An electron-bifurcating hydrogenase-like activity associated with mitochondrial Complex I under hypoxia in vascular plants

Molecular hydrogen (H2) is produced by plants under hypoxia and has been implicated in stress acclimation, yet its enzymatic source in vascular plants remains unknown because canonical hydrogenases are absent from angiosperm genomes. Here, using thermodynamic modeling, kinetic analysis, pharmacological perturbation, substrate-feeding assays, metabolite profiling, and cross-species comparison, we identify a mitochondrial origin for hypoxic H2 production in plants and define its biochemical requirements. Our data reveal a hydrogenase-like activity that obligatorily couples to Complex I Fe-S/quinone branch turnover and exhibits the hallmark of flavin-based electron bifurcation, a mechanism that cannot be explained by simple flavin over-reduction. The rotenone paradox, in which blockade of the N2-to-ubiquinone step abolishes rather than enhances H2 production, constitutes the most diagnostic evidence for this mechanism. This activity is promoted by convergent NADH- and succinate-supplying pathways, requires protonmotive force and continued ubiquinone re-oxidation through alternative oxidase, and is favored under micro-oxic and acidic conditions. The biochemical properties of this activity are consistent with a model in which a plant-specific Complex I assembly intermediate, termed CI*, provides the catalytic platform through an FMN-centered, N1a-assisted electron-bifurcation mechanism. Attempts to reconstitute H2 production from isolated Complex I subcomplexes have not yielded activity, consistent with the models prediction that this function requires an intact mitochondrial membrane system. H2-producing activity localized predominantly to mitochondria and was conserved across phylogenetically diverse vascular plants. Together, these findings reveal a previously unrecognized electron-bifurcating activity associated with a plant Complex I assembly intermediate. They provide a mechanistic framework for understanding endogenous H2 emission under hypoxia, a phenomenon first reported over 60 years ago but never explained, and identify mitochondrial redox flexibility as a potential target for improving tolerance to flooding and other oxygen-limiting stresses.