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J. Biol. Chem., Vol. 280, Issue 38, 33035-33044, September 23, 2005

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Reactivities of Quinone-free DsbB from Escherichia coli^*

Kenji Inaba, Yoh-hei Takahashi, and Koreaki Ito1

From the Institute for Virus Research, Kyoto University, and CREST of Japan Science and Technology Agency, Kyoto 606-8507, Japan

DsbB is a disulfide oxidoreductase present in the Escherichia coli plasma membrane. Its cysteine pairs, Cys⁴¹–Cys⁴⁴ and Cys¹⁰⁴–Cys¹³⁰, facing the periplasm, as well as the bound quinone molecules play crucial roles in oxidizing DsbA, the protein dithiol oxidant in the periplasm. In this study, we characterized quinone-free forms of DsbB prepared from mutant cells unable to synthesize ubiquinone and menaquinone. While such preparations lacked detectable quinones, previously reported lauroylsarcosine treatment was ineffective in removing DsbB-associated quinones. Moreover, DsbB-bound quinone was shown to contribute to the redox-dependent fluorescence changes observed with DsbB. Now we reconfirmed that redox potentials of cysteine pairs of quinone-free DsbB are lower than that of DsbA, as far as determined in dithiothreitol redox buffer. Nevertheless, the quinone-free DsbB was able to oxidize 40% of DsbA in a 1:1 stoichiometric reaction, in which hemi-oxidized forms of DsbB having either disulfide are generated. It was suggested that the DsbB-DsbA system is designed in such a way that specific interaction of the two components enables the thiol-disulfide exchanges in the "forward" direction. In addition, a minor fraction of quinone-free DsbB formed the DsbA-DsbB disulfide complex stably. Our results show that the rapid and the slow pathways of DsbA oxidation can proceed up to significant points, after which these reactions must be completed and recycled by quinones under physiological conditions. We discuss the significance of having such multiple reaction pathways for the DsbB-dependent DsbA oxidation.

Received for publication, June 7, 2005 , and in revised form, July 11, 2005.

^* This work was supported by PRESTO (to K. Inaba) and CREST (to K. Ito), Japan Science and Technology Agency and grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan and by its National Project on Protein Structural and Functional Analyses (to K. Ito). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: 81-75-751-4015; Fax: 81-75-771-5699; E-mail: kito{at}virus.kyoto-u.ac.jp.

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