Bovine Heart Mitochondrial Complex Research Articles

Respiratory complex II: ROS production and the kinetics of ubiquinone reduction

Bovine heart mitochondrial respiratory complex II generates ROS, mostly as superoxide, at the rate of about 20% of that detected during simultaneous operation of complex I and II when oxidation of ubiquinol is prevented by myxothiazol. ROS generating activity at different fumarate/succinate concentrations ratio implies that an enzyme component with a midpoint potential 40mV more positive than that of fumarate/succinate couple is the donor for one-electron reduction of oxygen. This suggests that the iron-sulfur cluster(s) is(are) involved in superoxide formation. Complex II-mediated ROS production exhibits a maximum at low (about 50μM) succinate concentration and gradually declines to zero activity upon further increase of the substrate. This phenomenology is explained and kinetically modeled to suggest a ping-pong mechanism of ROS generating activity where only dicarboxylate free reduced enzyme is oxidized by oxygen. The succinate:quinone reductase activity catalyzed by purified succinate:ubiquinone reductase also exhibits a ping-pong mechanism where only dicarboxylate free enzyme is oxidized by added quinone. Together these data suggest long distance interaction between the succinate (fumarate) binding and ubiquinone (ubiquinol) reactive sites.

Site-specific chemical modifications of the quinone binding cavity of bovine heart mitochondrial complex I

Site-specific chemical modifications of the quinone binding cavity of bovine heart mitochondrial complex I

バンレイシ科アセトゲニン類の合成とミトコンドリアｃｏｍｐｌｅｘ Ｉに対する阻害活性

Annonaceous acetogenins are a large family of natural polyketides. So far, more than 430 related compounds have been isolated. These compounds exhibit a number of interesting biological activities including cytotoxic, antitumor, pesticidal, anti-infective properties. From a view point of mechanism of action, they are among the most potent of the known inhibitors of complex I (NADH-ubiquinone oxidoreductase) in mitochondrial electron transfer system. We report here the asymmetric synthesis of mono-tetrahydrofuran (mono-THF) acetogenins, non-tetrahydrofuran acetogenins, and tetrahydropyran acetogenins. We also wish to report structure-activity relationship (SAR) study of the inhibitory action with bovine heart mitochondrial complex I.

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

125I-labeled ( trifluoromethyl)phenyl diazirinyl 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 (Q 2) and 2-azido-Q 2 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.

Characterization of the Inhibitor Binding Site in Mitochondrial NADH−Ubiquinone Oxidoreductase by Photoaffinity Labeling Using a Quinazoline-Type Inhibitor

The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex I, we synthesized a photoreactive azidoquinazoline {[(125)I]-6-azido-4-(4-iodophenethylamino)quinazoline, [(125)I]AzQ}, in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [(125)I]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of approximately 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [(125)I]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [(125)I]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.

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.

Total synthesis of 27-hydroxy-bullatacin and its C-15 epimer, and studies on their inhibitory effect on bovine heart mitochondrial complex I functions

The total synthesis of 27-hydroxybullatacin and its C-15 epimer has been achieved using rhenium(VII) oxides-mediated and Co(modp) 2-catalyzed oxidative cyclization (OC), diastereoselective alkynylation, Brown's enantioselective allylboration, and Grubbs' cross metathesis as the key reactions. The inhibitory effect of these compounds on the complex I function, as determined by using bovine submitochondrial particles, was in low nanomolar range.

Crucial Structural Factors and Mode of Action of Polyene Amides as Inhibitors for Mitochondrial NADH−Ubiquinone Oxidoreductase (Complex I)

