Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

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.     

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.     

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum

The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ(9)) and rhodoquinone-9 (RQ(9)) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ(2)) and ubiquinone-2 (UQ(2)) reduction. Structure-activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ(2) and NADH-UQ(2) oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ(2) and UQ(2), respectively. The pH profiles of both activities differed remarkably; NADH-RQ(2) oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ(2) oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ(2) and UQ(2) as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.     

Amiloride inhibition of the proton-translocating NADH-quinone oxidoreductase of mammals and bacteria

The proton-translocating NADH-quinone oxidoreductase in mitochondria (complex I) and bacteria (NDH-1) was shown to be inhibited by amiloride derivatives that are known as specific inhibitors for Na(+)/H(+) exchangers. In bovine submitochondrial particles, the effective concentrations were about the same as those for the Na(+)/H(+) exchangers, whereas in bacterial membranes the inhibitory potencies were lower. These results together with our earlier observation that the amiloride analogues prevent labeling of the ND5 subunit of complex I with a fenpyroximate analogue suggest the involvement of ND5 in H(+) (Na(+)) translocation and no direct involvement of electron carriers in H(+) (Na(+)) translocation.     

Kinetic characterization of mitochondrial complex I inhibitors using annonaceous acetogenins.

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.     

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.     

Enzymatic oxidation of mercury vapor by erythrocytes

The formation of glutathione radicals, the evolution of nascent oxygen or the peroxidatic reaction with catalase complex I are considered as possible mechanisms for the oxidation of mercury vapor by red blood cells. To select among these, the uptake of atomic mercury by erythrocytes from different species was studied and related to their various activities of catalase (hydrogenperoxide : hydrogen-peroxide oxidoreductase, EC 1.11.1.6) and glutathione peroxidase (glutathione : hydrogen-peroxide oxidoreductase, EC 1.11.1.9). A slow and continuous infusion of diluted H2O2 was used to maintain steady concentrations of complex I. 1% red cell supsensions were found most suitable showing high rates of Hg uptake and yielding still enough cells for subsequent determinations. The results indicate that the oxidation of mercury depends upon the H2O2-generation rate and upon the specific acticity of red-cell catalase. The oxidation occurred in a range of the catalase-H2O2 reaction where the evolution of oxygen could be excluded. Compounds reacting with complex I were shown to be effective inhibitors of the mercury uptake. GSH-peroxidase did not participate in the oxidation but rather, was found to inhibit it by competing with catalase for hydrogen peroxide. These findings support the view that elemental mercury is oxidized in erythrocytes by a peroxidatic reaction with complex I only.     

The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.     

The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction

Iron–sulfur cluster N2 of complex I (proton pumping NADH:quinone oxidoreductase) is the immediate electron donor to ubiquinone. At a distance of only ～ 7 Å in the 49-kDa subunit, a highly conserved tyrosine is found at the bottom of the previously characterized quinone binding pocket. To get insight into the function of this residue, we have exchanged it for six different amino acids in complex I from Yarrowia lipolytica. Mitochondrial membranes from all six mutants contained fully assembled complex I that exhibited very low dNADH:ubiquinone oxidoreductase activities with n-decylubiquinone. With the most conservative exchange Y144F, no alteration in the electron paramagnetic resonance spectra of complex I was detectable. Remarkably, high dNADH:ubiquinone oxidoreductase activities were observed with ubiquinones Q₁ and Q₂ that were coupled to proton pumping. Apparent K_m values for Q₁ and Q₂ were markedly increased and we found pronounced resistance to the complex I inhibitors decyl-quinazoline-amine (DQA) and rotenone. We conclude that Y144 directly binds the head group of ubiquinone, most likely via a hydrogen bond between the aromatic hydroxyl and the ubiquinone carbonyl. This places the substrate in an ideal distance to its electron donor iron–sulfur cluster N2 for efficient electron transfer during the catalytic cycle of complex I.     

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.     

Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.     

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.     

Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I)

Neither the route of electron transport nor the sites or mechanism of superoxide production in mitochondrial complex I has been established. We examined the rates of superoxide generation (measured as hydrogen peroxide production) by rat skeletal muscle mitochondria under a variety of conditions. The rate of superoxide production by complex I during NADH-linked forward electron transport was less than 10% of that during succinate-linked reverse electron transport even when complex I was fully reduced by pyruvate plus malate in the presence of the complex III inhibitor, stigmatellin. This asymmetry was not explained by differences in protonmotive force or its components. However, when inhibitors of the quinone-binding site of complex I were added in the presence of ATP to generate a pH gradient, there was a rapid rate of superoxide production by forward electron transport that was as great as the rate seen with reverse electron transport at the same pH gradient. These observations suggest that quinone-binding site inhibitors can make complex I adopt the highly radical-producing state that occurs during reverse electron transport. Despite complete inhibition of NADH: ubiquinone oxidoreductase activity in each case, different classes of quinone-binding site inhibitor (rotenone, piericidin, and high concentrations of myxothiazol) gave different rates of superoxide production during forward electron transport (the rate with myxothiazol was twice that with rotenone) suggesting that the site of rapid superoxide generation by complex I is in the region of the ubisemiquinone-binding sites and not upstream at the flavin or low potential FeS centers.     

Kinetic microplate-based assays for inhibitors of mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase.

