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" iron-sulfur 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.
Highlights
The cytochrome2 bc1 complex (EC 1.10.2.2) is found on the inner membrane of mitochondria and GM61904
QH2 is oxidized at the quinol oxidase (Qo) site, with one electron being transferred through the high potential chain to reduce cyt c, and the other electron is transferred through the low potential chain, to reduce a quinoid species (Q or SQ, depending on the state of the two-electron gate) at the quinol reductase (Qi) site
We found that the activation energies for the uninhibited Q-cycle and superoxide production in the presence of antimycin A (AA) were nearly identical within the noise (Fig. 4)
Summary
Yeast Strains—Yeast strains with site-directed mutations in the Rieske subunit of the cyt bc complex [22, 33, 34] were grown in a synthetic mixture of amino acids excluding tryptophan (Sigma) and with dextrose (Fisher) as the carbon source. The kinetic traces were initiated before the addition of enzyme (0.5–20 nM cyt b) to allow for the subtraction of background cyt c reduction (or background superoxide production) by free decylubiquinol. For the superoxide production experiments, the cyt bc complex was treated with a saturating (20 M) concentration of AA (Sigma). For measuring superoxide production in the presence of 20 M AA, we used the Amplex Red assay (product number 12212; Molecular Probes, Eugene, OR). This assay was found to be much more sensitive than the cyt c reduction experiments
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