Thermodynamic and Structural Stability of Cytochrome c Oxidase from Paracoccus denitrificans

The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically. In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III. Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67 degrees C, while subunit III is less stable and denatures 20 degrees C earlier. Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III. Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity. The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved. The amount of native alpha-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation. Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein. The calorimetric behavior of subunit III does not fully conform to that expected for a highly alpha-helical membrane protein. The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured