Hybrid ubiquinone: novel inhibitor of mitochondrial complex I

We synthesized novel ubiquinone analogs by hybridizing the natural ubiquinone ring (2,3-dimethoxy-5-methyl-1,4-benzoquinone) and hydrophobic phenoxybenzamide unit, and named them hybrid ubiquinones (HUs). The HUs worked as electron transfer substrates with bovine heart mitochondrial succinate-ubiquinone oxidoreductase (complex II) and ubiquinol-cytochrome c oxidoreductase (complex III), but not with NADH-ubiquinone oxidoreductase (complex I). With complex I, they acted as inhibitors in a noncompetitive manner against exogenous short-chain ubiquinones irrespective of the presence of the natural ubiquinone ring. Elongation of the distance between the ubiquinone ring and the phenoxybenzamide unit did not recover the electron accepting activity. The structure/activity study showed that high structural specificity of the phenoxybenzamide moiety is required to act as a potent inhibitor of complex I. These findings indicate that binding of the HUs to complex I is mainly decided by some specific interaction of the phenoxybenzamide moiety with the enzyme. It is of interest that an analogous bulky and hydrophobic substructure can be commonly found in recently registered synthetic pesticides the action site of which is mitochondrial complex I.     

Mode of inhibitory action of Deltalac-acetogenins, a new class of inhibitors of bovine heart mitochondrial complex I

We have revealed that Deltalac-acetogenins, a new class of inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), act differently from ordinary inhibitors such as rotenone and piericidin A [Ichimaru et al. (2005) Biochemistry 44, 816-825]. Since a detailed study of these unique inhibitors might provide new insight into the terminal electron transfer step of the enzyme, we further characterized their inhibitory action using the most potent Deltalac-acetogenin derivative (compound 1). Unlike ordinary complex I inhibitors, 1 had a dose-response curve for inhibition of the reduction of exogenous short-chain ubiquinones that was difficult to explain with a simple bimolecular association model. The inhibitory effect of 1 on ubiquinol-NAD(+) oxidoreductase activity (reverse electron transfer) was much weaker than that on NADH oxidase activity (forward electron transfer), indicating a direction-specific effect. These results suggest that the binding site of 1 is not identical to that of ubiquinone and the binding of 1 to the enzyme secondarily (or indirectly) disturbs the redox reaction of ubiquinone. Using endogenous and exogenous ubiquinone as an electron acceptor of complex I, we investigated the effect of 1 in combination with different ordinary inhibitors on the superoxide production from the enzyme. The results indicated that the level of superoxide production induced by 1 is significantly lower than that induced by ordinary inhibitors probably because of fewer electron leaks from the ubisemiquinone radical to molecular oxygen and that the site of inhibition by 1 is downstream of that by ordinary inhibitors. The unique inhibitory action of hydrophobic Deltalac-acetogenins may be closely associated with the dynamic function of the membrane domain of complex I.     

Exploring the binding site of acetogenin in the ND1 subunit of bovine mitochondrial complex I

¹²⁵I-labeled (trifluoromethyl)phenyldiazirinyl acetogenin, [¹²⁵I]TDA, a photoaffinity labeling probe of acetogenin, photo-cross-links to the ND1 subunit of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) with high specificity [M. Murai, A. Ishihara, T. Nishioka, T. Yagi, and H. Miyoshi, (2007) The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I, Biochemistry 46 6409-6416.]. To identify the binding site of [¹²⁵I]TDA in the ND1 subunit, we carried out limited proteolysis of the subunit cross-linked by [¹²⁵I]TDA using various proteases and carefully analyzed the fragmentation patterns. Our results revealed that the cross-linked residue is located within the region of the 4th to 5th transmembrane helices (Val144-Glu192) of the subunit. It is worth noting that an excess amount of short-chain ubiquinones such as ubiquinone-2 (Q₂) and 2-azido-Q₂ suppressed the cross-linking by [¹²⁵I]TDA in a concentration-dependent way. Although the question of whether the binding sites for ubiquinone and different inhibitors in complex I are identical remains to be answered, the present study provided, for the first time, direct evidence that an inhibitor (acetogenin) and ubiquinone competitively bind to the enzyme. Considering the present results along with earlier photoaffinity labeling studies, we propose that not all inhibitors acting at the terminal electron transfer step of complex I necessarily bind to the ubiquinone binding site itself.     

Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide

Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.     

Origin of selective inhibition of mitochondrial complex I by pyridinium-type inhibitor MP-24.

Positively charged pyridiniums are unique inhibitors to probe the structural and functional properties of the ubiquinone reduction site of bovine heart mitochondrial complex I. In this study, we synthesized a series of neutral as well as pyridinium analogues of MP-24 (N-methyl-4-[2-methyl-2-(p-tert-butylbenzyl)propyl]pyridinium), a selective inhibitor of one of the two proposed binding sites of these pyridinium-type inhibitors of complex I (H. Miyoshi et al., J. Biol. Chem. 273 (1998) 17368-17374), to elucidate the origin of its selectivity. Inhibitory potencies of all neutral and pyridinium analogues with tetraphenylboron (TPB(-)), which forms an ion-pair with pyridiniums, were comparable, although the degrees of selective inhibition by pyridiniums without TPB(-) were entirely different. In contrast to MP-24, the dose-response curves of nonselective pyridiniums and all neutral analogues were not affected by incubation conditions. These results strongly suggested that the process of the inhibitor passage to the binding sites is responsible for the selective inhibition.     

Exploring the binding pocket of quinone/inhibitors in mitochondrial respiratory complex I by chemical biology approaches

ABSTRACT

NADH-quinone oxidoreductase (respiratory complex I) is a key player in mitochondrial energy metabolism. The enzyme couples electron transfer from NADH to quinone with the translocation of protons across the membrane, providing a major proton-motive force that drives ATP synthesis. Recently, X-ray crystallography and cryo-electron microscopy provided further insights into the structure and functions of the enzyme. However, little is known about the mechanism of quinone reduction, which is a crucial step in the energy coupling process. A variety of complex I inhibitors targeting the quinone-binding site have been indispensable tools for mechanistic studies on the enzyme. Using biorationally designed inhibitor probes, the author has accumulated a large amount of experimental data characterizing the actions of complex I inhibitors. On the basis of comprehensive interpretations of the data, the author reviews the structural features of the binding pocket of quinone/inhibitors in bovine mitochondrial complex I.     

Exploring the ubiquinone binding cavity of respiratory complex I

Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Production of reactive oxygen species by complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli and comparison to the enzyme from mitochondria

The generation of reactive oxygen species by mitochondrial complex I (NADH:ubiquinone oxidoreductase) is considered a significant cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Defining its mechanism is important for the formulation of causative connections between complex I defects and pathological effects. Oxygen is probably reduced at two sites in complex I, one associated with NADH oxidation in the mitochondrial matrix and the other associated with ubiquinone reduction in the membrane. Here, we study complex I from Escherichia coli, exploiting similarities and differences in the bacterial and mitochondrial enzymes to extend our knowledge of O2 reduction at the active site for NADH oxidation. E. coli and bovine complex I reduce O2 at essentially the same rate, with the same potential dependence (set by the NAD (+)/NADH ratio), showing that the rate-determining step is conserved. The potential dependent rate of H2O2 production does not correlate to the potential of the distal [2Fe-2S] cluster N1a in E. coli complex I, excluding it as the point of O2 reduction. Therefore, our results confirm previous proposals that O2 reacts with the fully reduced flavin mononucleotide. Assays for superoxide production by E. coli complex I were prone to artifacts, but dihydroethidium reduction showed that, upon reducing O2, it produces approximately 20% superoxide and 80% H2O2. In contrast, bovine complex I produces 95% superoxide. The results are consistent with (but do not prove) a specific role for cluster N1a in determining the outcome of O2 reduction; possible reaction mechanisms are discussed.     

