Reaction Mechanism of Superoxide Generation during Ubiquinol Oxidation by the Cytochrome bc₁ Complex

Abstract

In addition to its main functions of electron transfer and proton translocation, the cytochrome bc₁ complex (bc₁) also catalyzes superoxide anion (O₂^˙̄) generation upon oxidation of ubiquinol in the presence of molecular oxygen. The reaction mechanism of superoxide generation by bc₁ remains elusive. The maximum O₂^˙̄ generation activity is observed when the complex is inhibited by antimycin A or inactivated by heat treatment or proteinase K digestion. The fact that the cytochrome bc₁ complex with less structural integrity has higher O₂^˙̄-generating activity encouraged us to speculate that O₂^˙̄ is generated inside the complex, perhaps in the hydrophobic environment of the Q_P pocket through bifurcated oxidation of ubiquinol by transferring its two electrons to a high potential electron acceptor, iron-sulfur cluster, and a low potential heme b_L or molecular oxygen. If this speculation is correct, then one should see more O₂^˙̄ generation upon oxidation of ubiquinol by a high potential oxidant, such as cytochrome c or ferricyanide, in the presence of phospholipid vesicles or detergent micelles than in the hydrophilic conditions, and this is indeed the case. The protein subunits, at least those surrounding the Q_P pocket, may play a role either in preventing the release of O₂^˙̄ from its production site to aqueous environments or in preventing O₂ from getting access to the hydrophobic Q_P pocket and might not directly participate in superoxide production.

Introduction

It has long been recognized that during mitochondrial respiration, there is a continuous release of electrons from the electron transfer chain to react with molecular oxygen to form a superoxide anion (O₂^˙̄) (1–3). The generated O₂^˙̄ is subsequently dismutated to H₂O₂ spontaneously or by the action of superoxide dismutases (4). Isolated mitochondria in state 4 generate 0.6–1.0 nmol of H₂O₂/min/mg of protein, accounting for about 2% O₂ uptake under physiological conditions (5). Production of O₂^˙̄ during mitochondrial respiration is closely related to mitochondrial coupling efficiency. More O₂^˙̄ is produced when the membrane potential of mitochondria is high (6, 7). In the past, most information concerning mitochondrial O₂^˙̄ generation sites was obtained from studies using intact heart mitochondria with selected electron transfer inhibitors by measuring the H₂O₂ concentration in the suspending medium (8, 9).

Two segments of the respiratory chain have been demonstrated to be responsible for the generation of O₂^˙̄ from oxygen. One is located at the NADH-Q³ oxidoreductase (complex I), and the other is at the cytochrome bc₁ complex (ubiquinol-cytochrome c oxidoreductase). Production of O₂^˙̄ by complex I is either via auto-oxidation of the flavine radical in NADH dehydrogenase (10) or via a bound ubisemiquinone radical (11) or the center N-2 (12) of the complex. It was recently suggested (13) that the reversed electron transport through complex I produced more O₂^˙̄ than the forward transport. Two redox components of the bc₁ complex, ubisemiquinone at the Q_P site (8) and the reduced cytochrome b₅₆₆ (9, 14), have been implicated as electron donors for molecular oxygen to generate O₂^˙̄. The production of O₂^˙̄ by the bc₁ complex is greatly enhanced when the complex is inhibited by antimycin (14–16).

