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Water-Soluble, Recombinant Cu_A-Domain of the Cytochrome ba_3 Subunit II from Thermus thermophilus

Slutter, Claire E. and Sanders, Donita and Wittung, Pernilla and Malmström, Bo G. and Aasa, Roland and Richards, John H. and Gray, Harry B. and Fee, James A. (1996) Water-Soluble, Recombinant Cu_A-Domain of the Cytochrome ba_3 Subunit II from Thermus thermophilus. Biochemistry, 35 (11). pp. 3387-3395. ISSN 0006-2960. doi:10.1021/bi9525839.

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Recently, the genes of cytochrome ba_3 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_A) domain of cytochrome ba_3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The Cu_A-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 Cu_A site in cytochrome ba_3 is a binuclear center. The absorption spectrum of the Cu_A site, free of the heme interference in cytochrome ba_3, is similar to the spectra of other soluble fragments from the aa_3-type oxidase of Paracoccus denitrificans [Lappalainen, P., et al. (1993) J. Biol. Chem. 268, 26416−26421] and the caa_3-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) 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 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 aa_3 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 (T_m ≈ 80 °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.

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Wittung, Pernilla0000-0003-1058-1964
Gray, Harry B.0000-0002-7937-7876
Additional Information:© 1996 American Chemical Society. Received October 31, 1995; Revised Manuscript Received January 16, 1996. This work was supported by NIH Grants GM35342 (J.A.F.), GM16424 (J.H.R.), and DK19038 (H.B.G.), predoctoral training Grant GM07616 (C.E.S.), and the Swedish Natural Science Research Council (B.G.M.). We are grateful for the following assistance:  The synthesis of PCR probes was done by Dr. Nicole Levy of the Biopolymer Synthesis and Analysis Resource Center at CalTech. The electrospray mass spectrometry was carried out by Dr. Gary Suizdak in the Mass Spectroscopy Facility at the Scripps Research Institute with support from the Lucille P. Markey Charitable Trust and a NIH Shared Instrumentation Grant RR07273-01. Dr. Andrew Thomson provided a manuscript prior to publication. Dr. Matthias Wilmanns provided structural coordinates of CyoA prior to publication. Dr. David B. Goodin recorded some of the EPR spectra and provided essential advice on obtaining the mass spectra. Dr. Andresz Pastuszyn carried out the quantitative amino acid analyses and the N-terminal amino acid sequencing. Mr. Ron LaBorde carried out the ICP-MS analyses. Mr. Patrik Pettersson carried out the TXRF analyses. Mr. Harold Kochounian carried out the DNA sequencing. Dr. Stephen Mayo and Mr. J. Luo assisted with obtaining the CD data. Portions of this work were done while J.A.F. was a Visiting Scientist at Göteborgs Universitet in April 1995, and he thanks T.V., R.A., and B.G.M. for their generous hospitality during that time.
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Swedish Natural Science Research CouncilUNSPECIFIED
Lucille P. Markey Charitable TrustUNSPECIFIED
Issue or Number:11
Record Number:CaltechAUTHORS:20150504-122517025
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Official Citation:Water-Soluble, Recombinant CuA-Domain of the Cytochrome ba3 Subunit II from Thermus thermophilus Claire E. Slutter, Donita Sanders, Pernilla Wittung, Bo G. Malmström, Roland Aasa, John H. Richards, Harry B. Gray, and James A. Fee Biochemistry 1996 35 (11), 3387-3395 DOI: 10.1021/bi9525839
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:57199
Deposited By: George Porter
Deposited On:04 May 2015 20:46
Last Modified:10 Nov 2021 21:09

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