Kinetic microplate-based assays for both mitochondrial NADH:ubiquinone oxidoreductase (complex I) and succinate:cytochrome c oxidoreductase using insect submitochondrial particles as the source of the enzyme activities have been developed. These assays have been used to design high-throughput screens for inhibitors of these mitochondrial electron transfer activities to assess their intrinsic in vitro efficacies as potential pesticides. These methods can be used to test up to 60 compounds per day without the use of automated sample handling and diluting technology. The accuracy, specificity, and reproducibility of the microplate methods compared well with conventional spectrophotometer-based assays.     

NADH-Ubiquinone oxidoreductase: substrate-dependent oxygen turnover to superoxide anion as a function of flavin mononucleotide

Bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) catalyzed NADH- and ubiquinone-1-dependent oxygen (O2) turnover to hydrogen peroxide that was stimulated by piericidin A and superoxide dismutase (SOD), but was insensitive to antimycin A, myxothiazol, and potassium cyanide. The extent of O2 consumption as a function of ubiquinone-1 did not correlate with piericidin A-sensitive rates of ubiquinone reduction. Decylubiquinone did not stimulate O2 consumption, but did initiate an SOD-sensitive cytochrome c reduction when complex I was isolated away from ubiquinol-cytochrome c oxidoreductase. Rates and extent of O2 turnover (ROS production) and ubiquinone reduction were higher than previously reported for submitochondrial particles (SMP) and isolated complex I. This ROS production was shown to co-isolate with complex I flavin.     

Topology of superoxide production from different sites in the mitochondrial electron transport chain

We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria.     

The ND5 subunit was labeled by a photoaffinity analogue of fenpyroximate in bovine mitochondrial complex I

Fenpyroximate is a potent inhibitor of the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I). We synthesized its photoaffinity analogue [(3)H](trifluoromethyl)phenyldiazirinylfenpyroximate ([(3)H]TDF). When bovine heart submitochondrial particles (SMP) were illuminated with UV light in the presence of [(3)H]TDF, radioactivity was mostly incorporated into a 50 kDa band. There was a good correlation between radioactivity labeling of the 50 kDa band and inhibition of the NADH oxidase activity, indicating that a 50 kDa protein is responsible for the inactivation of complex I. Blue native gel electrophoresis of the [(3)H]TDF-labeled SMP revealed that the majority of radioactivity was found in complex I. Analysis of the complex I band on an SDS gel showed a major peak of radioactivity at approximately 50 kDa. There are three subunits in complex I that migrate in this region: FP51K, IP49K, and ND5. Further analysis using the 2D gel electrophoresis implied that the labeled protein was the ND5 subunit. Labeling of the ND5 subunit was stimulated by NADH/NADPH but was prevented by various complex I inhibitors. Amiloride derivatives that are known to be inhibitors of Na(+)/H(+) antiporters also diminished the labeling. In agreement with the protective effect, we observed that the amiloride derivatives inhibited NADH-ubiquinone-1 reductase activity but not NADH-K(3)Fe(CN)(6) reductase activity in bovine SMP. These results suggest that the ND5 subunit is involved in construction of the inhibitor- and quinone-binding site(s). Furthermore, it seems likely that the ND5 subunit may participate in H(+)(Na(+)) translocation in coupling site 1.     

In vivo complementation of complex I by the yeast Ndi1 enzyme. Possible application for treatment of Parkinson disease

Recent studies suggest that dysfunction of the NADH-quinone oxidoreductase (complex I) is associated with a number of human diseases, including neurodegenerative disorders such as Parkinson disease. We have shown previously that the single subunit rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae mitochondria can restore NADH oxidation in complex I-deficient mammalian cells. The Ndi1 enzyme is insensitive to complex I inhibitors such as rotenone and 1-methyl-4-phenylpyridinium ion, known as a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test the possible use of the NDI1 gene as a therapeutic agent in vivo, we chose a mouse model of Parkinson disease. The NDI1-recombinant adeno-associated virus particles (rAAV-NDI1) were injected unilaterally into the substantia nigra of mice. The animals were then subjected to treatment with MPTP. The degree of neurodegeneration in the nigrostriatal system was assessed immunohistochemically through the analysis of tyrosine hydroxylase and glial fibrillary acidic protein. It was evident that the substantia nigra neurons on the side used for injection of rAAV-NDI1 retained a high level of tyrosine hydroxylase-positive cells, and the ipsilateral striatum exhibited significantly less denervation than the contralateral striatum. Furthermore, striatal concentrations of dopamine and its metabolites in the hemisphere that received rAAV-NDI1 were substantially higher than those of the untreated hemisphere, reaching more than 50% of the normal levels. These results indicate that the expressed Ndi1 protein elicits resistance to MPTP-induced neuronal injury. The present study is the first successful demonstration of complementation of complex I by the Ndi1 enzyme in animals.     

Selective inhibition of NADH-CoQ oxidoreductase (complex I) of rat brain mitochondria by arachidonic acid

The effects of arachidonic acid on the enzyme complexes in the electron transport system were investigated using submitochondrial particles from rat brain. Arachidonic acid irreversibly inhibited NADH-CoQ oxidoreductase (complex I) activity, but had no effect on the activities of succinate-CoQ oxidoreductase (complex II), CoQH2-cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV), ATPase (complex V), glutamate dehydrogenase, and malate dehydrogenase up to 50 microM. The inhibition was dose-dependent with an IC50 value of 110 nmol/mg protein. The Lineweaver-Burk plot revealed that the inhibition by arachidonic acid was noncompetitive against CoQ with a Ki value of 33 microM and uncompetitive against NADH with a Ki value of 22 microM.