Natural antibiotic polyene amides such as myxalamides are potent inhibitors of mitochondrial complex I. Because of the significant instability of this series of compounds due to an extended pi-conjugation skeleton, a detailed characterization of their inhibitory action has not been performed. To elucidate the action mechanism as well as binding manner of polyene amides with complex I, identification of the roles of each functional group in the inhibitory action is needed. We here synthesized a series of amide analogues and carried out structure-activity studies with bovine heart mitochondrial complex I. With respect to the left-hand portion, the natural pi-conjugation skeleton common to many natural products is not required for the inhibition and can be substituted with a simpler substructure such as a conjugated diene. The geometry and shape of the left-hand portion were shown to be important for the inhibition, suggesting that this portion may bind to a narrow hydrophobic pocket in the enzyme rather than merely partitioning into the lipid membrane phase. Concerning the right-hand portion of the inhibitor, the presence of the 2-methyl, amide NH, and (S)-1'-methyl groups was crucial for the activity, suggesting that both methyl groups neighboring the amide group finely adjust the hydrogen-bonding ability of the amide group. In contrast, modifications of the 2'-OH group did not significantly influence the activity, suggesting that the role of this functional group is not to serve as a hydrogen bond donor to the enzyme but to act as a hydrophilic anchor directing the right-hand portion at or near the membrane surface. Detailed characterization of the action mechanism indicated that the polyene amides share a common binding domain with other complex I inhibitors, though their binding position (or manner) within the domain may differ considerably from that of other inhibitors.

Synthesis of Murisolin (Ia), (15R, 16R, 19R, 20S)‐Murisolin A (Ib), and (15R, 16R, 19S, 20S)‐16,19‐cis‐Murisolin (Ic) and Their Inhibitory Action with Bovine Heart Mitochondrial Complex I.

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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.

The NDUFB11 gene is not a modifier in Leber hereditary optic neuropathy

Over 95% of Leber hereditary optic neuropathy (LHON) cases are due to mutations in mitochondrial DNA-encoded subunits of NADH:ubiquinone oxidoreductase (E.C.1.6.5.3., complex I). A recessive X-linked susceptibility gene that acts synergistically with the primary mtDNA mutation to produce visual loss is suggested by the high male-to-female ratio among LHON patients. The ESSS protein is a recently isolated subunit of bovine heart mitochondrial complex I. We revisited the genomic sequence of NDUFB11, the human homolog mapping to chromosome Xp11.23, and identified two mRNA isoforms showing different expression profiles in human tissues. Cultured skin fibroblasts from four LHON patients showed a pattern of expression similar to normal controls. Moreover, NDUFB11 did not seem to influence risk and age at onset of visual loss in a total of 65 individuals from 35 Italian LHON families. Also, the gene was not affected in 11 children with a severe encephalopathy associated with decreased complex I activity in skeletal muscle.

Effect of stereochemistry of Δlac-acetogenins on the inhibition of mitochondrial complex I (NADH-ubiquinone oxidoreductase)

Δlac-Acetogenins are a new type of inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). We synthesized a series of Δlac-acetogenins in which the stereochemistry around the hydroxylated tetrahydrofuran (THF) ring moiety was systematically modified, and examined their inhibitory effect on complex I. The present results revealed that the inhibitory effects of the bis-THF ring analogs are much more potent than those of the mono-THF ring analogs and that the stereochemistry around the bis-THF ring moiety significantly influences the inhibitory effect. The profiles of the structure–activity relationship observed for Δlac-acetogenins were entirely different from those for natural-type acetogenins.

Synthesis of Murisolin, (15R, 16R, 19R, 20S)‐Murisolin A, and (15R, 16R, 19S, 20S)‐16,19‐cis‐Murisolin and Their Inhibitory Action with Bovine Heart Mitochondrial Complex I

The asymmetric total synthesis of murisolin, (15R, 16R, 19R, 20S)-murisolin A, and (15R, 16R, 19S, 20S)-16,19-cis-murisolin was performed by using an epoxy alcohol as a versatile chiral building block for synthesizing the stereoisomers of mono-THF annonaceous acetogenins. The inhibitory activity of these murisolin compounds was examined with bovine heart mitochondrial complex I, and they showed almost the same activity.

Mode of Inhibitory Action of Δlac-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.

Function of the alkyl side chains of Δlac-acetogenins in the inhibitory effect on mitochondrial complex I (NADH-ubiquinone oxidoreductase)

We synthesized a series of Δlac-acetogenins in which the two alkyl side chains were systematically modified, and examined their inhibitory effect on bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). The results revealed that the physicochemical properties of the side chains, such as the balance of hydrophobicity and the width (or bulkiness) of the chains, are important structural factors for a potent inhibitory effect of amphiphilic Δlac-acetogenins. This is probably because such properties decide the precise location of the hydrophilic bis-THF ring moiety in the enzyme embedded in the inner mitochondrial membrane.

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