The mechanism of superoxide production by the antimycin-inhibited mitochondrial Q-cycle

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.

FIGURE 1.

Effect of substrate supply on superoxide production and steady-state reduction of cytochrome b₅₆₆ in the Q_o site of complex III. Stigmatellin-sensitive superoxide production (measured as hydrogen peroxide production) of isolated skeletal muscle mitochondria in the presence of 2 μm antimycin A is shown. a, 4 μm rotenone plus 5 mm succinate with subsequent additions of malonate as shown. b, 4 μm rotenone plus succinate at the concentrations shown. c, equimolar glutamate plus malate each at the concentration shown. All data were corrected for the rates remaining after addition of 1 μm stigmatellin and for matrix H₂O₂ losses through glutathione-dependent peroxidases (see under “Experimental Procedures”) and are means ± S.E. (n = 4–5). b₅₆₆ measurements were conducted in parallel to those above. Conditions are as follows: 2 μm antimycin A and 4 μm rotenone plus 5 mm succinate with subsequent additions of malonate as shown (d), 4 μm rotenone plus succinate at the concentrations shown (e), or equimolar glutamate plus malate each at the concentration shown (f). All data were corrected for the contribution from cytochrome b₅₆₂ to the 566–575 nm wavelength pair (see “Experimental Procedures”) and are means ± S.E. (n = 4).

FIGURE 2.

Relationship between superoxide production rate from the Q_o site of complex III and the reduction state of cytochrome b₅₆₆. Hydrogen peroxide production rates with each substrate from Fig. 1, a–c, are plotted against the appropriate percent reduction of cytochrome b₅₆₆ from Fig. 1, d–f. Data are means ± S.E. (n = 4–5).

FIGURE 3.

Relationship between superoxide production rate from the Q_o site of complex III and the reduction state of cytochrome b₅₆₆ in the presence of ATP. Filled symbols, superoxide production rate and cytochrome b₅₆₆ reduction were measured as in Fig. 1, a and d, respectively, except that 2 mm ATP was present, and the total concentration of succinate plus malonate was kept constant at 5 mm while altering their ratio. Data are means ± S.E. (n = 4). Open symbols, data for malonate titration of succinate as substrate, taken from Fig. 2, for comparison.

FIGURE 4.

Relationships between the steady-state reduction of cytochromes b₅₆₂ and b₅₆₆ in the absence and presence of ATP. Cytochrome b₅₆₆ was measured at 566–575 nm with correction for cytochrome b₅₆₂ by subtracting 50% of the signal at 561–569 nm. Cytochrome b₅₆₂ was measured at 561–569 nm. a, ATP absent, conditions as Fig. 1e. b, 2 mm ATP present, conditions as Fig. 3. Solid curves indicate the best fits of Equation 3, plotted with the relevant values of E_dif^m, see “Experimental Procedures.”

FIGURE 5.

Kinetic model of the Q_o site of complex III. The cubic model of the Q_o site describes the eight possible states of the Q_o site (quinone-binding site empty or occupied by Q, QH₂, or SQ; cytochrome b₅₆₆ oxidized (right or blue face of cube) or reduced (left or yellow face of cube)) and the reactions that connect them and contribute to superoxide formation in the presence of antimycin A. Rate equations (reactions 1–9 in Table 1) were written for each step of the model. For normal Q-cycle activity with cytochrome b₅₆₆ mostly oxidized (blue arrows), pool QH₂ binds to an open site (top right) in reaction 1 in Table 1, and one electron is donated to the high potential electron transport chain in reaction 5 to form a semiquinone (bottom right). If semiquinone accumulates, it can react with oxygen to form superoxide by reaction 9 in Table 1 (or it leaves the Q_o site then reacts with oxygen to form superoxide, not shown). The second electron is donated to cytochrome b₅₆₆ in reaction 6 in Table 1, forming Q (top left). In the absence of antimycin, cytochrome b₅₆₆ is reoxidized by cytochrome b₅₆₂; Q is released by reaction 2, and the Q-cycle proceeds. In the presence of antimycin, cytochrome b₅₆₆ cannot be reoxidized through the low potential chain once cytochrome b₅₆₂ is reduced, so Q is released by reaction 4 in Table 1. The next QH₂ to enter binds to the reduced complex (reaction 3 in Table 1) and generates a semiquinone by reaction 7. This semiquinone can be reduced to QH₂ by cytochrome b₅₆₆ in reaction 8 (Table 1), or it can be reoxidized by forming superoxide in reaction 9. However, if Q enters instead of QH₂ (reaction 4 in Table 1), it can reoxidize cytochrome b₅₆₆ in reaction 6, forming semiquinone.

FIGURE 6.

Model-generated fits to the data in Fig. 2. The experimental data (open symbols) were converted to moles of superoxide per s per mol of bc₁ complex. The antimycin-inhibited Q-cycle model (Fig. 5) was solved in steady state by numerical optimization of the rate constants within the ranges specified in Table 1, in four separate trials with different constraints, to give the best fit to the data and the known thermodynamic relationships between Q and the b hemes. The black squares show the best fit for each trial; superoxide production rates were reevaluated with finer steps of QH₂/Q ratios using the best fit rate constants. The criteria for goodness of fit are shown in Table 2. a, Trial 1. Rate constants were held to strict Q-cycle ranges. b, Trial 2. As trial 1, but rate constants for semiquinone formation (k₅ and k₇) were allowed to vary within a wide range. c, Trial 3. As trial 2 but rate constants for reaction 6 were allowed to depend on the reduction state of cytochrome b₅₆₂ (see Table 1). d, rate constants from trial 3 used to fit the data in Fig. 3 with ATP present. In this case, the model used the b₅₆₂ reduction profile shown in Fig. 4b instead of that in Fig. 4a to calculate reaction 6 rate constants. e, Trial 4. As trial 3, but the model simultaneously fit the data sets from Figs. 2 and 3 using the relevant b reduction profiles from Fig. 4, a and b, to generate a single set of rate constants that defined both data sets.

FIGURE 7.

Model-generated prediction of the percentage of bc₁ complexes occupied by semiquinone under different conditions. Rate constants from the best fit in trial 3 applied without (a) and with (b) ATP added were used to calculate the steady-state concentrations of different forms of complex III at different imposed QH₂/Q ratios. Colors correspond to Fig. 5 as follows: yellow represents semiquinone bound to the complex with cytochromes b₅₆₆ and b₅₆₂ reduced; dark blue represents semiquinone bound to the complex with b₅₆₆ reduced and b₅₆₂ oxidized; light blue represents semiquinone bound to the complex with b₅₆₆ oxidized and b₅₆₂ reduced, and purple represents semiquinone bound to the complex with b₅₆₆ and b₅₆₂ oxidized. The top contour corresponds to the total concentration of semiquinone, which is proportional to superoxide production. The thickness of each band within the peak describes the proportion of each semiquinone species contributing to the total.