In continuing our study of the structural and functional relationship of the cytochrome bc₁ complex, it is important to understand the reaction mechanism of superoxide generation in this complex. The fact that antimycin inhibits the electron transfer activity of the cytochrome bc₁ complex and stimulates the O₂^˙̄-generating activity, together with the observation that both activities have a similar activation energy, led investigators to believe that both activities share, at least, a common intermediate (17). According to the Q-cycle mechanism (18–20), during the catalytic reaction of the cytochrome bc₁ complex, Q-H₂ undergoes bifurcated oxidation by transferring its two electrons, sequentially or simultaneously (concerted), to the iron-sulfur cluster (ISC) of the iron-sulfur protein (ISP) subunit and heme b_L of the cytochrome b subunit. In the sequential mechanism (21–23), ubiquinol transfers its first electron to the ISC to become low potential ubisemiquinone that contains a free electron and reduces heme b_L instantly. The lack of a functional ubisemiquinone at the Q_P site (21, 24, 25) undermines this mechanism substantially, although some radicals have been reported under abnormal conditions (26–28). Recently, more compelling evidence against the existence of the semiquinone radical at the Q_P site has been reported (29). No such radical in a heme b_L knock-out mutant complex is detected upon reduction by quinol. Because heme b_L is the designated electron acceptor of the semiquinone radical at the Q_P site in the sequential Q-cycle mechanism, one would expect to see the accumulation of this intermediate when the acceptor is not available, but this is not the case. In the concerted mechanism (29–31), no semiubiquinone is formed, and two electrons of Q-H₂ are transferred simultaneously to ISC and heme b_L. This bifurcated electron transfer reaction provides a basis for the high efficiency of the bc₁ complex and is done inside the cytochrome b subunit buried in the membrane bilayer. It is thus expected that any compromise in the structural integrity of cytochrome b should lead to a decrease in the electron transfer efficiency and to an increase in the production of superoxide. The observation that mutants lacking heme b_L or heme b_H, respectively, show little electron transfer activity but have high superoxide-generating activity (25) is consistent with the idea that the structural integrity of cytochrome b is required for normal electron transfer activity but not for superoxide generation.

Herein we report a systematic comparison of the electron transfer and O₂^˙̄-generating activities in various cytochrome bc₁ complexes, such as the complexes with varying numbers of supernumerary subunits,⁴ or with different extents of heat inactivation or proteinase K digestion to see whether or not the intact protein components of the complex are required for O₂^˙̄-generating activity. We also determined the effect of ubiquinol, phospholipid vesicles, detergent micelles, cytochrome c, and ferricyanide concentration on O₂^˙̄ generation. Based on the results obtained, we establish that an electron donor (ubiquinol, a high potential electron acceptor), ISC, cytochrome c, or ferricyanide and a hydrophobic environment are required for O₂^˙̄ production. We formulated a working hypothesis for the reaction mechanism of O₂^˙̄ production in the bc₁ complex.

EXPERIMENTAL PROCEDURES

Materials

Cytochrome c (horse heart, type III), acetylated cytochrome c, and superoxide dismutase were purchased from Sigma. Proteinase K was purchased from Invitrogen. N-Dodecyl-d-maltopyranoside (DM) and N-octyl-d-gluocopyranoside (OG) were obtained from Anatrace. N,N-Dimethyldodecylamine N-oxide (LDAO) was obtained from Sigma. Nickel nitrilotriacetic acid gel and a QIAprep spin miniprep kit were obtained from Qiagen. 2-Methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazol[1,2-α] pyrazin-3-one, hydrochloride (MCLA) was obtained from Molecular Probes, Inc. 2,3-Dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinol(Q₀C₁₀BrH₂) was prepared as reported previously (33). All other chemicals were of the highest purity commercially available.

Enzyme Preparations and Activity Assays

Chromatophores, intracytoplasmic membrane, and the His₆-tagged cytochrome bc₁ complexes, wild type (34) and mutants (25, 35), were prepared as reported previously. Bovine heart mitochondrial cytochrome bc₁ complex was prepared according to the method developed in our laboratory (36, 37).

To assay the cytochrome bc₁ complex activity, purified complexes were diluted with 50 mm Tris-Cl, pH 8.0, containing 200 mm NaCl and 0.01% DM to a final concentration of cytochrome c₁ of 1 μm. Appropriate amounts of the diluted samples were added to 1 ml of assay mixture containing 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 300 μm EDTA, 100 μm cytochrome c, and 25 μm Q₀C₁₀BrH₂. Because Q₀C₁₀BrH₂ is easily auto-oxidized at neutral or higher pH, the stock solution is kept in 95% ethanol containing 1 mm HCl and diluted in the buffer before use. Activities were determined by measuring the reduction of cytochrome c (the increase of absorbance at 550 nm) in a Shimadzu UV 2101 PC spectrophotometer at 23 °C, using a millimolar extinction coefficient of 18.5 for the calculation. The non-enzymatic oxidation of Q₀C₁₀BrH₂, determined under the same conditions in the absence of the enzyme, was subtracted from the assay.

