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Originally published In Press as doi:10.1074/jbc.M507616200 on August 8, 2005

J. Biol. Chem., Vol. 280, Issue 41, 34654-34660, October 14, 2005
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The Respiratory Substrate Rhodoquinol Induces Q-cycle Bypass Reactions in the Yeast Cytochrome bc1 Complex

MECHANISTIC AND PHYSIOLOGICAL IMPLICATIONS*

Jonathan L. Cape{ddagger}, Jeff R. Strahan§, Michael J. Lenaeus§, Brook A. Yuknis§, Trieu T. Le§, Jennifer N. Shepherd§1, Michael K. Bowman{ddagger}, and David M. Kramer{ddagger}

From the {ddagger}Institute of Biological Chemistry, Washingston State University, Pullman, Washington 99164-6340, §Department of Chemistry, Gonzaga University, Spokane, Washington 99258-0005, and Structural Biology and Microimaging, Battelle Northwest Laboratory, Richland, Washington 99352-0999

Received for publication, July 13, 2005 , and in revised form, August 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mitochondrial cytochrome bc1 complex catalyzes the transfer of electrons from ubiquinol to cyt c while generating a proton motive force for ATP synthesis via the "Q-cycle" mechanism. Under certain conditions electron flow through the Q-cycle is blocked at the level of a reactive intermediate in the quinol oxidase site of the enzyme, resulting in "bypass reactions," some of which lead to superoxide production. Using analogs of the respiratory substrates ubiquinol-3 and rhodoquinol-3, we show that the relative rates of Q-cycle bypass reactions in the Saccharomyces cerevisiae cyt bc1 complex are highly dependent by a factor of up to 100-fold on the properties of the substrate quinol. Our results suggest that the rate of Q-cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the quinol oxidase site of the enzyme. We conclude that normal operation of the Q-cycle requires a fairly narrow window of redox potentials with respect to the quinol substrate to allow normal turnover of the complex while preventing potentially damaging bypass reactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytochrome (cyt)2 bc1 complex (EC 1.10.2.2 [EC] ) functions as the ubiquinol:cytochrome c oxidoreductase in the electron transport chains of mitochondria and bacteria, where it couples the energy released from the oxidation of ubiquinol (UQH2) by high potential electron carriers (e.g. cyt c) to establish a proton motive force used to drive ATP synthesis (1, 2). Although the cyt bc1 complex usually conserves energy across the membrane, deleterious side reactions can occur that bypass normal electron flow in the enzyme, robbing the cell of energy and potentially producing reactive oxygen species (3–7). To deal with this problem, the cyt bc1 complex has apparently developed strict control mechanisms to prevent side reactions of ubiquinone/ubiquinol (UQ (1)/UQH2; see Fig. 1) reaction intermediates (3, 6, 8–10).3

The turnover mechanism of the cyt bc1 complex is best described by the modified Q-cycle (11, 12). The key feature of this mechanism is the bifurcated oxidation of UQH2 at the quinol oxidase (Qo) site of the enzyme that sends one UQH2 electron to a high potential chain (composed of the Rieske 2Fe2S cluster and cyt c1), whereas the other electron reduces a low potential chain (cyt bL and bH) and is eventually recycled back into the Q pool through UQ reduction at a quinone reductase (Qi) site on the opposite side of the membrane. In most models UQH2 oxidation proceeds by the initial reduction of the Rieske 2Fe2S cluster (13–15), which produces an unstable semiquinone (SQ) intermediate localized to the Qo site (SQo), which then reduces the low potential chain and UQ at the Qi site, leaving a labile UQ in the Qo pocket.

Certain conditions such as mutation (16–18), imposition of a high proton motive force (17, 19, 20), or inhibitor treatment (3–5, 7) cause interruption of electron flow from SQo to the low potential chain and can lead to a large fraction of reactions that bypass the normal Q-cycle. Most Q-cycle bypass reactions are thought to result from the buildup of a highly reactive SQo species, although some are suggested to occur via reduced cyt bL (18); some of these reactions include (3, 4, 7) 1) double reduction of the high potential chain by SQo, 2) re-oxidation of reduced cyt bL by SQo, Q, or O2, 3) oxidation of SQo by O2, and 4) direct reduction of cyt c by a labile SQo that leaves the Qo site. Although each of these reactions is thermodynamically favorable in comparison to the Q-cycle, they are typically not detected during uninhibited turnover of the complex, leading to the hypothesis that the cyt bc1 complex has evolved a mechanism to steer its reactive intermediate toward productive, rather than harmful, reactions.

Several variants of the Q-cycle have been proposed to explain the prevention of bypass reactions, most of which impart strict constraints on the chemical and thermodynamic properties of the substrate QH2 and its proposed initial oxidation product, SQo (6, 8, 11–14, 21–26). Previous studies (27–29) have suggested a link between these thermodynamic properties and the overall turnover rate of the bc1 complex, but it is unclear how the intrinsic redox properties of the QH2 substrate and the influence of the protein environment on these properties work together to enforce normal electron flow in the Q-cycle while also preventing bypass reactions. We suggest that both these factors tailor QH2 oxidation in the Qo site to minimize the concentration of reactive intermediates that can participate in these bypass reactions, which can be achieved through the use of a substrate whose oxidation results in a less reducing redox poise in the Qo site (i.e. by preventing the accumulation of either SQo or reduced cyt bL).



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  		FIGURE 1.
Representative structures of UQ and RQ.

 
In this work we use synthetic analogues of the quinone substrates UQ and rhodoquinone (RQ (2), see Fig. 1) to test boundary conditions on how the intrinsic thermodynamic properties of the substrate and its associated SQ stability influence the rate of Q-cycle bypass reactions. RQ is used in certain eukaryotes (30–36) and prokaryotes (37, 38) to shuttle electrons from the NADH dehydrogenase to a fumarate reductase (31, 36, 39–41). This alternative electron transport chain is used during anoxia to maintain cellular redox balance by using fumarate as a terminal electron acceptor instead of oxygen. Although RQ is chemically similar to UQ (a OCH3 substituent on the ring is replaced by NH2), the electron donating nature of its amino group gives it substantially more reducing power than its counterpart UQ (42–44), thus offering the opportunity to probe the efficiency of the Q-cycle under conditions where the relative stability of SQ species are modulated in both the Qo and Qi sites.

