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

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 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.

Flexibility of the metal-binding region in apo-cupredoxins

“…The soluble fragment herein studied comprises only the soluble cupredoxin domain of subunit II, where the transmembrane helix is not part of the purified protein. This fragment retains the structure observed in the whole oxidase (32,45) and it is competent for ET with its biological redox partner (46,47). Given that residues 1-42 are absent in our protein construct, residue numbering starts at 43 in the results presented here, following that employed in the X-ray structure of oxidized holoCu A [Protein Data Bank (PDB) ID 2CUA] (32).…”

“…Samples of azurin and apo-and holoCu A proteins were uniformly labeled with 15 N or 13 C and 15 N (Cambridge Isotope Laboratories, Inc.) and produced as described elsewhere (40,47,64). The apoCu A protein was expressed and purified in the absence of copper, whereas azurin was purified as Cu(II)-azurin and the apoprotein was obtained by dialysis against 50 mM KCN, 200 mM Tris·HCl pH 8.5, followed by several dialysis steps without the complexing agent.…”

Loop recognition and copper-mediated disulfide reduction underpin metal site assembly of Cu A in human cytochrome oxidase

“…S1). Removal of this transmembrane stretch has allowed the expression of soluble COX II domains from several bacterial oxidases (30,31). Among them, the soluble fragment from Thermus thermophilus COX II (Tt COX II hereafter) is the only one stable for long periods of time and under different conditions (32,33).…”

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

“…Because the His tag (coded on the parent vector) is not used for purification, nor is its presence in the protein desirable, it was cleaved off in vivo by co-transforming pRK603 into this E. coli strain (21), thus providing constitutive expression of the TEV-protease. Cells were grown on minimal medium containing 40 g/liter glycerol, 7.5 g/liter K 2 HPO 4 , 5.3 g/liter NaH 2 PO 4 -H 2 0, 2 g/liter NH 4 Cl, 1 g/liter glucose, 1 mM MgSO 4 , 0.1 mM CaCl 2 , 10 ml/liter trace element solution 1 (22), 0.2 M thiamin, 100 g/ml ampicillin, 25 g/ml kanamycin, in a 10-liter New Brunswick Microferm fermentor. Cells were induced with 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside at an A 600 of ϳ4.0.…”

“…The Cu A fragment of the T. thermophilus ba 3 cytochrome-c oxidase was expressed and purified as described previously (4). The cytochrome c 552 soluble domain of P. denitrificans was expressed in E. coli according to the published protocol (23).…”