The cytochrome bc1 and related complexes are essential energyconserving components of mitochondrial and bacterial electron transport chains. They orchestrate a complex sequence of electron and proton transfer reactions resulting in the oxidation of quinol, the reduction of a mobile electron carrier, and the translocation of protons across the membrane to store energy in an electrochemical proton gradient. The enzyme can also catalyze substantial rates of superoxide production, with deleterious physiological consequences. Progress on understanding these processes has been hindered by the lack of observable enzymatic intermediates. We report the first direct detection of a semiquinone radical generated by the Q o site using continuous wave and pulsed EPR spectroscopy. The radical is a ubisemiquinone anion and is sensitive to both specific inhibitors and mutations within the Qo site as well as O2, suggesting that it is the elusive intermediate responsible for superoxide production. Paramagnetic interactions show that the new semiquinone species is buried in the protein, probably in or near the Qo site but not strongly interacting with the 2Fe2S cluster. The semiquinone is substoichiometric, even with conditions optimized for its accumulation, consistent with recently proposed models where the semiquinone is destabilized to limit superoxide production. The discovery of this intermediate provides a critical tool to directly probe the elusive chemistry that takes place within the Qo site.electron transfer ͉ free radical ͉ photosynthesis ͉ reactive oxygen species ͉ respiration T he cytochrome (cyt) bc 1 , b 6 f, and related complexes, collectively termed cyt bc complexes, are essential components of the respiratory and photosynthetic electron transport chains in mitochondria, many bacteria, and chloroplasts (1-3). These complexes oxidize quinol and reduce one-electron redox carriers while generating an electrochemical gradient of protons, termed the proton motive force (pmf ), which drives the synthesis of ATP and other bioenergetic processes. The natural substrate is ubiquinol (UQH 2 ) in the case of the mitochondrial and bacterial cyt bc 1 complexes, and the mobile carrier is cyt c in mitochondria or photosynthetic bacteria. The general mechanistic framework for the cyt bc complexes is the Q-cycle, first proposed by Mitchell (4-6) and modified by many others (e.g., refs. 7-14).In ''standard'' versions of the Q-cycle (2, 9, 15) a unique bifurcated oxidation of QH 2 occurs in the Q o site, located on the positively charged side (p-side) of the membrane. An initial single electron transfer to the ''Rieske'' 2Fe2S cluster produces a free radical semiquinone (SQ) intermediate (the anionic form or the neutral form, depending on the exact sequence of electron and proton transfers). The Rieske 2Fe2S cluster is the first in a series of carriers, termed the ''high potential chain,'' which in mitochondria and certain bacteria includes cyt c 1 followed by a soluble (or mobile) cyt c. Under normal conditions, the SQ intermediate ...

(18)O-isotope-labeling studies have led to the conclusion that there exist two major pathways for water oxidation catalyzed by dimeric ruthenium ions of the general type cis, cis-[L2Ru(III)(OH2)]2O(4+). We have proposed that both pathways involve concerted addition of H and OH fragments derived from H 2O to the complexes in their four-electron-oxidized states, i.e., [L2Ru(V)(O)]2O(4+), ultimately generating bound peroxy intermediates that decay with the evolution of O2. The pathways differ primarily in the site of addition of the OH fragment, which is either a ruthenyl O atom or a bipyridine ligand. In the former case, water addition is thought to give rise to a critical intermediate whose structure is L2Ru(IV)(OH)ORu(IV)(OOH)L2(4+); the structures of intermediates involved in the other pathway are less well defined but may involve bipyridine OH adducts of the type L2Ru(V)(O)ORu(IV)(OH)(L(*)OH)L(4+), which could react further to generate unstable dioxetanes or similar endoperoxides. Published experimental and theoretical support for these pathways is reviewed within the broader context of water oxidation catalysis and related reactions reported for other diruthenium and group 8 monomeric diimine-based catalysts. New experiments that are designed to probe the issue of bipyridine ligand "noninnocence" in catalysis are described. Specifically, the relative contributions of the two pathways have been shown to correlate with substituent effects in 4,4'- and 5,5'-substituted bipyridine complexes in a manner consistent with the formation of a reactive OH-adduct intermediate in one of the pathways, and the formation of OH-bipyridine adducts during catalytic turnover has been directly confirmed by optical spectroscopy. Finally, a photosensitized system for catalyzed water oxidation has been developed that allows assessment of the catalytic efficiencies of the complex ions under neutral and alkaline conditions; these studies show that the ions are far better catalysts than had previously been assumed based upon reported catalytic parameters obtained with strong oxidants in acidic media.

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Current competing models for the two-electron oxidation of quinol (QH2) at the cytochrome bc1 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 (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 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 an unusual and unexpected primary deuterium kinetic isotope effect on its Arrhenius activation energy (DeltaGTS), where DeltaGTS for the protiated form is larger than that for the deuterated form. The same behavior 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. 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 bc1 complex.