Digestion of the Cytochrome bc₁ Complex by Proteinase K

A stock solution of proteinase K, 3%, was made in 10 mm Tris-HCl, pH 7.5, containing 20 mm CaCl and 50% glycerol. Two μl of proteinase K solution was added into 200 μl of cytochrome bc₁ complex (200 μm cyt b) in 50 mm Tris-HCl, pH 8.0, containing 200 mm NaCl and 0.01% DM. The mixture was incubated at room temperature. The electron transfer and O₂^˙̄-generating activities were measured during the course of incubation until all the electron transfer activity was diminished. The digested bc₁ was then subjected to SDS-PAGE to confirm that all the subunits were digested.

Preparation of Phospholipid Vesicles

Phospholipid vesicles were prepared by the cholate dialysis method (38). Asolectin was dissolved in chloroform and dried as a thin film against the tube by flushing with nitrogen gas while the tube was rotating. The phospholipid was then suspended in 50 mm potassium/sodium phosphate buffer, pH 7.4, containing 1% sodium cholate. The mixture was subjected to sonification intermittently for 30 min until the solution become clear and then dialyzed against the same buffer overnight, with three changes of buffer.

Determination of Superoxide Production

Superoxide production was determined by measuring the chemiluminescence of MCLA-O₂^˙̄ adduct (39) in an Applied Photophysics stopped-flow reaction analyzer SX.18MV-R (Leatherhead, UK) by leaving the excitation light off and registering light emission (40, 41). Reactions were carried out at 23 °C by mixing 1:1 of solutions A and B. For the determination of O₂^˙̄ production by the native, heat-inactivated, or proteinase K-digested cytochrome bc₁ complexes, Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 1 mm EDTA, 1 mm NaN₃, 0.1% bovine serum albumin, 0.01% DM, and 5.0 μm cytochrome bc₁. Solution B contains 125 μm Q₀C₁₀BrH₂ and 4 μm MCLA in the same buffer. Once the reaction started, the produced fluorescence, in voltage, was consecutively monitored for 2 s. One volt from the Applied Photophysics stopped-flow reaction analyzer SX.18MV-R equals the chemiluminescence (maximum peak height of light intensity) generated by 0.5 unit of xanthine oxidase using 100 μm hypoxanthine as a substrate.

Because MCLA is a neutral, relatively non-polar molecule, it is possible that the efficiency of O₂^˙̄ production detected by chemiluminescence may be affected by the presence of different detergent micelles, thus complicating the determination of the micelle effect of different detergents on superoxide production. To avoid this possible complication, superoxide productions in the presence of different detergent, micelles were compared by measuring the reduction of acetylated cytochrome c (42) because different detergent micelles do not show significant effect on superoxide production by xanthine oxidase and hypoxanthine determined by the acetylated cytochrome c method. Reduction of acetylated cytochrome c was followed by the increase of absorption at 550 nm in the same stopped-flow reaction analyzer in the normal way. A millimolar extinction coefficient of 18.5 was used for the concentration calculation. Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 8.0, 10 μm acetylated cytochrome c, and various amounts of detergents. Solution B contains 250 μm Q₀C₁₀BrH₂ in 0.5 mm Na⁺/K⁺ phosphate buffer, pH 4, in the presence or absence of 300 units/ml superoxide dismutase.

RESULTS AND DISCUSSION

The Inverse Relationship between Superoxide-generating and Electron Transfer Activities in the Cytochrome bc₁ Complex

Table 1 summarizes the electron transfer and O₂^˙̄-generating activities in various bc₁ complex preparations. The bovine heart mitochondrial complex has 11 protein subunits. This complex has the highest electron transfer activity and lowest superoxide-generating activity among the complexes tested. The Rhodobacter sphaeroides complex, which contains four protein subunits (three core subunits and one supernumerary subunit), has only about one-twelfth of the electron transfer activity of the bovine complex but has about six times the O₂^˙̄-generating activity of the bovine enzyme. When the only supernumerary subunit (subunit IV) is deleted from the R. sphaeroides wild-type complex, the resulting three-subunit core complex (RsΔIV) has only a fraction of the electron transfer activity of the wild-type complex but has about four times the O₂^˙̄-generating activity. When the three-subunit core complex is reconstituted with subunit IV, the electron transfer activity increases, and the O₂^˙̄-generating activity decreases to the same level as those in the wild-type, four-subunit complex.

