Mitochondria are the site where most of the energy from food is converted into adenosine triphosphate (ATP). This process is taking place at the inner membrane (IM) of mitochondria, and is called oxidative phosphorylation, and results in the establishment of a proton motive force (pmf). The proton motive force is a combination of a proton difference over the mitochondrial IM and a charge difference. The ATP is then synthesized by the ATP synthase, which is utilizing the pmf for this process. The IM of mitochondria has many invaginations, which are called cristae. The enzymes of the respiratory chain are mainly located at the flat sheet part, while the ATP synthase is located at the rim of the cristae. The hypothesis arises whether the cristae membrane would serve as a proton sink for the ATP synthase, due to the curved shape of the cristae. We aimed at answering this hypothesis by attaching a pH-sensitive green fluorescent protein (pHluorin2) at different locations within the mitochondria. The study revealed that there is no substantial pH difference across the IM of yeast mitochondria and that the cristae are not functioning as a proton sink, but rather its main function is to provide an optimal environment, for coupled enzymatic activity. The second project investigated the importance of the mobile electron carrier; cytochrome c (cyt c) of its ability to freely diffuse along with the IM. Cyt c is the electron carrier between the bc₁ complex and cytochrome c oxidase of the respiratory chain. It is also involved in programmed cell death (apoptosis) of higher eukaryotes, where its release from mitochondria initiates apoptosis. As its role in yeast apoptosis is not entirely clear, we created a yeast strain where cyt c was tethered to the IM, in a background strain that was devoid of the mobile cyt c. Interestingly, the level of apoptosis was higher in the yeast strain with the non-mobile cyt c, which indicated that cyt c release in yeast is not a necessary step to initiate apoptosis. The strain with the IM tethered cyt c had also higher levels of reactive oxygen species (ROS), shorter life span, alterations of the mitochondrial network in comparison to the wild type strain. Despite not showing any major alterations in the respiratory chain, the mutant yeast strain had elevated oxygen consumption, indicating a compensatory mechanism, which could have caused the elevated ROS levels which ultimately induced apoptosis. Maintaining a steady level of ATP is crucial for the cell, and one such mechanism in higher eukaryotes is the creatine phosphate shuttle system, by the enzyme; creatine kinase. Creatine kinase is catalyzing the phosphorylation of creatine in mitochondria, and the phosphocreatine is transported out to the cytosol, where the cytoplasmic isoform of the enzyme is regenerating ATP from the phosphocreatine. A yeast strain was created to express the mitochondrial creatine kinase, which could serve as a strategy in industrially relevant yeast strains, to circumvent ATP levels to drop during the production processes. To gain an understanding of the importance of cyt c diffusion, its relevance for the respiratory chain, the yeast strain from the second project was modified so that it was fused to the bc₁ complex. The strain showed a functional respiratory chain, and further work will provide insights into the diffusion of the respiratory complexes and their interaction with the IM.

Respiratory processes for cellular energy conversion are carried out by the membrane-associated enzymes of the electron transfer chain (ETC). In recent years there has been emerging data showing that the members of the ETC organize into higher-level assemblies called supercomplexes (SCs) whose functional relevance is not yet fully understood. SCs composed of complexes III (cytochrome (cyt.) bc₁ complex) and IV (cyt. c oxidase) are the most common. The small electron-carrier protein cyt. c shuttles electrons between complexes III and IV. In mitochondria-like ETCs cyt. c is present only in a soluble form, while in some bacteria it has additional membrane-anchored analogs or is fused to complex III forming the cyt. cc subunit, as in actinobacteria.

We determined the structure of the obligate III/IV SC from actinobacterium Mycobacterium (M.) smegmatis with cryo-electron microscopy. The structure showed that the distances between the co-factors of the SC are short enough for electron transfer with the catalytically relevant rates. Complexes III and IV within the SC were intertwined. In particular, the entrance to the D-proton pathway of complex IV was shielded by a loop of the QcrB subunit of complex III, possibly influencing proton uptake characteristics. Furthermore, superoxide dismutase was shown to be an integral part of the M. smegmatis SC, which might have a functional role in the energy conservation by the SC.

With the goal to unravel the structure-function relationships between complexes III and IV in the actinobacterial SCs, we investigated the charge transfer kinetics in SCs on a single-turnover time scale. Using time-resolved spectroscopic techniques we have determined the rates of electron and proton transfer upon oxidation of reduced SCs of M. smegmatis and another actinobacterium Corynebacterium glutamicum. Electron transfer from cyt. cc in complex III to the primary redox center Cu_A in complex IV was not rate-limiting for the SC turnover. In contrast to the canonical complex IV, certain reaction steps in SC were not pH-dependent and proton uptake kinetics through the D-pathway of complex IV was altered showing much slower kinetics. This suggests that the QcrB loop of complex III, which blocks the entrance to the D-pathway, modulates the kinetics of proton uptake in complex IV. 

In another study, we showed the existence of a non-obligate supercomplex in the alfa-proteobacterium Rhodobacter (R.) sphaeroides. This SC was purified and characterized biochemically. We concluded that complexes III and IV interact via the membrane-anchored version of cyt. c (MA-cyt. c), which is expressed in the bacterium in addition to the soluble variant. MA-cyt. c most likely plays a central role in forming the SC in R. sphaeroides by functionally connecting complexes III and IV.

In addition to being an electron shuttle, in eukaryotes cyt. c participates in apoptosis. We investigated the consequences of anchoring the cyt. c to the membrane, similar to MA-cyt. c in R. sphaeroides, in a single-cell eukaryote Saccharomyces cerevisiae, thereby not allowing the release of cyt. c from the intermembrane space of mitochondria during the induced apoptosis.

The work presented in this thesis increases our understanding of the general function-structure relationships of respiratory SCs and might have applications in potential drug development.