The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Characterization of the 9.5-kDa ubiquinone-binding protein of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

A small polypeptide subunit of the NADH:ubiquinone reductase (complex I) from Neurospora crassa has been identified by photoaffinity labeling to participate in the binding of ubiquinone [Heinrich, H., & Werner, S. (1992) Biochemistry (preceding paper in this issue)]. This polypeptide is further characterized by its primary structure and by an assessment of its localization within complex I. A lambda gt11 cDNA expression library was screened using a specific antibody directed against this individual subunit of complex I. Two groups of clones, coding for polypeptide subunits of the appropriate apparent molecular weight, were isolated. One group was shown to contain the relevant recombinants. The derived amino acid sequence for the 9.5-kDa ubiquinone-binding polypeptide shows a similarity with a putative ubiquinol-binding subunit (also a 9.5-kDa polypeptide) from complex III of bovine heart [Usui, S., Yu, L., & Tu, C.-A. (1990) Biochemistry 29, 4618-4626]. The polypeptide has a hydrophobic stretch of a sufficient length to span the membrane. It resists against extraction with NaBr or Na2CO3, and therefore probably is buried in the so-called hydrophobic membrane portion of complex I. This nuclearly-encoded subunit lacks a typical cleavable presequence and is imported into isolated mitochondria by a membrane potential-dependent process.     

Functional properties of the alternative NADH:ubiquinone oxidoreductase from E. coli through comparative 3-D modelling

The alternative NADH:ubiquinone oxidoreductase (NDH-2) from Escherichia coli is a membrane protein playing a prominent role in respiration by linking the reduction of NADH to the quinone pool. Remote sequence similarity reveals an evolutionary relation between alternative NADH:quinone oxidoreductases and the SCOP-family "FAD/NAD-linked reductases". We have created a structural model for NDH-2 from E. coli through comparative modelling onto a template from this family. Combined analysis of our model and sequence conservation allowed us to include the cofactor FAD and the substrate NADH in atomic detail. Furthermore, we propose the most plausible orientation of NDH-2 relative to the membrane and specify a region of the protein potentially involved in ubiquinone binding.     

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.     

Benzothiadiazole inhibits mitochondrial NADH:ubiquinone oxidoreductase in tobacco

An inducer of acquired disease resistance in plants, benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester, exhibited direct, concentration-dependent inhibition of the NADH:ubiquinone oxidoreductase activity of complex I of the mitochondrial electron transport chain of cultured tobacco cells. The complex I activity was less sensitive to inhibition by salicylic acid, an endogenous activator of acquired disease resistance. Using a dichlorodihydrofluorescein assay, it was found that benzothiadiazole, salicylic acid and the complex I inhibitor rotenone, increased reactive oxygen species production within cells in a concentration-dependent manner. The results indicate that both benzothiadiazole and salicylic acid affect the mitochondria of treated plant cells and result in increased production of reactive oxygen species. The biochemical basis of this response could be related to the inhibition of the NADH:ubiquinone oxidoreductase activity of complex I that results in channelling of electrons via complex II, with concomitant higher levels of superoxide production.     

Mitochondrial dynamics in human NADH:ubiquinone oxidoreductase deficiency

Mitochondrial NADH:ubiquinone oxidoreductase or complex I (CI) is a frequently affected enzyme in cases of mitochondrial disorders. However, the cytopathological mechanism of the associated pediatric syndromes is poorly understood. Evidence in the literature suggests a connection between mitochondrial metabolism and morphology. Previous quantitative analysis of mitochondrial structure in cultured fibroblasts of 14 patients revealed that mitochondria were fragmented and/or less branched in patients with severe CI deficiency. These patient cells also displayed greatly increased levels of reactive oxygen species (ROS) and marked aberrations in mitochondrial and cellular Ca²⁺/ATP handling upon hormone stimulation. Here, we discuss the interrelationship between these parameters and demonstrate that the hormone-induced increase in mitochondrial Ca²⁺ and ATP concentration, as well as the rate of cytosolic Ca²⁺ removal, are not related to mitochondrial length and/or degree of branching, but decrease as a function of the number of mitochondria per cell. This suggests that the amount of mitochondria, and not their shape, is important for Ca²⁺-induced stimulation of mitochondrial ATP generation to feed cytosolic ATP-demanding processes.     

Inhibition of mitochondrial NADH:ubiquinone oxidoreductase by ethoxyformic anhydride

The NADH:ubiquinone, but not the NADH:ferricyanide, reductase activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) is inhibited by incubation of the enzyme at pH 6.0 and 0 degree C with ethoxyformic anhydride (EFA), and the inhibition is partially reversed by subsequent incubation of EFA-treated complex I with hydroxylamine. These results and spectral changes of EFA-treated complex I in the u.v. region are consistent with modification of essential histidyl or tyrosyl residues between the primary NADH dehydrogenase and the site of ubiquinone reduction. Treatment of complex I with EFA in the presence of high concentrations of Seconal or Demerol did not protect against EFA inactivation, suggesting that the site of EFA modification may not be the same as the inhibiton sites of Seconal and Demerol. However, the presence of NADH during incubation of complex I with EFA greatly enhanced the inhibition rate, indicating that the reduced conformation of complex I is more susceptible to attack by EFA.     

