Water-soluble, Recombinant CuA-Domain of the Cytochrome ba3 Subunit II from Thermus Thermophilus. Claire E. Slutter, Donita Sanders, Pernilla Wittung, Bo G. Malmstrom, Roland Aasa, John H. Richards, Harry B. Gray, and James A Fee. Biochemistry 35 (1996) 3387-

SUMMARY: Recently, the genes of cytochrome ba3 from Thermus thermophilus (Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358), a homologue of the heme-copper oxidase family, have been cloned. We report here the expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome oxidase, 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 heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3 oxidase of Paracoccus denitrificans, [Lappalainen et al. (1993) J. Biol. Chem. 268, 26416-26421] and the caa3 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 predictions from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structures reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al., (1995) Nature 376, 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 ~ 80C) 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.