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1. The aerobic incubation of iron-deficient Torulopsis utilis cells for 12h under non-growing conditions results in the recovery by mitochondria of the previously absent site 1 energy conservation and sensitivity to piericidin A. 2. The recovery of piericidin A sensitivity but not site 1 is prevented by the presence of cycloheximide (100mug/ml) in the medium used for aerobic incubation of the cells. Rotenone sensitivity behaved similarly. 3. Chloramphenicol, erythromycin and tetracycline were without effect on the recovery of site 1 and piericidin A sensitivity. 4. Inclusion of (59)Fe in the growth medium can be used as the basis for a highly sensitive assay for non-haem iron. 5. Iron-limited growth of T. utilis lowers the concentration of both non-haem iron and acid-labile sulphide of submitochondrial particles by over 20-fold compared with the ;normal' situation with iron-supplemented glycerol-limited growth. 6. Increases in the non-haem iron and acid-labile sulphide concentrations of submitochondrial particles occur when site 1 and piericidin A sensitivity are recovered. The increase is approximately halved by the presence of cycloheximide. 7. The non-haem iron of T. utilis submitochondrial particles does not exchange with added iron. 8. Continuous culture of T. utilis at the transition between glycerol- and iron-limitation results in cells where mitochondria possess site 1 energy conservation but lack piericidin A sensitivity. 8. It is concluded, in contrast with widely held views to the opposite, that energy conservation at site 1 does not require electron flow to proceed through a piericidin A- or rotenone-sensitive route. 9. Restriction of the iron supplied to growing T. utilis to a concentration just above that required for growth limitation demonstrates that a 10- to 20-fold decrease of the ;normal' non-haem iron concentration of both cells and mitochondria is without effect on the growth yield per unit of carbon source. Submitochondrial particles prepared from such iron-restricted but otherwise functionally normal cells have a non-haem iron concentration of about 0.5-0.8ng-atom/mg of protein. It is concluded that the concentration of iron-sulphur protein required for normal function by the respiratory chain is close to the concentrations of cytochromes and flavoproteins.

1. Mitochondria prepared from Torulopsis utilis grown in a chemostat with iron-limited growth were found to lack energy conservation but not electron flow in that segment of the respiratory chain leading from intramitochondrial NADH to the cytochromes [i.e. the site 1 segment (Lehninger, 1964)]. 2. Site 1 energy conservation was present in mitochondria prepared from cells grown under conditions of limitation by glycerol, ammonium and magnesium. Phosphate-limited growth resulted in mitochondrial preparations without respiratory control. 3. Mitochondria from cells grown under conditions of iron limitation were insensitive to the respiratory inhibitor piericidin A, whereas sensitivity was present in mitochondria prepared from glycerol-, ammonium-, magnesium- or phosphate-limited cells. 4. These observations are considered to provide indirect evidence for a role of non-haem iron in the mechanism of energy conservation and also piericidin A sensitivity in T. utilis mitochondria. 5. A readily constructed and inexpensive pH-measuring and -controlling circuit is described for use with continuous-culture apparatus.

1. A spectroscopic resolution has been made of the components contributing to the ;iron-flavoprotein' trough extending from 450 to 520nm in the reduced-minus-oxidized difference spectrum of submitochondrial particles of Torulopsis utilis. 2. Seven components were identified other than cytochrome b, ubiquinone and succinate dehydrogenase. On the basis of the effects of iron- and sulphate-limited growth of cells on their subsequently derived electron-transport particles, and also by consideration of analytical measurements of the concentration of FMN, FAD, non-haem iron and acid-labile sulphide in the electron-transport particles in relation to the magnitude of the spectroscopic changes, it was possible to identify five of these components as follows: species 1a, the flavin of NADH dehydrogenase ferroflavoprotein; species 1b, the iron-sulphur component of NADH dehydrogenase ferroflavoprotein; species 1', the flavin of an NADPH dehydrogenase; species 2, an iron-sulphur or ferroflavoprotein component; species 3, the flavin of l-3-glycerophosphate dehydrogenase. Two additional components were a fluorescent flavoprotein, probably lipoamide dehydrogenase, and a b-type cytochrome reducible by NADH or NADPH but not reoxidizable by the respiratory chain. 3. Species 1b and 2 were undetectable in electron-transport particles from iron- or sulphate-limited cells, but could be recovered in vivo under non-growing conditions. 4. The recovery in vivo of species 2 but not species 1b was inhibited by cycloheximide. 5. The recovery of species 1b correlates with the recovery of site 1 conservation. 6. The recovery of species 1b with species 2 correlates with the recovery of piericidin A sensitivity. 7. Evidence is presented for an NADPH dehydrogenase distinct from NADH dehydrogenase. The oxidation of NADH and NADPH by the respiratory chain is sensitive to piericidin A, and an iron-sulphur protein common to both pathways (species 2) is suggested as the piericidin A-sensitive component. 8. The approximate E'(0) (pH7.0) values of species 1 (a and b, low potential) and species 2 (high potential) indicate that site 1 energy conservation occurs between the levels of species 1 (a and b) and species 2.

1. Conditions have been established for the sulphate-limited growth of Torulopsis utilis in continuous culture. 2. Mitochondria prepared from sulphate-limited cells lack both piericidin A sensitivity and the first energy-conservation site (site 1). Sensitivity to antimycin A or cyanide and the second and third energy-conservation sites were apparently unaffected by sulphate-limited growth. 3. Aerobic incubation for 8h of sulphate-limited cells with a low concentration of sulphate (50mum or less) resulted in the recovery of mitochondrial piericidin A sensitivity and site 1. The use of higher concentrations of sulphate (250mum or more) still resulted in the recovery of mitochondrial piericidin A sensitivity and site 1, but also resulted in the appearance of a non-phosphorylating oxidase, which mediated oxidation of the respiratory chain at about the level of cytochrome b in an antimycin A- and cyanide-insensitive manner. Both this alternative route and the conventional normal route of respiration were shown to coexist and to intercommunicate at the level of cytochrome b. 4. Low-temperature spectroscopy failed to identify any new respiratory component to explain the alternative route. 5. The apparent affinity of the alternative route for oxygen was similar to that for the conventional route through cytochrome oxidase, namely half-maximal activity at 0.1mum-oxygen or less. 6. The non-haem iron concentration of submitochondrial particles was unaffected by sulphate limitation, whereas the acid-labile sulphide concentration was lowered tenfold. Marked increases (between four- and 30-fold) in the acid-labile sulphide concentration of submitochondrial particles were observed in sulphate-limited cells after aerobic incubation with various concentrations of sulphate. The lowest increase (fourfold) was observed without added sulphate, the highest (30-fold) with 1.0mm added sulphate. 7. The ratio of non-haem iron to acid-labile sulphide in submitochondrial particles varied with different growth conditions from a maximum of 15.0 to a minimum of 0.72. It is suggested that analytical measurements of non-haem iron are an inadequate guide to the concentration of iron-sulphur protein in complex systems. 8. The effects of sulphate-limited growth on site 1 and piericidin sensitivity are interpreted to indicate a role for iron-sulphur protein in these properties. 9. The aerobic incubation of sulphate-limited cells with cycloheximide resulted in the recovery by mitochondria of site 1 but not of piericidin sensitivity. 10. The appearance of the alternative route for cyanide- and antimycin-A (but not piericidin A-) insensitive respiration on incubating sulphate-limited cells with sulphate concentrations higher than 250mum indicates that the alternative route involves an iron-sulphur protein.