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 A 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 A 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.
Brasseur
Gaël
G
Laboratoire de Bioénergétique et Ingénierie des Protéines, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. brasseur@ibsm.cnrs-mrs.fr
Lemesle-Meunier
Danielle
D
Reinaud
Floriane
F
Meunier
Brigitte
B
D001665:Binding Sites; D002384:Catalysis; D002455:Cell Division; D003580:Cytochromes / Q000737:chemistry; D045303:Cytochromes b / Q000737:chemistry; D003575:Cytochromes c1 / Q000737:chemistry; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000235:genetics*; D004583:Electrons; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D003580:Cytochromes; D007506:Iron-Sulfur Proteins; D045303:Cytochromes b; D003575:Cytochromes c1; D014450:Electron Transport Complex III; D010100:Oxygen