TABLE 1.

Comparison of electron transfer and superoxide-generating activities of various cytochrome bc₁ complexes

Rsbc₁, wild-type, 4 subunit R. sphaeroides bc₁; RsΔIV, Rsbc₁ lacking subunit IV; Cyt b(H198N), Rsbc₁ lacking heme b_L; Cyt b(H111N), Rsbc₁ lacking heme b_H; XO, xanthine oxidase. Each data point represents an average of four experiments.

Preparation	Activities
	Electron transfer (μmol of cytochrome c reduced/min/nmol of cytochrome c)	O2˙̄ generation (milliunits of XO/nmol of cytochrome c)
		−Antimycin	+Antimycin
Mitochondrial bc1	43.0 ± 0.3	15 ± 2	19 ± 2
Rsbc1	3.5 ± 0.2	91 ± 2	300 ± 2
RsΔIV	0.8 ± 0.2	325 ± 2	546 ± 5
Cyt b(H198N)	0.4 ± 0.2	510 ± 5	510 ± 5
Cyt b(H111N)	0.3 ± 0.2	500 ± 4	510 ± 6

The differential scanning calorimetric studies indicate that the order of thermal stability among these complexes is: beef > wild-type R. sphaeroides complex = reconstituted complex > RsΔIV complex (data not shown). Therefore, the electron transfer activity of the bc₁ complex is in direct proportion to the structural integrity of the complex, whereas the O₂^˙̄-generating activity has an inverse relationship with the structural integrity of the complex. In other words, the electron transfer activity is inversely proportional to the O₂^˙̄-generating activity in the bc₁ complex.

The finding that the cytochrome bc₁ complex with less structural integrity has higher O₂^˙̄-generating activity encouraged us to speculate that O₂^˙̄ is generated inside the complex, perhaps in the hydrophobic environment of the Q_P pocket, and that the protein subunits, at least those surrounding the Q_P pocket, may play a role either in preventing the release of O₂^˙̄ from its production site to aqueous environments or in preventing O₂ from getting access to the hydrophobic Q_P pocket.

The Superoxide Anion-generating Activity in the Heat-inactivated Cytochrome bc₁ Complex

If the above speculation is correct, then one should see an increase in O₂^˙̄ generation in the complex with denatured protein subunits. To test this speculation, the wild-type R. sphaeroides bc₁ complex was incubated at 37 °C to inactivate the complex, and the electron transfer and O₂^˙̄ generation activities were measured during the course of incubation. As the incubation time increases, the electron transfer activity decreases, whereas the O₂^˙̄-generating activity increases. Fig. 1 shows the relationship between the electron transfer activity and the O₂^˙̄-generating activity during the heat inactivation process. Maximum O₂^˙̄-generating activity is obtained when more than 90% of the electron transfer activity is abolished. This result indicates that the production of O₂^˙̄ during quinol oxidation by bc₁ complex does not require the presence of an intact complex. The impairment of the structural integrity of the complex by the heat denaturalization of the protein subunits leads to the increase in the accessibility of molecular oxygen to the hydrophobic environment of the Q_P pocket to generate O₂^˙̄ and to facilitate the release of the produced O₂^˙̄ to the aqueous medium.

FIGURE 1.

The relationship between the electron transfer activity and superoxide generation during temperature inactivation of cytochrome bc₁ complex. 200 μl of cytochrome bc₁ complex (200 μm cyt b) in 50 mm Tris-Cl, pH 8.0, containing 200 mm NaCl and 0.01% DM was incubated at 37 °C. At different time intervals, samples were withdrawn and determined for superoxide production (shown as voltage) and electron transfer activities (shown as relative electron transfer activity of the untreated complex; 100% activity is equal to 3.5 μmol of cytochrome c reduced/min/nmol of bc₁ complex). Electron transfer activity and the superoxide production were measured as described under “Experimental Procedures.” Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 1 mm EDTA, 1 mm NaN₃, 0.1% bovine serum albumin, 0.01% DM, and 5.0 μm incubated cytochrome bc₁. Solution B was the same as Solution A with bc₁ complex being replaced with 125 μm Q₀C₁₀BrH₂ and 4 μm MCLA. Each data point represents an average of four experiments. Error bars indicate S.D.

