Thermal inactivation of electron-transport functions and F 0F 1-ATPase activities

Abstract

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30°C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 ± 1.0°C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F 0F 1 catalyzed ATP/P i exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 ± 1.0°C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 ± 1.0°C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F 0F 1 persists to 61.0 ± 1.0°C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 ± 2.0°C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F 0F 1 vesicles also exhibit different inactivation temperatures for the ATP/P i exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 ± 1.0°C, but the latter at 58.0 ± 2.0°C. Differential scanning calorimetry also detects biphasic transitions of F 0F 1, with similar temperatures of denaturation (49.0 and 54.0°C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F 0F 1 complex can be done. (2) The ATP synthesis activity of the F 0F 1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10°C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4–5°C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site. (3) Protein complexes of the mitochondrial energy-transducing membrane are metastable, i.e., they are in a high-energy state under aerobic environments. These complexes are susceptible to oxidation (consuming 250 nmol O 2 per mg protein) and as a consequence dissipate heat. Whether this exothermic energy is relevant to the in vivo ATP synthesis remains unclear.