Abstract
<jats:p>Although molecular hydrogen (H2) ameliorates a wide range of pathological conditions, how animal cells, which lack the metal hydrogenases that dissociate the nonpolar, exceptionally strong H–H bond, activate H2 has remained unresolved. Using first-principles calculations (unrestricted B3LYP-D3BJ/def2-SVP/CPCM) on active-site clusters of the Complex I Q-chamber (PDB 7QSK) and the Complex III Qo site (PDB 1PP9), we examined whether semiquinone can dissociate H2 in the buried, low-dielectric enzyme interior. At both sites the anionic semiquinone Q•- had no pathway for cleaving H–H, and only the neutral QH•, protonated by nearby residues, opened a homolytic hydrogen atom transfer (HAT) pathway with a well-defined transition state and a bound product. The spin density on the transferring hydrogen shifts continuously from nearly 0 in the reactant to +0.99 for free H•, confirming at the electronic level a true homolytic HAT, neither proton nor hydride transfer. The reaction energies (+23 to +30 kcal/mol) agree with a bond-dissociation-free-energy (BDFE) comparison showing QH• to be a spin-allowed hydrogenatom abstractor about 30 kcal/mol superior to superoxide, and thus central to the reaction. However, this reactive QH• arises only under hyper-reductive pressure, where quinone-pool overreduction and a high membrane potential coincide to occupy and protonate the buried semiquinone site; H2 dissociation is therefore established only when this hyper-reductive-pressure reaction-site environment is present, as a necessary condition. Because H2 has among the strongest σ bonds, the barrier is high and sharp, and quantum tunnelling is required for the reaction to proceed at body temperature. Crossover-temperature analysis places Complex I (316.9 K) on the tunnelling-dominated side and Complex III (255 K) on the thermal side, so the two sites straddle body temperature, with a predicted primary kinetic isotope effect of kH/kD ≈ 6–7. The Marcus inverted region we previously observed in solution is an experimental fingerprint of this tunnelling. Together, these results reframe H2 medicine from classical antioxidant scavenging to a quantum-tunnelling-assisted, reductive-pressure-conditioned conditional hydrogenase activity, and position it within quantum biology.</jats:p>