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

The therapeutic effects of molecular hydrogen (H 2 ), 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 H 2 -medated redox modulation, particularly in mitochondrial reverse electron transfer (RET)-driven superoxide (O 2 • - ) generation, remain unclear. Here we show that under membrane-less in-solution conditions, H 2 modulates O 2 • - 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; KO 2 ) systems combined with the O 2 • - -specific chemiluminescent probe, 2-methyl-6-p-methoxyethynyl-imidazopyrazinone (MPEC), we observed bell-shaped and U-shaped O 2 • - kinetics as a function of H 2 . In Q-free assays, O 2 • - appeared to activate H 2 , yielding a clear bell-shaped kinetic profile compatible with tunneling-assisted electron transfer from H 2 to O 2 • - . When Q was present, distinct U-shaped profiles emerged, consistent with Q• - -mediated electron buffering followed by H 2 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 (QH 2 ). Collectively, these in-solution data support a metal-free pathway for H 2 participation in Q redox cycling that is compatible with tunneling-assisted electron transfer under defined in vitro conditions. These findings demonstrate the chemical feasibility of H 2 -driven Q reduction in-solution; the in vivo relevance remains to be determined.