| Abstract
| - We have previously reported that mutant strains of Rhodobacter capsulatus that have alanineinsertions (+nAla mutants) in the hinge region of the iron sulfur (Fe−S) containing subunit of the bc1complex have increased redox midpoint potentials (Em) for their [2Fe2S] clusters. The alteration of theEm in these strains, which contain mutations far from the metal binding site, implied that the localenvironment of the metal center is indirectly altered by a change in the interaction of this subunit with thehydroquinone oxidizing (Qo) site [Darrouzet, E., Valkova-Valchanova, M., and Daldal, F. (2002) J. Biol.Chem.277, 3464−3470]. Subsequently, the Em changes have been proposed to be predominantly due toa stronger or more stabilized hydrogen bonding between the reduced [2Fe2S] cluster and the Qo siteinhabitant ubiquinone (Q) [Shinkarev, V. P., Kolling, D. R. J., Miller, T. J., and Crofts, A. R. (2002)Biochemistry41, 14372−14382]. To further investigate this issue, Fe−S protein-Q interactions weremonitored by electron paramagnetic resonance (EPR) spectroscopy and the findings indicated that thewild type and mutant proteins interactions with Q are similar. Moreover, when the Qpool was chemicallydepleted, the Em of the [2Fe2S] cluster in mutant bc1 complexes remained more positive than a similarlytreated native enzyme (e.g., the [2Fe2S] Em of the +2Ala mutant was 55 mV more positive than the wildtype). These data suggest that the increased Em of the [2Fe2S] cluster in the +nAla mutants is in part dueto the cluster's interaction with Q, and in part to additional factors that are independent of hydrogenbonding to Q. One such factor, the possibility of a different position of the Fe−S at the Qo site of themutant proteins versus the native enzyme, was addressed by determining the orientation of the [2Fe2S]cluster in the membrane using EPR spectroscopy. In the case of the +2Ala mutant, the [2Fe2S] clusterorientation in the absence of inhibitor is different than that seen in the native enzyme. However, the+2Ala mutant cluster shared a similar orientation with the native enzyme when both samples were exposedto either stigmatellin or myxothiazol. In addition, Qpool extracted membranes of +2Ala mutant exhibitedfewer overall orientations, with the predominant one being more similar to that observed in the non-Q-depleted membranes of the +2Ala mutant than the Q-depleted membranes of a wild-type strain. Therefore,additional component(s) that are independent of Qo site inhabitants and that originate from the newlyobserved orientations of the [2Fe2S] clusters in the +nAla mutants also contribute to the increased midpointpotentials of their [2Fe2S] clusters. While the molecular basis of these components remains to bedetermined, salient implications of these findings in terms of Qo site catalysis are discussed.
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