In certain organisms (nematodes (45), parasitic helminths (35, 46), mussels and oysters (35), and the photosynthetic bacterium Rhodospirillum rubrum (34)), RQ is co-localized in the mitochondrial or plasma membrane with UQ (32, 34, 47). This occurs even under aerobic conditions where biosynthesis of RQ is down-regulated but not entirely absent, providing the potential for the interaction of RQ or RQH2 with the cyt bc1 complex. The use of the synthetic substrates ubiquinone-3 (UQ3) and rhodoquinone-3 (RQ3), where n = 3 isoprene units (instead of 9 or 10 in the natural substrates), allow us to probe the Q-cycle and bypass reactions as well as to measure the relevant redox potentials of the electrochemical couples involved in these reactions. From these results we predict the potential impact due to bypass of the Q-cycle in organisms that contain a mixed UQ/RQ pool (32, 37, 48) and test the relationship between the rates of bypass reactions and the thermodynamic properties of these two substrates.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals for RQ3 Synthesis—All reactions were performed under a positive pressure of Ar with minimal exposure to light. Diethyl ether, ethyl acetate, hexanes, methanol, and tetrahydrofuran were purchased from Fisher (ACS-certified grade). Tetrahydrofuran was distilled from sodium benzophenone ketyl, and methanol was dried over 3-Å molecular sieves. Mesyl chloride, sodium azide, sodium sulfate, triethylamine, and triphenylphosphine were purchased from Aldrich. Thin layer chromatography was performed on Whatman (F254) 0.25-mm coated silica gel plates and visualized using 254-nm light or by heating samples with vanillin stain. Column chromatography was carried out using 230–245 mesh silica gel from Bodman Scientific.

5-Farnesyl-2-mesyl-3-methoxy-6-methyl-1,4-benzoquinone (4)—Demethylubiquinone-3 (3, 200 mg, 0.54 mmol) was dissolved in tetrahydrofuran (5 ml) and cooled to -20 °C before treating with triethylamine (0.082 ml, 0.59 mmol). The color of the solution changed from red to purple upon deprotonation. Mesyl chloride (0.046 ml, 0.59 mmol) was then added dropwise, and the reaction was allowed to warm to room temperature. By thin layer chromatography, the reaction was complete after 3 h (3, Rf = 0.5; 4, Rf = 0.8 in 6:4 hexanes:ethyl acetate). The reaction was quenched with H2O (5 ml) and extracted three times with diethyl ether (Et2O, 15 ml total). The organic layers were combined and rinsed with brine (10 ml). The organic layers were dried over Na2SO4 and concentrated in vacuo to yield compound 4, a yellow solid (162 mg, 73% yield). 4: 1H NMR (300 MHz, CDCl3): {delta} 5.05 (m, 2H), 4.89 (t, 1H, J = 6.9 Hz), 4.20 (s, 3H), 3.47 (s, 3H), 3.19 (d, 2H, J = 7.1 Hz), 1.97 (s, 3H), 1.83 (m, 8H), 1.62 (m, 12H); 13C NMR (75 MHz, CDCl3): 182.6, 182.4, 149.0, 142.3, 139.5, 138.2, 135.3, 132.6, 131.3, 124.3, 123.8, 118.2, 61.5, 40.4, 39.7, 29.7, 26.7, 26.4, 25.7, 25.5, 17.6, 16.3, 16.0, 12.0; IR (thin film on NaCl): 2924, 2853, 1670, 1654, 1615, 1448, 1371, 1262, 1176, 970, 789 cm-1. High resolution mass spectrum calculated for C24H34O6SNa (M++Na) was 473.1968; found was 473.1919.

2-Azido-5-farnesyl-3-methoxy-6-methyl-1,4-benzoquinone (5)—The mesylate 4 (200 mg, 0.44 mmol) was dissolved in methanol (MeOH, 3.7 ml) and treated with sodium azide (NaN3, 58 mg, 0.89 mmol). The solution turned from a reddish-orange color to a reddish-brown color after 5 min. The reaction was allowed to proceed for 1.5 h before quenching with H2O (5 ml). The mixture was extracted three times with ethyl acetate (15 ml total), and the organic layers were washed with brine (10 ml), dried over Na2SO4, and concentrated in vacuo. Flash chromatography (9:1 hexanes:ethyl acetate) afforded 5, a reddish-orange solid (58 mg, 33% yield). Unreacted mesylate was recovered during chromatography. 5: 1H NMR (300 MHz, CDCl3): {delta} 5.06 (m, 2H), 4.91 (t, 1H, J = 6.8 Hz), 4.07 (s, 3H), 3.19 (d, 2H, J = 7.0 Hz), 2.06 (s, 3H), 1.94 (m, 8H), 1.62 (m, 12H); 13C NMR (75 MHz, CDCl3): 183.8, 181.7, 144.8, 142.7, 139.1, 137.9, 135.2, 131.3, 126.9, 124.3, 123.8, 118.5, 61.4, 39.7, 39.6, 26.8, 26.4, 25.7, 25.3, 17.6, 16.3, 16.0, 12.1; IR (thin film on NaCl): 2920, 2346, 2114, 1654, 1602, 1443, 1219, 1146 cm-1; high resolution mass spectrum calculated for C23H31N3O3Na (M++Na)was 420.2258 found was 420.2244.

2-Amino-5-farnesyl-3-methoxy-6-methyl-1,4-benzoquinone (RQ3, 7)—Compound 5 (50 mg, 0.13 mmol) was dissolved in tetrahydrofuran (1 ml), and triphenylphosphine (33 mg, 0.13 mmol) was added. The solution immediately turned dark purple, and the reaction was allowed to proceed for 2 h. The reaction was quenched with H2O (2 ml), and a standard ethyl acetate/brine work-up was performed to provide 6. Compound 6 was hydrolyzed by slow gravity elution on SiO2 gel (9:1 hexane:ethyl acetate) to provide pure 7 (27 mg, 64% for 2 steps). 6: 1H NMR (300 MHz, CDCl3): {delta} 7.66 (m 15H, 5.06 (m, 2H), 4.99 (t, 1H, J = 6.8 Hz), 3.76 (s, 3H), 3.17 (d, 2H, J = 7.0 Hz), 2.06 (s, 3H), 1.94 (m, 8H), 1.62 (m, 12H). 7: 1H NMR (300 MHz, CDCl3): {delta} 5.06 (m, 2H), 4.99 (t, 1H, J = 6.8 Hz), 4.68 (broad s, 2H), 3.86 (s, 3H), 3.18 (d, 2H, J = 7.0 Hz), 2.03 (m, 8H), 1.96 (s, 3H), 1.67 (m,12H); 13C NMR (75 MHz, CDCl3): {delta} 185.1, 180.8, 143.4, 137.2, 136.1, 135.8, 135.1, 134.7, 131.2, 124.3, 123.9, 119.3, 60.1, 39.7 (2C), 26.7, 26.5, 25.7, 25.3, 17.6, 16.3, 16.0, 11.6; IR (thin film on NaCl): 3485, 3362, 2918, 2849, 1652, 1606, 1468, 1440, 1375, 1302, 1206 cm-1; high resolution mass spectrum calculated for C23H34NO3 (M++1) was 372.2533; found was 372.2519.

