Kinetic Characterization of Mitochondrial Complex I Inhibitors Using Annonaceous Acetogenins

Archives of Biochemistry and Biophysics Vol. 369, No. 1, September 1, pp. 119 –126, 1999 Article ID abbi.1999.1343, available online at http://www.idealibrary.com on

Kinetic Characterization of Mitochondrial Complex I Inhibitors Using Annonaceous Acetogenins Jose´ R. Tormo, M. Carmen Gonza´lez, Diego Cortes, and Ernesto Estornell* ,1 Departament de Farmacologia, Farmacogno´sia, i Farmacodina`mia and *Departament de Bioquı´mica i Biologia Molecular, Facultat de Farma`cia, Universitat de Vale`ncia. Avgda. Vicent Andre´s Estelle´s s/n, E-46100 Burjassot (Vale`ncia), Spain

Received February 24, 1999, and in revised form June 8, 1999

The NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial respiratory chain is by far the largest and most complicated of the proton-translocating enzymes involved in the oxidative phosphorylation. Many clues regarding the electron pathways from matrix NADH to membrane ubiquinone and the links of this process with the translocation of protons are highly controversial. Different types of inhibitors become valuable tools to dissect the electron and proton pathways of this complex enzyme. Therefore, further knowledge of the mode of action of complex I inhibitors is needed to understand the underlying mechanism of energy conservation. This study presents for the first time a detailed exploration of the inhibitory action of the Annonaceous acetogenins, the most powerful inhibitors of the mammalian enzyme, taking as the head-series rolliniastatin-1, rolliniastatin-2, and corossolin. Despite their close chemical resemblance, each of them inhibits the complex I with different kinetic features reflecting differential binding to the enzyme. © 1999 Academic Press Key Words: mitochondrial complex I; NADH-ubiquinone oxidoreductase; Annonaceous acetogenins; respiratory inhibitor.

The mitochondrial NADH:ubiquinone oxidoreductase, also known as respiratory complex I, is by far the largest and most complicated protein complex of the inner mitochondrial membrane involved in the oxidative phosphorylation. Due to its complex nature, many clues regarding the electron pathways from matrix NADH to membrane ubiquinone and the links of this process with the translocation of protons across the 1 To whom correspondence should be addressed. Fax: 134-963864917. E-mail: [email protected].

0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

inner membrane are highly controversial (1–5). In this regard, different types of inhibitors become useful tools for the elucidation of structural and mechanistic aspects in order to understand how the enzyme works. However, the transmembrane part of the complex I, where ubiquinone is reduced and a electrochemical gradient is generated (1–5), is inhibited by a great variety of compounds with very different structures (6, 7). The most recent hypotheses on the complex I mechanism, although with discrepancies regarding both the redox and proton-ejection mechanisms, are coincident to establish three ubiquinone reaction sites within the membrane domain of the enzyme (3–5). In parallel, inhibitors of complex I have been classified into two or three functional groups (6 –12) according to the mechanistic models. However, due to the different experimental techniques related with the ubiquinone substrate used, the kinetic features of each type of inhibitor remain ambiguous. Some of the most powerful complex I inhibitors are Annonaceous acetogenins (6, 10, 13). They form a wide group of more than 250 natural products isolated from several species of the Annonaceae family. Their general structure consists of an aliphatic backbone of 35–37 carbons with a terminal g-lactone, a central nucleus frequently containing one or two tetrahydrofuran rings, and several hydroxyl groups at different positions (14, 15). Contrary to the usual complex I inhibitors, the Annonaceous acetogenins are large and less rigid structures due to the long alkyl chain that separates the two functional moieties (the terminal g-lactone and the tetrahydrofuran core). These unusual structural characteristics as well as their strong inhibitory potency make these molecules a very interesting group with which to investigate unsolved aspects of complex I function. Furthermore, previous reports have suggested that Annonaceous acetogenins could act in different ways to inhibit complex I (3, 6, 10, 119

