Abstract
It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. These findings were reinvestigated in the present study. The parameters and properties followed during these transitions were sensitivity of NADH oxidation to piericidin, presence or absence of coupling site 1, EPR signals appearing on reduction with NADH or dithionite, the specific activities of NADH oxidase, NADH-ferricyanide reductase, and NADH-5-hydroxy-1,4-naphthoquinone (juglone) reductase, and the kinetic behavior of NADH dehydrogenase in the ferricyanide assay. Monitoring the rates of oxidation of NADH in submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations. It was found that on gradually increasing the concentration of iron during continuous culture (transition from ironlimited to iron- and substrate-limited growth), as well as on aeration of iron-limited cells, coupling site 1, piericidin sensitivity, NADH-ferricyanide activity, and iron-sulfur centers 1 and 2 increased concurrently, with concomitant decline of NADH-juglone reductase activity. Cycloheximide prevented all these changes. Iron-sulfur centers 3 plus 4 underwent relatively little increase during these transitions. It is concluded that in both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piercidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement. No evidence was found for the development of coupling site 1 without the appearance of piericidin sensir th
Highlights
In submitochondrial particles with artificial oxidants, observing the kinetics of the ferricyanide assay, and measuring the concentration of iron-sulfur centers elicited by EPR permitted ascertaining the type of NADH dehydrogenase present and its relative concentration in different experimental situations
Relatively little increase during these transitions. In both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piericidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement, No evidence was found for the development of coupling site 1 without the appearance of piericidin sensitivity
It may be noted that the degree of inhibition of the oxidation of malat#e plus pyruvate or of ethanol, at any incoming iron concentration, coincides with the inhibition of NADH oxidation in ETP, from the same cells
Summary
It has been reported that cells of Candida utilis, grown in continuous culture under iron-limited conditions, develop site 1 phosphorylation, without the appearance of piericidin sensitivity and without changes in the iron-sulfur centers of NADH dehydrogenase, on aeration in the presence of cycloheximide, as well as on increasing the supply of iron during growth. In both of these experimental conditions a replacement of the type of NADH dehydrogenase present in exponential phase cells by that characteristic of stationary phase cells occurs and that the appearance of site 1 phosphorylation, piericidin sensitivity, and iron-sulfur centers 1 plus 2, all associated with the latter enzyme, is a consequence of this replacement, No evidence was found for the development of coupling site 1 without the appearance of piericidin sensitivity It has been reported [2] that Candida utilis cells grown in continuous culture at the transition between iron-limited and substrate-limited growth possess energy coupling site 1 but lack pkricidin sensitivity.
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