Purification of the Cyt bc1 Complex—Yeast (Saccharomyces cerevisiae) cyt bc1 complex was isolated from store-bought bakers' yeast using the protocol of Ljungdahl et al. (49) with modifications (4). The concentration of the cyt bc1 complex was determined by the ferricyanide-ascorbate-dithionite absorbance difference spectra using published values of the extinction coefficients (49).

Steady State Turnover Measurements—Steady state turnover was measured after the reduction of cyt c using an extinction coefficient of 17 mM-1 cm-1 for the 550-nm - 542-nm difference absorption (3, 4). Just before measurement, synthetic substrate, Q3H2 (UQ3H2 or RQ3H2), was mixed in a cuvette containing 50 µM cyt c and 1 mM NaN3 in reaction buffer (50 mM MOPS, 50 mM Tricine, and 100 mM KCl at pH 8.0). In previous work (3, 4) we used only MOPS as a buffer but have now included Tricine to ensure a high buffering capacity in the pH range 8.0 and above; this additional component did not cause any measurable changes in the activity of the complex. Cyt bc1 (1–3 nM) was added and mixed in the cuvette after collection of 20 s of background cyt c reduction. Turnover numbers were calculated by subtracting the background cyt c reduction rate from the initial rate observed after the addition of cyt bc1. Measurement of Q-cycle bypass reactions under partially inhibited conditions was performed by the addition of 30 µM antimycin-A (AA) to the cuvette before Q3H2 addition and mixing. Cyt bc1 (20–40 nM) was used for bypass reaction measurements involving UQ3H2 due to the very low rate of this reaction (1–5 s-1). All experiments were repeated once with the addition of stigmatellin as a control, which completely abolished activity in all cases.



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  		SCHEME 1.
Synthesis of RQ3. Reaction conditions: a, mesyl chloride, triethylamine, tetrahydrofuran tetrahydrofuran, -20 °C; b, NaN3, MeOH; c, triphenylphosphine, tetrahydrofuran; d, 8:2 hexanes:ethyl acetate, SiO2.

 
Measurement of Superoxide Production—Under partially inhibited conditions, i.e. conditions where AA blocks reduction of Q at the Qi site, the intermediate SQo can reduce oxygen to superoxide, which then reduces cyt c directly in a diffusion-limited reaction (4). Thus, the rate of superoxide production from the Qo site can be determined by taking the difference in rates of AA-resistant cyt c reduction with and without added manganese superoxide dismutase (SOD). In most cases the addition of SOD inhibits the rate of cyt c reduction by 25–50%; the residual non-SOD-inhibitable cyt c reduction is caused by other Q-cycle bypass reactions such as the direct reduction of cyt c by SQo or double reduction of the high potential chain (4). A typical superoxide production assay measuring cyt c reduction was performed by the addition of 300 units of manganese SOD to the steady state assay mixture described above.

The Extent of Cyt b Reduction in the Steady State—Measurement of the reduction state of both cyt b hemes during steady state turnover was performed by mixing 30 µM Q3H2 into a cuvette containing 265 nM cyt bc1 and 5 µM cyt c. The low concentration of cyt c was used to slow depletion of the substrate through normal turnover and background cyt c reduction, which allowed us to observe steady state turnover of the complex over a timescale of minutes. The reaction was followed using the 561-nm minus 578-nm wavelength pair using an extinction coefficient of 26 mM-1 cm-1 for total cyt b. Saturating concentrations of AA and stigmatellin were added as needed. Sodium azide was omitted from the assay medium to allow re-oxidation of cyt c by the cyt c oxidase, which is a trace impurity in our preparations of cyt bc1 complex. The reduction state of the cyt c pool was measured optically to verify that the complex had actually reached the steady state. Re-oxidation of reduced cyt c after an initial spike in the concentration (up to 300 nM) brought the steady state reduced cyt c concentration to ~60 nM. Over this time, the extent of cyt b reduction was nearly constant after the initial increase in reduction, suggesting rapid establishment of steady state conditions in the low potential chain.

Cyclic Voltammetry and Determination of Redox Potentials—Voltammetric measurements of the Q/QH2 and couples for UQ3 and RQ3 were performed in aqueous solution under an Ar atmosphere at 23 °C using a BAS C50-W potentiostat with an Ag/AgCl reference electrode, glassy carbon working electrode, and Pt wire auxiliary electrode. The working electrode was washed with dilute H2SO4, repeatedly rinsed with distilled water, and then polished with alumina before all measurements. Determination of was performed in unbuffered solution (100 mM KCl) as described in Shim and Park (50) at pH > 5.0. Aqueous values were determined at several pH values above pH 5.0 to ensure that the E1/2 and {Delta}E values were independent of pH and, thus, reflect a pure electron transfer process. The two-electron Q/QH2 potential was measured in 50 mM phosphate, 100 mM KCl buffer at pH 7.0. E1/2 values for each couple were taken as the average of the cathodic and anodic peaks at half height. All potentials given in the text are referenced to the standard hydrogen electrode.

We used experimentally determined values for the and Q/QH2 couples of UQ3 and RQ3 in aqueous solution to estimate reasonable values of the aqueous stability constant, KS, and Em values for the various redox couples contributing to KS, according to Equation 1 (51–53),

(Eq. 1)
where pKa,SQ is the pKa for deprotonation of QH·. The pKa,SQ for UQ was previously determined to be 5.6 in aqueous solution using pulse radiolysis (54). We used a calibrated computational technique using a combination of density functional theory calculations and thermodynamic cycles to estimate the pKa of RQ3H·; the details of these calculations are to be presented in a forthcoming manuscript.4 This pKa was found to be similar to that of UQH·, 5.0–5.6.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synthesis of RQ3 and UQ3—A novel and efficient synthesis of RQ3 was developed. The farnesylated analogue of demethylubiquinone (3), a known biosynthetic precursor of UQ (55–57), was prepared from fumagatin using an improved method that utilized farnesyltrimethyl stannane and BF3-O(CH2CH3)2 followed by oxidation with Ag2O (Scheme 1) (58–60). Compound 3 was converted to the mesylate 4 using mesyl chloride and triethylamine in a 77% yield (38). The mesylate was then treated with NaN3 to generate the azide 5 via a nucleophilic addition-elimination reaction (62). This reaction produced a 33% yield of azide; however, unreacted starting material could be recovered. Reduction of the azide with triphenylphosphine formed an iminophosphorane 6, which was not readily hydrolyzed during an aqueous work-up as previously reported (63, 64). However, treatment of 6 with dilute acid or slow elution through a silica gel column provided pure RQ3 (7) in a 64% yield (two steps). UQ3 was prepared using published procedures (55).

