Current competing models for the two-electron oxidation of quinol (QH(2)) at the cytochrome b(C1), complex and related complexes impose distinct requirements for the reaction intermediate. At present, the intermediate species of the enzymatic oxidation process have not been observed or characterized, probably due to their transient nature. Here, we use a biomimetic oxidant, excited-state Ru(bpy)(2)(pbim)(+) (bpy = 2,2'-dipyridyl, pbim = 2-(2-pyridyl)benzimidazolate) in an aprotic medium to probe the oxidation of the ubiquinol analogue, 2,3-dimethoxy-5-methyl-1,4-benzoquinol (UQH(2)-0), and the plastoquinol analogue, trimethyl-1,4-benzoquinol (TMQH(2)-0), using time-resolved and steady-state spectroscopic techniques. Despite its simplicity, this system qualitatively reproduces key features observed during ubiquinol oxidation by the mitochondrial cytochrome b(C1) complex. Comparison of isotope-dependent activation properties in the native and synthetic systems as well as analysis of the time-resolved direct-detection electron paramagnetic resonance signals in the synthetic system allows us to conclude that (1) the initial and rate-limiting step in quinol oxidation, both in the biological and biomimetic systems, involves electron and proton transfer, probably via a proton-coupled electron-transfer mechanism, (2) a neutral semiquinone intermediate is formed in the biomimetic system, and (3) oxidation of the QH center dot/QH(2) couple for UQH(2)-0, but not TMQH(2)-0, exhibits an unusual and unexpected primary deuterium kinetic isotope effect on its Arrhenius activation energy (Delta G(TS)), where Delta G(TS) for the protiated form is larger than that for the deuterated form. The same behavior is observed during steady-state turnover of the cyt b(C1) complex using ubiquinol, but not plastoquinol, as a substrate, leading to the conclusion that similar chemical pathways are involved in both systems. The synthetic system is an unambiguous n = 1 electron acceptor, and it is thus inferred that sequential oxidation of ubiquinol (by two sequential n = 1 processes) is more rapid than a truly concerted (n = 2) oxidation in the cyt b(C1) complex.