Mitochondrial NADH-ubiquinone Oxidoreductase Research Articles

Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs Mitochondrial Function in Cardiac Myocytes Exposed to High Glucose

Increased nuclear protein O-linked beta-N-acetylglucosamine glycosylation (O-GlcNAcylation) mediated by high glucose treatment or the hyperglycemia of diabetes mellitus contributes to cardiac myocyte dysfunction. However, whether mitochondrial proteins in cardiac myocytes are also submitted to O-GlcNAcylation or excessive O-GlcNAcylation alters mitochondrial function is unknown. In this study, we determined if mitochondrial proteins are O-GlcNAcylated and explored if increased O-GlcNAcylation is linked to high glucose-induced mitochondrial dysfunction in neonatal rat cardiomyocytes. By immunoprecipitation, we found that several mitochondrial proteins, which are members of complexes of the respiratory chain, like subunit NDUFA9 of complex I, subunits core 1 and core 2 of complex III, and the mitochondrial DNA-encoded subunit I of complex IV (COX I) are O-GlcNAcylated. By mass spectrometry, we identified that serine 156 on NDUFA9 is O-GlcNAcylated. High glucose treatment (30 mm glucose) increases mitochondrial protein O-GlcNAcylation, including those of COX I and NDUFA9 which are reduced by expression of O-GlcNAcase (GCA). Increased mitochondrial O-GlcNAcylation is associated with impaired activity of complex I, III, and IV in addition to lower mitochondrial calcium and cellular ATP content. When the excessive O-GlcNAc modification is reduced by GCA expression, mitochondrial function improves; the activity of complex I, III, and IV increases to normal and mitochondrial calcium and cellular ATP content are returned to control levels. From these results we conclude that specific mitochondrial proteins of cardiac myocytes are O-GlcNAcylated and that exposure to high glucose increases mitochondrial protein O-GlcNAcylation, which in turn contributes to impaired mitochondrial function.

L-Galactono-1,4-lactone Dehydrogenase Is Required for the Accumulation of Plant Respiratory Complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.

Synthesis and Characterization of New Piperazine-Type Inhibitors for Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

The mode of action of Deltalac-acetogenins, strong inhibitors of bovine heart mitochondrial complex I, is different from that of traditional inhibitors such as rotenone and piericidin A [Murai, M., et al. (2007) Biochemistry 46 , 6409-6416]. As further exploration of these unique inhibitors might provide new insights into the terminal electron transfer step of complex I, we drastically modified the structure of Deltalac-acetogenins and characterized their inhibitory action. In particular, on the basis of structural similarity between the bis-THF and the piperazine rings, we here synthesized a series of piperazine derivatives. Some of the derivatives exhibited very potent inhibition at nanomolar levels. The hydrophobicity of the side chains and their balance were important structural factors for the inhibition, as is the case for the original Deltalac-acetogenins. However, unlike in the case of the original Deltalac-acetogenins, (i) the presence of two hydroxy groups is not crucial for the activity, (ii) the level of superoxide production induced by the piperazines is relatively high, (iii) the inhibitory potency for the reverse electron transfer is remarkably weaker than that for the forward event, and (iv) the piperazines efficiently suppressed the specific binding of a photoaffinity probe of natural-type acetogenins ([ (125)I]TDA) to the ND1 subunit. We therefore conclude that the action mechanism of the piperazine series differs from that of the original Deltalac-acetogenins. The photoaffinity labeling study using a newly synthesized photoreactive piperazine ([ (125)I]AFP) revealed that this compound binds to the 49 kDa subunit and an unidentified subunit, not ND1, with a frequency of approximately 1:3. A variety of traditional complex I inhibitors as well as Deltalac-acetogenins suppressed the specific binding of [ (125)I]AFP to the subunits. The apparent competitive behavior of inhibitors that seem to bind to different sites may be due to structural changes at the binding site, rather than occupying the same site. The meaning of the occurrence of diverse inhibitors exhibiting different mechanisms of action is discussed in light of the functionality of the membrane arm of complex I.

Synthesis of pyranicin and its deoxygenated analogues and their inhibitory action with bovine heart mitochondrial complex I

Total synthesis of pyranicin and its deoxygenated analogues was achieved using Cl 2Pd(CH 3CN) 2 catalyzed diastereoselective cyclization of the allylic ester as the key step. The inhibitory activity of these compounds for mitochondrial NADH–ubiquinone oxidoreductase (complex I) was poorer than those of ordinary mono-THF acetogenins such as annonacin.

