The Amino-Terminal Portion of the Rieske Iron-Sulfur Protein Contributes to the Ubihydroquinone Oxidation Site Catalysis of the Rhodobacter capsulatus bc1 Complex

Abstract

The Rieske iron-sulfur (Fe-S) protein subunit of bc₁ 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_o site of the bc₁ complex (UQ/UQH₂ 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 bc₁ 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 bc₁ 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_o site occupancy. In addition, they contained substoichiometric amounts of the Fe-S protein with respect to the other subunits of the bc₁ 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_o site of the bc₁ complex but also for the properties of its [2Fe-2S] cluster.