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16, 17), a surprising observation taking into account the very close chemical resemblance among them. This opens an interesting perspective to elucidate some intrinsic characteristics of both the inhibitor and the ubiquinone binding environment within the complex. The present work studies the mode of action of the Annonaceous acetogenins taking as the head series the already known rolliniastatin-1 and rolliniastatin-2 (10, 14, 16, 17), the most potent inhibitors of bovine complex I, and the new one, corossolin (14, 15). Despite their close chemical resemblance, herein we demonstrate that each one inhibits the complex I in a different manner, and, thus, they can be taken as models of three classes of specific inhibitors defined by the different kinetic features. Differences involve titration-curve shapes, potency against catalytic measurements of complex I activity, Michaelis–Menten kinetics, and kinetic displacement by rotenone. MATERIALS AND METHODS Annonaceous acetogenins. Annonaceous acetogenins were isolated and purified from the plant material by standard procedures involving partition with organic solvents, silicagel chromatography, and semi-preparative HPLC (see (14, 15) for review). Rolliniastatin-1 and rolliniastatin-2 were extracted from Rollinia membranaceae seeds (16, 17). Corossolin was isolated from Annona glabra seeds. All Annonaceous acetogenins were judged pure by chromatographic, 1H and 13C NMR, and MS spectroscopy criteria. Ethanolic solutions were evaluated by UV spectrophotometry. Rolliniastatin-1 and rolliniastatin-2 are both adjacent bis-tetrahydrofuranic acetogenins that differ only by the stereochemistry, whereas corossolin belongs to the group of the mono-tetrahydrofuranic acetogenins. Chemical structures and configurations are given in Fig. 1. Other reagents. Rotenone, antimycin A, decylubiquinone, and other biochemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The commercial inhibitors and decylubiquinone were kept at 220°C as ethanolic solutions in the dark, and their titer was determined spectrophotometrically as previously described (10, 18). Salts and solvents were purchased from Merck (Darmstadt, Germany) and Panreac (Barcelona, Spain). Preparation of beef-heart submitochondrial particles. Inverted submitochondrial particles (SMP) 2 from beef heart were obtained by extensive ultrasonic disruption of frozen–thawed mitochondria in such a way as to produce open membrane fragments where permeability barriers to substrates were lost. After ultracentrifugation they were finally resuspended in 250 mM sucrose, 10 mM Tris–HCl buffer, pH 7.4, and stored at 280°C (19). Complex I content of submitochondrial particles was estimated as previously described (10, 19). The optimized preparation of the SMP allowed us to obtain samples with a very constant complex I content (45.8 6 0.3 pmol/mg) and complex I activity. Inhibitor titrations. Stock solutions (2 mM in absolute ethanol) of the Annonaceous acetogenins used in this study (Fig. 1) were prepared and kept in the dark at 220°C. Appropriate dilutions (5–50 mM) were made before the titrations. Beef-heart SMP were diluted to 0.5 mg/ml in sucrose–Tris buffer and treated with 300 mM NADH to activate complex I (18). Increasing concentrations of the ethanolic solution of each inhibitor were added to this preparation, with about

2

Abbreviations used: SMP, submitochondrial particles; DB, decylubiquinone.

FIG. 1. Chemical structure of the three Annonaceous acetogenins used in this study. Rolliniastatin-1 and rolliniastatin-2 are both adjacent bis-tetrahydrofuranic acetogenins that differ only by the configuration of the bis-tetrahydrofuranic core of the molecule. Corossolin is a mono-tetrahydrofuranic acetogenin with a hydroxyl group at C-10 instead the hydroxyl group at C-4 near the methyla,b-unsaturated-g-lactone moiety of the first aliphatic tail.

