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


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.


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 (TM = 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.

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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Unknown, Unknown
Thesis Committee:
  • Unknown, Unknown
Defense Date:8 February 1996
Record Number:CaltechTHESIS:06092017-092206826
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10331
Deposited By: Benjamin Perez
Deposited On:09 Jun 2017 17:39
Last Modified:16 Apr 2021 22:28

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