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.