Dynamic Function of the Spacer Region of Acetogenins in the Inhibition of Bovine Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

Studies of the action mechanism of acetogenins, the most potent and structurally unique inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), are valuable in characterizing the inhibitor binding site in this enzyme. Our previous study deepened our understanding of the dynamic function of the spacer region of bis-THF acetogenins [Abe, M., et al. (2005) Biochemistry 44, 14898-14906] but, at the same time, posed new important questions. First, while the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings) span a distance shorter than that of the extended 13 carbon atoms [-(CH 2) 13-], what is the apparent optimal length of the spacer for the inhibition of 13 carbon atoms? In other words, what is the functional role of the additional methylene groups? Second, why was the inhibitory potency of the mono-THF derivative, but not the bis-THF derivative, drastically reduced by hardening the spacer covering 10 carbon atoms into a rodlike shape [-CH 2-(C identical withC) 4-CH 2-]? This study was designed not only to answer these questions but also to further disclose the dynamic functions of the spacer. We here synthesized systematically designed acetogenins, including mono- and bis-THF derivatives, and evaluated their inhibitory effects on bovine complex I. With regard to the first question, we demonstrated that the additional methylenes enhance the hydrophobicity of the spacer region, which may be thermodynamically advantageous for bringing the polar gamma-lactone ring into the membrane-embedded segment of complex I. With regard to the second question, we observed that a decrease in the flexibility of the spacer region is more adverse to the action of the mono-THF series than that of the bis-THF series. As a cause of this difference, we suggest that for bis-THF derivatives, one of the two THF rings, being adjacent to the spacer, is capable of working as a pseudospacer to overcome the remarkable decrease in the conformational freedom and/or the length of the spacer. Moreover, using photoresponsive acetogenins that undergo drastic and reversible conformational changes with alternating UV-vis irradiation, we provided further evidence that the spacer region is free from steric congestion arising from the putative binding site probably because there is no receptor wall for the spacer region.

Synthesis of Pyranicin and Its Inhibitory Action with Bovine Heart Mitochondrial Complex I

Total synthesis of pyranicin was achieved using Cl2Pd(CH3CN)2-catalyzed diastereoselective cyclization of the allylic ester as the key step. The inhibitory activity of this compound for mitochondrial NADH-ubiquinone oxidoreductase (complex I) was slightly poorer than that of ordinary mono-THF acetogenins such as cis-solamin.

Synthesis, determination of the absolute configuration of tonkinelin, and inhibitory action with bovine heart mitochondrial complex I

The first synthesis of two possible diastereomers of tonkinelin was achieved. By comparison of the optical rotation of two candidates of tonkinelin and the natural compound, it is suggested that the absolute configuration of natural tonkinelin is likely to be (17 S,18 S). The inhibitory activity of these compounds was examined with bovine heart mitochondrial NADH–ubiquinone oxidoreductase. These compounds showed remarkably weak inhibitory activity compared to ordinary acetogenins such as bullatacin.

RNA Silencing of Mitochondrial m-Nfs1 Reduces Fe-S Enzyme Activity Both in Mitochondria and Cytosol of Mammalian Cells

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.

The MELAS mutations 3946 and 3949 perturb the critical structure in a conserved loop of the ND1 subunit of mitochondrial complex I

The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.

Cytotoxicity of 17 tetrahydroisoquinoline derivatives in SH-SY5Y human neuroblastoma cells is related to mitochondrial NADH–ubiquinone oxidoreductase inhibition

Since the first report that 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine induces parkinsonism, various kinds of low-molecular-weight neurotoxins, such as tetrahydroisoquinoline derivatives, have been identified as possible Parkinson's disease-inducing substances. In the present study, we measured four parameters of 17 tetrahydroisoquinoline derivatives, i.e., cytotoxicity in SH-SY5Y human neuroblastoma cells, inhibitory activity towards mitochondrial NADH–ubiquinone oxidoreductase (complex I), affinity for dopamine transporter, and 1-butanol–H 2O partition coefficient (as an index of lipophilicity). Six of the derivatives showed comparatively strong inhibitory activity towards complex I (IC 50 values < 100 μM) and five of them were cytotoxic to SH-SY5Y cells (TC 50 values < 200 μM). Some of these compounds are endogenous. We found good correlations between cytotoxicity and complex I inhibitory activity, but not between cytotoxicity and affinity for dopamine transporter. Since cytotoxicity to SH-SY5Y neuroblastoma cells was related to inhibitory activity towards mitochondrial complex I, complex I inhibition is likely to be involved, at least in part, in the mechanism of TIQ derivative-induced cell death. Uptake of most of these compounds seems to be dependent on lipophilicity, rather than active transport via dopamine transporter.

