Comp. Biochem. Physiol. Vol. 74B, No. 3, pp. 567 to 571, 1983 0305-0491/83/030567-05503.00/0 Printed in Great Britain. © 1983PergamonPress Ltd ADAPTATION OF TERMINAL RESPIRATION PATHWAYS OF TETRAHYMENA PYRIFORMIS ST TO GROWTH WITH ADDED IRON OR CARBONYL CYANIDE m-CHLOROPHENYLHYDRAZONE MARK D. UN1TT*, ROBERTI. SCOTTand DAVID LLOYD Department of Microbiology, University College, Newport Road, Cardiff CF2 1TA, Wales, UK Abstract--1. Tetrahymena pyriformis grown in medium which contained 0.6/~g/ml iron possesses at least three pathways of terminal respiration which are inhibited by cyanide, azide and salicylhydroxamicacid respectively. 2. Mid-exponential phase cultures grown in the presence of 5.6/~g/ml iron and glucose showed the same respiratory properties in terms of respiration rate and inhibitor sensitivity as cultures grown in the absence of added iron and glucose. 3. The respiration of mid-exponential phase cultures adapted to growth in the presence of 2.5/~M- carbonyl cyanide m-chlorophenylhydrazone was incompletely inhibited by a combination of cyanide, azide and salicylhydroxamic acid. 4. The residual respiration accounted for between 25 and 35~o of the total, indicating synthesis of a novel terminal oxidase or gross modification of existing terminal respiratory pathways in the presence of the uncoupler of energy conservation. 5. The sole functional cytochrome oxidase present in organisms grown in the absence or presence of added iron and glucose or after adaptation to uncoupler was cytochrome a6zo; no other CO-reacting haemoprotein was detected in photochemical action spectra. INTRODUCTION Many eukaryotic micro-organisms show a residual respiration in the presence of cyanide, azide or carbon monoxide, even when these inhibitors are present in sufficiently high concentrations to inhibit cytochrome c oxidase completely (Lloyd, 1974). Such inhibitor studies on protozoa have demonstrated the presence of at least two terminal oxidases, apart from cyto- chrome c oxidase, in the trypanosome Trypanosoma mega (Hill & Degn, 1977) and in the soil amoeba Acanthamoeba castellanii (Lloyd et al., 1979). Respiration of the ciliated protozoon Tetrahymena pyriformis has several unusual features and has been the subject of study since Baker & Baumberger (1941) noted its atypical a-type cytochromes. Studies of the effects of respiratory inhibitors on intact T. pyriformis have shown that the process of terminal oxidation in this protozoon can be resolved into five distinct path- ways, terminating in at least three oxidases (Lloyd et al., 1980). Pathway 1 is inhibited by azide, cyanide, CO and H2S. A second major pathway is inhibited by much higher cyanide concentrations and by H2S. Pathway 3 is H2S-sensitive but cyanide-insensitive, while a fourth pathway is inhibited only by high con- centrations of cyanide (1-2 mM). Cytochrome %20 is kinetically competent to act as a terminal oxidase (Turner et al., 1971), reacts with cyanide and CO (Lloyd & Chance, 1972), and is probably the terminal oxidase for pathway 1. Pathways 2, 3 and 4 may ter- minate at novel alternative oxidases or involve modi- * Present address: Department of Biochemistry, Univer- sity College, Cathays Park, Cardiff CF1 1XL, Wales, UK Abbreviations: CCCP, Carbonyl cyanide m-chlorophe- nylhydrazone; SHAM, Salicylhydroxamicacid. fled forms of component 1. Pathway 5 is unaffected by azide, cyanide, CO or H2S but is sensitive to inhi- bition by salicylhydroxamic acid (SHAM). The oxi- dase sensitive to inhibition by SHAM has recently been demonstrated in T. pyriformis by Eichel & Stearns (1977), and it has been suggested that it is extremely active in media supplemented with metal ions (Stearns et al., 1978). T. pyriformis can become physiologically adapted to grow in medium containing 10 - 4 M cyanide (McCashland, 1955). Euglena gracilis, adapted to growth in the presence of 10-SM 2,4-dinitrophenol (Kahn, 1974), becomes resistant to other uncouplers of energy conservation. Crithidia fasciculata has also been adapted to growth in the presence of carbonyl- cyanide m-chlorophenylhydrazone (CCCP) (Kutzman & Roberts, 1978). The resistance reported in these organisms is apparently not a result of mutant selec- tion. In both T. pyriformis (McCashland & Andresen, 1963) and E. gracilis (Kahn, 1973), resistance is readily reversible by culturing the cells in the absence of the stressing agent. In the present paper, we describe the effects of azide, cyanide and SHAM on the respiration of intact organisms cultured in media supplemented with iron and in the presence of CCCP, and show that cyto- chrome a620 is the sole cytochrome oxidase irrespec- tive of growth conditions. EXPERIMENTAL METHOD Maintenance and 9rowth of oroanisms Tetrahymena pyriformis ST was maintained and grown axenically on a basal medium composed of: 2% (w/v) pro- teose peptone (Difco) and 0.1% (w/v) liver digest (Oxoid) with shaking at 30°C, exactly as described previously 567