Thermodynamic Characterization of UQ3 and RQ3—Cyclic voltammetry of UQ3 in unbuffered aqueous solution (50) at pH > 5 exhibited quasi-irreversible cathodic and anodic peaks ({Delta}E = 190 mV). The lack of complete reversibility likely reflects adsorption of UQ3 to the electrode surface. Rigorous cleaning of the electrode surface did not affect the reversibility of these peaks. The E1/2 value obtained from this measurement was -23 mV (see TABLE ONE), which is consistent with a previous report of E1/2 = -30 mV for the UQ couple in 80% aqueous ethanol (65). The couple for RQ3 also exhibited quasi-irreversible voltammetric peaks ({Delta}E = 109 mV), although to a much lesser extent than UQ3, with E1/2 = -170 mV.


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  		TABLE ONE
Thermodynamic properties of UQ and RQ redox and protonation reactions Values were determined as described under "Experimental Procedures." Electrochemical midpoint potentials are expressed against the standard hydrogen electrode.

 
As an additional assurance that the E1/2 values measured here are reasonable estimates of the actual equilibrium potentials, we compared the (Q/QH2) couple of RQ3 with the previously published polarographic value. Our result (TABLE ONE), E1/2 = -45 mV, is in reasonable agreement with the previously published value of -65 mV (44). Measurement of the E1/2 for the UQ3 (Q/QH2) couple was not possible due to complete irreversibility of the cathodic peak, resulting in the absence of an anodic peak under these conditions.

Normal Turnover and Q-cycle Bypass Reactions Induced by UQ3H2 and RQ3H2—TABLE TWO summarizes the results from steady state turnover of the cyt bc1 complex driven by UQ3H2 and RQ3H2 under uninhibited (Fig. 2 and TABLE TWO, row 1) and AA-inhibited (TABLE TWO, row 2) conditions. Cyt c reduction observed under AA-inhibited conditions results from partial turnover of the complex and one or more reactions that bypass the Q-cycle and directly reduce cyt c. Using UQ3H2 as a substrate, the addition of 30 µM AA decreased the rate of cyt bc1 turnover from 143 to 3 s-1, consistent with previous results (3, 4, 7). On the other hand, the use of RQ3H2 exhibited an uninhibited turnover rate substantially lower than UQ3H2, 76 s-1, and exhibited a relative insensitivity to AA treatment, 88 s-1.


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  		TABLE TWO
Substrate-dependent properties of cyt bc1 turnover All kinetic measurements were performed at 23 °C with the isolated cyt bc1 complex as described under "Experimental Procedures." The velocity of superoxide production is calculated from the difference between Vmax (AA-inhibited) and Vmax (AA-inhibited + SOD).

 
We verified that AA did indeed bind to the Qi site of the complex under these conditions by observing the red-shifted absorbance spectrum of cyt bH induced by AA binding in the presence of RQ3 (66) (data not shown). Further titration of RQ3 into the AA-treated complex did not reverse the red-shifted spectrum of cyt bH, showing that RQ3 does not displace AA during the assay.

A large degree of curvature in the kinetic traces with RQ3H2 was observed, suggesting that the true initial rate of cyt c reduction was obscured during the manual mixing time. We thus repeated the AA-induced bypass reaction experiments using stopped flow. Indeed, as shown in TABLE ONE, the rate of UQ3H2 AA-resistant turnover was 2.3 s-1, consistent with steady state measurements, whereas the rate of RQ3H2 AA-resistant turnover was very high, at 207 s-1, a difference in bypass rates of more than 100-fold. Even this rate might be limited by the 0.2-s time resolution of our spectrophotometer.



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  		FIGURE 2.
Representative kinetic assays of cyt bc1 with either UQ3H2 or RQ3H2. Assays were performed as described under "Experimental Procedures." Both traces shown here were collected using either 70 µM UQ3H2 (open circles)or30 µM RQ3H2 (filled squares) with 4 nM cyt bc1 and 50 µM cyt c. The background rate of cyt c reduction by the substrate alone is subtracted for simplicity. UQH2 always reached the full extent of cyt c reduction, whereas RQH2 did not (see "Normal Turnover and Q-cycle Bypass Reactions Induced by UQ3H2 and RQ3H2").

 
Both RQ3H2 and UQ3H2 exhibited similar apparent Km values with the bc1 complex, 10 and 8 µM for UQ3H2 and RQ3H2, respectively. These Km values are similar to those measured previously for the isolated yeast bc1 complex by various groups (7, 67–69).

Partial Inhibition of the Q-cycle by RQ3H2/RQ3—The curvature in the steady state kinetic traces described above suggested some form of product inhibition (Fig. 2). We first tested for inhibition of Q reduction at the Qi site induced by RQ3 or UQ3. Typically, efficient oxidation of cyt b by Q at the Qi site results in only a small accumulation of reduced cyt b in the low potential chain under steady state conditions, whereas blockage of cyt b reoxidation at the Qi site with inhibitors results in a substantial accumulation (30–50%) of reduced cyt b (10). The data shown in Fig. 3 confirm these observations in our system using UQ3H2 as substrate. Approximately 10% of total cyt b was reduced under uninhibited conditions, whereas 52% went reduced in the presence of AA. In contrast, when RQ3H2 was used (Fig. 3), 77% of cyt b went reduced under uninhibited conditions, suggesting that the re-oxidation of cyt b by RQ3 was slowed with respect to its reduction. The addition of AA in the presence of RQ3H2 resulted in eventual reduction of nearly all (92%) of cyt b. The statistically significant overall lower accumulation of cyt b during RQ3H2 oxidation in the absence of AA probably indicates that reduction of RQ3 at the Qi site occurs, albeit at a lower rate than with UQ3.



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  		FIGURE 3.
Steady state measurement of cyt b reduction in the presence of UQ3H2 and RQ3H2. Measurement of the total cyt b reduction state under steady state conditions in the cyt bc1 complex is described under "Experimental Procedures." Data were determined in triplicate, and results were consistent between two different preparations of the cyt bc1 complex. Error bars represent the S.D. of the measurement. Assays were performed using 265 nM bc1 complex (yielding 530 nM total cyt b heme) using either 30 µM RQ3H2 or UQ3H2 with 5 µM cyt c used as an acceptor to the complex in all assays. AA (30 µM) was added as needed, and sodium azide was omitted to allow re-oxidation of cyt c by residual cyt c oxidase. Cyt b reduction was followed using the 561 - 578 nm wave-length pair. Inset, time-based absorbance traces during the steady state measurement for RQ3H2 + AA (A), RQ3H2 with no AA (B), UQ3H2 + AA (C), and UQ3H2 with no AA (D). The slow autooxidation of cyt b shown in the inset for traces A, B, and C is due to substrate depletion after several minutes through background cyt c reduction and QH2 autooxidation.

