Exploring interactions between the 49 kDa and ND1 subunits in mitochondrial NADH-ubiquinone oxidoreductase (complex I) by photoaffinity labeling

Quinazolines are strong inhibitors of NADH-ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Using a photoreactive quinazoline, [(125)I]AzQ, and bovine heart submitochondrial particles (SMPs), we demonstrated previously that [(125)I]AzQ binds at the interface of the 49 kDa and ND1 subunits in complex I; it labeled a site in the N-terminal (Asp41-Arg63) region of the 49 kDa subunit, suggesting that this region contacts the ND1 subunit [Murai, M., et al. (2009) Biochemistry 48, 688-698]. The labeled region of ND1 could not be identified because it is highly hydrophobic, and the SMPs did not yield sufficient amounts of labeled protein. Here, we describe how photoaffinity labeling of isolated complex I by [(125)I]AzQ yielded sufficient material for identification of the labeled region of the ND1 subunit. The inhibition of the isolated enzyme by AzQ is comparable to that of SMPs. Our results reveal that the labeled site in ND1 is between Asp199 and Lys262, mostly likely in the third matrix loop that connects the fifth and sixth transmembrane helices. Thus, our results reveal new information about the interface between the hydrophilic and hydrophobic domains of complex I, a region that is thought to be important for ubiquinone reduction and energy transduction.     

Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy

The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol-cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient.     

Bis-THF motif of acetogenin binds to the third matrix-side loop of ND1 subunit in mitochondrial NADH-ubiquinone oxidoreductase

Natural acetogenins are among the most potent inhibitors of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I). Our photoaffinity labeling study suggested that the hydroxylated bis-THF ring moiety of acetogenins binds at "site A" in the third matrix-side loop connecting the fifth and sixth transmembrane helices in the ND1 subunit [Kakutani et al. (2010) Biochemistry 49, 4794-4803]. Nevertheless, since this proposition was led using a photoreactive Δlac-acetogenin derivative, it needs to be directly verified using a natural acetogenin-type probe. We therefore conducted photoaffinity labeling using a photoreactive natural acetogenin mimic ([(125)I]diazinylated natural acetogenin, [(125)I]DANA), which has a small photolabile diazirine group, in place of a hydroxy group, attached to the bis-THF ring moiety. Analysis of the photocross-linked protein in bovine heart submitochondrial particles unambiguously revealed that [(125)I]DANA binds to the membrane subunit ND1 with high specificity. The photocross-linking was completely blocked in the presence of just a 5-fold excess of bullatacin, indicating that [(125)I]DANA is an excellent mimic of natural acetogenins and hence binds to the site that accommodates natural products. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by different proteases and their combinations indicated that the cross-linked residue is predominantly located at the supposed site A in the third matrix-side loop.     

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association.

1. The NADH-ubiquinone oxidoreductase complex (Complex I) and the ubiquinol-cytochrome c oxidoreductase complex (Complex III) combine in a 1:1 molar ratio to give NADH-cytochrome c oxidoreductase (Complex I-Complex III). 2. Experiments on the inhibition of the NADH-cytochrome c oxidoreductase activity of mixtures of Complexes I and III by rotenone and antimycin indicate that electron transfer between a unit of Complex I-Complex III and extra molecules of Complexes I or III does not contribute to the overall rate of cytochrome c reduction. 3. The reduction by NADH of the cytochrome b of mixtures of Complexes I and III is biphasic. The extents of the fast and slow phases of reduction are determined by the proportion of the total Complex III specifically associated with Complex I. 4. Activation-energy measurements suggest that the structural features of the Complex I-Complex III unit promote oxidoreduction of endogenous ubiquinone-10.     

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 approximately 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.     

The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Restoration of ubiquinone-pool behaviour.

1. In the inner mitochondrial membrane, dehydrogenases and cytochromes appear to act independently of each other, and electron transport has been proposed to occur through a mobile pool of ubiquinone-10 molecules [Kröger & Klingenberg (1973) Eur. J. Biochem. 34, 358--368]. 2. Such behaviour can be restored to the interaction between purified Complex I and Complex III by addition of phospholipid and ubiquinone-10 to a concentrated mixture of the Complexes before dilution. 3. A model is proposed for the interaction of Complex I with Complex III in the natural membrane that emphasizes relative mobility of the Complexes rather than ubiquinone-10. Electron transfer occurs only through stoicheiometric Complex I-Complex III units, which, however, are formed and re-formed at rates higher than the rate of electron transfer.     

EPR studies of iron-sulfur clusters in isolated subunits and subfractions of NADH-ubiquinone oxidoreductase

The thermodynamic and EPR characteristics of the iron-sulfur clusters of NADH-ubiquinone oxidoreductase have been examined in various subfractions and subunits of the enzyme. These were obtained by fragmentation of the enzyme with chaotropic agents and detergent and salt fractionation. We provide evidence for the presence of three tetranuclear clusters and five or six binuclear clusters, accounting well for the chemically determined iron content of this enzyme (22-24 atoms/molecule of FMN). Some of the clusters can be identified with EPR-detectable species in intact NADH-ubiquinone oxidoreductase and, by combining information on subunit topography and spin-spin interactions between redox centers in the native enzyme, we propose a tentative scheme for the spatial organization of these iron-sulfur clusters in the enzyme and in the membrane.     

The antihypertensive drug carvedilol inhibits the activity of mitochondrial NADH-ubiquinone oxidoreductase

A study is presented on the interaction of carvedilol with mitochondria isolated from several rat organs. It is shown that carvedilol causes a moderate uncoupling effect under non phosphorylating succinate supported respiration of intact mitochondria, as well as a marked inhibition of coupled respiration with NAD-dependent substrates. The inhibitory effect was also found in the bovine heart purified Complex I as well as in experiments with mitochondrial particles, where the individual redox segments of the respiratory chain were analysed. It is also shown that carvedilol, though exhibiting an intrinsic scavenger activity, caused reactive oxygen species to be produced as a consequence of its inhibitory effect on the steady-state respiration. Under these conditions the pro-oxidant activity of carvedilol appears to prevail over its scavenging activity, and a net generation of ROS is promoted.     

