The anaerobic fungus Caecomyces churrovis produces H₂ via a non-bifurcating NADH-dependent enzyme complex

Hydrogenosomes are mitochondrion-derived organelles that produce ATP and H₂ to support energy metabolism in anaerobic eukaryotes. H₂ production allows reoxidation of reduced cofactors generated during fermentative metabolism; however, the metabolic mechanisms for H₂ production in anaerobic eukaryotes remain incompletely understood. In particular, it remains unclear whether anaerobic fungi (AF) hydrogenosomes use a ferredoxin-dependent pathway or a distinct mechanism to regenerate NAD(P)⁺ and link electron transfer to H₂ formation. Here, by combining genomic search, proteomic analysis, and enzymology, we reveal the molecular mechanism for H₂ production in the AF Caecomyces churrovis. Our enzyme assays on the organelle fraction of C. churrovis revealed the activity of H₂:NAD⁺ oxidoreductase but not pyruvate:ferredoxin oxidoreductase, which is usually linked to H₂ formation. We identified genes encoding [FeFe] hydrogenase (Hyd) and NADH dehydrogenase subunits E and F (NuoE and NuoF) in C. churrovis and confirmed their expression in the isolated hydrogenosomal fractions by proteomic analysis. Combining the individually purified enzymes, we found Hyd and NuoEF proteins formed H₂ directly from NADH independently of ferredoxin, functioning as a non-bifurcating NADH-dependent enzyme rather than an electron-bifurcating enzyme known from anaerobic prokaryotes. We identified homologs of hydrogenosomal NuoE, NuoF, and Hyd in many other AF, indicating this pathway is commonly shared among the AF. This work demonstrates the existence of a non-bifurcating NADH-dependent enzyme complex for H₂ production in eukaryotes. Moreover, this complex could potentially be exploited as a target for controlling AF H₂ production and altering fungal metabolism.

H₂ production is a prominent feature of anaerobic energy metabolism, yet our understanding of eukaryotic mechanisms remains limited. Anaerobic fungi (AF) are key decomposers of lignocellulose and contribute to hydrogen flux in anaerobic environments. Although it has been more than 40 years since the H₂ production in Neocallimastix was first reported, the molecular mechanism for hydrogenosomal H₂ production and redox balance remains unclear. We demonstrate that AF produce H₂ from NADH utilizing a non-bifurcating NADH-dependent enzyme complex rather than an electron-bifurcating, ferredoxin-dependent variant. We show that this enzyme complex is conserved across multiple AF lineages and thus demonstrate the occurrence of a non-bifurcating NADH-dependent enzyme in eukaryotes. This discovery expands our understanding of eukaryotic hydrogenosomal metabolism, reveals a previously unknown strategy for redox balancing, and highlights potential targets for manipulating H₂ production. These insights have broad implications for microbial energy metabolism, anaerobic ecosystems, and bioengineering of H₂-producing systems.