Superoxide-and Semiquinone-Linked Activation of Molecular Hydrogen in Metal-Catalyst-Free solution

Toru Ishibashi^1*Enjuro Harunari²Genki Ishihara³Tetsushi Niiyama⁴Mami Noda- Urata⁴Nobuaki Komori⁵

• ¹Anicom Specialty Medical Institute Inc., Tokyo, Japan
• ²Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural Universitybiote, Toyama, Japan
• ³12-pharmacy, 5-33-36-3F Minamitoriyama, Setagaya-ku, Tokyo 157-0062, Japan, Tokyo, Japan
• ⁴Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, Fukuoka, Japan
• ⁵Anicom Senshin Iryo Kenkyujo Kabushiki Kaisha, Shinjuku, Japan

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 semiquinone radicals (Q•⁻), 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 in vitro conditions. These findings demonstrate the chemical feasibility of H₂-driven Q reduction in-solution; the in vivo relevance remains to be determined.