The amino-terminal portion of the Rieske iron-sulfur protein contributes to the ubihydroquinone oxidation site catalysis of the Rhodobacter capsulatus bc(1) complex

The Rieske iron-sulfur (Fe-S) protein subunit of bet complexes contains in its carboxyl-terminal part two highly conserved hexapeptide motifs (box I and box II) that include the four amino acid ligands of its [2Fe-2S] cluster. In the preceding paper [Liebl, U,, Sled, V,, Brasseur, G., Ohnishi, T., & Daldal, F. (1997) Biochemistry 36, 11675-11684], the effects of mutations at two of the nonliganding residues [threonine (T) 134 and leucine (L) 136 in the Rhodobacter capsulatus Rieske Fe-S protein] of box I have been described, In this work, interactions between the occupants of the Q(0) site of the bc(1) complex (UQ/ UQH(2) and the inhibitors stigmatellin and myxothiazol), and the [2Fe-2S] cluster of the Rieske Fe-S protein were probed by isolating photosynthesis-proficient (Ps(+)) revertants of the Ps(-) mutants L136R, -H, -D and -G. These revertants contained either a single substitution at the original position 136 or an additional mutation located in the amino-terminal part of the Fe-S protein at either position 44 or 46, The same-site revertants L136A and -Y grew well under photosynthetic conditions and contained highly active bc(1) complexes but exhibited modified EPR spectra both in the presence and in the absence of stigmatellin. Unexpectedly, they were highly resistant to stigmatellin (Sti(R)) and hypersensitive to myxothiazol (Myx(HS)) in vivo, demonstrating for the first time that mutations located in the Fe-S subunit confer resistance to stigmatellin. The [2Fe-2S] cluster of the same-site revertants responded weakly to the Q(pool) redox state and had redox midpoint potential (E-m7) values (around 265 mV) lower than those of their wild type counterpart (about 310 mV). On the other hand, the second-site revertants L136H/V44L, L136G/V44F, and L136G/A46T, -V, or -P supported photosynthetic growth poorly, were Sti(R) and Myx(HS), and contained barely active bc(1) complexes. Like the same-site revertants, they exhibited modified EPR spectra both in the presence and in the absence of stigmatellin and had perturbed Q(0) site occupancy. In addition, they contained substoichiometric amounts of the Fe-S protein with respect to the other subunits of the bc(1) complex. The E-m7 values of the [2Fe-2S] cluster of these double mutants were lower (around 245 mV) than that of the wild type strain but appreciably higher than those of their Ps(-) parents (about 200 mV for L136G). In order to define the molecular nature of the suppression mediated by the second-site mutations, the single mutants V44L and -F and A46T and -V were constructed in the absence of the original mutations at position 136. These mutants behaved like a wild type strain with respect to their Ps(+) growth ability, inhibitor sensitivity, EPR spectra of their [2Fe-2S] cluster, and response to stigmatellin or to the Q(pool) redox state. But surprisingly, the E-m7 values of their [2Fe-2S] cluster were much higher (about 385 mV) than that of a wild type strain. These findings demonstrated for the first time that the amino-terminal part of the Rieske Fe-S protein encompassing residues 44 and 46 is important not only for the structure and function of the Q(0) site of the bc(1) complex but also for the properties of its [2Fe2S] cluster.