Anaerobic derivates of mitochondria and peroxisomes in the free-living amoeba Pelomyxa schiedti revealed by single-cell genomics

Pelomyxa schiedti is a free-living amoeba belonging to the group Archamoebae, which encompasses anaerobes bearing mitochondrion-related organelles (MROs) - hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica. Anaerobic peroxisomes, another adaptation to anaerobic lifestyle, were identified only recently in M. balamuthi. We found evidence for both these organelles in the single-cell-derived genome and transcriptome of P. schiedti, and corresponding vesicles were tentatively revealed in electron micrographs. In silico reconstructed MRO metabolism seems similar to that of M. balamuthi harboring respiratory complex II, electron-transferring flavoprotein, partial TCA cycle running presumably in reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, glycine cleavage system, and sulfate activation pathway. The cell disposes with an expanded set of NIF enzymes for iron sulfur cluster assembly, but their localization remains unclear. Quite contrary, out of 67 predicted peroxisomal enzymes, only four were reported also in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Other putative functions of peroxisomes could be pyridoxal 5′-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Future experimental evidence is necessary to define functions of this surprisingly enzyme-rich anaerobic peroxisome.

Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°′ ∼−450 mV) using NADH (E°′ −320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°′ −80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family

ABSTRACT Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes. IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.