Fenpyroximate binds to the interface between PSST and 49 kDa subunits in mitochondrial NADH-ubiquinone oxidoreductase

Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003) Biochemistry 42, 746-754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1-15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010) Nature 465, 441-445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[(125)I]-4-azidophenyl fenpyroximate ([(125)I]APF) and [(125)I]-3-azido-5-iodobenzyl fenpyroximate ([(125)I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that [(125)I]APF and [(125)I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [(125)I]APF and [(125)I]AIF are located in the Ser43-Arg66 (PSST) and Asp160-Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I.     

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.     

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.     

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

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

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.     

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.     

Relation of superoxide generation and lipid peroxidation to the inhibition of NADH-Q oxidoreductase by rotenone, piericidin A, and MPP+.

The addition of NADH to submitochondrial particles inhibited by agents which interrupt electron transport from NADH-Q oxidoreductase (Complex I) to Q10 (rotenone, piericidin A, and MPP+) results in superoxide formation and lipid peroxidation. A study of the quantitative relations now shows that oxyradical formation does not appear to be the direct result of the inhibition. Although tetraphenyl boron (TPB) greatly enhances the inhibition by MPP+, it has no effect on O2. formation or lipid peroxidation. When submitochondrial particles completely inhibited by rotenone or piericidin A are treated with bovine serum albumin to remove spuriously bound inhibitor molecules without affecting those bound at the specific inhibition site, NADH-Q activity remains inhibited and lipid peroxidation occurs but superoxide formation ceases. Thus oxyradical formation may be the result of the binding of inhibitors at sites in the membrane other than those related to the inhibition of electron transport.     

The Origin of Cluster N2 of the Energy-Transducing NADH–Quinone Oxidoreductase: Comparisons of Phylogenetically Related Enzymes

NADH–quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient ( ). Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.     

The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I

The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[125I](trifluoromethyl)phenyldiazirinylacetogenin, [125I]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [125I]TDA, radioactivity was predominantly incorporated into an approximately 30 kDa band on a SDS gel. Blue native gel electrophoresis of the [125I]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at approximately 30 kDa. Immnoprecipitation of the [125I]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [125I]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of Deltalac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of Deltalac-acetogenins differs from that of traditional inhibitors.     

Evidence that the blockade of mitochondrial respiration by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) involves binding at the same site as the respiratory inhibitor, rotenone

It has been postulated that 1-methyl-4-phenylpyridinium (MPP+) blocks mitochondrial respiration by combining at the same site as rotenone, a potent inhibitor of NADH oxidation in mitochondria, known to act at the junction of NADH dehydrogenase and coenzyme Q (CoQ). The present experiments show that MPP+ and two of its analogs indeed act in a concentration dependent manner to prevent the binding of [14C]-rotenone to submitochondrial particles (ETP) and significantly decrease the inhibition of electron transport caused by rotenone. It therefore appears that MPP+ binds at the same site as rotenone or an adjacent site, supporting the hypothesis that its neurotoxic action is due to the inhibition of mitochondrial respiration.     

Quinone binding and reduction by respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.     

Interaction of 1-Methyl-4-Phenylpyridinium Ion (MPP+) and Its Analogs with the Rotenone/Piericidin Binding Site of NADH Dehydrogenase

Nigrostriatal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease results from the inhibition of mitochondrial respiration by 1-methyl-4-phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4'-analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4'-heptyl-MPP+, being 6 microM. The 4'-analogs compete with rotenone for the binding site in a concentration-dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4'-propyl-MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.     

Spin labeling of the Escherichia coli NADH ubiquinone oxidoreductase (complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron microscopy revealed the two-part structure of the complex with a peripheral arm involved in electron transfer and a membrane arm most likely involved in proton translocation. It was proposed that the quinone binding site is located at the joint of the two arms. Most likely, proton translocation in the membrane arm is enabled by the energy of the electron transfer reaction in the peripheral arm transmitted by conformational changes. For the detection of the conformational changes and the localization of the quinone binding site, we set up a combination of site-directed spin labeling and EPR spectroscopy. Cysteine residues were introduced to the surface of the Escherichia coli complex I. The spin label (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate (MTSL) was exclusively bound to the engineered positions. Neither the mutation nor the labeling had an effect on the NADH:decyl-ubiquinone oxidoreductase activity. The characteristic signals of the spin label were detected by EPR spectroscopy, which did not change by reducing the preparation with NADH. A decyl-ubiquinone derivative with the spin label covalently attached to the alkyl chain was synthesized in order to localize the quinone binding site. The distance between a MTSL labeled complex I variant and the bound quinone was determined by continuous-wave (cw) EPR allowing an inference on the location of the quinone binding site. The distances between the labeled quinone and other complex I variants will be determined in future experiments to receive further geometry information by triangulation.     

