Effects of Temperature on Cytochrome Oxidase Activity in Solubilized Form and in Lipid Vesicle Systems1

Abstract

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break T_b) at about 20°C when assayed in a medium containing Emasol. The activation energies above and below 20°C were 9.3 (E_H) and 18.9 kcal/mol (E_L), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature T_t=40°C), dimyristoyl phosphatidylcholine (DMPC, T_t=23°C) and dioleoyl phosphatidylcholine (DOPC, T_t=−22°C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36°C with E=22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with T_t=23°C. E_b and E_L were 6.6 and 15.8 kcal/mol, espectively. The plot for the oxidase-DMPC also showed a break (T_b=26°C) with E_H=6.6 and E_L=26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the “hot” and “cold” conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.