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Reports in the literature indicate that during the exponential phase of growth of Candida utilis NADH oxidation is insensitive to rotenone, that rotenone sensitivity is acquired during the transition to the late stationary phase and is again lost on catabolite repression. The acquisition and loss of rotenone sensitivity appears to be accompanied by similar changes in Site 1 phosphorylation but does not appear to be reflected in the rate of oxidation of NADH (by mitochondria) or of NAD-linked substrates (by mitochondria or whole cells). In the present paper evidence is presented that these fluctuations in sensitivity to inhibitors of NADH oxidation reflect the presence of different types of inner membrane-bound NADH dehydrogenases in different phases of growth. Thus inner membrane preparations from exponential phase cells contain an NADH dehydrogenase which reacts equally well with ferricyanide and juglone as electron acceptor, appears to be very labile, and lacks EPR signals corresponding to iron-sulfur Centers 1 and 2, whereas a new species, probably an iron-sulfur protein, with resonances at g|| = 2.01, and g = 1.92 in the reduced state, is present. This species is not significantly reduced by NADH. In corresponding preparations from late stationary phase cells NADH-ferricyanide activity is high, juglone reductase activity is low, and the enzyme is stable and exhibits the EPR signals of iron-sulfur Centers 1 and 2, whereas the EPR signals of iron-sulfur Centers 3 + 4 change very little on transition from exponential to stationary phase cells. There is also a decrease in cytochrome concentration. Most prominent among these is a b-type cytochrome (g = 2.54; 2.23; 1.87) which decreases 2- to 3-fold. The EPR detectable species with g|| = 2.01 and g = 1.92 in the reduced state is no longer detected. On catabolite repression of late stationary phase cells there is an 80 to 90% decline in NADH-ferricyanide activity, of iron-sulfur Centers 1 and 2, a 50 to 60% decrease of Centers 3 + 4, and an increase in a b cytochrome, but the specific activity in NADH-juglone reductase and NADH oxidase assays increases, the enzyme becomes once again labile, and the EPR detectable species with g|| = 2.01 and g = 1.92 appears on reduction with dithionite. All these changes are prevented by cycloheximide. The data suggest that sensitivity to piericidin A and coupling to energy conservation Site 1 are properties of the type of NADH dehydrogenase present in late stationary phase cells but not in exponential phase or of catabolite-repressed cells.

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