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Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc1) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O2-reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form.