 
In addition to functioning as a Qi site inhibitor, Fig. 4 demonstrates that RQ3 also interferes with UQ3H2 binding to the Qo site. The addition of up to 20 µM RQ3 both increased the apparent Km for UQ3H2, from 8.5 to 13.7 µM, and lowered the apparent Vmax, from 98 to 84 s-1, for UQ3H2 oxidation. These results are consistent with RQ3 acting as a weak mixed competitive inhibitor of the bc1 complex.

Superoxide Production Resulting from Q-cycle Bypass Dramatically Increases Using RQ3H2 as a Substrate—The SOD sensitivity of AA-resistant cyt c reduction was used to estimate the fraction of bypass reactions resulting in superoxide production. The initial rate of AA-resistant UQ3H2-induced turnover resulting in superoxide production was 1.0 s-1, consistent with previous measurements (3, 4, 7), whereas the same measurements performed with RQ3H2 resulted in an astonishing superoxide production rate of 29.5 s-1. The ~30-fold increase in rates for RQ3H2-induced superoxide production relative to UQ3H2 (see TABLE TWO, row 5) correlates well with the overall increased rate of bypass reactions when this substrate is used (see TABLE TWO, row 3), which also increases by a factor of ~30, suggesting that about 66% of all QH2 oxidations result in the reduction of oxygen to superoxide (3, 4, 7) under partially inhibited conditions regardless of the substrate used (TABLE TWO, row 6).

We observed complex (or strongly multi-phasic) cyt c reduction kinetics, probably reflecting a progressive onset of product inhibition (see "Partial Inhibition of the Q-cycle by RQ3H2/RQ3"). Because the curvature was apparent even at short reaction times, we suggest that our measured rates of RQ3H2-induced superoxide production represent a lower limit. Unfortunately, even with presteady state kinetics using stopped-flow, it is not presently possible to distinguish between the first two non-inhibited turnovers (which result in cyt bH and bL reduction) and the subsequent true bypass reactions.



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  		FIGURE 4.
Mixed competitive inhibition of the cyt bc1 complex by RQ3. UQ3H2 was titrated from 2.5 to 60 µM with 2 nM cyt bc1 complex in the presence of 0 µM (filled squares), 10 µM (open circles), and 20 µM (open triangles) RQ3. Measurements were also performed using decyl-UQH2, a synthetic UQH2 analogue, with similar results (not shown). All activity was demonstrated to be stigmatellin-sensitive (data not shown). Inset, replot of extrema in double reciprocal form showing the decrease in Vmax and increase in Km upon increasing [RQ] from 0 µM (filled squares) to 20 µM (open circles).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The Thermodynamic Properties of RQ and UQ and Predictions for Reactivity—Our electrochemical data (TABLE ONE) show that, as expected, the redox potential of the E(QH·/QH2) for RQH2 is substantially more negative than the same couple for UQH2 (by nearly 200 mV). We predict that the low potential of the RQH·/RQH2 couple will result in increased concentrations of rhodosemiquinone (RSQ) in the Qo site due to an increase in the equilibrium constant for RSQ formation by over 3 orders of magnitude, thus increasing the rate of bypass reactions. Similarly, the aqueous values of E(RQ3/RQ3H2) and are ~150 mV lower than the corresponding UQ species, likely decreasing the equilibrium constant for reduction of RQ at the Qi site by more than 2 orders of magnitude. We, thus, predict that the slow oxidation of the low potential chain during steady state turnover will lead to an AA-like inhibition of the complex, again having the effect of increasing the rate of bypass reactions. Consistent with these predictions, we observe that the rates of bypass reactions and superoxide production are drastically increased (more than 100-fold, see TABLE TWO) when RQ3H2 is used as a substrate instead of UQ3H2.

The SQ stability constant, KS, is often used alone as a qualitative predictor of SQ reactivity and steady state concentration for many Qo site models (6, 8, 11, 70). Despite the clear differences between these substrates in our estimates for the one-electron couples, E(QH·/QH2) and , the estimated semiquinone stability constants (TABLE ONE) for RSQ and USQ differ by only a factor of 2, with RSQ (KS = 1 x 10-7) slightly more stable than USQ (KS = 5.3 x 10-8). The semiquinone stability constant by itself is, thus, an incomplete predictor of reactivity and SQ concentration and must be used in conjunction with the overall two-electron Q/QH2 couple to derive the one electron couples, so that meaningful predictions can be made.

It is important to note that the in situ potentials of the Q/QH2 species in the Qo and Qi sites are likely to differ substantially from the aqueous potentials measured and estimated here due to the unique electrostatic environment of the protein and dielectric effects (71–74). Interestingly, the two Q binding sites in the complex likely exhibit disparate effects on the stability of the SQ species; the Qi site has been shown to stabilize the SQ (74, 75), whereas some models suggest the Qo site greatly destabilizes the SQ (11–13, 70). Nevertheless, it is reasonable to assume that the changes in the redox potentials of the RQ species in the Qo pocket will parallel those of the UQ species so that the differences measured in solution will persist in the Qo pocket.

RQ/RQH2 Alters the Redox Poise of the bc1 Complex to Favor Bypass Reactions—Turnover of the cyt bc1 complex using RQ3H2 as a substrate exhibited three features distinguishing it from that with UQ3H2 (Fig. 2); 1) upon manual mixing of substrate into reaction buffer (with a mixing time of ~2 s) the initial rate of uninhibited turnover (76 s-1) was about half that with UQ3H2 (143 s-1), 2) the kinetic trace exhibited a high degree of curvature as the reaction proceeded, lowering the steady state rate over time, and 3) unlike UQ3H2 oxidation, the addition of AA had little effect on the initial rate of turnover.

We suggest that all of these observations can be explained by product inhibition at both the Qo and Qi sites. This interpretation is supported by the "backup" of reducing equivalents in the low potential chain (Fig. 3) when RQ3H2 is used as a substrate and the mixed inhibition observed from RQ3 using UQ3H2 as a substrate (see Fig. 4).

