Features of Assembly and Mechanism of Yeast Mitochondrial Ubiquinol:Cytochrome C Oxidoreductase
Several deletion mutants of S. cerevisiae, deficient in one of the subunits of the yeast mitochondrial ubiquinol: cytochrome c oxidoreductase, contain subnormal amounts of various subunits, due to proteolytic breakdown as consequence of improper assembly. Only deletion of the 17 kDa subunit or the Fe-S protein does not have any significant effect on assembly. It is concluded that the complex is assembled from various subcomplexes of which the subcomplex consisting of cytochrome b, the 11 kDa subunit and the 14 kDa subunit is sensitive to proteolytic breakdown in the absence of any of the two core proteins. The Fe-S protein has to be integrated into this subcomplex to become proteinase-resistent. Cytochrome b is absent in the 11 and 14 kDa0 mutants, and present at very low amounts in the 40 and 44 kDa0 mutants.
Using digitonin for selective disruption of the outer and both outer and inner membrane of the yeast mitochondria the topology of the various subunits is investigated. It is concluded that the 11 kDa subunit can be digested from the intermembrane space, while the 14 kDa can be disrupted only from the matrix. The core proteins, especially the 40 kDa subunit, protect this subunit from proteolytic breakdown.
Studies with embellin, fluorescamin and dicyclohexylcarbodiimide show that one of the two antimycin-binding sites in the dimeric complex can be occupied without any effect on the rate of electron transfer from exogenous quinol to cytochrome c, while the reduction of cytochrome b by exogenous quinol via centre i is fully blocked. It is concluded that in steady-state electron transfer the reaction between cytochrome b and exogenous quinone (at centre i) is not involved. The relation of these phenomena with the known effect of e.g. dicyclohexylcarbodiimide on the proton translocation is briefly discussed.
KeywordsElectron Transfer Core Protein Dimeric Complex Proton Translocation Submitochondrial Particle
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