QO site deficiency can be compensated by extragenic mutations in the hinge region of the iron-sulfur protein in the bc1 complex of Saccharomyces cerevisiae

The mitochondrial bc(1) complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c. The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd(1) amphipathic helix in the quinol-oxidizing (Q(o)) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the Q(o) site. An intragenic suppressor mutation was selected (A144F+F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 Angstrom from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc(1) complex. They were located about 20 Angstrom from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c(1) during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd(1) helix in the quinol-oxidizing Q(o) site and the ISP via the flexible hinge region and that fine-tuning of the Q(o) site catalysis can be achieved by subtle changes in the linker domain of the ISP.