5 min incubation on ice between each addition (10). Maximal ethanol concentration never exceeded 2% of the volume and control activity was not affected by the ethanol concentration used in the assays. After each addition of inhibitor, NADH:ubiquinone oxidoreductase or NADH oxidase was measured. The enzymatic activities were assayed at 22°C in 50 mM potassium phosphate buffer, pH 7.4, 1 mM EDTA with the SMP diluted to 6 mg/ml in the cuvette (0.28 6 0.01 nM complex I). NADH:ubiquinone oxidoreductase was measured with 75 mM NADH and 30 mM decylubiquinone (DB) as a soluble short-chain analogue of ubiquinone in the presence of 2 mM antimycin and 2 mM potassium cyanide to block any reaction downstream the complex I (20). NADH oxidase was measured as the aerobic oxidation of 75 mM NADH in the absence of the quinone substrate and other inhibitors of the respiratory chain. Data from four titrations under the same conditions were pulled and fitted for graphics. IC 50s were taken as the final compound concentrations in the assay

MITOCHONDRIAL COMPLEX I INHIBITION KINETICS TABLE I

IC 50 of Rolliniastatin-1, Rolliniastatin-2, Corossolin, and Rotenone for NADH Oxidase and NADH:ubiquinone (NADH:DB) Oxidoreductase in Bovine Heart Submitochondrial Particles IC 50 (nM)

Inhibitor

NADH oxidase

NADH:DB oxidoreductase

Ratio

Rolliniastatin-1 Rolliniastatin-2 Corossolin Rotenone

0.62 6 0.05 0.51 6 0.03 1.26 6 0.18 5.1 6 0.9

0.75 6 0.04 0.61 6 0.04 6.2 6 0.4 28.8 6 1.5

1.2 1.2 4.9 5.6

Note. Data are means 6 SD from four determinations for each product. NADH:ubiquinone oxidoreductase was determined with decylubiquinone (DB) as the ubiquinone analogue. Final complex I content was 0.28 nM. Control activity was approximately 0.95 mmol min 21 mg 21 for NADH oxidase and 0.55 mmol min 21 mg 21 for NADH: ubiquinone oxidoreductase.

cuvette that yielded 50% inhibition of complex I activity. Data from individual titrations were used to assess the means and standard deviations. Linear fittings of the double-reciprocal and Dixon plots were calculated by least square regression analysis. The symbol thickness in the figures was selected to cover the maximal error for each data point. The linear regression coefficient of all calculated lines was at least 0.988 (.0.995 for most experiments). Extrapolation intercepts were calculated with less than 5% error.

RESULTS

Table I shows the IC 50 values of rolliniastatin-1, rolliniastatin-2, and corossolin against the NADH oxidase and NADH:ubiquinone (NADH:DB) oxidoreductase activities. Complex I content was kept in all experiments at a final concentration of 0.28 6 0.01 nM and, thus, it was expected that the observed IC 50 values approached the true K i in these conditions (6). However, the extremely high potent inhibition for both rolliniastatin-1 and rolliniastatin-2 leads to a possible underestimation of differences because IC 50 values for close-to-stoichiometric inhibitors are often not proportional to affinities. In any case, values are comparable between them and with previous reports (6, 10, 16, 17). Rolliniastatin-1 has been previously considered the most potent inhibitor of complex I (6, 10), although we have found that rolliniastatin-2 was slightly more potent under our conditions. Corossolin was less potent than both rolliniastatins, although it was found to be fourfold more potent than rotenone against the NADH oxidase activity. The same approximate pattern was observed against the NADH:ubiquinone oxidoreductase activity measured with DB as the artificial electron acceptor. Interestingly, rolliniastatin-1 and rolliniastatin-2 inhibited the DB-induced NADH oxidation with approximately the same IC 50 as that for the

