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J. Biol. Chem., Vol. 281, Issue 43, 32310-32317, October 27, 2006

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The Quinone Binding Site in Escherichia coli Succinate Dehydrogenase Is Required for Electron Transfer to the Heme b^*

Quang M. Tran, Richard A. Rothery, Elena Maklashina, Gary Cecchini, and Joel H. Weiner¹

From the Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 473 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada and the Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, California 94121 and Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California 94143

We have examined the role of the quinone-binding (Q_P) site of Escherichia coli succinate:ubiquinone oxidoreductase (succinate dehydrogenase) in heme reduction and reoxidation during enzyme turnover. The SdhCDAB electron transfer pathway leads from a cytosolically localized flavin adenine dinucleotide cofactor to a Q_P site located within the membrane-intrinsic domain of the enzyme. The Q_P site is sandwiched between the [3Fe-4S] cluster of the SdhB subunit and the heme b₅₅₆ that is coordinated by His residues from the SdhC and SdhD subunits. The intercenter distances between the cluster, heme, and Q_P site are all within the theoretical 14 Å limit proposed for kinetically competent intercenter electron transfer. Using EPR spectroscopy, we have demonstrated that the Q_P site of SdhCDAB stabilized a ubisemiquinone radical intermediate during enzyme turnover. Potentiometric titrations indicate that this species has an E_m,8 of 60 mV and a stability constant (K_STAB) of 1.0. Mutants of the following conserved Q_P site residues, SdhC-S27, SdhC-R31, and SdhD-D82, have severe consequences on enzyme function. Mutation of the conserved SdhD-Y83 suggested to hydrogen bond to the ubiquinone cofactor had a less severe but still significant effect on function. In addition to loss of overall catalysis, these mutants also affect the rate of succinate-dependent heme reduction, indicating that the Q_P site is an essential stepping stone on the electron transfer pathway from the [3Fe-4S] cluster to the heme. Furthermore, the mutations result in the elimination of EPR-visible ubisemiquinone during potentiometric titrations. Overall, these results demonstrate the importance of a functional, semiquinone-stabilizing Q_P site for the observation of rapid succinate-dependent heme reduction.

Received for publication, August 7, 2006 , and in revised form, August 30, 2006.

^* This work was funded by the Canadian Institutes of Health Research, National Institutes of Health Grant GM61606, and the Department of Veterans Affairs. Infrastructure funding was provided by the Canada Foundation for Innovation. 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 Holds a Canada Research Chair in Membrane Biochemistry. To whom correspondence should be addressed. Tel.: 780-492-2761; Fax: 780-492-0886; E-mail: joel.weiner{at}ualberta.ca.

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				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]

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