The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinolcytochrome c reductase; cyt. bc 1 ) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc 1 . In the present study, we have investigated the kinetics of ligand binding, the singleturnover reaction of CytcO with O 2 , and the linked cyt. bc 1 -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. bc 1 -CytcO supercomplex. Furthermore, in the rcf1Δ and rcf2Δ strains, the single-turnover reaction of CytcO with O 2 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. bc 1 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. bc 1 and CytcO.cytochrome c oxidase | electron transfer | membrane protein | cytochrome aa 3 | cytochrome bc 1

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.

Respiratory supercomplex factor (Rcf) 1 is a membrane-bound protein that modulates the activity of cytochrome c oxidase (CytcO) in Saccharomyces cerevisiae mitochondria. To investigate this regulatory mechanism, we studied the interactions of CytcO with potassium cyanide (KCN) upon removal of Rcf1. While the addition of KCN to the wild-type mitochondria results in a full reduction of heme a, with the rcf1Δ mitochondria, a significant fraction remains oxidized. Upon addition of ascorbate in the presence of O and KCN, the reduction level of hemes a and b was a factor of ~ 2 larger with the wild-type than with the rcf1Δ mitochondria. These data indicate that turnover of CytcO was less blocked in rcf1Δ than in the wild-type mitochondria, suggesting that Rcf1 modulates the structure of the catalytic site.

Kinetic methods used to investigate electron and proton transfer within cytochrome c oxidase (CytcO) are often based on the use of light to dissociate small ligands, such as CO, thereby initiating the reaction. Studies of intact mitochondria using these methods require identification of proteins that may bind CO and determination of the ligand-binding kinetics. In the present study we have investigated the kinetics of CO-ligand binding to S. cerevisiae mitochondria and cellular extracts. The data indicate that CO binds to two proteins, CytcO and a (yeast) flavohemoglobin (yHb). The latter has been shown previously to reside in both the cell cytosol and the mitochondrial matrix. Here, we found that yHb resides also in the intermembrane space and binds CO in its reduced state. As observed previously, we found that the yHb population in the mitochondrial matrix binds CO, but only after removal of the inner membrane. The mitochondrial yHb (in both the intermembrane space and the matrix) recombines with CO with τ≅270ms, which is significantly slower than observed with the cytosolic yHb (main component τ≅1.3ms). The data indicate that the yHb populations in the different cell compartments differ in structure.

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