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,Joel H Weiner

doi:10.1074/jbc.m607476200

Abstract

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 b(556) 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 A 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 approximately 60 mV and a stability constant (K(STAB)) of approximately 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.

Highlights

Escherichia coli encodes a tetrameric enzyme, succinate dehydrogenase2, that is responsible for the oxidation of succinate to fumarate during aerobic growth
We have examined the role of the quinone-binding (QP) site of Escherichia coli succinate:ubiquinone oxidoreductase in heme reduction and reoxidation during enzyme turnover
All mutants and wild-type enzymes were expressed in E. coli DW35, which contains a deletion in the frdABCD operon and an inactivated sdhCDAB operon to eliminate any chromosomally encoded enzyme contributing to the activity

Summary

Introduction

Escherichia coli encodes a tetrameric enzyme, succinate dehydrogenase (succinate:ubiquinone oxidoreductase; SQR), that is responsible for the oxidation of succinate to fumarate during aerobic growth. SdhA contains a redox active flavin adenine dinucleotide (FAD) at its dicarboxylic acid binding site that cycles via an EPR-visible flavin semiquinone between the FAD and FADH2 redox states during catalytic succinate oxidation [7] The electrons from this reaction are sequentially transferred through SdhB, the iron-sulfur protein subunit, via a series of cofactors, a [2Fe-2S] cluster, a [4Fe-4S] cluster, and a [3Fe-4S] cluster, to a quinone binding site (QP site) located at the interface of the SdhB, SdhC, and SdhD subunits. The edge-to-edge distances from the [3Fe-4S] cluster to the Qp site and the nearest heme propionate are 6.8 and 8.3 Å, respectively These distances are well within the limit for effective electron tunneling of 14 Å [13], and it is unknown what role the heme may have in the electron transfer pathway, as electrons are fully capable of traveling directly to a ubiquinone bound at the Qp site. The structure of the B. subtilis SQR membrane domain is radically different compared with the E. coli SQR

Methods

Results

Conclusion