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

The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (Cu_A) domain. The Cu_a domain ofParacoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cu_a domain in pET11d vector was expressed inE. coli BL21 (DE3). The results showed that the Cu_a domain was expressed mostly in inclusion bodies and the Cu_a domain protein synthesized inE. 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 nm 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 β-sheet structure. The fluorescence spectra showed that its maximal excitation wavelength and maximal emission wavelength are at 280 and 345 nm, respectively.