The notion that an intact protein subunit structure is not required for O₂^˙̄-generating activity of the bc₁ complex is further supported by the observation that mutants H198N and H111N, which lack heme b_L and heme b_H, respectively, have very little electron transfer activity but show O₂^˙̄-generating activity equal to that of the antimycin-treated wild-type complex (25) (Table 1). The increased rates of O₂^˙̄ formation when in the heme b_L knock-out complex is strong evidence that although the heme b_L may react with oxygen when it is present, a O₂^˙̄ can also be formed by a route other than by reaction with the heme b_L. The loss of either heme b_L or heme b_H would be expected to have a strong impact on the overall structural integrity of the bc₁ complex, leading to the distortion of the Q_P pocket environment and presumably increasing the accessibility of O₂ to the site. It is as expected that the incubation of these two heme b-lacking mutant complexes at 37 °C to denature the protein subunits does not further increase the O₂^˙̄-generating activity because the structural integrity of these two complexes has already been deteriorated by mutation.

The Superoxide Anion-generating Activity in Proteinase K-digested Complex

To further confirm that superoxide-generating activity is independent of the presence of an intact protein structure, the bc₁ complex was subjected to proteinase K digestion and electron transfer, and superoxide-generating activities were measured during the course of digestion. As shown in Fig. 2, the electron transfer activity diminishes, whereas the O₂^˙̄-generating activity increases as the digestion time increases. Maximum superoxide-generating activity is observed when electron transfer activity is completely abolished. SDS-PAGE analysis of the proteinase K-digested complex reveals no intact subunits of cytochromes b, c₁, or ISP (Fig. 3). The largest peptide band presence in the digested complex has an apparent molecular mass of less than 7 kDa. These results further support our suggestion that the intact protein components of the complex or the intact complex play no direct role in O₂^˙̄ generation. A Western blotting experiment using anti-ISP indicated that at least a part of this peptide band is derived from ISP. In other words, the ISC detected by EPR is housed in this peptide.

FIGURE 2.

Activity tracings of the electron transfer and O₂^˙̄ generation during the course of proteinase K digestion of the complex. 200 μl of cytochrome bc₁ complex (200 μm cyt b) in 50 mm Tris-Cl, pH 8.0, containing 200 mm NaCl and 0.01% DM was incubated with 60 μg of proteinase K at 37 °C. At the indicated time intervals, samples were withdrawn and determined for superoxide production (Xs) and electron transfer (open circles) activities. Each data point represents an average of four experiments. Error bars indicate S.D.

FIGURE 3.

SDS-PAGE of the cytochrome bc₁ complex and its proteinase K-digested products. Lane 1, intact wild-type cytochrome bc₁. Lane 2, standard polypeptides. Lane 3, proteinase K-treated wild-type complex. Sub. IV, subunit IV.

If this notion is correct, then what remaining elements in the proteinase K-digested complex contributed to superoxide production from Q-H₂? A hydrophobic environment and a high potential electron acceptor ISC in the digested system could contribute to the superoxide formation in the presence of molecular oxygen. The presence of intact ISC in the proteinase K-digested complex was confirmed by the presence of EPR signals of ISC in the digested complex (data not shown). Thus, it is possible that ISC serves as a high potential electron acceptor and oxygen serves as a low potential electron acceptor during bifurcated oxidation of Q-H₂ in a hydrophobic environment of the Q_P pocket to produce superoxide. Because there are no free metal ions in the purified cytochrome bc₁ complex and the iron in the heme and iron-sulfur cluster is not released during heat treatment or proteinase digestion, as indicated by absorption and EPR spectra of the tested samples, the possibility that the observed O₂^˙̄ production is due to free iron can be eliminated.

