Supplementary File 3 from Adaptation to life on land at high O₂ via transition from ferredoxin-to NADH-dependent redox balance

Pyruvate : ferredoxin oxidoreductase (PFO) and iron only hydrogenase ([Fe]-HYD) are common enzymes among eukaryotic microbes that inhabit anaerobic niches. Their function is to maintain redox balance by donating electrons from food oxidation via ferredoxin (Fd) to protons, generating H₂ as a waste product. Operating in series, they constitute a soluble electron transport chain of one-electron transfers between FeS clusters. They fulfil the same function—redox balance—served by two electron-transfers in the NADH- and O₂-dependent respiratory chains of mitochondria. Although they possess O₂-sensitive FeS clusters, PFO, Fd and [Fe]-HYD are also present among numerous algae that produce O₂. The evolutionary persistence of these enzymes among eukaryotic aerobes is traditionally explained as adaptation to facultative anaerobic growth. Here, we show that algae express enzymes of anaerobic energy metabolism at ambient O₂ levels (21% v/v), Chlamydomonas reinhardtii expresses them with diurnal regulation. High O₂ environments arose on Earth only some approximately 450 million years ago. Gene presence/absence and gene expression data indicate that during the transition to high O₂ environments and terrestrialization, diverse algal lineages retained enzymes of Fd-dependent one-electron-based redox balance, while the land plant and land animal lineages underwent irreversible specialization to redox balance involving the O₂-insensitive two-electron carrier NADH.