AUTHOR=Ishibashi Toru , Harunari Enjuro , Ishihara Genki , Niiyama Tetsushi , Noda-Urata Mami , Komori Nobuaki 

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

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=Volume 12 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2025.1680812

DOI=10.3389/fmolb.2025.1680812

ISSN=2296-889X

ABSTRACT=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 in vitro conditions. These findings demonstrate the chemical feasibility of H2-driven Q reduction in-solution; the in vivo relevance remains to be determined.