Generation of Superoxide Anion upon Oxidation of Ubiquinol by Cytochrome c or Ferricyanide in the Presence of Phospholipid Vesicles

Although the addition of ferricytochrome c to the proteinase K-digested complex can increase its superoxide production, oxidation of ubiquinol by ferricytochrome c in the aqueous solution at neutral pH is a very slow reaction with a reduction rate constant K₁ of 0.24/s, and little O₂^˙̄ formation is detected, suggesting that a hydrophobic environment provided by the digested complex is required for superoxide production. Similar results were obtained when potassium ferricyanide was used to replace ferricytochrome c.

To confirm that a hydrophobic environment is needed for O₂^˙̄ production, varying amounts of phospholipid vesicles were added to the mixture containing constant amounts of ubiquinol and cytochrome c, and the superoxide anion production was measured by the reduction of acetylated cytochrome c method. In the presence of phospholipid vesicles, the rate of the formation of O₂^˙̄ is proportional to the amount of vesicles added up to 0.3% (Fig. 4). This result clearly indicates that the formation of O₂^˙̄ takes place in hydrophobic environments.

FIGURE 4.

Phospholipid vesicle concentration-dependent superoxide formation under constant amounts of cytochrome c and ubiquinol. The superoxide generation was measured by the reduction of superoxide dismutase-sensitive acetylated cytochrome c reduction as described under “Experimental Procedures.” Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 10.0 μm acetylated cytochrome c, and different concentrations of asolectin vesicle. Solution B contains 0.5 mm Na⁺/K⁺ phosphate buffer, pH 4, 250 μm Q-H₂ in the presence or absence of 300 units/ml superoxide dismutase. Each data point represents an average of four experiments. Error bars indicate S.D.

Detergents Facilitate Superoxide Generation by QH₂ and Cytochrome c

If the hydrophobic environment in the bilayer of phospholipid vesicles can facilitate O₂^˙̄ generation, one would expect that a micelle solution of detergent should do the same. To test the effects of detergents on the O₂^˙̄ production by Q-H₂ and cytochrome c, various detergents, non-ionic (OG, DM), anionic (sodium cholate (SC) and deoxycholate (DOC)), and cationic (LDAO), were used to substitute phospholipid vesicles. The detergent micelle solution (or phospholipid vesicles), which facilitates superoxide anion production, does not prevent dismutation of the superoxide anion generated by hypoxanthine/xanthine oxidase. Therefore, the superoxide generation-facilitating activity observed is real and not due to the decrease of dismutation. Fig. 5 shows the effect of detergent concentration on the superoxide production during quinol oxidation by cytochrome c. As expected, little O₂^˙̄ generation is observed when the concentration of detergent used is below its critical micelle concentration because no hydrophobic environment is available. When the concentration of detergent used is higher than its critical micelle concentration, the O₂^˙̄ production rate increases as the detergent micelle concentration in the system increases. Interestingly, SC or DOC is much more effective in promoting superoxide generation than OG, DM, or LDAO. The critical micelle concentrations for LDAO, OG, DM, DOC, and SC are 1, 25, 0.15, 1.33, and 3 mm (32), respectively, which do not appear to correlate with their abilities to facilitate O₂^˙̄ generation. However, it is very apparent that detergent with a negatively charged head group such as SC or DOC can provide a better environment for O₂^˙̄ generation than neutral (OG or DM) or cationic (LDAO) detergents can. There are two simple explanations for the negatively charged detergents to have a better efficiency in promoting O₂^˙̄ production during the oxidation of quinol. First, the negative charges on the micelle surface may facilitate deprotonation of the 1-hydroxyl group of quinol. Second, the negatively charged detergent micelle surface may attract the positively charged ferricytochrome c better than the neutral or positively charged micelles. Thus, the high potential electron acceptor, ferricytochrome c, has a better accessibility to quinol, which is located near the surface of the detergent micelles. When potassium ferricyanide is used as the high potential electron acceptor, the effect on O₂^˙̄ generation by the difference in the charge of the micelle surface is less apparent.

FIGURE 5.