Cryo-electron microscopy reveals how acetogenins inhibit mitochondrial respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a crucial enzyme in energy metabolism, captures the redox potential energy from NADH oxidation/ubiquinone reduction to create the proton motive force used to drive ATP synthesis in oxidative phosphorylation. High-resolution single-particle electron cryo-EM analyses have provided detailed structural knowledge of the catalytic machinery of complex I, but not of the molecular principles of its energy transduction mechanism. Although ubiquinone is considered to bind in a long channel at the interface of the membrane-embedded and hydrophilic domains, with channel residues likely involved in coupling substrate reduction to proton translocation, no structures with the channel fully occupied have yet been described. Here, we report the structure (determined by cryo-EM) of mouse complex I with a tight-binding natural product acetogenin inhibitor, which resembles the native substrate, bound along the full length of the expected ubiquinone-binding channel. Our structure reveals the mode of acetogenin binding and the molecular basis for structure–activity relationships within the acetogenin family. It also shows that acetogenins are such potent inhibitors because they are highly hydrophobic molecules that contain two specific hydrophilic moieties spaced to lock into two hydrophilic regions of the otherwise hydrophobic channel. The central hydrophilic section of the channel does not favor binding of the isoprenoid chain when the native substrate is fully bound but stabilizes the ubiquinone/ubiquinol headgroup as it transits to/from the active site. Therefore, the amphipathic nature of the channel supports both tight binding of the amphipathic inhibitor and rapid exchange of the ubiquinone/ubiquinol substrate and product.     

The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor

NADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [³H]benzophenone-asimicin ([³H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [³H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS-PAGE) of [³H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding.     

Structural basis for the quinone reduction in the bc1 complex: a comparative analysis of crystal structures of mitochondrial cytochrome bc1 with bound substrate and inhibitors at the Qi site

Cytochrome bc(1) is an integral membrane protein complex essential to cellular respiration and photosynthesis. The Q cycle reaction mechanism of bc(1) postulates a separated quinone reduction (Q(i)) and quinol oxidation (Q(o)) site. In a complete catalytic cycle, a quinone molecule at the Q(i) site receives two electrons from the b(H) heme and two protons from the negative side of the membrane; this process is specifically inhibited by antimycin A and NQNO. The structures of bovine mitochondrial bc(1) in the presence or absence of bound substrate ubiquinone and with either the bound antimycin A(1) or NQNO were determined and refined. A ubiquinone with its first two isoprenoid repeats and an antimycin A(1) were identified in the Q(i) pocket of the substrate and inhibitor bound structures, respectively; the NQNO, on the other hand, was identified in both Q(i) and Q(o) pockets in the inhibitor complex. The two inhibitors occupied different portions of the Q(i) pocket and competed with substrate for binding. In the Q(o) pocket, the NQNO behaves similarly to stigmatellin, inducing an iron-sulfur protein conformational arrest. Extensive binding interactions and conformational adjustments of residues lining the Q(i) pocket provide a structural basis for the high affinity binding of antimycin A and for phenotypes of inhibitor resistance. A two-water-mediated ubiquinone protonation mechanism is proposed involving three Q(i) site residues His(201), Lys(227), and Asp(228).     

Q-site inhibitor induced ROS production of mitochondrial complex II is attenuated by TCA cycle dicarboxylates

The impact of complex II (succinate:ubiquinone oxidoreductase) on the mitochondrial production of reactive oxygen species (ROS) has been underestimated for a long time. However, recent studies with intact mitochondria revealed that complex II can be a significant source of ROS. Using submitochondrial particles from bovine heart mitochondria as a system that allows the precise setting of substrate concentrations we could show that mammalian complex II produces ROS at subsaturating succinate concentrations in the presence of Q-site inhibitors like atpenin A5 or when a further downstream block of the respiratory chain occurred. Upon inhibition of the ubiquinone reductase activity, complex II produced about 75% hydrogen peroxide and 25% superoxide. ROS generation was attenuated by all dicarboxylates that are known to bind competitively to the substrate binding site of complex II, suggesting that the oxygen radicals are mainly generated by the unoccupied flavin site. Importantly, the ROS production induced by the Q-site inhibitor atpenin A5 was largely unaffected by the redox state of the Q pool and the activity of other respiratory chain complexes. Hence, complex II has to be considered as an independent source of mitochondrial ROS in physiology and pathophysiology.     

