Abstract
The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The Cua domain ofParacoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cua domain in pET11d vector was expressed inE. coli BL21 (DE3). The results showed that the Cua domain was expressed mostly in inclusion bodies and the Cua 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 Cua 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.
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Li, L., Song, A., Xie, Y. et al. Expression, purification and characterization of the soluble CuA domain of cytochrome c oxidase ofParacoccus versutus . Chin.Sci.Bull. 46, 1608–1611 (2001). https://doi.org/10.1007/BF02900617
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DOI: https://doi.org/10.1007/BF02900617