Effect of detergents on superoxide generation under constant amounts of cytochrome c and ubiquinol. The superoxide generation was measured by the reduction of superoxide dismutase-sensitive acetylated cytochrome c reduction as described under “Experimental Procedures.” Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 8.0, 10 μm acetylated cytochrome c, and different concentrations of detergents (LDAO, DM, OG, SC, and DOC). Solution B contains 0.5 mm Na⁺/K⁺ phosphate buffer, pH 4, 250 μm Q-H₂ in the presence or absence of 300 units/ml superoxide dismutase. Each data point represents an average of four experiments. Error bars indicate S.D.

Superoxide Anion Generation Is High Potential Oxidant- and Ubiquinol Concentration-dependent

Generation of O₂^˙̄ requires an electron donor ubiquinol and a high potential oxidant such as ISC, cytochrome c, or ferricyanide as an electron acceptor. To test the cytochrome c concentration dependence of the O₂^˙̄ generation, various concentrations of cytochrome c were added to a reaction mixture containing 25 μm Q-H₂ and 6 mm sodium cholate. As seen in Fig. 6 in the curve with open circles, it is clear that the O₂^˙̄ production is proportional to the concentration of cytochrome c. In Fig. 6, the curve with Xs shows the effect of ferricyanide concentration on the O₂^˙̄ generation. Varying concentrations of ferricyanide were added to a reaction mixture containing 25 μm Q-H₂ and 6 mm sodium cholate. Like cytochrome c, the O₂^˙̄ production is proportional to the concentration of ferricyanide added but with five times more efficiency than that of cytochrome c.

FIGURE 6.

High potential oxidant (cytochrome c or ferricyanide) concentration-dependent superoxide generation under a constant amount of ubiquinol. The superoxide production was measured as described under “Experimental Procedures.” The curve with open circles represents cytochrome c, and the curve with Xs represents ferricyanide. Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 6 mm sodium cholate, and a different concentration of cytochrome c or ferricyanide. Solution B contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 6 mm sodium cholate, 50 μm Q-H₂, and 4 μm MCLA. Each data point represents an average of four experiments. Error bars indicate S.D.

The effect of the Q-H₂ concentration on superoxide generation was also studied. Under a constant concentration of cytochrome c (2.5 or 25 μm) and sodium cholate (6 mm), O₂^˙̄ production increases when Q-H₂ concentration increases (Fig. 7). When 2.5 μm cytochrome c is used, maximum O₂^˙̄ production is obtained when 25 μm Q-H₂ is used. When 25 μm cytochrome c is used, maximum O₂^˙̄ production is not reached until the Q-H₂ concentration in the system is about 125 μm or higher (data not shown). These results indicate that at a given concentration of Q-H₂, O₂^˙̄ production is dependent on the concentration of cytochrome c used, confirming the results shown in Fig. 6.

FIGURE 7.

Quinol concentration-dependent superoxide production under a constant amount of sodium cholate. The superoxide production was measured as described under “Experimental Procedures.” Solution A contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, and 5 μm (open circles) or 50 μm (Xs) of cytochrome c. Solution B contains 100 mm Na⁺/K⁺ phosphate buffer, pH 7.4, 4 μm MCLA, 12 mm sodium cholate, and different concentrations of Q-H₂. Each data point represents an average of four experiments. Error bars indicate S.D.

Reaction Mechanism of Superoxide Anion Generation by the Cytochrome bc₁ Complex

Based on the results obtained that O₂^˙̄ is produced during Q-H₂ oxidation by cytochrome c or ferricyanide in the presence of phospholipid vesicles or a detergent micelle and that the O₂^˙̄ is produced by a heat-inactivated or proteinase K-digested complex, a reaction mechanism for O₂^˙̄ production in the bc₁ complex is proposed. In this proposed mechanism, four elements are directly involved in O₂^˙̄ generation. These are: a hydrophobic environment, a high potential electron acceptor, a low potential electron acceptor, and an electron donor.