The Qi site inhibition by RQ3/RQ3H2 can be explained by two possible, non-exclusive models, both of which are consistent with the predictions based on solution thermodynamics given in the previous section. First, using a pure thermodynamic argument, RQH2 is a considerably stronger reductant (~150 mV) than UQH2, allowing for the establishment of a more reducing cyt b redox poise. Likewise, the lower redox potential of the RQ/RQ. couple should make it a poor substrate for the Qi site, effectively "trapping" electrons in the cyt b chain. Second, using a kinetics argument, RQH2, RQ, or even RSQ could act as inhibitors of or slow substrates for the Qi site. The simplest interpretation is that both the kinetic and thermodynamic properties of RQ/RQH2 result in a more reduced cyt b chain, leading to a buildup of reactive intermediates in the Qo site, possibly an SQ, which increases the rate of superoxide production and other bypass reactions (TABLE TWO). These data offer an explanation for previous findings that RQH2 oxidation by the cyt bc1 complex in R. rubrum chromatophores is insensitive to AA (34) (i.e. it is likely that the bc1 complex is already partially inhibited by RQ).

The Physiological Impact of a Mixed Q Pool in RQ-producing Organisms—The results discussed above beg the question, How do organisms that use both RQ/RQH2 and UQ/UQH2 in their quinone pools prevent deleterious side reactions at the cyt bc1 complex? For example, Euglena gracilis possesses both RQ/RQH2 and UQ/UQH2 simultaneously (although RQ synthesis is down-regulated under aerobic conditions, residual RQ persists for extended periods, with a ratio of UQ:RQ approaching 3:1 under fully aerobic conditions) (32). Simultaneous accumulation of RQ and UQ has also been observed in certain nematodes (45), parasitic helminths (35, 46), mussels and oysters (35), and the photosynthetic bacterium R. rubrum (34). The presence of the two quinones is apparently physiologically necessary for rapid transitions between aerobic and anaerobic respiration in these organisms. Our model system, S. cerevisiae, does not synthesize RQ, but we suggest that if the cyt bc1 complex in these RQ-containing organisms is similar to that in yeast, then co-mingling of RQ and UQ in cyt bc1-accessible compartments should result in production of large amounts of superoxide and other unwanted bypass reactions.

We consider three possible mechanisms by which this superoxide production could be prevented in these organisms. First, the cyt bc1 complexes from RQ-containing organisms might have different substrate specificities. Sequence alignments of cyt b proteins between organisms known to contain mixed RQ/UQ pools (Mytilus edilus, R. rubrum) and UQ-only species (chicken, yeast) did not reveal any discernable trends in conserved residues known to be involved in Qo and Qi site binding (not shown), making this possibility seem unlikely. Nevertheless, we are currently testing this possibility directly by characterizing the cyt bc1 complexes from several RQ-containing organisms. Another possibility is that the mitochondrial inner membrane or bacterial plasma membranes are compartmentalized either into separate membrane systems or domains (76, 77) that contain separate electron transfer chains. A third possibility is that the cyt bc1 complex and/or fumarate reductase are under regulatory control to prevent the co-existence of RQH2 and an active cyt bc1 complex in the same bioenergetic membrane.

Implications for the Function (and Dysfunction) of the Cyt bc1 Complexes—This work shows that the propensity of the bc1 complex to participate in Q-cycle bypass reactions can be greatly modified by changing the thermodynamic properties of the substrates Q and QH2, particularly those that affect the steady state concentration of reactive intermediates in the Qo site. In principle either SQo or cyt bL could be responsible for the reduction of oxygen during bypass reactions, although it has been argued that cyt bL is not sufficiently reducing to act as an effective O2 reductant (4). Our data show that the total amount of reduced cyt b does accumulate to a larger extent with RQ3H2 than with UQ3H2, but in most Q-cycle models the accumulation of reduced low potential chain components would also imply an increase in the concentration of SQo. Alternatively, RSQ might be very labile relative to USQ in the Qo site, thus allowing it to more readily escape and collide with O2.

In principle, both thermodynamic and kinetic factors will influence the amount of reactive intermediates formed in the steady state, i.e. how much of the intermediate is formed, how fast it is formed, and how fast it is consumed. All of these factors will be influenced by the redox and binding properties of SQo/QH2 and Q/SQ couples, which in turn are determined by the structural properties of the substrate and the binding site.

We propose that the Q-cycle requires a fairly narrow window of substrate thermodynamic (redox and binding) properties to ensure a low steady state concentration of reactive intermediates while allowing for rapid Q-cycle turnover. UQ/UQH2 seems to be particularly well suited to this task in comparison to RQ/RQH2. Intriguingly, this "matching" of the quinone/quinol redox properties with those of the cyt bc1 complex appears to be well conserved; menaquinol-oxidizing cyt bc-type complexes have electron carriers with redox potentials about 150 mV lower than those in UQH2-oxidizing complexes, perfectly matching the 150-mV lower potential of the substrate Q/QH2 couple (61, 78–80).


   FOOTNOTES
 
* This work was supported by National Science Foundation CAREER Award CHE-0135091 (to J. N. S.), Research Corporation Cottrell College Science Award CC5171 (to J. N. S.), National Institutes of Health Grant GM61904 (to M. K. B.), and United States Department of Energy Grant DE-FG02-04ER15559 (to D. M. K.). Part of this work was performed at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Chemistry, Gonzaga University, 502 E. Boone Ave., Spokane, WA 99258-0005. Tel.: 509-323-6628; Fax: 509-323-5804; E-mail: shepherd{at}gonzaga.edu.

2 The abbreviations used are: cyt, cytochrome; UQH2,ubiquinol; UQ, ubiquinone; USQ, ubisemiquinone; QH2, quinol; Q, quinone; Qo, quinol oxidase site; Qi, quinone reductase site; SQ, semiquinone; SQo, Qo site semiquinone; RQH2, rhodoquinol; RQ, rhodoquinone; RSQ, rhodosemiquinone; UQ3, ubiquinone-3; RQ3, rhodoquinone-3; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; AA, antimycin-A; SOD, superoxide dismutase. Back

