PNNL

Abstract

Current competing models for the two-electron oxidation of quinol (QH2) at the cytochrome bc1 complex and related complexes have different 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, Ru(bpy)2(pbim)(PF6)2 (bpy = 2,2’-dipyridyl, pbim = 2-(2-benzimidazolate)pyridine) in an aprotic medium to probe the oxidation of the ubiquinol analogue, 2,3-dimethoxy-5-methyl-1,4-benzoquinol (UQH2-0), an the plastoquinol analogue, trimethyl-1,4-benzoquinol (TMQH2-0), using time-resolved and steady state spectroscopic techniques. This system qualitatively reproduces key features observed during ubiquinol oxidation by the mitochondrial cytochrome bc1 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•/QH2 couple for UQH2-0, but not TMQH2-0, exhibits a non-classical primary deuterium kinetic isotope effect on its Arrhenius activation energy (?GTS), where ?GTS for the protiated form is larger than for the deuterated form. The same behavior is observed during steady state turnover of the cyt bc¬1 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 bc¬1 complex.