Mitochondrial cytochrome c oxidase (COX) is the primary site of cellular oxygen consumption and is essential for aerobic energy generation in the form of ATP. Human COX is a copper-heme A hetero-multimeric complex formed by 3 catalytic core subunits encoded in the mitochondrial DNA and 11 subunits encoded in the nuclear genome. Investigations over the last 50 years have progressively shed light into the sophistication surrounding COX biogenesis and the regulation of this process, disclosing multiple assembly factors, several redox-regulated processes leading to metal co-factor insertion, regulatory mechanisms to couple synthesis of COX subunits to COX assembly, and the incorporation of COX into respiratory supercomplexes. Here, we will critically summarize recent progress and controversies in several key aspects of COX biogenesis: linear versus modular assembly, the coupling of mitochondrial translation to COX assembly and COX assembly into respiratory supercomplexes.

Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.

In most eukaryotes, the mitochondrion is the main organelle for the formation of iron-sulfur (FeS) clusters. This function is mediated through the iron-sulfur cluster assembly machinery, which was inherited from the α-proteobacterial ancestor of mitochondria. In Archamoebae, including pathogenic Entamoeba histolytica and free-living Mastigamoeba balamuthi, the complex iron-sulfur cluster machinery has been replaced by an e-proteobacterial nitrogen fixation (NIF) system consisting of two components: NifS (cysteine desulfurase) and NifU (scaffold protein). However, the cellular localization of the NIF system and the involvement of mitochondria in archamoebal FeS assembly are controversial. Here, we show that the genes for both NIF components are duplicated within the M. balamuthi genome. One paralog of each protein contains an amino-terminal extension that targets proteins to mitochondria (NifS-M and NifU-M), and the second paralog lacks a targeting signal, thereby reflecting the cytosolic form of the NIF machinery (NifS-C and NifU-C). The dual localization of the NIF system corresponds to the presence of FeS proteins in both cellular compartments, including detectable hydrogenase activity in Mastigamoeba cytosol and mitochondria. In contrast, E. histolytica possesses only single genes encoding NifS and NifU, respectively, and there is no evidence for the presence of the NIF machinery in its reduced mitochondria. Thus, M. balamuthi is unique among eukaryotes in that its FeS cluster formation is mediated through two most likely independent NIF machineries present in two cellular compartments.hydrogenosome | mitosome | free-living protist

SUMMARYThe mitochondrial respiratory chain is organized in a dynamic set of supercomplexes (SCs). The COX7A2L protein is essential for mammalian SC III2+IV assembly. However, its function in respirasome (SCs I+III2+IVn) biogenesis remains controversial. To unambiguously determine the COX7A2L role, we generated COX7A2L-knockout (COX7A2L-KO) HEK293T and U87 cells. COX7A2L-KO cells lack SC III2+IV but have enhanced complex III steady-state levels, activity, and assembly rate, normal de novo complex IV biogenesis, and delayed respirasome formation. Nonetheless, the KOs have normal respire some steady-state levels, and only larger structures (SCs I1–2+III2+IV2-n or megacomplexes) were undetected. Functional substrate-driven competition assays showed normal mitochondrial respiration in COX7A2L-KO cells in standard and nutritional-, environmental-, and oxidative-stress-challenging conditions. We conclude that COX7A2L establishes a regulatory checkpoint for the biogenesis of CIII2 and specific SCs, but the COX7A2L-dependent MRC remodeling is essential neither to maintain mitochondrial bioenergetics nor to cope with acute cellular stresses.

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi. We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named “anaerobic” peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.