Reversible Dimerization of Cytochrome Oxidase in Detergent Solution as Detected by Dynamic Light Scattering

Reversible changes in the quaternary structure of eukaryotic cytochrome c oxidase (CcO) are presumably involved in the enzyme activity regulation. Conventional methods for determining quaternary states are relatively complex. We applied dynamic light scattering (DLS) to estimate the size of CcO in micelles and identified different oligomeric forms in dynamic equilibrium. Thin-layer gel filtration and non-denaturing gel electrophoresis revealed CcO forms with different molecular masses. CcO quaternary structure was analyzed by cryo-electron microscopy. Prokaryotic cytochrome oxidase served as a control. Increasing protein concentration, cholate treatment, and acidification shifted the equilibrium toward larger micellar structures, suggesting CcO oligomerization. Protein dilution, dodecyl maltoside at high concentrations, alkalinization, and treatment with Triton X-100 reversed this process. The apparent size of prokaryotic cytochrome oxidase micelles remained constant regardless of enzyme concentration and presence of cholate. The apparent molecular masses of CcO in diluted and concentrated preparations corresponded to monomers and dimers, respectively. The amphipol-stabilized CcO forms, both monomeric and the cholate-containing dimeric, retained their characteristic sizes across various enzyme concentrations. Given that the DLS-based identification of the CcO oligomeric states was confirmed by three independent methods mentioned above, DLS appears to be a convenient tool for characterizing CcO quaternary structures in solution and monitoring their interconversion.