Abstract
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.