Light Control of Mitochondrial Complex I Activity by a Photoresponsive Inhibitor

We recently developed a new class of inhibitors of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), named Deltalac-acetogenin [Ichimaru et al. (2005) Biochemistry 44, 816-825]. The inhibitory potency of Deltalac-acetogenin is remarkably affected by the molecular shape of the alkyl side chains. We speculated that if the shape of the side chains can be changed by the trans-cisphotoisomerization of the azobenzene unit that is introduced into the chain moiety, the inhibitory effect could be switched on and off in a reversible manner. Such a photoresponsive inhibitor may allow rapid, remote, and noninvasive control of complex I activity. Therefore, we here synthesized Deltalac-acetogenin (3) possessing an azobenzene unit in the side chains. (1)H NMR, HPLC, and UV-visible absorption analyses indicated that the azobenzene unit in 3 is rapidly and reversibly trans-cis isomerized by photoirradiation in chloroform and ethanol. The inhibitory effect of trans,trans-3 on complex I activity in submitochondrial particles was more potent than that of cis,cis-3. When 3 was applied at the nanomolar level to complex I, the inhibitory effect was reversibly reduced and enhanced by alternating irradiation by UV and visible light, respectively. The present study gives a positive clue to the light control of complex I activity.

Synthesis of (4 R,15 R,16 R,21 S)- and (4 R,15 S,16 S,21 S)-rollicosin, squamostolide, and their inhibitory action with bovine heart mitochondrial complex I

A convergent stereoselective synthesis of (4 R,15 R,16 R,21 S)- and (4 R,15 S,16 S,21 S)-rollicosin and squamostolide was accomplished via a Pd-catalyzed cross-coupling reaction. The inhibitory activity of these compounds was examined with bovine heart mitochondrial NADH-ubiquinone oxidoreductase. These compounds showed a remarkably weak inhibitory activity compared to ordinary acetogenins such as bullatacin. Our results indicate that to maintain potent inhibitory effect, the hydroxylated lactone cannot substitute for the hydroxylated mono- or bis-THF rings with a long alkyl chain that can be seen in ordinary acetogenins.

Synthesis of photolabile .DELTA.lac-acetogenin for photoaffinity labeling of mitochondrial complex I

Δlac-Acetogenins are a novel type of inhibitor acting at the terminal electron transfer step of mitochondrial NADH-ubiquinone oxidoreductase (complex I). To identify the binding site of Δlac-acetogenins by photoaffinity labeling, we synthesized a photolabile Δlac-acetogenin that possesses a biotin probe to enable the detection and the isolation of the labeled peptide without the use of a radioisotope. The photolabile Δlac-acetogenin synthesized in this study elicited potent inhibition of bovine heart mitochondrial complex I at the nanomolar level. © Pesticide Science Society of Japan

Synthesis of non-THF analogs of acetogenin toward simplified mimics

Acetogenin analogs in which the bis-adjacent THF ring was replaced with an enantioselectively synthesized 1,2-cyclopentanediol bis-ether skeleton were synthesized to obtain simplified mimics, and their inhibitory effect on mitochondrial NADH–ubiquinone oxidoreductase (complex I ) was examined. The results clearly demonstrate that the 1,2-cyclopentanediol bis-ether motif can substitute for the bis-THF ring while maintaining very potent inhibitory activity at the nanomolar level.

Import and orientation of the MWFE protein in mitochondrial NADH-ubiquinone oxidoreductase

The MWFE subunit of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a small, essential membrane protein of 70 amino acids that is made in the cytosol, imported into mitochondria, and assembled without further proteolytic processing. The experiments identify the first ∼30 amino acids as a minimal mitochondrial targeting sequence, and establish its orientation in the inner membrane and in complex I. This sequence has a highly conserved glutamate at position 4, which is not typical of a mitochondrial targeting signal. However, it is not essential for MWFE function. Within this sequence there is also a ‘stop-transfer’ signal. The membrane anchor cannot be replaced by that from another subunit within complex I.

Cover Picture: Total Synthesis of 34‐Hydroxyasimicin and Its Photoactive Derivative for Affinity Labeling of the Mitochondrial Complex I (Chem. Eur. J. 9/2004)

The cover picture shows representative plants of the Annonaceae plants (background), a family of tropical trees and shrubs that accommodates over 2300 species. More than 350 acetogenins have already been isolated from 37 species in this family, many of which have shown to be potent cytotoxic, antitumor, antimalarial, immunosuppressing, pesticidal, and antifeedant agents. It has been previously proposed that the cellular target of many Annonaceous acetogenins is the mitochondrial NADH–ubiquinone oxidoreductase (Complex I). The electron microscopic three‐dimensional reconstructions of Complex I (lower right corner) from N. crassa (wire frame) and the homologous E. coli complex (surface rendered) show that the two complexes have similar structures. The cytoplasmic domain for E. coli is colored in gold and the membrane domain in blue. The surfaces of the membrane are shown schematically in red (T. Friedrich et al., Biochim. Biophys. Acta 1998, 1365, 215). All acetogenins shown in the picture were previously synthesized by the Keinan–Sinha group. The asymmetric total synthesis of 34‐acyloxy‐34‐tritio‐asimicin (bottom) was achieved by means of a convergent synthetic strategy. This photoaffinity labeling agent is designed to identify the target protein subunit in Complex I. For more details see the article by E. Keinan, S. C. Sinha et al. on p. 2149 ff.