3 D. M. Kramer, J. L. Cape, I. Forquer, and M. K. Bowman, manuscript in preparation. Back

4 J. L. Cape, D. M. Kramer, and M. K. Bowman, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Catherine Clarke, Sabeeha Merchant, Jeff Cronk, and Isaac Forquer for insightful and stimulating discussion regarding this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Berry, E. A., Guergova-Kuras, M., Huang, L. S., and Crofts, A. R. (2000) Annu. Rev. Biochem. 69, 1005-1075[CrossRef][Medline] [Order article via Infotrieve]
  2. Crofts, A. R. (2004) Annu. Rev. Physiol. 66, 689-733[CrossRef][Medline] [Order article via Infotrieve]
  3. Kramer, D. M., Roberts, A. G., Muller, F., Cape, J., and Bowman, M. K. (2004) Methods Enzymol. 382, 21-45[CrossRef][Medline] [Order article via Infotrieve]
  4. Muller, F., Crofts, A. R., and Kramer, D. M. (2002) Biochemistry 41, 7866-7874[CrossRef][Medline] [Order article via Infotrieve]
  5. Sun, J., and Trumpower, B. L. (2003) Arch. Biochem. Biophys. 419, 198-206[CrossRef][Medline] [Order article via Infotrieve]
  6. Osyczka, A., Moser, C. C., Daldal, F., and Dutton, P. L. (2004) Nature 427, 607-612[CrossRef][Medline] [Order article via Infotrieve]
  7. Muller, F. L., Roberts, A. G., Bowman, M. K., and Kramer, D. M. (2003) Biochemistry 42, 6493-6499[CrossRef][Medline] [Order article via Infotrieve]
  8. Osyczka, A., Moser, C. C., and Dutton, L. (2005) Trends Biochem. Sci. 30, 176-182[CrossRef][Medline] [Order article via Infotrieve]
  9. Kramer, D. M., Cape, J., and Bowman, M. K. (2005) in Photosynthesis: Fundamental Aspects to Global Perspectives (Est, A. V. D., and Bruce, D., eds) Vol. 1, pp. 424-427, ACG Publishing, Lawrence, KS
  10. Forquer, I., Cape, J. L., Forquer, I., Bowman, M. K., and Kramer, D. M. (2005) in Photosynthesis: Fundamental Aspects to Global Perspectives (van der Est, A., and Bruce, D., eds) Vol. 1, pp. 430-432, ACG Publishing, Lawrence, KS
  11. Crofts, A. R., and Wang, Z. (1989) Photosynth. Res. 22, 69-87[CrossRef]
  12. Crofts, A. R., Shinkarev, V. P., Kolling, D. R., and Hong, S. (2003) J. Biol. Chem. 278, 36191-36201[Abstract/Free Full Text]
  13. Crofts, A. R., Guergova-Kuras, M., Kuras, R., Ugulava, N., Li, J., and Hong, S. (2000) Biochim. Biophys. Acta 1459, 456-466[Medline] [Order article via Infotrieve]
  14. Crofts, A. R. (2004) Biochim. Biophys. Acta 1655, 77-92[Medline] [Order article via Infotrieve]
  15. Cape, J., Kramer, D. M., and Bowman, M. K. (2005) J. Am. Chem. Soc. 127, 4208-4215[CrossRef][Medline] [Order article via Infotrieve]
  16. Fisher, N., and Meunier, B. (2001) Eur. J. Biochem. 268, 1155-1162[Medline] [Order article via Infotrieve]
  17. Muller, F. (2000) J. Am. Aging Assoc. 23, 227-253
  18. Gong, X., Yu, L., Xia, D., and Yu, C. A. (2004) J. Biol. Chem. 280, 9251-9257
  19. Shinkarev, V. P., Crofts, A. R., and Wraight, C. A. (2001) Biochemistry 40, 12584-12590[CrossRef][Medline] [Order article via Infotrieve]
  20. Zhang, L., Yu, L., and Yu, C. A. (1998) J. Biol. Chem. 273, 33972-33976[Abstract/Free Full Text]
  21. Link, T. A. (1997) FEBS Lett. 412, 257-264[CrossRef][Medline] [Order article via Infotrieve]
  22. Covian, R., Gutierrez-Cirlos, E. B., and Trumpower, B. L. (2004) J. Biol. Chem. 279, 15040-15049[Abstract/Free Full Text]
  23. Zhang, L., Li, Z., Quinn, B., Yu, L., and Yu, C. A. (2002) Biochim. Biophys. Acta 1556, 226-232[Medline] [Order article via Infotrieve]
  24. Crofts, A. R., Shinkarev, V. P., Dikanov, S. A., Samoilova, R. I., and Kolling, D. (2002) Biochim. Biophys. Acta 1555, 48-53[Medline] [Order article via Infotrieve]
  25. Trumpower, B. L. (2002) Biochim. Biophys. Acta 1555, 166-173[Medline] [Order article via Infotrieve]
  26. Snyder, C. H., Gutiererrez-Cirlos, E. B., and Trumpower, B. L. (2000) J. Biol. Chem. 275, 13535-13541[Abstract/Free Full Text]
  27. Gu, L. Q., Yu, L., and Yu, C. A. (1990) Biochim. Biophys. Acta 1015, 482-492[Medline] [Order article via Infotrieve]
  28. Gu, L. Q., Yu, L., and Yu, C. A. (1989) J. Biol. Chem. 264, 4506-4512[Abstract/Free Full Text]
  29. Snyder, C. H., and Trumpower, B. L. (1999) J. Biol. Chem. 274, 31209-31216[Abstract/Free Full Text]
  30. Beach, D. H., Mueller, J. F., and Holz, G. G., Jr. (1980) Mol. Biochem. Parasitol. 1, 269-278[Medline] [Order article via Infotrieve]
  31. Fioravanti, C. F., and Kim, Y. (1988) Mol. Biochem. Parasitol. 28, 129-134[CrossRef][Medline] [Order article via Infotrieve]
  32. Hoffmeister, M., van der Klei, A., Rotte, C., van Grinsven, K. W., van Hellemond, J. J., Henze, K., Tielens, A. G., and Martin, W. (2004) J. Biol. Chem. 279, 22422-22429[Abstract/Free Full Text]
  33. Ozawa, H., Sato, M., Natori, S., and Ogawa, H. (1969) Experientia (Basel) 25, 484-485[CrossRef]
  34. Okayama, S., Yamamoto, N., Nishikawa, K., and Horio, T. (1968) J. Biol. Chem. 243, 2995-2999[Abstract/Free Full Text]
  35. Van Hellemond, J. J., Klockiewicz, M., Gaasenbeek, C. P., Roos, M. H., and Tielens, A. G. (1995) J. Biol. Chem. 270, 31065-31070[Abstract/Free Full Text]
