Expression, purification and characterization of the soluble CuA domain of cytochrome c oxidase of Paracoccus versutus

The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CUA) domain. The Cu-A domain of Paracoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cu-A domain in pET11d vector was expressed in E. coli BL21 (DE3). The results showed that the Cu-A domain was expressed mostly in inclusion bodies and the Cu-A domain protein synthesized in E. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved In urea, dialyzed and recombined with Cu+/Cu2+ and purified by the Q-sepharose fast flow anion-exchange column and Sephadex G-75 gel filtration column, the soluble purple-colored protein, which shows a single band In electrophoresis, was obtained. The UV-visible absorption spectrum Of Cu-A domain showed that there are intense band at 478 nm and a shoulder peak at 530 nm, and two weak bands at 360 and 806 run respectively, which can be assigned to the charge transfer and the Interactions of obitals of Cu-S and Cu-Cu in the mixed-valence binuclear metal center (Cu2S2R2). The far-UV CD spectrum indicated that this domain is predominantly in beta -sheet structure. The fluorescence spectra showed that its maximal excitation wavelength and maximal emission wavelength are at 280 and 345 nm, respectively.