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

Abstract

Abstract Molecular hydrogen (H2) evolution by higher plants under oxygen limitation was reported more than six decades ago, yet its origin, biochemical basis and physiological significance have remained unresolved. Here, we localize this activity to a mitochondrial, Complex I-associated electron-transfer system. Mitochondrial fractions from etiolated Vigna radiata hypocotyls accumulated H2 under hypoxia and mildly acidic conditions. H2 accumulation was nearly abolished by rotenone and was sensitive to perturbations of Complex II, quinone-pool turnover, terminal oxidases and protonmotive force. NADH-generating substrates and succinate-fumarate supplementation stimulated H2 accumulation, while metabolic profiling linked H2 production to succinate and NADH accumulation. Cumulative H2 production exceeded the measured NADH pool by approximately two orders of magnitude, requiring sustained NADH regeneration. These findings support a division-of-labour model involving coexisting mature Complex I and CI*, a Complex I assembly or remodeling state. TCA-cycle dehydrogenation and succinate-driven reverse electron transfer (RET) through mature Complex I replenish the matrix NADH pool, whereas CI* consumes NADH through an H2-evolving branch, regenerating NAD. This model identifies a potential conditional redox function for CI* and an electron outlet coupled to continuous NADH cycling. Unlike mammalian RET, which can generate pronounced reactive oxygen species (ROS), diversion of reducing equivalents into H2 could attenuate the corresponding ROS signature, potentially helping explain the elusive physiological evidence for plant RET. The data establish a RET-compatible supply route, not direct reverse electron flux. Structural and computational analyses support an FMN-Fe-S branched mechanism whose catalytic steps remain unverified. More broadly, Complex I assembly may not merely build the respiratory machinery; under specific physiological conditions, its intermediate states may reshape electron fate, exposing latent hydrogenase-related chemistry associated with Complex I's evolutionary ancestry.