  36. Yamashita, T., Ino, T., Miyoshi, H., Sakamoto, K., Osanai, A., Nakamaru-Ogiso, E., and Kita, K. (2004) Biochim. Biophys. Acta 1608, 97-103[Medline] [Order article via Infotrieve]
  37. Gimenez-Gallego, G., Ramirez-Ponce, M. P., Lauzurica, P., and Ramirez, J. M. (1982) Eur. J. Biochem. 121, 343-347[Medline] [Order article via Infotrieve]
  38. Miyadera, H., Hiraishi, A., Miyoshi, H., Sakamoto, K., Mineki, R., Murayama, K., Nagashima, K. V., Matsuura, K., Kojima, S., and Kita, K. (2003) Eur. J. Biochem. 270, 1863-1874[Medline] [Order article via Infotrieve]
  39. Kita, K., Takamiya, S., Furushima, R., Ma, Y. C., Suzuki, H., Ozawa, T., and Oya, H. (1988) Biochim. Biophys. Acta 935, 130-140[Medline] [Order article via Infotrieve]
  40. Tielens, A. G., and Van Hellemond, J. J. (1998) Biochim. Biophys. Acta 1365, 71-78[Medline] [Order article via Infotrieve]
  41. van Hellemond, J. J., van der Klei, A., van Weelden, S. W., and Tielens, A. G. (2003) Phil. Trans. R. Soc. Lond. B. 358, 205-213[CrossRef][Medline] [Order article via Infotrieve]
  42. Moore, H. W., and Folkers, K. (1966) J. Am. Chem. Soc. 88, 567-570[CrossRef]
  43. Moore, H. W., and Folkers, K. (1965) J. Am. Chem. Soc. 87, 1409-1410[CrossRef][Medline] [Order article via Infotrieve]
  44. Erabi, T., Higuti, T., Kakuno, T., Yamashita, J., Tanaka, M., and Horio, T. (1975) J. Biochem. (Tokyo) 78, 795-801[Abstract/Free Full Text]
  45. Takamiya, S., Matsui, T., Taka, H., Murayama, K., Matsuda, M., and Aoki, T. (1999) Arch. Biochem. Biophys. 371, 284-289[CrossRef][Medline] [Order article via Infotrieve]
  46. Van Hellemond, J. J., Luijten, M., Flesch, F. M., Gaasenbeek, C. P., and Tielens, A. G. (1996) Mol. Biochem. Parasitol. 82, 217-226[CrossRef][Medline] [Order article via Infotrieve]
  47. Sato, M., and Ozawa, H. (1969) J. Biochem. (Tokyo) 65, 861-867[Abstract/Free Full Text]
  48. Allen, P. C. (1973) Exp. Parasitol. 34, 211-219[CrossRef][Medline] [Order article via Infotrieve]
  49. Ljungdahl, P. O., Pennoyer, J. D., Robertson, D. E., and Trumpower, B. L. (1987) Biochim. Biophys. Acta 891, 227-241[Medline] [Order article via Infotrieve]
  50. Shim, Y. B., and Park, S. M. (1997) J. Electroanal. Chem. 425, 201-207[CrossRef]
  51. Roginsky, V. A., Pisarenko, L. M., Bors, W., and Michel, C. (1999) J. Chem. Soc. Perkin Trans. II 871-876
  52. Trumpower, B. L. (1981) J. Bioenerg. Biomembr. 13, 1-24[CrossRef][Medline] [Order article via Infotrieve]
  53. Hendler, R. W., Bunow, B., and Rieske, J. S. (1985) J. Bioenerg. Biomembr. 17, 51-64[CrossRef][Medline] [Order article via Infotrieve]
  54. Kantilal, P. B., and Willson, R. L. (1973) J. Chem. Soc. Faraday Trans. I 814-825
  55. Poon, W. W., Barkovich, R. J., Hsu, A. Y., Frankel, A., Lee, P. T., Shepherd, J. N., Myles, D. C., and Clarke, C. F. (1999) J. Biol. Chem. 274, 21665-21672[Abstract/Free Full Text]
  56. Olson, R. E., and Rudney, H. (1983) Vitam. Horm. 40, 1-43[Medline] [Order article via Infotrieve]
  57. Gibson, F. (1973) Biochem. Soc. Trans. 1, 317-326
  58. Marbois, B., Gin, P., Faull, K. M., Poon, W. W., Lee, P. T., Strahan, J., Shepherd, J. N., and Clarke, C. F. (2005) J. Biol. Chem. 280, 20231-20238[Abstract/Free Full Text]
  59. Glerum, D. M., Koerner, T. J., and Tzagoloff, A. (1995) J. Biol. Chem. 270, 15585-15590[Abstract/Free Full Text]
  60. Naruta, Y. (1980) J. Org. Chem. 45, 4097-4104[CrossRef]
  61. Brasseur, G., Bruscella, P., Bonnefoy, V., and Lemesle-Meunier, D. (2002) Biochim. Biophys. Acta 1555, 37-43[Medline] [Order article via Infotrieve]
  62. Sakamoto, K., Nomura, K., and Miyoshi, H. (2002) J. Pest Sci. 27, 147-149
  63. Vaultier, N., Knouzi, N., and Carrié, R. (1983) Tetrahedron Lett. 24, 763-764[CrossRef]
  64. Nagarajan, S., and Ganem, B. (1987) J. Org. Chem. 52, 5044-5046[CrossRef]
  65. Bauscher, M., and Manele, W. (1992) J. Phys. Chem. 96, 11101-11108[CrossRef]
  66. Gutierrez-Cirlos, E. B., and Trumpower, B. L. (2002) J. Biol. Chem. 277, 1195-1202[Abstract/Free Full Text]
  67. Brasseur, G., Lemesle-Meunier, D., Reinaud, F., and Meunier, B. (2004) J. Biol. Chem. 279, 24203-24211[Abstract/Free Full Text]
  68. Brasseur, G., Di Rago, J. P., Slonimski, P. P., and Lemesle-Meunier, D. (2001) Biochim. Biophys. Acta 1506, 89-102[Medline] [Order article via Infotrieve]
  69. Palsdottir, H., Lojero, C. G., Trumpower, B. L., and Hunte, C. (2003) J. Biol. Chem. 278, 31303-31311[Abstract/Free Full Text]
  70. Hong, S., Ugulava, N., Kuras-Guergova, M., and Crofts, A. R. (1999) J. Biol. Chem. 274, 33931-33944[Abstract/Free Full Text]
  71. Takahashi, E., and Wraight, C. A. (1992) Biochemistry 31, 855-866[CrossRef][Medline] [Order article via Infotrieve]
  72. Beroza, P., Fredkin, D. R., Okamura, M. Y., and Feher, G. (1995) Biophys. J. 68, 2233-2250[Abstract/Free Full Text]
  73. Knox, P. P., Zakharova, N. I., Seifullina, N. H., Churbanova, I. Y., Mamedov, M. D., and Semenov, A. Y. (2004) Biochemistry (Mosc) 69, 890-896[CrossRef][Medline] [Order article via Infotrieve]
  74. Ohnishi, T., and Trumpower, B. L. (1980) J. Biol. Chem. 255, 3278-3284[Free