Mitochondrial NADH:ubiquinone reductase: complementary DNA sequence of the import precursor of the bovine 75-kDa subunit

The 75-kDa subunit of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria is its largest subunit and is a component of the iron-sulfur (IP) fragment of the enzyme. It is encoded in nuclear DNA and is imported into the organelle. Protein sequences have been determined at the N-terminus of the intact protein and on fragments generated by partial cleavage with cyanogen bromide and with Staphylococcus aureus protease V8. Parts of these data have been used to design two mixtures of oligonucleotides 17 bases long, containing 192 and 256 different sequences, which have been synthesized and used as hybridization probes for identification of cognate cDNA clones. Two different but overlapping clones have been isolated, and the sequences of the cloned DNAs have been determined. Together they code for a precursor of the 75-kDa subunit of complex I. The mature protein is 704 amino acids in length, has a calculated molecular mass of 75,961 daltons, and contains no segments of sequence that could be folded into hydrophobic alpha-helixes of sufficient length to span the inner membrane of the mitochondrion. Its precursor has an N-terminal extension of 23 amino acids to specify its import into the mitochondrion from the cytoplasm. Seventeen cysteine residues are dispersed throughout the 75-kDa subunit; some of them are close to each other in the sequence in three separate groups and, by analogy with other iron-sulfur proteins, could be involved in iron-sulfur clusters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca(2+) movements and the association of abnormal Ca(2+) homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.     

Mitochondrial NADH:ubiquinone oxidoreductase alterations are associated with endometriosis

Genetic alterations and aberrant expression of ‘mitochondrial membrane complex I’ (MMC-I) underlie several complex human disorders, but no reports are documented to date in endometriosis. Sequencing of mitochondrially encoded MMC-I subunits revealed 72 mutations of which 2 missense (G10398A; A13603A/G) mutations and 1 synonymous (T10400C) mutation showed higher prevalence in patients. In silico functional analysis predicted A13603A/G, a novel heteroplasmy as a ‘damaging variant’. Our results indicate higher endometriosis risk for haplotype ‘10398A/10400C/13603AG’ and haplogroup ‘N’. Immunohistochemical analysis revealed elevated MMC-I expression in eutopic endometria of patients compared to controls. In conclusion, MMC-I alterations may constitute an inheritable risk factor for endometriosis.     

Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I)

Three-dimensional structures of NADH:ubiquinone oxidoreductase (or complex I) from the respiratory chain of mitochondria and bacteria have been recently studied by electron microscopy. The low-resolution structures all reveal a characteristic L shape for complex I; however, some of the differences among these structures may have important implications for the location of the functional elements of complex I, for example, the ubiquinone-binding site.     

Mitigation of NADH: ubiquinone oxidoreductase deficiency by chronic Trolox treatment

Deficiency of mitochondrial NADH:ubiquinone oxidoreductase (complex I), is associated with a variety of clinical phenotypes such as Leigh syndrome, encephalomyopathy and cardiomyopathy. Circumstantial evidence suggests that increased reactive oxygen species (ROS) levels contribute to the pathogenesis of these disorders. Here we assessed the effect of the water-soluble vitamin E derivative Trolox on ROS levels, and the amount and activity of complex I in fibroblasts of six children with isolated complex I deficiency caused by a mutation in the NDUFS1, NDUFS2, NDUFS7, NDUFS8 or NDUFV1 gene. Patient cells displayed increased ROS levels and a variable decrease in complex I activity and amount. For control cells, the ratio between activity and amount was 1 whereas for the patients this ratio was below 1, indicating a defect in intrinsic catalytic activity of complex I in the latter cells. Trolox treatment dramatically reduced ROS levels in both control and patient cells, which was paralleled by a substantial increase in the amount of complex I. Although the ratio between the increase in activity and amount of complex I was exactly proportional in control cells it varied between 0.1 and 0.8 for the patients. Our findings suggest that the expression of complex I is regulated by ROS. Furthermore, they provide evidence that both the amount and intrinsic activity of complex I are decreased in inherited complex I deficiency. The finding that Trolox treatment increased the amount of complex I might aid the future development of antioxidant treatment strategies for patients. However, such treatment may only be beneficial to patients with a relatively small reduction in intrinsic catalytic defect of the complex.     

NADH/NAD+ interaction with NADH: ubiquinone oxidoreductase (complex I)

The quantitative data on the binding affinity of NADH, NAD(+), and their analogues for complex I as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. The redox-dependency of the nucleotide binding and the reductant-induced change of FMN affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of complex I.     

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.     

Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM. NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM. Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP. The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency. The effect of pH on Vmax was studied. The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.     

Identification of two tetranuclear FeS clusters on the ferredoxin-type subunit of NADH:ubiquinone oxidoreductase (complex I)

The proton-translocating NADH:ubiquinone oxidoreductase of respiratory chains (complex I) contains one flavin mononucleotide and five EPR-detectable iron-sulfur clusters as redox groups. Because of the number of conserved motifs typical for binding iron-sulfur clusters and the high content of iron and acid-labile sulfide of complex I preparations, it is predicted that complex I contains additional clusters which have not yet been detected by EPR spectroscopy. To search for such clusters, we used a combination of UV/vis and EPR spectroscopy to study complex I from Neurospora crassa and Escherichia coli adjusted to distinct redox states. We detected a UV/vis redox difference spectrum characterized by negative absorbances at 325 and 425 nm that could not be assigned to the known redox groups. Redox titration was used to determine the pH-independent midpoint potential to be -270 mV, being associated with the transfer of two electrons. Comparison with UV/vis difference spectra obtained from complex I fragments and related enzymes showed that this group is localized on subunit Nuo21.3c of the N. crassa or NuoI of the E. coli complex I, respectively. This subunit (the bovine TYKY) belongs to a family of 8Fe-ferredoxins which contain two tetranuclear iron-sulfur clusters as redox groups. We detected EPR signals in a fragment of complex I which we attribute to the novel FeS clusters of complex I.