A small isoform of NADH:ubiquinone oxidoreductase (complex I) without mitochondrially encoded subunits is made in chloramphenicol-treated Neurospora crassa

In mitochondria of Neurospora crassa grown in the presence of chloramphenicol a small form of NADH:ubiquinone reductase is made in place of the normal electron-transfer-complex I. This smaller enzyme has a molecular mass of approximately 350 kDa and consists of (at least) 13 different subunits which are all synthesized in the cytoplasm. The complex I which is normally found in Neurospora has a molecular mass of approximately 700 kDa and consists of around 30 different subunits, of which at least six are made in the mitochondria. Immunoblotting and peptide mapping suggest that the subunits of the small enzyme are homologous to subunits of the large enzyme, one subunit might even be identical. The small and the large NADH:ubiquinone reductases have the same high-affinity binding site for NADH but the two enzymes differ in the affinity and inhibitor sensitivity of the ubiquinone-binding site. The possibility is discussed that the small NADH:ubiquinone reductase is primitive isoform of complex I.     

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

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

Na+ translocation by the NADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae

Complex I is the site for electrons entering the respiratory chain and therefore of prime importance for the conservation of cell energy. It is generally accepted that the complex I-catalysed oxidation of NADH by ubiquinone is coupled specifically to proton translocation across the membrane. In variance to this view, we show here that complex I of Klebsiella pneumoniae operates as a primary Na+ pump. Membranes from Klebsiella pneumoniae catalysed Na+-stimulated electron transfer from NADH or deaminoNADH to ubiquinone-1 (0.1-0.2 micromol min-1 mg-1). Upon NADH or deaminoNADH oxidation, Na+ ions were transported into the lumen of inverted membrane vesicles. Rate and extent of Na+ transport were significantly enhanced by the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP) to values of approximately 0.2 micromol min-1 mg-1 protein. This characterizes the responsible enzyme as a primary Na+ pump. The uptake of sodium ions was severely inhibited by the complex I-specific inhibitor rotenone with deaminoNADH or NADH as substrate. N-terminal amino acid sequence analyses of the partially purified Na+-stimulated NADH:ubiquinone oxidoreductase from K. pneumoniae revealed that two polypeptides were highly similar to the NuoF and NuoG subunits from the H+-translocating NADH:ubiquinone oxidoreductases from enterobacteria.     

ND3 and ND4L subunits of mitochondrial complex I, both nucleus encoded in Chlamydomonas reinhardtii, are required for activity and assembly of the enzyme

Made of more than 40 subunits, the rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. In vascular plants, fungi, and animals, at least seven complex I subunits (ND1, -2, -3, -4, -4L, -5, and -6; ND is NADH dehydrogenase) are coded by mitochondrial genes. The role of these highly hydrophobic subunits in the enzyme activity and assembly is still poorly understood. In the unicellular green alga Chlamydomonas reinhardtii, the ND3 and ND4L subunits are encoded in the nuclear genome, and we show here that the corresponding genes, called NUO3 and NUO11, respectively, display features that facilitate their expression and allow the proper import of the corresponding proteins into mitochondria. In particular, both polypeptides show lower hydrophobicity compared to their mitochondrion-encoded counterparts. The expression of the NUO3 and NUO11 genes has been suppressed by RNA interference. We demonstrate that the absence of ND3 or ND4L polypeptides prevents the assembly of the 950-kDa whole complex I and suppresses the enzyme activity. The putative role of hydrophobic ND subunits is discussed in relation to the structure of the complex I enzyme. A model for the assembly pathway of the Chlamydomonas enzyme is proposed.     

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH:ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide:oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.     

Post-translational Modifications near the Quinone Binding Site of Mammalian Complex I

Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N^G and ω-N^G′ nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.