Molecular defects of NADH-ubiquinone oxidoreductase (Complex I) in mitochondrial diseases

Defects in Complex I of the mitochondrial respiratory chain have been identified in 38 patients. The clinical and laboratory features are reviewed and the results of recently devised strategies aimed at characterizing the primary molecular and genetic abnormalities are presented. Although not exhaustive, these studies have provided a molecular basis for the contention that defects in Complex I may have their origin in nuclear or in mitochondrial genes.     

A spin label study of the lipid boundary layer of mitochondrial NADH-ubiquinone oxidoreductase

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I) is a lipoprotein enzyme containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin. Enzyme preparations containing endogenous cardiolipin and a range of either soyabean PC or dimyristoylphosphatidylcholine (DMPC) concentrations have been made. Using a spin-labelled fatty acid, two probe environments differing in mobility have been shown to be present. The fatty acid probe has a relative binding constant (or partition coefficient between lipid and protein) of unity. The boundary layer or lipid annulus reported by the probe has a value of approx. 300 lipid molecules per molecule of enzyme FMN in preparations containing soyabean PC, or DMPC above the phase transition temperature of the latter. In soyabean PC-replaced enzyme the apparent size of the boundary layer is independent of temperature between 30 degrees C and 14 degrees C but shows a modest increase to about 400 lipid molecules per molecule of FMN between 14 degrees C and 2 degrees C. Complex I replaced with high concentrations of DMPC gives non-linear Arrhenius plots of NADH-ubiquinone oxidoreductase activity. The results of the ESR experiments show that both boundary layer and bulk lipid must be motionally restricted for this to occur. Thus, the change in activity is probably not caused by an effect exerted directly on the catalytic activity of the enzyme but is more likely due to restriction of free diffusion of ubiquinone to its site of reduction.     

Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.     

Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling.

NADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[(3)H]pyridaben ([(3)H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP(+)) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP(+), stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I.     

Riboflavin enhances the assembly of mitochondrial cytochrome c oxidase in C. elegans NADH-ubiquinone oxidoreductase mutants

Mitochondrial respiratory chain dysfunction is responsible for a large variety of early and late-onset diseases. NADH-ubiquinone oxidoreductase (complex I) defects constitute the most commonly observed mitochondrial disorders. We have generated Caenorhabditis elegans strains with mutations in the 51 kDa active site subunit of complex I. These strains exhibit decreased NADH-dependent respiration and lactic acidosis, hallmark features of complex I deficiency. Surprisingly, the mutants display a significant decrease in the amount and activity of cytochrome c oxidase (complex IV). The metabolic and reproductive fitness of the mutants is markedly improved by riboflavin. In this study, we have examined how the assembly and activity of complexes I and IV are affected by riboflavin. Our results reveal that the mutations result in variable steady-state levels of different complex I subunits and in a significant reduction in the amount of COXI subunit. Using native gel electrophoresis, we detected assembly intermediates for both complexes I and IV. Riboflavin promotes the assembly of both complexes, resulting in increased catalytic activities. We propose that one primary pathogenic mechanism of some complex I mutations is to destabilize complex IV. Enhancing complex I assembly with riboflavin results in the added benefit of partially reversing the complex IV deficit.     

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.     

Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase

Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.     

An analysis of the polypeptide composition of bovine heart mitochondrial NADH-ubiquinone oxidoreductase by two-dimensional polyacrylamide-gel electrophoresis.

Purified preparations of Complex I (NADH-ubiquinone oxidoreductase) from bovine heart mitochondria may be resolved into 26 polypeptides by two-dimensional analysis combining isoelectric focusing and polyacrylamide-gel electrophoresis in sodium dodecyl sulphate. Similar analyses of the fragments obtained from chaotropic resolution of the enzyme show that each of these fragments contains a distinct and non-overlapping set of polypeptides. Evidence that the polypeptides seen in the intact enzyme are true constituents comes from analyses of immunoprecipitates obtained by allowing Complex I or solubilized submitochondrial particles to react with antisera directed against the whole enzyme and a subfragment of the enzyme.     

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.     

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

We synthesized a series of Deltalac-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 Deltalac-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.     

The role of the ESSS protein in the assembly of a functional and stable mammalian mitochondrial complex I (NADH-ubiquinone oxidoreductase).

The ESSS protein is a recently identified subunit of mammalian mitochondrial complex I. It is a relatively small integral membrane protein (122 amino acids) found in the beta-subcomplex. Genomic sequence database searches reveal its localization to the X-chromosome in humans and mouse. The ESSS cDNA from Chinese hamster cells was cloned and shown to complement one complementation group of our previously described mutants with a proposed X-linkage. Sequence analyses of the ESSS cDNA in these mutants revealed chain termination mutations. In two of these mutants the protein is truncated at the C-terminus of the targeting sequence; the mutants are null mutants for the ESSS subunit. There is no detectable complex I assembly and activity in the absence of the ESSS subunit as revealed by blue native polyacrylamide gel electrophoresis (BN/PAGE) analysis and polarography. Complex I activity can be restored with ESSS subunits tagged with either hemagglutinin (HA) or hexahistidine (His6) epitopes at the C-terminus. Although, the accumulation of ESSS-HA is not dependent upon the presence of mtDNA-encoded subunits (ND1-6,4 L), it is incorporated into complex I only in presence of compatible complex I subunits from the same species.