Abstract
Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bell-shaped relationship to the reduction state of cytochrome b(566), suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q(o)). Imposition of a membrane potential changed the relationships between superoxide production and b(566) reduction and between b(562) and b(566) redox states, suggesting that b(562) reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q(o) site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b(566) to depend on the b(562) redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b(566) was reduced, and reduction of b(562) stabilized the semiquinone when b(566) was oxidized. Thus, superoxide production at Q(o) depends on the reduction states of b(566) and b(562) and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.
Highlights
None oxidoreductase [5,6,7]
The sensitivity to stigmatellin defined superoxide generated in the Qo site of complex III, with the reasonable assumption that no other superoxide-generating site changed redox state when stigmatellin was added under these conditions
Instead, during titration with glutamate plus malate in the absence of rotenone. These results show that saturating substrate concentrations do not lead to maximal superoxide production from the Qo site of complex III; instead, the results are consistent with superoxide generation being maximal at an intermediate Q reduction state
Summary
Mitochondria—Mitochondria were isolated from hind limb skeletal muscle of female Wistar rats aged 5– 8 weeks by differential centrifugation [18, 19]. H2O2 production rates were determined by measurement of Amplex UltraRed fluorescence (Invitrogen) in a Shimadzu RF5301-PC spectrofluorometer (Kyoto, Japan) at the wavelength couple excitation ϭ 530 nm and emission ϭ 580 nm. All assays contained 50 M Amplex UltraRed, 5 units of horseradish peroxidase/ml, and 12 units of superoxide dismutase/ml. Superoxide production from the Qo site was defined by sensitivity to the Qo site inhibitor, stigmatellin; all data in this study refer to rates that were inhibitable by stigmatellin, with all stigmatellin-insensitive H2O2 production subtracted before further calculations. The best current estimate of the topology of superoxide production by complex III is that 63% is directed to the matrix [21], based on the empirical observation of 50% matrix-directed superoxide production [22] and correcting for matrix glutathione peroxidase activity. For the modeling (see below) the redox state of b562 (b5%6r2ed) was calculated as a function of b566 redox state (b5%6r6ed in %) by assuming thermodynamic equilibrium between the two b hemes (Equation 2)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
