| Abstract
| - Current competing models for the two-electron oxidation of quinol (QH2) at the cytochrome bc1complex and related complexes impose distinct requirements for the reaction intermediate. At present, theintermediate species of the enzymatic oxidation process have not been observed or characterized, probablydue 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 theubiquinol analogue, 2,3-dimethoxy-5-methyl-1,4-benzoquinol (UQH2-0), and the plastoquinol analogue,trimethyl-1,4-benzoquinol (TMQH2-0), using time-resolved and steady-state spectroscopic techniques.Despite its simplicity, this system qualitatively reproduces key features observed during ubiquinol oxidationby the mitochondrial cytochrome bc1 complex. Comparison of isotope-dependent activation properties inthe native and synthetic systems as well as analysis of the time-resolved direct-detection electronparamagnetic 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 protontransfer, probably via a proton-coupled electron-transfer mechanism, (2) a neutral semiquinone intermediateis formed in the biomimetic system, and (3) oxidation of the QH•/QH2 couple for UQH2-0, but not TMQH2-0,exhibits an unusual and unexpected primary deuterium kinetic isotope effect on its Arrhenius activationenergy (ΔGTS), where ΔGTS for the protiated form is larger than that for the deuterated form. The samebehavior is observed during steady-state turnover of the cyt bc1 complex using ubiquinol, but not plastoquinol,as a substrate, leading to the conclusion that similar chemical pathways are involved in both systems. Thesynthetic system is an unambiguous n = 1 electron acceptor, and it is thus inferred that sequential oxidationof ubiquinol (by two sequential n = 1 processes) is more rapid than a truly concerted (n = 2) oxidation inthe cyt bc1 complex.
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