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

• Published in: Biochemistry (Easton) Vol. 35; no. 11; pp. 3387 - 3395
• Main Authors: SLUTTER, Claire E., SANDERS, Donita, WITTUNG, Pernilla, MALMSTRöM, Bo G., AASA, Roland, RICHARDS, John H., GRAY, Harry B., FEE, James A.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 19.03.1996
• Subjects: Amino Acid Sequence; Base Sequence; Binding Sites; Copper - chemistry; Cytochrome b Group - chemistry; DNA Primers - chemistry; Electron Spin Resonance Spectroscopy; Electron Transport Complex IV - chemistry; Metalloproteins - chemistry; Molecular Sequence Data; Protein Structure, Secondary; Recombinant Proteins; Solubility; Thermus thermophilus - enzymology; Water - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9525839

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 Parachccus 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) cn(-1)), and a weaker band at 363 nm (1300M(-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 beta-sheet, in agreement with the recent X-ray structure 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 approximately 80 degrees 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.