The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 220.127.116.11) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluoresence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the proteimlipid molar ratio indicates that cytochrome c oxidase prevents 99 ± 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 °C and characterized by an enthalpy change of 550 ± 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 °C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes ~10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process. Deconvolution analysis of the heat capacity function indicates that the overall curve is composed of at least four sequential melting steps. The calorimetric experiments have been complemented with thermal gel electrophoresis experiments directed to identify the enzyme subunits in the main melting steps. According to these experiments, the first melting step, for the membrane-reconstituted enzyme, at 52 °C, involves primarily subunit III. This step is followed by two closely spaced melting steps at 61 and 64 °C involving the bulk of the enzyme complex. According to the thermal gel analysis experiments, subunits I, II, IV, VII, and VIII melt between 60 and 65 °C. The melting of subunits V and VIb could not be detected by gel electrophoresis, and their melting temperatures could not be assigned.
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