Total synthesis of 34-hydroxyasimicin and its photoactive derivative for affinity labeling of the mitochondrial complex I.

The asymmetric total synthesis of the 34-hydroxyasimicin and its 3-(4-benzoylphenyl)propionate ester was achieved by means of a convergent synthetic strategy. This ester, which contains eight asymmetric centers, represents the first photoaffinity-labeling agent that is derived from an Annonaceous acetogenin. The key transformations in the synthesis include the Sharpless asymmetric dihydroxylation reaction, the Wittig olefination reaction, an oxidative cyclization reaction with rhenium(vii) oxide, the Williamson etherification reaction, and a palladium-catalyzed cross-coupling reaction. Use of the target molecule for photoaffinity-labeling studies of bovine mitochondrial NADH-ubiquinone oxidoreductase (Complex I) may shed light on the structure/function of this intricate enzyme and on the origin of the high antitumor activity exhibited by the Annonaceous acetogenins.

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 (O 2) 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 O 2 consumption as a function of ubiquinone-1 did not correlate with piericidin A-sensitive rates of ubiquinone reduction. Decylubiquinone did not stimulate O 2 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 O 2 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.

A single-subunit NADH-quinone oxidoreductase renders resistance to mammalian nerve cells against complex I inhibition.

Numerous studies suggest that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Development of methods to correct complex I defects seems important. We have previously shown that the single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1P) can work as a replacement for complex I in mammalian cells. Using a recombinant adeno-associated virus vector carrying the NDI1 gene, we now demonstrated that the Ndi1 enzyme was successfully expressed in the dopaminergic cell lines rat PC12 and mouse MN9D. The cells expressing the Ndi1 protein were resistant to known inhibitors of complex I, such as rotenone and pyridaben. In addition, the NDI1-transduced cells were still capable of morphological maturation as examined by induction of neurite outgrowth. Also, it was possible to infect the cells after the maturation. The expressed Ndi1 protein was located both in cell bodies and in neurites and was functionally active. It is conceivable that the NDI1 gene will be a promising tool in the treatment of neurodegenerative conditions caused by complex I inhibition.

Role of antioxidant defences in the species-specific response of isolated atria to menadione

In previous works we demonstrated that 2-methyl-1,4-naphthoquinone (menadione) causes a marked increase in the force of contraction of guinea pig and rat isolated atria. This inotropic effect was significantly higher in the guinea pig than in the rat and was strictly related to the amount of superoxide anion (O 2 − ), generated as a consequence of cardiac menadione metabolism through mitochondrial NADH-ubiquinone oxidoreductase. The present study was designed to further elucidate the basis of these quantitatively different positive inotropic responses. To this purpose, we measured O 2 − and hydrogen peroxide (H 2O 2) produced by mitochondria isolated from guinea pig and rat hearts in the presence of 20 μM menadione. Moreover, we evaluated the menadione detoxification activity (DT-diaphorase) and the antioxidant defences of guinea pig and rat hearts, namely their GSH/GSSG content, Cu/Zn- and Mn-dependent superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx) activities. Our results indicate that DT-diaphorase activity and glutathione levels were similar in both animal species. By contrast, guinea pig mitochondria produced greater amounts of O 2 − and H 2O 2 than those of rat heart. This is probably due to both the higher Mn-SOD activity (2.93±0.02 vs . 1.95±0.06 units/mg protein; P<0.05) and to the lower Gpx activity (10.09±0.30 vs. 32.67±1.02 units/mg protein; P<0.001) of guinea pig mitochondria. A lower CAT activity was also observed in guinea pig mitochondria (2.40±0.80 vs . 6.13±0.20 units/mg protein; P<0.01). Taken together, these data provide a rational explanation for the greater susceptibility of guinea pig heart to the toxic effect of menadione: because of the greater amount of O 2 − generated by the quinone and the higher mitochondrial Mn-SOD activity, guinea pig heart is exposed to more elevated concentrations of H 2O 2 that is less efficiently detoxified, because of lower Gpx and CAT levels of mitochondria.