The ubihydroquinone:cytochrome c oxidoreductase (cyt bc 1) contains two catalytically active domains, termed the hydroquinone oxidation (Q o) and quinone reduction (Q i) sites, which are distant from each other by over 30 Å. Previously, we have reported that binding of inhibitors to the Q i site on one (n) side of the energy-transducing membrane changes the local environment of the iron-sulfur (Fe/ S) protein subunit residing in the Q o site on the other (p) side of the lipid bilayer [Cooley, J. W., Ohnishi, T., and Daldal, F. (2005) Biochemistry 44, 10520-10532]. These findings best fit a model whereby the Q o and Q i sites of the cyt bc 1 are actively coupled in spite of their distant locations. Because the Fe/S protein of the cyt bc 1 undergoes a large-scale (macro) domain movement during catalysis, we examined various macromobility-defective Fe/S subunit mutants to assess the role of this motion on the coupling of the active sites and also during the multiple turnovers of the enzyme. By monitoring the changing environments of the Fe/S protein [2Fe-2S] cluster upon addition of Q i site inhibitors in selected mutants, we found that the Q o-Q i site interactions manifest differently depending on the ability of the Fe/S protein to move between the cytochrome b and cytochrome c 1 subunits of the enzyme. In the presence of antimycin A, an immobile Fe/S protein mutant exhibited no changes in its EPR spectra. In contrast, mobility-restricted mutants showed striking alterations in the EPR line shapes and revealed two discrete subpopulations in respect to the [2Fe-2S] cluster environments at the Q o site. These findings led us to conclude that the mobility of the Fe/S protein is involved in its response to the occupancy of the Q i site by different molecules. We propose that the heterogeneity seen might reflect the distinct responses of the two Fe/S proteins at the Q o sites of the dimeric enzyme upon the occupancy of the Q i sites and discuss it in terms of the function of the dimeric cyt bc 1 during its multiple turnovers.
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