Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae

Abstract

The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinolcytochrome c reductase; cyt. bc1) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc1. In the present study, we have investigated the kinetics of ligand binding, the singleturnover reaction of CytcO with O2, and the linked cyt. bc1-CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1δ and rcf2δ, respectively). The data show that in the rcf1δ and rcf2δ strains, in a significant fraction of the population, ligand binding occurs over a time scale that is ∼100-fold faster (τ ≅ 100 μs) than observed with the wild-Type mitochondria (τ ≅ 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc1CytcO supercomplex. Furthermore, in the rcf1δ and rcf2δ strains, the single-Turnover reaction of CytcOwithO2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electrontransfer bridge from cyt. bc1 to CytcO via a tightly bound cyt. c. We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc1 and CytcO.