The α1-Helix in the Extra-Membranous Domain Contributes to the Stability of the Iron–Sulfur Protein of the Cytochrome bc1Complex

Previously, we reported that several charged amino acids located in the α1–β4 loop of the iron–sulfur protein are required to maintain the stability of the protein and its assembly into the cytochrome bc1complex. The current study extends this analysis to several amino acids localized in the single α-helix present in the extra-membranous domain of the protein. Three charged and two uncharged residues in the α1-helix were mutated and used to transform yeast cells (JPJ1) lacking the iron–sulfur protein gene. Mutants V132L and H124L grew at half the rate of the wild type in medium containing glycerol/ethanol, while E125Q grew more slowly than the wild type. The rates of growth of T122A and E128Q were identical to that of the wild-type cells. Activity of the cytochrome bc1complex was decreased 70, 40, and 80% in mutants H124L, E125Q, and V132L, respectively, while the activity of T122A and E128Q was decreased 30 and 20% relative to the wild type. Western blotting experiments revealed that the content of the iron–sulfur protein was decreased in mutants H124L and V132L; however, no decrease in the content of the iron–sulfur protein was observed in the other mutants. These results suggest that several amino acids located in the α1-helix of the protein are important in maintaining the stability and proper assembly of the iron–sulfur protein in the bc1complex.