Modification of flow‐force relationships by external ATP in yeast mitochondria

Abstract

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP.ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP.At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions.However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase was measured under the three conditions mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system. The general concept that the proton electrochemical potential difference, A f i H + , acts as the coupling intermediate between proton pumps embedded in energy-transducing membrane [l] has been largely questioned. Indeed, certain data have been interpreted as being inconsistent with the view that A f i H + is either the only flux-controlling parameter or the real free-energy intermediate (see [2, 31 for reviews); of these data following should be mentioned. a) Inhibition of the respiratory rate is accompanied by an identical inhibition of ATP synthesis under conditions where the protonmotive force does not vary. b) No unique relationship has been established between a flux and any of its associated thermodynamic strengths, i.e. respiratory rate and protonmotive force (dp = Aj.&+/F). The relationships depend on the way in which the force varies (ATP synthesis, proton leak etc.).c) The dG/AfiH+ ratio at static head increases quasi-exponentially when A jiH + decreases.The first two of these results were first interpreted as indicating allosteric links between the respiratory chain and ATPase [4-81. The third result has, however, led to the proposal that A $ H + is not, in fact, the direct free-energy intermediate between respira...

Regulation of Cytochrome c Oxidase by Adenylic Nucleotides. Is Oxidative Phosphorylation Feedback Regulated by its End‐Products?

“…On isolated yeast mitochondria, a spectroscopic and kinetic control analysis of various steady states led to the identi cation of ATPactivating site(s) on the cytosolic side of complex IV, possibly borne by the ATP-binding protein, subunit VIa. ATP activation increases the response of the enzyme toward the associated thermodynamic forces, decreases its ux control coef cient (64,78) and is modulated, at least, by the external ionic composition (78). This may explain the discrepancies noted in the literature concerning the effect of external adenylic nucleotides on isolated and reconstituted COX (37,43,44).…”

“…Indeed, control of respiration exerted by COX was low when the respiratory rate was varied by ATP synthesis, whereas it increased as a function of the respiratory rate with uncoupler in the absence of ATP. However, ATP addition decreased the control coefcient under uncoupling conditions to reach the same value as measured during ATP synthesis (64).…”

“…In isolated mitochondria, we measured the protonmotive force and cytochrome reduction levels under different respiratory steady states sustained by ethanol respiration (64). We observed that in yeast mitochondria, as in rat liver mitochondria (60,61), there is more than one relationship between the respiratory rate and the size of 1p, when the respiratory rate is modi ed by either ADP plus Pi or by an uncoupler.…”

“…This is also in agreement with the ATP inhibition of the enzyme on the puri ed mutant COX at high ionic strength (77, see also 80). In contrast to the wild-type, the ATP effect on the subunit VIa null mutant is prevented by an inhibition of the ADP/ATP carrier, indicating that the ATP-binding site responsible for the inhibition is situated on the matrix side of the mutant COX, whereas ATP activation occurs on the cytosolic side of the wild-type COX (64,77). Moreover, experiments done with a nonhydrolyzabl e ATP analog, on one hand, and a nonselective inhibitor of protein kinases, on the other, suggest that protein phosphorylations are probably not involved in the kinetic effect of ATP on the respiratory rate of wild-type and subunit VIa null mutant ( Table 2).…”

“…These results point to changes in the kinetic behavior of the cytochrome oxidase of mutant and wild-type mitochondria with respect to ATP regulation. Indeed, the pattern of cytochrome a C a3 redox state and the ux control analysis point to an activation of the wild-type COX by ATP (64,77), on one hand, and an inhibition by ATP of the COX lacking subunit VIa, on the other. For each steady state, we estimated the thermodynamic driving force across COX (i.e., 2 £ 1E h ¡ n £ 1p), assuming that the complex vectorially pumps one proton across the membrane per electron transferred from cytochrome c to O 2 .…”