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integrated NADH oxidase activity. These values were in the range described previously for both Annonaceous acetogenins in assays with other ubiquinone analogues such as undecylubiquinone, taking into account the protein dependence of the titration (10). On the contrary, corossolin was significantly less effective to inhibit this activity, giving an IC 50 approximately five-fold higher than that for the NADH oxidase and, thus, following the same loss of potency than rotenone. IC 50 values observed with this ubiquinone analogue are generally larger than those obtained in the NADH oxidase assay (6, 10, 17, 21), although it was not the case for both rolliniastatins. Despite the difficulty in correlating IC 50 values with inhibitor affinities, these data indicate that the inhibitory action of corossolin differs from that of both rolliniastatins. Tentatively it seems that corossolin behavior is closer to that of rotenone than that of the other two acetogenins. Figure 2 shows the titration curves of the three Annonaceous acetogenins and rotenone. This figure has been included to note some unusual features that reveal differences among inhibitors. Rolliniastatin-1 and rolliniastatin-2 fully inhibited the aerobic oxidation of NADH at low inhibitor concentration. However, corossolin needed to reach a greater concentration to achieve full inhibition, showing, as rotenone, a slower decay of the complex I activity beyond the IC 50 (not completely shown in the graphic). Maximal inhibition of the DB-induced NADH oxidation by corossolin was not complete, even at relatively high concentration of inhibitor, but it was in the same range than that of rotenone in accordance with previous reports (e.g., 22 and references therein). Note that neither rotenonenor piericidin-insensitive activity was subtracted from the 100% activity as it is commonly practiced. Thus, the greater extent of the rolliniastatins inhibition is observed. In fact, acetogenins further inhibited the activity according to previous observations (10, 22), with rolliniastatin-1 being slightly more effective than rolliniastatin-2. Moreover, the shape of the titration curves was different. Typical hyperbolic curves were given by corossolin and rolliniastatin-2 (the last one approaching the linearity against the NADH oxidase activity) and also by rotenone. Nevertheless, rolliniastatin-1 consistently gave an apparent sigmoidal curve against both activities. Sigmoidicity in the titration curve of both rolliniastatin-1 and other related acetogenins was first observed recently (16) when they were titrated at a low mitochondrial protein content and very low inhibitor concentrations in the initial points. Sigmoidal titration curves have been observed for other complex I inhibitors that have been proposed to bind two different sites of the enzyme (11, 23). A dual inhibition of complex I by rolliniastatin-1 has also been previously suggested (10, 14, 16), although it has not been clearly characterized.

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action of rotenone and corossolin were more evident. Nevertheless, rolliniastatins were coincident despite the different shape of the titration curves. However, these results can not rule out the previously suggested possibility (10, 16) that rolliniastatin-1 may act by a dual mechanism overlapping with both rolliniastatin-2 and rotenone (and also with corossolin). To define this overlapping, each inhibitor was titrated in the presence of each other and in the presence of increasing amounts of rotenone against the NADH: ubiquinone oxidoreductase activity. Figure 4 shows the elaborated Dixon plots in the presence of rotenone, selected as the most common inhibitor of complex I. The plots deviated from the straight line at high inhibitor concentration and, thus, only the best-fit linear

FIG. 2. Titration of Annonaceous acetogenins against NADH oxidase and NADH:ubiquinone oxidoreductase in bovine heart submitochondrial particles. Mitochondrial protein concentration was 6 mg/ml (0.28 nM complex I). Control activity was approximately 0.95 mmol min 21 mg 21 for NADH oxidase and 0.55 mmol min 21 mg 21 for NADH:decylubiquinone oxidoreductase in the presence of 2 mM antimycin. Data were obtained from four determinations for each product.

