Sequential dissociation of subunits from bovine heart cytochrome c oxidase by urea

  • Sedlák E
  • Robinson N
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Abstract

The quaternary stability of purified, detergent-solubilized, cytochrome c oxidase (CcO) was probed using two chemical denaturants, urea and guanidinium chloride (GdmCl). Each chaotrope induces dissociation of five subunits in a concentration-dependent manner. These five subunits are not scattered over the surface of CcO but are clustered together in close contact at the dimer interface. Increasing the concentration of urea selectively dissociates subunits from CcO in the following order: VIa and VIb, followed by III and VIIa, and finally Vb. After incubation in urea for 10 min at room temperature, the sigmoidal dissociation transitions were centered at 3.7, 4.6, and 7.0 M urea, respectively. The secondary structure of CcO was only minimally perturbed, indicating that urea causes disruption of subunit interactions without urea-induced conformational changes. Incubation of CcO in urea for 120 min produced similar results but shifted the sigmoidal dissociation curves to lower urea concentrations. Incubation of CcO with increasing concentrations of GdmCl produces an analogous effect; however, the GdmCl-induced dissociation of subunits occurs at lower concentrations and with a narrower concentration range. Thermodynamic parameters for each subunit dissociation were evaluated from the sigmoidal dissociation data by assuming a single transition from bound to dissociated subunit. The free energy change accompanying urea-induced dissociation of each subunit ranged from 18.0 to 29.7 kJ/mol, which corresponds to 0.32-0.59 kJ/mol per 100 A(2) of newly exposed solvent-accessible surface area. These values are 30-50-fold smaller than previously reported for the unfolding of soluble or membrane proteins.

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