Volume 52, number I FEBSLETTERS March 1975 zyxwvutsr THE DEVELOPMENT OF MITOCHONDRIAL IRON-SULFUR PROTEINS DURING RESPIRATORY ADAPTATION OF SACCHAROMYCES CARLSBERGENSIS Tomoko OHNISHI zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Department of Biophysics and physical Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania I91 74 USA and Trevor G. CARTLEDGE and David LLOYD Department of Microbiology. University College, Newport Road, Cardiff, W ales, UK Received 10 January 1975 1. Introduction The process of respiratory adaptation, which occurs when suspensions of anaerobically-grown yeast cells are aerated [ 11, provides a convenient system for studies of the biosynthesis of respiratory components and their integration into functional mitochondrial electron transport chains [2-41. The development of the respiratory chain and of other mitochondrial enzyme activities, as well as formation of highly organized mitochondria, has been studied in detail in Saccharomyces carlsbergensis [5,6] ; we now present data on the time-course of appearance of iron-sulfur centers in this system as detected in EPR spectra. Submitochondrial particles from aerobically-grown S. cerevisiae [7,8] or S. carlsbergensis [9] show no EPR signals associated with the NADH dehydiogenase segment of the respiratory chain, either at 77°K or at temperatures below 30°K, i.e. identical spectra were obtained when either NADH or succinate plus glycerol l-phosphate were used as reductants [lo]. The ab- sence of the NADH dehydrogenase iron-sulfur centers simplifies the interpretation of EPR signals from other iron-sulfur centers which appear as respiratory adapta- tion progresses. In mammalian or avian systems, three different iron-sulfur centers [designated as Centers S-l, S-2 and S-3 (1 l-13)] in the succinate dehydrogenase segment and two (Rieske’s iron-sulfur center and ‘Center 5’) in the cytochrome b- cl region of the respiratory chain have so far been identified [IO]. 90 2. Materials and methods S. carlsbergensis (N.C.Y.C. 74s) was grown under strictly anaerobic conditions in a complex growth medium in the presence of 10% glucose, Tween 80 and ergosterol [ 141. Respiratory adaptation was carried out in the absence of net cell growth [ $61. Cells were harvested and sphaeroplasts prepared as previously described [ 15,161. Preparation of submitochondrial particles from these, and from aerobically-grown S. cerevisiae cells, was by the method of Ohnishi et al. [9]. EPR spectra were obtained with a Varian E4 spectrometer at temperatures below 77”K, which were obtained by cooling samples with a stream of cold helium gas derived from boiling liquid helium. Temperature measurement was performed with a thermocouple (gold-cobalt alloy-versus-platinum) placed just below samples. All preparations were kept frozen in liquid nitrogen and thawed anaerobi- cally before use. The half-reduction potentials of iron-sulfur centers were measured potentiometri- tally [ 17,181 using the chamber designed to permit anaerobic transfer of aliquots directly into EPR sample tubes which were then rapidly frozen in liquid isopentane at 113°K. Oxidation-reduction mediators employed were diaminodurol, phenazine methosulphate, phenazine ethosulfate, duroquinone, pyocyanine, resorufin, 2-OH-naphthoquinone, phenosafranine, benzyl viologen and methyl viologen. North-Holland Publishing Company - Amsterdam