Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking.

The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.     

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.     

Identification of the dicyclohexylcarbodiimide-binding subunit of NADH-ubiquinone oxidoreductase (Complex I)

The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and this inhibition correlates with incorporation of radioactivity from [14C]DCCD into a Complex I subunit of Mr 29,000 (Yagi, T. (1987) Biochemistry 26, 2822-2828). Resolution of [14C]DCCD-labeled Complex I in the presence of NaClO4 showed that the labeled Mr 29,000 subunit was in the hydrophobic fraction of the enzyme. This fraction, which contains greater than 17 unlike polypeptides, was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the Mr 29,000 subunit, containing bound [14C]DCCD, was isolated and purified. The amino acid composition and partial sequence of this subunit corresponded to those predicted from the mitochondrial DNA for the product of the mtDNA gene designated ND-1. The identity of the Mr 29,000 subunit with the ND-1 gene product was further confirmed by immunoblotting and immunoprecipitation experiments, using the hydrophobic fraction of [14C]DCCD-labeled Complex I and antiserum to a C-terminal undecapeptide synthesized on the basis of the human mitochondrial ND-1 nucleotide sequence. Thus, it appears that the DCCD-binding subunits of the respiratory chain Complexes I, III, and IV and in certain organisms the DCCD-binding subunit of the ATP synthase complex (Complex V) are all mtDNA products.     

The organization of NADH dehydrogenase polypeptides in the inner mitochondrial membrane.

The organization of the constituent polypeptides of mitochondrial NADH dehydrogenase was studied by using two membrane-impermeable probes, diazobenzene[35S]sulphonate and lactoperoxidase-catalysed radioiodination. The incorporation of label into the subunits of the isolated enzyme was compared with that obtained with enzyme immunoprecipitated from labelled mitochondria or inverted submitochondrial particles. On the basis of accessibility to these two labels, we divide the polypeptides of Complex I into five groups: those that are apparently buried in the enzyme, those that are accessible to labelling in the isolated enzyme but not in the membrane, those that are exposed on the cytoplasmic face of the membrane, those that are exposed on the matrix face and finally those that are exposed on both faces and are therefore transmembranous. We conclude that NADH dehydrogenase is asymmetrically organized across the inner mitochondrial membrane.     

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.     

A reductase/isomerase subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of the complex.

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.     

Specific modification of a Na+ binding site in NADH:quinone oxidoreductase from Klebsiella pneumoniae with dicyclohexylcarbodiimide

The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases Na+) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na+-translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N'-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for Na+ binding. At low Na+ concentrations (0.6 mM), DCCD inhibited both quinone reduction and Na+ transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM Na+, NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [14C]DCCD was prevented, indicating that Na+ and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue of the ND1 subunit from mitochondrial complex I. It is proposed that Na+ binds to the NuoH subunit during NADH-driven Na+ transport by NDH-1.     

The NADH-binding subunit of respiratory chain complex I is nuclear-encoded in plants and identified only in mitochondria

In higher plants, genes for subunits of respiratory chain complex I (NADH:ubiquinone oxidoreductase) have so far been identified solely in organellar genomes. At least nine subunits are encoded by the mitochondrial DNA and 11 homologues by the plastid DNA. One of the 'key' components of complex I is the subunit binding the substrate NADH. The corresponding gene for the mitochondrial subunit has now been cloned and identified in the nuclear genome from potato ( Solanum tuberosum ). The mature protein consists of 457 amino acids and is preceded by a mitochondrial targeting sequence of 30 amino acids. The protein is evolutionarily related to the NADH-binding subunits of complex I from other eukaryotes and is well conserved in the structural domains predicted for binding the substrate NADH, the FMN and one iron-sulphur cluster. Expression examined in different potato tissues by Northern blot analysis shows the highest steady-state mRNA levels in flowers.
Precursor proteins translated in vitro from the cDNA are imported into isolated potato mitochondria in a ΔΨ-dependent manner. The processed translation product has an apparent molecular mass of 55 kDa, identical to the mature protein present in the purified plant mitochondrial complex I. However, the in-vitro translated protein is not imported into isolated chloroplasts. To further investigate whether the complex I-like enzyme in chloroplasts contains an analogous subunit for binding of NAD(P)H, different plastid protein fractions were tested with a polyclonal antiserum directed against the bovine 51 kDa NADH-binding subunit. In none of the different thylakoid or stroma protein fractions analysed were specific crossreactive polypeptides detected. These results are discussed particularly with respect to the structure of a potential complex I in chloroplasts and the nature of its acceptor site.     

One- and two-electron reduction of ubiquinone homologs by NADH- dehydrogenase preparations from the mitochondrial respiratory chain

The mechanism of ubiquinone homologs reduction by different preparations of mitochondrial NADH dehydrogenase: complex I within submitochondrial particles, isolated NADH-ubiquinone oxidoreductase and soluble low molecular weight NADH dehydrogenase, has been investigated. It has been shown that NADH oxidation via the rotenone-insensitive reaction is associated with one-electron reduction of low molecular weight ubiquinone homologs (Q0, Q1, Q2) to semiquinone with subsequent fast oxidation of the latter by atmospheric oxygen to form a superoxide radical. The two-electron ubiquinone reduction to quinol in the rotenone-sensitive reaction is unaccompanied by the semiquinone release from the enzyme active center into the surrounding solution.     