In the intact cytochrome bc₁ complex, the protein subunits of the complex directly involved in the superoxide production are ISP, which houses a high potential electron acceptor ISC, and cytochrome b, which provides a hydrophobic environment, a Q_P pocket, and a low potential electron acceptor heme b_L. Based on the concerted Q-cycle mechanism (29–31), quinol undergoes bifurcated oxidation in the Q_P pocket by simultaneously transferring its two electrons to ISC and heme b_L. The molecular oxygen can also receive a hydrogen from quinol to produce a protonated superoxide (O₂H), which generates O₂^˙̄ upon deprotonation. It is also likely that the reduced heme b_L can transfer an electron to the molecular oxygen to form O₂^˙̄, particularly when the oxidant (heme b_H) of reduced heme b_L is limited or unavailable, such as in the presence of antimycin A (Fig. 8A). According to this proposed O₂^˙̄ generation mechanism, the generation of only a fraction of O₂^˙̄ during quinol oxidation catalyzed by intact bc₁ complex may result from: (i) the limited accessibility of molecular oxygen to the Q_P pocket, which is surrounded by structured protein subunits; (ii) molecular oxygen competing unfavorably with heme b_L for the electron during bifurcated ubiquinol oxidation, thus ensuring that few electrons are readily available for oxygen to react with; and (iii) the fact that the O₂^˙̄ being generated within the Q_P pocket may not easily escape to the aqueous medium.

FIGURE 8.

The schematic depiction of bifurcated oxidation of ubiquinol at Q_P pocket (A) and the proposed location of Q₀C₁₀BrH₂ in the phospholipid vesicle (B) or in the detergent micelle (C). Stig., stigmatellin; Myxo, myxothiazol; AA, antimycin A; IMS, the mitochondrial intermembrane space; TM, the transmembrane region.

Although intact protein subunits of the bc₁ complex are not directly involved in O₂^˙̄ generation, they form a barrier for the Q_P pocket to limit accessibility of molecular oxygen to the pocket. Destruction of protein structural integrity by heme b_L or b_H deletion, heat inactivation, or proteinase K digestion leads to a loosening of the structural integrity of the Q_P pocket to facilitate the molecular oxygen to get access to the Q_P pocket and to ease the release of produced O₂^˙̄ to the aqueous medium.

In addition, it is possible that in the cytochrome bc₁ complex, an oxygen molecule is located between the 4-HO⁻ group of Q-H₂ and heme b_L to mediate the electron transfer between them in the hydrophobic environment of the Q_P pocket. The poor hydrogen-bonding nature of the oxygen molecule seems to work against this speculation. However, if this were the case, in a hydrophobic environment, one would expect to see a higher presteady state reduction rate of cytochrome b by ubiquinol in the presence of oxygen than in the absence of it. Our preliminary results seem to support this speculation. Further investigation on the role of oxygen in the reduction of cytochrome b by ubiquinol is currently in progress in our laboratory.

In the protein-free system, our results appear to suggest that the benzoquinol ring of Q-H₂ is located at or near the surface of the lipid vesicles or detergent micelles with its 1-hydroxy group extended into the water phase and the 4-hydroxy group together with its alkyl side chain located inside the bilayer or micelle (Fig. 8, B and C). When the ISC is used as an electron acceptor, we speculate that a hydrogen is transferred from the 1-hydroxyl group to the N-3 of the imidazole ring of histidine residue, which is a ligand of the ISC. At the same time, a hydrogen is transferred concurrently from the 4-hydroxy group to a molecule of oxygen, which is dissolved inside the lipid bilayer of vesicles or in the hydrophobic interior of detergent micelles, to generate a protonated superoxide (HO₂), which then diffuses to the water phase to become a superoxide anion upon deprotonation.

The high potential electron acceptor ISC can be substituted with cytochrome c or ferricyanide. In this case, we surmise that the 1-hydroxy group of ubiquinone is deprotonated and releases a proton to the water phase before the electron is transferred to ferricyanide or cytochrome c. At the same time, the 4-hydroxy group transfers its hydrogen atom to a molecular oxygen.

In the phospholipid vesicles or detergent micelle systems, we suggest that the electron transfer between cytochrome c and Q-H₂ takes place at the surface of the lipid bilayer and that the transfer between Q-H₂ and O₂ occurs concurrently inside the bilayer. This suggestion is consistent with the fact that the solubility of oxygen is much higher in the hydrophobic environment than that in the aqueous medium and that the rate of reduction of cytochrome c by Q-H₂ is much lower under anaerobic conditions than when in the presence of oxygen.