Effect of ubiquinones and their analogs on the respiratory chain enzyme activity of Candida guilliermondii yeasts

The effect of ubiquinones with different length of their chain (CoQ0, CoQ1, CoQ2, CoQ6, CoQ9) and their synthetic analogues (analogues of ubiquinone-1, hexahydroubiquinone-4, monophytylquinone, diphytylquinone, triphytylquinone) on the activity of ubiquinone dependent enzyme systems was studied in mitochondrial fractions from the yeast Candida guilliermondii. All of the ubiquinone homologues studied activated these systems. The synthetic analogues of ubiquinone nonspecifically inhibited the activity of NADH2-oxidase system. The inhibition was reversible when CoQ0 and CoQ1, but not CoQ6 and CoQ9, were added to the system. In the succinate-CoQ-reductase system, the inhibition caused by the analogues of ubiquinone was eliminated when all of the tested homologues were added to the system. In contrast to other analogues of ubiquinone, hexahydroubiquinone-4 was an inhibitor for the NADH2-oxidase system and an activator for the succinate-CoQ-reductase system, and eliminated the inhibiting action of other ubiquinone analogues in this system. Similar action of ubiquinone homologues was shown in the elimination of the inhibition of ubiquinone dependent systems caused by the specific inhibitors of electron transport, viz. rotenone and antimycin A.     

On the mechanism of rotenone-insensitive reduction of quinones by mitochondrial NADH:ubiquinone reductase. The high affinity binding of NAD+ and NADH to the reduced enzyme form

NADH acts as an incomplete competitive inhibitor for 5,8-dioxy-1,4-naphtoquinone during its rotenone-insensitive reduction by mitochondrial NADH:ubiquinone reductase. NAD+ and ADP-ribose act as incomplete mixed-type inhibitors. Ki of NAD+ and NADH towards quinone are about one order less than towards ferricyanide. The bimolecular rate constant of the reduction of the enzyme by NADH in the quinone reductase reaction is about 2 times less than that of ferricyanide reductase reaction. These data indicate that the reduction site of 5,8-dioxy-1,4-naphtoquinone is close to NAD+/NADH and ferricyanide binding site. It seems that during the steady-state reduction of ferricyanide and 5,8-dioxy-1,4-naphtoquinone these oxidizers react with NADH:ubiquinone reductase reduced to different extents.     

Exploring the inhibitor binding pocket of respiratory complex I

Numerous hydrophobic and amphipathic compounds including several detergents are known to inhibit the ubiquinone reductase reaction of respiratory chain complex I (proton pumping NADH:ubiquinone oxidoreductase). Guided by the X-ray structure of the peripheral arm of complex I from Thermus thermophilus we have generated a large collection of site-directed mutants in the yeast Yarrowia lipolytica targeting the proposed ubiquinone and inhibitor binding pocket of this huge multiprotein complex at the interface of the 49-kDa and PSST subunits. We could identify a number of residues where mutations changed I(50) values for representatives from all three groups of hydrophobic inhibitors. Many mutations around the domain of the 49-kDa subunit that is homologous to the [NiFe] centre binding region of hydrogenase conferred resistance to DQA (class I/type A) and rotenone (class II/type B) indicating a wider overlap of the binding sites for these two types of inhibitors. In contrast, a region near iron-sulfur cluster N2, where the binding of the n-alkyl-polyoxyethylene-ether detergent C(12)E(8) (type C) was exclusively affected, appeared comparably well separated. Taken together, our data provide structure-based support for the presence of distinct but overlapping binding sites for hydrophobic inhibitors possibly extending into the ubiquinone reduction site of mitochondrial complex I.     

