Conference Agenda

Mycobacterium smegmatis is a gram-positive Actinobacterium and a close relative of the important pathogen M. tuberculosis. The M. smegmatis respiratory chain is branched at the level of the menaquinol pool, with the final oxygen reduction step being carried out either by the bcc-aa₃ supercomplex [1] or the cyt. bd quinol oxidase [2].

For the bcc-aa₃ supercomplex we have investigated the mechanistic details and showed that the bcc QcrB-loop that covers the entry to the D-pathway in the aa₃ oxidase has important consequences [3] for the proton transfer characteristics of the complex in comparison to canonical aa₃ oxidases. Also, out of the supercomplex, the aa₃ does not regain canonical properties. We will discuss the implications of these results.

For the cytochrome bd, interestingly the M. smegmatis genome contains an additional AppBC gene cluster, coding for a putative second bd protein. We have expressed and purified this bd-II (as well as the bd-I) and we can show that it contains the same heme components as the bd-I; hemes b⁵⁵⁸, b⁵⁹⁵ and a heme d, but that its substrate preferences and catalytic properties are different from the M. smegmatis bd-I. The possible advantages of these differences for the bacterium will be discussed.

[1] B. Wiseman, R.G. Nitharwal, O. Fedotovskaya, J. Schäfer, H. Guo, Q. Kuang, S. Benlekbir, D. Sjöstrand, P. Ädelroth, J.L. Rubinstein, P. Brzezinski, M. Högbom, Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis, Nat. Struct. Mol. Biol. 25(12) (2018) 1128-1136.

[2] W. Wang, Y. Gao, Y. Tang, X. Zhou, Y. Lai, S. Zhou, Y. Zhang, X. Yang, F. Liu, L.W. Guddat, Q. Wang, Z. Rao, H. Gong, Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels, Nat. Commun. 12(1) (2021) 4621.

[3] S. Krol, O. Fedotovskaya, M. Högbom, P. Ädelroth, P. Brzezinski, Electron and proton transfer in the M. smegmatis III(2)IV(2) supercomplex, Biochim. Biophys. Acta 1863(7) (2022) 148585.

A comprehensive understanding of the pathophysiological role of the mitochondrial respiratory chain (MRC) hinges upon a well-grounded model explaining how its biogenesis is regulated. The lack of a coherent framework to elucidate the modes and mechanisms governing the formation of MRC complexes and supercomplexes (SCs) impedes progress in the field. Initially proposed as a means to account for the co-existence of individual MRC complexes and their association into SCs, the plasticity model gained traction due to its simplicity. However, accumulating data over recent years cast doubt on the universal validity of the plasticity model as originally proposed. Instead, a cooperative assembly model provides a broader explanation to the phenomena observed when studying MRC biogenesis in physiological and pathological settings. For example, only the cooperative assembly model can account for the existence of intermediate SC assembly stages, wherein specific submodules from different complexes associate, and can explain how structural deficiencies in one single MRC complex leads to combined MRC deficiency in patients. The role of the various COX7A protein isoforms will be discussed within this framework. These proteins act as regulatory check-points guiding distinct biogenetic pathways for complex IV assembly, either as an individual complex or integrated within a wide variety of respiratory SCs, thereby shaping the structural and functional diversity of the MRC.

Respiratory complexes (RCs) are intricate molecular assemblies, consisting of multiple subunits that arise from two distinct genomes. Within the total of 90 different subunits constituting RCs (44 in CI, 4 in CII, 11 in CIII, 14 in CIV, and 17 in CV), 13 are encoded by mitochondrial DNA (mtDNA), while the remaining 77 stem from nuclear DNA (nDNA). Interestingly, within those encoded by nDNA 3 are in the X-chromosome (Ndufa1, Cox7b, or Ndufb11), a feature conserved in mammals. The number of distinct components raises to 103 if we consider the possibility of assembling alternative isoforms encoded by paralogous genes in CIV and CV. The system is envisioned as an invariant structure between cells and individuals. However, the existence of alternative paralogs and the diploid nature and genetic diversity inherent to nuclear-encoded OxPhos genes (nOxPhos) has the potential to impact the structural and functional attributes of mitochondrial complexes and supercomplexes. The respiratory complexes in the mitochondrial inner membrane can be found in monomer, homodimer, hetero-multimer (also called supercomplexes) in different combinations. This convoluted organization is universal and can be found in fungus, plants, and animals. The functional relevance of this associations has been a question of controversy with apparently conflicting results among different studies since the discovery of supercomplexes twenty-three years ago. Despite of the controversy several agreements have been achieved. Both, free complexes and supercomplexes co-exist. Both free complexes and supercomplexes can participate in a functional electron transport chain. The plasticity model of organization of the mitochondrial respiratory chain postulate that a whole free or whole super assembled electron transport chain should allow a respiratory competent mitochondrion and therefore be compatible with life. Up to know this postulated could not be confirmed experimentally. Here we will discuss the physiological implications that the genetic heterogenicity impose in the physiology of the OxPhos system

Respiratory chain supercomplexes are suggested to affect OXPHOS function in physiology and disease. Unfortunately, the available data are mostly correlative and based on in vitro studies of cell lines. To critically test the in vivo role of supercomplexes, we recently generated homozygous knock-in mice with drastically reduced levels of supercomplex I/III₂ and respirasomes (supercomplex I/III₂/IV). These mice have apparently normal function of the individual respiratory chain complexes and no obvious impairment of physiology. These results argue that the formation of respirasomes is not critically important for basal bioenergetics and physiology in mammals. In another study, we developed highly specific, allosteric inhibitors of the mitochondrial RNA polymerase (POLRMT) to treat cancer by impairing tumour metabolism in preclinical models. Unexpectedly, treatment with inhibitors of mitochondrial transcription (IMTs) leads to a rapid normalization of body weight, reversal of hepatosteatosis, and restoration of normal glucose tolerance in mice with diet-induced obesity. The IMT treatment causes a severe reduction of OXPHOS capacity concomitant with marked upregulation of fatty acid oxidation in the liver, as determined by bioenergetics, proteomics, and metabolomics analyses. This rewiring of metabolism caused by reduced mtDNA expression in the liver provides a novel principle for drug treatment of obesity and obesity-related pathology.