Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

Abstract

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q o ) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" ironsulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q o site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q o site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O 2 reduction; 2) the Q o site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q o site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.The cytochrome (cyt) 2 bc 1 complex (EC 1.10.2.2) (cyt bc 1 complex) is found on the inner membrane of mitochondria and energy transducing membranes of many bacteria (1-3). It is structurally and functionally homologous to a taxonomically wide spread group, collectively referred to as "bc complexes," additionally consisting of the cyt b 6 f complex and the menaquinol oxidizing complexes in many bacteria (4 -6). In all well studied cases, cyt bc complexes couple the oxidation of a substrate quinol (QH 2 ) to the formation of a proton motive force across the energy transducing membrane in which the given complex resides, the energy of which is ultimately stored as ATP.Cytochrome bc 1 complexes contain four redox-active metal centers, arranged in two separate chains (2, 5, 7-9). The "high potential chain" consists of the Rieske iron-sulfur [2Fe2S] cluster (in the Rieske, or "iron-sulfur subunit," hereafter "the [2Fe2S] cluster"), and a c-type cyt, known in mitochondria and Gram-negative bacteria as cyt c 1 . The "low potential chain," which is liganded to the cyt b subunit of ubiquinol oxidizing complexes, consists of two b-type hemes, cyt b L and b H labeled for their relatively lower and higher electrochemical potentials. Crystal structures from several taxonomic sources (2, 8, 10, 11) suggest a well conserved placement of the cofactors throughout the various cyt bc ...

A semiquinone intermediate generated at the Q o site of the cytochrome bc 1 complex: Importance for the Q-cycle and superoxide production

“…In some models the SQ is thermodynamically stabilized (45), thus lowering its reactivity with O 2 ; in other models it is specifically destabilized (3,36), limiting its reactivity with O 2 by lowering its steady-state concentration. Some models posit that the SQ is shielded from O 2 within the Q o site (3), while access to the site or its reactivity is proposed to be allosterically (46,47) or electrostatically (14,27) gated.…”

“…In preparations of isolated, AA-treated cyt bc 1 complex, we measure turnover numbers for superoxide of Ϸ10 s Ϫ1 (20,22,36,50). Assuming a simple second-order process with a maximum second-order rate constant for superoxide production of 10 8 M Ϫ1 ⅐s Ϫ1 (51) and air-saturated solutions, we predict a minimum steady-state concentration of SQ of Ϸ4 nM from 10 M cyt bc 1 complex; this concentration should be detectable by EPR.…”

Menaquinone as pool quinone in a purple bacterium

“…By contrast, the reasons why a ''UQ-type'' complex should not work with MKs are less obvious. From an energetic point of view, the increased difference between the low potential of MK and the cofactors of a UQ-adapted bc complex would result in a more favorable oxidation of MKH 2 to semiquinone at Q O , the rate-limiting step in the Q-cycle (32). One might therefore expect that MKcontaining organisms simply ''take advantage'' of this additional driving force and get along with a single, UQ-type bc 1 complex.…”

“…One might therefore expect that MKcontaining organisms simply ''take advantage'' of this additional driving force and get along with a single, UQ-type bc 1 complex. However, it has recently been shown that conservation of the Q-cycle driving force is critical to preventing deleterious superoxide production at the Q O site (32)(33)(34)(35). Substitution of the LP rhodoquinol (RQH 2 ) for UQH 2 in mitochondrial cyt.…”

Movement of the Iron−Sulfur Head Domain of Cytochrome bc1 Transiently Opens the Catalytic Qo Site for Reaction with Oxygen

“…This, however, may change when the impeded electron flow outbalances reducing equivalents in the two chains, which has long been known from the classic experiments with an inhibitor antimycin which demonstrated that blocking specifically the electron flow through the Q i site is sufficient to make the enzyme vulnerable to superoxide production (10, 11). These kinds of observations have raised an ongoing debate about whether cytochrome bc 1 does produce superoxide in living cells (12)(13)(14)(15)(16) (5,(17)(18)(19)(20)(21)(22)(23)(24). Answering those questions not only would bring clarity to many physiological and medical studies but also should improve our understanding of the molecular mechanisms of energy conservation supported by cytochrome bc 1 .…”