HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, X. Articles by Yu, C.-A. Search for Related Content PubMed PubMed Citation Articles by Yang, X. Articles by Yu, C.-A. Social Bookmarking                      What's this?

Volume 272, Number 15, Issue of April 11, 1997 pp. 9683-9689
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Resolution and Reconstitution of Succinate-Ubiquinone Reductase from Escherichia coli

(Received for publication, December 13, 1996, and in revised form, February 4, 1997)

From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078

A modified procedure is developed for isolation of highly purified succinate-ubiquinone reductase from Escherichia coli NM256 containing a cloned sdh operon in a multicopy plasmid. Succinate-ubiquinone reductase is solubilized from the membrane by polyoxyethylene-9-lauryl ether and purified by DEAE-Sepharose CL-6B column chromatography. The isolated reductase is resolved into a reconstitutively active, two-subunit succinate dehydrogenase and a two-subunit membrane anchoring protein fraction (the SdhC-SdhD fraction) by alkaline (pH 10.2) treatment of the reductase in the presence of 1 M urea, followed by DEAE-Sepharose CL-6B column chromatography under anaerobic conditions. Isolated succinate dehydrogenase and the SdhC-SdhD fraction alone show no succinate-ubiquinone reductase activity. However, when a given amount of the SdhC-SdhD fraction is mixed with varying amounts of succinate dehydrogenase or vice versa succinate-ubiquinone reductase activity increases as the amount of succinate dehydrogenase or the SdhC-SdhD fraction added increases. Maximum reconstitution is obtained when the weight ratio of succinate dehydrogenase to the SdhC-SdhD fraction reaches 5.26. This ratio is slightly higher than the calculated value of 3.37, obtained by assuming 1 mol of succinate dehydrogenase reacts with 1 mol of SdhC and SdhD. The isolated SdhC-SdhD fraction contains 35 nmol cytochrome b₅₅₆/mg protein. Unlike mitochondrial cytochrome b₅₆₀, the cytochrome b₅₅₆ is reducible by succinate in the isolated and complex forms. Furthermore, cytochrome b₅₅₆ in the isolated SdhC-SdhD fraction has absorption properties, carbon monoxide reactivity, and EPR characteristics similar to those of cytochrome b₅₅₆ in intact succinate-ubiquinone reductase, indicating that its heme environments are not affected by the presence of succinate dehydrogenase. However, the redox potential of cytochrome b₅₅₆ in the SdhC-SdhD fraction (22 mV) increases slightly when complexed with succinate dehydrogenase (34 mV). No hybrid succinate-ubiquinone reductase is formed from mitochondrial QPs (the membrane-anchoring protein fraction of bovine heart mitochondrial succinate-ubiquinone reductase) and E. coli succinate dehydrogenase or vice versa. However, the cytochrome b₅₅₆ in E. coli SdhC-SdhD fraction is reducible by succinate in the presence of mitochondrial succinate dehydrogenase, and the rate of cytochrome b₅₅₆ reduction correlates with the reconstitutive activity of the mitochondrial succinate dehydrogenase.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Q. M. Tran, R. A. Rothery, E. Maklashina, G. Cecchini, and J. H. Weiner Escherichia coli succinate dehydrogenase variant lacking the heme b PNAS, November 13, 2007; 104(46): 18007 - 18012. [Abstract] [Full Text] [PDF]

				X. Yang, L. Yu, D. He, and C.-A. Yu The Quinone-binding Site in Succinate-ubiquinone Reductase from Escherichia coli. QUINONE-BINDING DOMAIN AND AMINO ACID RESIDUES INVOLVED IN QUINONE BINDING J. Biol. Chem., November 27, 1998; 273(48): 31916 - 31923. [Abstract] [Full Text] [PDF]

				E. Maklashina, R. A. Rothery, J. H. Weiner, and G. Cecchini Retention of Heme in Axial Ligand Mutants of Succinate-Ubiquinone Oxidoreductase (Complex II) from Escherichia coli J. Biol. Chem., May 25, 2001; 276(22): 18968 - 18976. [Abstract] [Full Text] [PDF]