In any case, the rolliniastatin-1 apparent behavior differed from that of the rolliniastatin-2, suggesting that the mode of action of both bis-tetrahydrofuranic acetogenins could be nonequivalent in accordance with previous results (3, 10). On the other hand, corossolin and rotenone seemed to act again with high similarities. The type of inhibition with respect to decylubiquinone substrate could indicate the differences among the three Annonaceous acetogenins. Figure 3 shows the reciprocal plots for each inhibitor. Rolliniastatin-1 and rolliniastatin-2 were found to be uncompetitive inhibitors in accordance with previous assays using undecylubiquinone as substrate (10), whereas corossolin behaved as a noncompetitive inhibitor like rotenone. At this point, similarities between the mode of

FIG. 3. Reciprocal plots of the Michaelis–Menten kinetics of the NADH:decylubiquinone oxidoreductase in the presence of the inhibitors. Conditions were similar to those described in the legend to Fig. 2. Inhibitor concentrations were 0.6 nM for rolliniastatin-1 (F), 1.2 nM for rolliniastatin-2 (E), 1.7 nM for corossolin (Œ), and 18.5 nM for rotenone (‚).

MITOCHONDRIAL COMPLEX I INHIBITION KINETICS

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FIG. 4. Dixon plots of the three Annonaceous acetogenins in presence of rotenone. Relative activity refers to NADH:decylubiquinone oxidoreductase in the conditions given in the legend to Fig. 2. Rotenone fixed concentrations were 0 nM (■), 26 nM (F), and 96 nM (Œ).

correlation below the IC 50 for each evaluated inhibitor is shown. These conditions allow visualizing kinetic displacements between inhibitors (10, 13, 17). If binding of both inhibitors does not overlap, the plots give a common intercept at the x-axis (the apparent K i). Contrarily, if the two inhibitors are mutually exclusive, plots extrapolate at different intercepts. At a low inhibitor concentration, rolliniastatin-1 was displaced by rotenone (Fig. 4A) and also by corossolin and rolliniastin-2 (results not shown). Contrarily, both rotenone (Fig. 4B) and corossolin did not modify the K i for rolliniastin-2, but rolliniastatin-1 did (not shown). Corossolin was clearly displaced by both rotenone (Fig. 4C) and rolliniastatin-1 but not by rolliniastatin-2 (not shown). Data presented here confirm that rolliniastatin-2 binding is different from that of rotenone (6, 10, 12, 17), and thus, it explains the different inhibition pattern observed. Contrarily, binding of corossolin

completely overlaps with that of rotenone. The third Annonaceous acetogenin, rolliniastatin-1, may act by blocking simultaneously both sites, being mutually exclusive with each other inhibitor. If the sigmoidal titration curve of rolliniastatin-1 observed in Fig. 2 depends on a dual binding, and rolliniastatin-2 and corossolin (or rotenone) binding does not overlap, a combination of the two last inhibitors should mimic the rolliniastatin-1 behavior. Figure 5 shows the titration curves of mixtures of two inhibitors against the NADH oxidase activity. Each experiment was done by combining the two inhibitors in a relative amount approaching the IC 50 ratio to compensate for their differences in potency. As expected, combinations of rolliniastatin-2 with corossolin (1:2) and rotenone (1:10) gave sigmoidal titration curves (Figs. 5A and 5C). In fact, the shape of the curve was very similar to that of rolliniastatin-1 alone (see Fig. 2).

FIG. 5. Titrations of two mixed inhibitors against the NADH oxidase activity. Inhibitors were combined at a ratio approaching the IC 50 ratio to compensate for their differences in potency. Inhibitor concentration is given as total concentration of both inhibitors. A, rolliniastatin-2 plus corossolin (1:2) (F), B, corossolin plus rotenone (1:4) (E), C, rolliniastatin-2 plus rotenone (1:10) (■).

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TORMO ET AL. TABLE II

Functional Classification of the Annonaceous Acetogenins Main characteristics Functional type

Titration-curve shape

Rolliniastatin-2 type General acetogenin type Dual rolliniastatin-1 type

Hyperbolic Hyperbolic Sigmoidal

Type of Inhibition Uncompetitive Noncompetitive Uncompetitive

Exclusivity with rotenone No Yes Yes

Note. Classification is based on the inhibition kinetic features reported: titration-curve apparent shape, inhibition kinetics with decylubiquinone (DB) as the complex I final substrate, and kinetic displacement by rotenone. Rolliniastatin-1 is mutually exclusive with rolliniastatin-2, too.

