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.