold. AMP selectively stimulated the cyanide-insensitive respiration. Preincubation of Paramecium mitochondria with trace of ADP increased their cyanide sensitivity. This effect of ADP was prevented by uncouplers. 7. P O ratios in wild type mitochondria were close to 2 for pyruvate oxidation, between 1 and 2 for succinate oxidation and between 0.5 and 1 for ascorbate oxidation. In mitochondria of the mutant Cl1, P O ratios for pyruvate and succinate oxidation were lower by about one third in comparison to those of wild type mitochondria. Addition of cyanide decreased P O ratios with pyruvate and succinate as substrates for both types of mitochondria (30% for the mutant, 40-70% for the wild type). Ascorbate oxidation by mitochondria of the mutant was not coupled to ATP synthesis and did not generate a membrane potential. 8. H+ efflux dependent on O2 pulses was measured in the presence of increasing concentrations of cyanide. Using succinate as substrate, the H+ O ratio was 4 for wild type mitochondria and 1.5 for mitochondria of the mutant. The curve relating H+ efflux to added cyanide was biphasic in both cases. There was a rapid drop of H+ O to 30-50% of the original value when cyanide was added to 0.1 mM. Upon further addition of cyanide up to 1 mM, the H+ O ratio decreased slowly towards zero. 9. Under steady-state conditions of respiration, the percentage of oxidation of the respiratory carriers increased from ubiquinone to cytochrome aa3, except for the b-type cytochromes which remained more oxidized than cytochromes cc1. 10. The CO-inhibited respiration in mitochondria of the mutant Cl1 was partially photorestored by white light. As the only CO-reactive pigment in these mitochondria is a b-type cytochrome, the photorestoration data suggest that the CO-reactive cytochrome b may be autooxidizable. 11. The above data suggest that the respiratory chain of Paramecium mitochondria possesses three terminal oxidases, namely cytochrome aa3 (absent in mitochondria of the mutant Cl1), a CO-sensitive b-type cytochrome and a salicylic hydroxamic acid-sensitive oxidase. © 1979.