Heterocyclic analogues of squamocin as inhibitors of mitochondrial complex I. On the role of the terminal lactone of annonaceous acetogenins

Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.     

New evidence for the multiplicity of ubiquinone- and inhibitor-binding sites in the mitochondrial complex I

Determination of the number of ubiquinone- and inhibitor-binding sites in the mitochondrial complex I (NADH:ubiquinone oxidoreductase) is a controversial question with a direct implication for elaborating a suitable model to explain the bioenergetic mechanism of this complicated enzyme. We have used combinations of both selective inhibitors and common ubiquinone-like substrates to demonstrate the multiplicity of the reaction centers in the complex I in contrast with competition studies that have suggested the existence of a unique binding site for ubiquinone. Our results provide new evidence for the existence of at least two freely exchangeable ubiquinone-binding sites with different specificity for substrates, as well as for a different kinetic interaction of inhibitors with the enzyme.     

Effect of inhibitors on the ubiquinone binding capacity of the primary energy conversion site in the Rhodobacter capsulatus cytochrome bc(1) complex.

A key issue concerning the primary conversion (Q(O)) site function in the cytochrome bc(1) complex is the stoichiometry of ubiquinone/ubihydroquinone occupancy. Previous evidence suggests that the Q(O) site is able to accommodate two ubiquinone molecules, the double occupancy model [Ding, H., Robertson, D. E., Daldal, F., and Dutton, P. L. (1992) Biochemistry 31, 3144-3158]. In the recently reported crystal structures of the cytochrome bc(1) complex, no electron density was identified in the Q(O) site that could be ascribed to ubiquinone. To provide further insight into this issue, we have manipulated the cytochrome bc(1) complex Q(O) site occupancy in photosynthetic membranes from Rhodobacter capsulatus by using inhibitor titrations and ubiquinone extraction to modulate the amount of ubiquinone bound in the site. The nature of the Q(O) site occupants was probed via the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectra to modulation of Q(O) site occupancy. Diphenylamine (DPA) and methoxyacrylate (MOA)-stilbene are known Q(O) site inhibitors of the cytochrome bc(1) complex. Addition of stoichiometric concentrations of MOA-stilbene or excess DPA to cytochrome bc(1) complexes with natural levels of ubiquinone elicits the same change in the [2Fe-2S] cluster EPR spectra; the g(x)() resonance broadens and shifts from 1. 800 to 1.783. This is exactly the same signal as that obtained when there is only one ubiquinone present in the Q(O) site. Furthermore, addition of MOA-stilbene or DPA to the cytochrome bc(1) complex depleted of ubiquinone does not alter the [2Fe-2S] cluster EPR spectral line shapes, which remain indicative of one ubiquinone or zero ubiquinones in the Q(O) site, with broad g(x)() resonances at 1. 783 or 1.765, respectively. The results are quite consistent with the Q(O) site double occupancy model, in which MOA-stilbene and DPA inhibit by displacing one, but not both, of the Q(O) site ubiquinones.     

Inhibition of mitochondrial NADH:ubiquinone oxidoreductase by ethoxyformic anhydride

The NADH:ubiquinone, but not the NADH:ferricyanide, reductase activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) is inhibited by incubation of the enzyme at pH 6.0 and 0 degree C with ethoxyformic anhydride (EFA), and the inhibition is partially reversed by subsequent incubation of EFA-treated complex I with hydroxylamine. These results and spectral changes of EFA-treated complex I in the u.v. region are consistent with modification of essential histidyl or tyrosyl residues between the primary NADH dehydrogenase and the site of ubiquinone reduction. Treatment of complex I with EFA in the presence of high concentrations of Seconal or Demerol did not protect against EFA inactivation, suggesting that the site of EFA modification may not be the same as the inhibiton sites of Seconal and Demerol. However, the presence of NADH during incubation of complex I with EFA greatly enhanced the inhibition rate, indicating that the reduced conformation of complex I is more susceptible to attack by EFA.