Cytochrome c oxidase (Complex IV, CcO) is the terminal enzyme of the inner mitochondrial membrane that catalyzes electron transfer from reduced cytochrome c to molecular oxygen. The enzyme is composed of 13 non-identical protein subunits. We have used differential scanning calorimetry (DSC) to probe kinetic stability of detergent-solubilized CcO isolated from bovine heart. Specifically, we have focused on the effect of previously identified factors affecting the functional and structural properties of CcO such as tightly-bound cardiolipin (CL) and detergent [1,2]. Bovine heart CcO exhibited two large thermal transitions centered at 50 and 60oC. Both thermally induced processes are highly dependent on scan rate and associated with irreversible, endothermic processes. The analysis of thermal transition of CL-containing and CL-free CcO led to the following conclusion: (i) stability of CcO strongly depends on tightly-bound cardiolipin, delipidation destabilizes CcO, (ii) increasing of protein concentration resulted in protein stabilization, indicating concentration-dependent CcO dimerization, (iii) stability of CcO dimeric form is about 2 order of magnitude higher than its monomeric form at physiological temperatures. Moreover, CcO activity correlates with the presence of subunits VIIa and III, i. e. the same subunits which are destabilized by high hydrostatic pressure or by chemical denaturants, such as urea and guanidine hydrochloride .
This work was supported by the ESF 26110230097, Slovak Grant Agency VEGA, grants 2/0062/14 and 1/0521/12, and CELIM (316310) funded by 7FP EU (REGPOT).