Studies on the structure of NADH:ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur flavoprotein component

A catalytic component of the bovine mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a soluble NADH dehydrogenase iron-sulfur flavoprotein (FP). FP is composed of three subunits of Mr 51,000, 24,000, and 9,000, and contains FMN and two iron-sulfur clusters. Previous studies by others with the use of various chemical probes had suggested that, except for an access for NADH to the 51-kDa subunit, the FP polypeptides are buried within Complex I and shielded from the medium. In the present study, monospecific antibodies were raised to each of the three FP subunits, and used in conjunction with Complex I, submitochondrial particles (SMP), mitoplasts, and intact mitochondria as sources of antigens. Results of enzyme-linked immunosorbent assays and 125I-protein A labeling experiments indicated that epitopes from the 51-, 24-, and 9-kDa subunits of FP are exposed to the medium in Complex I and SMP, but not in mitoplasts and mitochondria. Appropriate enzymatic assays showed that none of the antibodies inhibited the NADH dehydrogenase activity of isolated FP or the NADH oxidase activity of SMP. These results have been discussed in relation to the structure of Neurospora Complex I deduced from membrane crystals of the isolated enzyme complex by Leonard et al. [K. Leonard, H. Haiker, and H. Weiss (1987) J. Mol. Biol. 194, 277-286].     

The polypeptide composition of the mitochondrial NADH: ubiquinone reductase complex from several mammalian species.

The polypeptide composition of isolated mitochondrial NADH:ubiquinone reductase (NADH dehydrogenase) is very similar to that of material immunoprecipitated from detergent-solubilized bovine heart submitochondrial particles by antisera to the holoenzyme. The specificity of the antisera for dehydrogenase polypeptides was determined by immunoblotting, which showed that antisera reacting with only a few proteins were able to immunoprecipitate all others in parallel. The polypeptide compositions of rat, rabbit and human NADH dehydrogenase were determined by immunoprecipitation of the enzyme from solubilized submitochondrial particles and proved to be very similar to that of the bovine heart enzyme, particularly in the high-Mr region. Further homologies in these and other species were explored by immunoblotting with antisera to the holoenzyme and monospecific antisera raised against iron-sulphur-protein subunits of the enzyme.     

A novel strong competitive inhibitor of complex I

Alkaline incubation of NADH results in the formation of a very potent inhibitor of complex I (NADH:ubiquinone oxidoreductase). Mass spectroscopy (molecular mass equal to 696) and absorption spectroscopy suggest that the inhibitor is derived from attachment of two oxygen atoms to the nicotinamide moiety of NADH. The inhibitor is competitive with respect to NADH with a K(i) of about 10(-8)M. The inhibitor efficiently suppresses NADH-oxidase, NADH-artificial acceptor reductase, and NADH-quinone reductase reactions catalyzed by submitochondrial particles, as well as the reactions catalyzed by either isolated complex I or the three subunit flavoprotein fragment of complex I.     

Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography.

In order to isolate the mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase EC 1.6.99.3) from wheat, we developed a one-step immunoaffinity procedure using antibodies raised against the NAD9 subunit. By native electrophoresis we showed that the antibodies are able to recognize the NAD9 subunit on the complex in its native form, therefore allowing the immunoaffinity chromatography. The complex retained on the column proved to be a functional complex I, since the preparation showed NADH:duroquinone and NADH:FeK3(CN)6 reductase activities which were inhibited by rotenone. The pattern of the protein subunits (about 30) eluted from the purified complex showed a high level of similarities with complex I purified from potato and broad bean by conventional techniques. Twelve subunits were identified by cross-reactions with antibodies against heterologous complex I subunits including mitochondrial- and nuclear-encoded proteins. In order to study the genetic origin of the subunits, we purified wheat complex I after in organello labelling of mitochondrial-encoded polypeptides. We found that no other complex I subunit than those corresponding to the nine mitochondrial nad genes sequenced so far, is encoded in the mitochondria of wheat.     

Studies on the structure of NADH: ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur protein component

Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of Mr 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490-496]. IP contains six subunits of Mr 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts.     

Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.     

Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [14C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [14C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity.     

cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.     

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.     

Reversible dissociation of flavin mononucleotide from the mammalian membrane-bound NADH: ubiquinone oxidoreductase (complex I)

Conditions for the reversible dissociation of flavin mononucleotide (FMN) from the membrane-bound mitochondrial NADH:ubiquinone oxidoreductase (complex I) are described. The catalytic activities of the enzyme, i.e. rotenone-insensitive NADH:hexaammineruthenium III reductase and rotenone-sensitive NADH:quinone reductase decline when bovine heart submitochondrial particles are incubated with NADH in the presence of rotenone or cyanide at alkaline pH. FMN protects and fully restores the NADH-induced inactivation whereas riboflavin and flavin adenine dinucleotide do not. The data show that the reduction of complex I significantly weakens the binding of FMN to protein thus resulting in its dissociation when the concentration of holoenzyme is comparable with K(d ( approximately 10(-8)M at pH 10.0).     

The role of glycine residues 140 and 141 of subunit B in the functional ubiquinone binding site of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na(+)-NQR with its electron acceptor, ubiquinone.