The yeast mitochondrial proteins Rcf1 and Rcf2 support the enzymology of the cytochrome oxidase complex and generation of the proton motive force.

The yeast mitochondrial proteins Rcf1 and Rcf2 are associated with a subpopulation of the cytochrome -cytochrome oxidase supercomplex and have been proposed to play a role in the assembly and/or modulation of the activity of the cytochrome oxidase (complex IV, CIV). Yeast mutants deficient in either Rcf1 or Rcf2 proteins can use aerobic respiration-based metabolism for growth, but the absence of both proteins results in a strong growth defect. In this study, using assorted biochemical and biophysical analyses of Rcf1/Rcf2 single and double null-mutant yeast cells and mitochondria, we further explored how Rcf1 and Rcf2 support aerobic respiration and growth. We show that the absence of Rcf1 physically reduces the levels of CIV and diminishes the ability of the CIV that is present to maintain a normal mitochondrial proton motive force (PMF). Although the absence of Rcf2 did not noticeably affect the physical content of CIV, the PMF generated by CIV was also lower than normal. Our results indicate that the detrimental effects of the absence of Rcf1 and Rcf2 proteins on the CIV complex are distinct in terms of CIV assembly/accumulation and additive in terms of the ability of CIV to generate PMF. Thus, the combined absence of Rcf1 and Rcf2 alters both CIV physiology and assembly. We conclude that the slow aerobic growth of the Rcf1/Rcf2 double null mutant results from diminished generation of mitochondrial PMF by CIV and limits the level of CIV activity required for maintenance of the PMF and growth under aerobic conditions.