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J. Biol. Chem., Vol. 281, Issue 37, 27662-27668, September 15, 2006

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The Iron-Sulfur Clusters in Escherichia coli Succinate Dehydrogenase Direct Electron Flow^*

From the Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 473 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b₅₅₆. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.

Received for publication, May 22, 2006 , and in revised form, July 21, 2006.

^* This work was supported in part by the Canadian Institutes of Health Research. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5.

¹ Supported by an Alberta Heritage Foundation for Medical Research graduate studentship.

² 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]