Water-Soluble, Recombinant CuA-Domain of the Cytochrome ba3 Subunit II from Thermus thermophilus

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Abstract
Recently, the genes of cytochrome ba3 from Thermus thermophilus [Keightley, J. A., et al. (1995) J. Biol. Chem.270, 20345−20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3 is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Paracoccus denitrificans [Lappalainen, P., et al. (1993) J. Biol. Chem.268, 26416−26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C., et al. (1994) FEBS Lett.340, 109−113]. There are intense bands at 480 nm (3100 M-1 cm-1) and 530 nm (3200 M-1 cm-1), a band in the near-IR centered at 790 nm (1900 M-1 cm-1), and a weaker band at 363 nm (1300 M-1 cm-1). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly β-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature376, 660−669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A.92, 11955−11959]. However, the thermostability of the fragment described here (Tm ≈ 80 °C) and the stable binding of copper over a broad pH range (pH 3−9) suggest this protein may be uniquely suitable for detailed physical-chemical study.