Superoxide- and semiquinone-linked activation of molecular hydrogen in metal-catalyst-free solution

The therapeutic effects of molecular hydrogen (H₂), particularly in ischemia-reperfusion (I/R) injury and deleterious inflammation, have been increasingly attributed to its modulation of redox balance. However, the precise molecular mechanisms underlying H₂-medated redox modulation, particularly in mitochondrial reverse electron transfer (RET)-driven superoxide (O₂•^-) generation, remain unclear. Here we show that under membrane-less in-solution conditions, H₂ modulates O₂•^- kinetics in ways consistent with a tunneling-assisted electron transfer involving SQ radicals, without catalytic metals or hydrogenases. Using enzymatic (xanthine oxidase/hypoxanthine; XO/Hx) and non-enzymatic (potassium superoxide; KO₂) systems combined with the O₂•^--specific chemiluminescent probe, 2-methyl-6-p-methoxyethynyl-imidazopyrazinone (MPEC), we observed bell-shaped and U-shaped O₂•^- kinetics as a function of H₂. In Q-free assays, O₂•^- appeared to activate H₂, yielding a clear bell-shaped kinetic profile compatible with tunneling-assisted electron transfer from H₂ to O₂•^-. When Q was present, distinct U-shaped profiles emerged, consistent with Q•^--mediated electron buffering followed by H₂ activation. Electron spin resonance (ESR) radical scavenging experiments and quantitative high-performance liquid chromatography (HPLC) analyses confirmed transient semiquinone-mediated redox cycling leading to the formation of ubiquinol (QH₂). Collectively, these in-solution data support a metal-free pathway for H₂ participation in Q redox cycling that is compatible with tunneling-assisted electron transfer under defined <i>in vitro</i> conditions. These findings demonstrate the chemical feasibility of H₂-driven Q reduction in-solution; the <i>in vivo</i> relevance remains to be determined.