Contrarily, combination of corossolin with rotenone (1:4) gave the typical hyperbolic curve (Fig. 5B). Similar results were obtained against the DB-induced NADH oxidation (data not shown), although sigmoidicity was more marked in agreement with the rolliniastatin-1 single behavior (see Fig. 2). Therefore, this sigmoidicity should be taken as a characteristic of the mode of action of this compound and another related inhibitors (16). DISCUSSION

Annonaceous acetogenins have been a part of complex I research since Weiss established their mode of action in 1991 (24). This large family of natural compounds includes the most potent inhibitors of the mammalian complex, but the most striking feature is their different characteristics of inhibition. We have shown that the three studied Annonaceous acetogenins, selected as representatives, have different kinetic characteristics as complex I inhibitors. Therefore, each one has been taken as a head series for a functional type. Table II summarizes those findings. The rolliniastatin-2 type includes only another one, the cherimolin-1 (17), at least at present. The general type includes corossolin and most of the acetogenins tested, characterized by showing the same behavior as rotenone, although with varying potency (6, 10, 13). The dual rolliniastatin-1 type is only represented today by this compound and it shares common properties with piericidin A (10). It is noteworthy that the inhibition kinetics of rolliniastatin-2 is controversial. Friedrich et al. (12) described a partial competitive inhibition with CoQ 2, and recently Miyoshi et al. (13) have reported a noncompetitive inhibition with CoQ 1 for this acetogenin (called annonin VI and bullatacin, respectively, in their papers). Therefore, it is likely that the used quinone acceptor varies the inhibition kinetics. Recent comprehensive reviews on the catalytic measurement of complex I (25) and its quinone specificity (26) have pointed out the difficulty in assessing a reliable assay for com-

plex I activity. The ubiquinone homologue CoQ 2 has been proved to be a poor substrate for mammalian complex I and, furthermore, a competitive inhibitor (19, 20, 22). The hydrophilic CoQ 1, although it elicits high rates of NADH oxidation (19, 20), reacts incompletely with complex I (22, 27), probably involving a nonphysiological site. However, the analogue DB, used in this study, yields lower activity with an apparent underevaluation of complex I, but its reaction with the enzyme seems to be more complete (19, 22, 27) or, at least, more physiological (26). Rolliniastatin-2, cherimolin-1, and rolliniastatin-1 have been found to be uncompetitive inhibitors with undecyl- and decylubiquinone (10, 17). Moreover, inhibition type was the same when CoQ 1 was used as the ubiquinone substrate (unpublished results). Instead, rotenone and rotenonelike acetogenins were always noncompetitive irrespective to the ubiquinone used for the assay. Differences in binding to the enzyme could explain the different kinetic features shown by each representative Annonaceous acetogenin (Table II), mainly inhibition kinetics. The noncompetitive pattern could be explained by a random independent binding of both the substrate and the inhibitor. However, rotenone and corossolin gave IC 50 values about five-fold higher in the NADH oxidoreductase assay (Table I). It indicates partial competition with the substrate in a way that the inhibitor cannot bind the enzyme–substrate complex, whereas the substrate can bind the enzyme–inhibitor complex. The increased amount of the quinone substrate within the mitochondrial membrane when the external analogue is added displaces the steady-state equilibrium to the enzyme–substrate complex, resulting in a higher inhibitor concentration to achieve the same inhibition degree. Contrarily, rolliniastatin-1 and rolliniastatin-2 were both uncompetitive inhibitors and yielded approximately the same IC 50 against both ways to measure complex I activity, indicating that the inhibitor biding site is probably different or, at least, not equivalent.

