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Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1beta subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1alpha, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP(+) oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.