Oxidative phosphorylation in mitochondria is performed by enzyme complexes and electron carriers that reside in the inner membrane. It is now generally accepted that these respiratory enzyme complexes assemble into larger so-called supercomplexes. However, it is presently not known why, under which conditions or how these supercomplexes form.

A number of factors of particular importance for the formation of supercomplexes have been identified, such as the Respiratory supercomplex factors (Rcf1 and Rcf2) and cardiolipin. The work presented in this thesis is focused on the characterization of cytochrome c oxidase (CytcO) in mitochondria from Saccharomyces cerevisiae strains in which these components have been removed, with a particular focus on Rcf1. First, we concluded that Rcf1 has an impact on the activity and ligand binding kinetics of CytcO, which upon genetic deletion of rcf1 leads to formation of sub-populations of CytcO with different functionality. Second, we noted that the ability of CytcO to oxidize cytochrome c (cyt. c) depends on the presence of Rcf1. Further, we observed that while CytcO in wild-type mitochondria displayed differences in the oxidation kinetics of cyt. c from horse heart or S. cerevisiae, with the Δrcf1 mitochondria these differences were lost. This observation suggested that Rcf1 interacts with cyt. c. Furthermore, the data showed that in CytcO from Δrcf1 mitochondria heme a₃ was altered while heme a was intact.

Using proteo-liposomes of different lipid composition and size we also investigated the influence of lipid head groups on the coupled activity of a quinol oxidase and ATP-synthase. Specifically, we addressed the question if protons are transferred between proton “producers” and “consumers” via lateral proton transfer along the membrane surface or via bulk water. Our data supported the principle of lateral proton transfer.

Lastly, we characterized the ligand binding of yeast flavohemoglobin and concluded that the flavohemoglobin has a population that resides in the intermembrane space of mitochondria, not only in matrix and cytosol as previously suggested.

Energy conversion from nutrients to ATP is a vital process in cells. The process, called oxidative phosphorylation (OXPHOS) is performed by a combination of membrane-bound proteins. These proteins have been studied in great detail in the past, however much is still unknown about how they interact with each other. Studying the OXPHOS proteins in their native environment can be difficult due to the complexity of living cells. By isolating parts of the OXPHOS system and inserting them into membrane-mimetic systems it is possible to investigate their functions in a controlled environment.

In the work presented here, we co-reconstituted several of these proteins into liposomes made from synthetic lipids. We demonstrated production of ATP at steady-state conditions with the ATP synthase, driven by proton pumping by cytochrome bo₃. Introduction of anionic lipids decreased the coupled activity and we could correlate this effect to weaker interactions between ATP synthase and cytochrome bo₃ in the membrane. We also reconstituted cytochrome c oxidase (CytcO) from Saccharomyces cerevisiae with Respiratory supercomplex factor 1 (Rcf1) into liposomes and submitochondrial particles (SMPs). Loss of Rcf1 has previously been found to result in a lower CytcO activity. We found that activity could be restored upon co-reconstitution of CytcO with Rcf1, but only after unfolding and re-folding of the latter, which shows that Rcf1 can adopt two configurations in the membrane.