MITOCHONDRIAL COMPLEX I INHIBITION KINETICS

The existence of multiple binding sites for complex I inhibitors was first suggested for piericidin A (28). Gluck et al. (11) demonstrated the existence of two inhibitor sites in complex I with different affinity for hydrophobic and hydrophilic methyl-phenylpyridinium derivatives. Simultaneously, Friedrich et al. (12) defined two classes of inhibitors (I and II) based on the type of inhibition. In parallel, at least two ubiquinone-reactive sites within the membrane domain of complex I were found (29, 30). A third site for complex I inhibitors, with strong resemblance with the center o of complex III, was further proposed (9, 18). According to those findings, the most recent mechanistic models for complex I, i.e., the dual Q-gate pump model by Degli Esposti and Ghelli (3), the redox-gated ligand conduction mechanism proposed by Brandt (4), and the reductant-induced oxidation model stated by Dutton et al. (5), propose three interaction sites for ubiquinone. Furthermore, Degli Esposti (6) has classified the complex I inhibitors in three classes, which bind three separate and functionally different ubiquinone-reaction sites. Nevertheless, the relationship among ubiquinone-reaction sites and the inhibitor-binding sites and the physical situation of the later remain unsolved. Recently, Okun et al. (31) have proposed an unique inhibitor binding domain for hydrophobic inhibitors of complex I based on competitive displacement studies with both fluorescent and radiolabeled dyes. In parallel, Schuler et al. (32) have further supported this idea by competitive photoaffinity labeling of the subunit PSST of the complex I, where it seems to reside in an unique inhibitor-binding site. However, this domain is wide enough to accommodate a great variety of structures acting as inhibitors with partially overlapping binding sites. Therefore, each inhibitor binding site should be interpreted as a limited area within this wide domain that could overlap other inhibitor biding areas. Our steady-state kinetic data can not conclusively discriminate whether inhibitor-binding sites are spatially separated or very close. However, they have shown that different compounds affect complex I function in different manners. In other words, different molecules interfere with different ubiquinone reactions (or different reaction steps) to yield differential kinetics. Nevertheless, taking into account the structure of the three compounds used in this study (see Fig. 1), our results could support the unique binding domain hypothesis. Shimada et al. (33) have shown that the tetrahydrofuran core of the Annonaceous acetogenins acts as an anchor to the polar groups of the membrane phospholipids, whereas the lactone ring is oriented at different levels in the membrane depending on the length of the alkyl chain. Therefore, this part of the molecule is likely involved in binding complex I. Both steric constraints due to the tetrahydrofuran-core stereochemis-

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try and polar groups placed along the alkyl chain could determine a different interaction with the enzyme to yield different kinetic characteristics. Activity studies by Miyoshi et al. (13) have shown that the proper length and flexibility of the alkyl chain spacer is essential for potent complex I inhibition, whereas the number of the tetrahydrofuran rings is not essential. However, we show that structural differences not only affect the potency but also the inhibition mechanism. In this way, binding of rolliniastatin-2-type acetogenins does not interfere with binding of corossolin, rotenone, and many other inhibitors. Instead, the binding site of the stereoisomer rolliniastatin-1 could be overlapped with both rolliniastatin-2 and rotenone binding sites, yielding the same uncompetitive kinetics as rolliniastatin-2 but with a sigmoidal titration curve similar to that obtained with a combination of the two other functional types. If there is a unique domain in complex I involved in binding ubiquinone and inhibitors (31, 32), it should perform a complicated reaction sequence that can be interfered in different ways by different compounds, even if they only differ by small chemical changes. In this sense, Annonaceous acetogenins could be very helpful to elucidate the still unknown events of the enzyme mechanism and to resolve the controversies of the functional models proposed up to date. ACKNOWLEDGMENTS This work has been supported by the Comisio´n Interministerial de Ciencia y Tecnologı´a, Proyectos I1D, Spain (CICYT, SAF97-0013). We also thank the Spanish Ministerio de Educacio´n y Cultura for a predoctoral fellowship grant to J.R.T. We acknowledge the valuable and critical comments by Dr. Mauro Degli Esposti (Monash University, Clayton, Victoria, Australia) to improve our work.

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