Overexpression and Characterization of the Copper A Domain from Cytochrome ba_3 of Thermus Thermophilus

• Main Author: Slutter, Claire Ellen
• Format: Others
• Language: en
• Published: 1996
• Online Access: https://thesis.library.caltech.edu/10331/8/Slutter_ce_1996.pdf; Slutter, Claire Ellen (1996) Overexpression and Characterization of the Copper A Domain from Cytochrome ba_3 of Thermus Thermophilus. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ded1-5k24. https://resolver.caltech.edu/CaltechTHESIS:06092017-092206826 <https://resolver.caltech.edu/CaltechTHESIS:06092017-092206826>

<p>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 (Cu_^ domain of cytochrome ba₃, downstream from the T7 RNA polymerase promoter in Escherichia coli. The Cu_^ domain is obtained in high yields as a watersoluble, thermostable, purple copper protein. The absorption spectrum of the Cu_^ 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 (3,100 M^-1 cm^-1) and 530 nm (3,200 M^-1 cm^-1), a band in the near-IR centered at 790 nm (1,900 M^-1 cm^-1) and a weaker band at 363 nm (1,300 M^-1 cm^-1). 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) Nature 376 660-669] and the engineered, purple CyoA protein [Wilmanns et al., Proc. Natl. Acad. Sci. USA, in press]. Soluble Cu_^ fragments from other terminal oxidases have been expressed; however, the thermostability of the fragment described here (T_M = 80 °C) and the stable binding of copper over a broad pH range (pH 3-9) makes this protein uniquely suitable for detailed physical-chemical study. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification all indicate that this protein contains two copper ions bound in a mixed-valence state, consistent with the prediction that the Cu_^ site in cytochrome ba₃ is a binuclear center.</p> <p>Flash photolysis has been used to initiate electron transfer from excited tris(2,2'bipyridyl) ruthenium(II) to the Cu_^ site of the soluble Thermus domain. Luminescence quenching of the excited state of the ruthenium(II) complex was observed at low protein concentrations (20-200 μM Cu_^ domain), with second-order kinetics and rate constants of 2.9 x 10⁹ M^-1s^-1 and 1.3 x 10⁹ M^-1s^-1 at low and high ionic strength, respectively, At high protein concentrations (>250 μM Cu_^) and low ionic strength, the quenching rate saturates due to ground-state complex formation; a first-order rate constant of 1.5 x 10⁵ s^-1 was estimated for ET in the complex.</p>