NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

Schuler, Franz; Yano, Takahiro; Bernardo, Salvatore Di; Yagi, Takao; Yankovskaya, Victoria; Singer, Thomas P.; Casida, J. E.

doi:10.1073/pnas.96.7.4149

Cited by 162 publications

(134 citation statements)

References 38 publications

(34 reference statements)

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“…This was, in part, based on photoaffinity labeling of NuoB from membranes of Paracoccus nitrificans and Thermus thermophilus with a 3 H-labeled pyridaben analog (31). In the same study, both rotenone and piericidin A prevented binding of the 3 H-labeled pyridaben analog (another pesticide) (31). This is compatible with the results of the present study, where compound A-resistant mutants had missense mutations in either the C terminus of nuoD or in nuoB.…”

Section: Discussionsupporting

confidence: 81%

“…In addition, they suggested that the binding site for the polar head of ubiquinone is located at the interface of NuoD and NuoB. This was, in part, based on photoaffinity labeling of NuoB from membranes of Paracoccus nitrificans and Thermus thermophilus with a 3 H-labeled pyridaben analog (31). In the same study, both rotenone and piericidin A prevented binding of the 3 H-labeled pyridaben analog (another pesticide) (31).…”

Section: Discussionmentioning

confidence: 95%

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Molecular Characterization of Benzimidazole Resistance in Helicobacter pylori

Mills

Yang

MacCormack

2004

Antimicrob Agents Chemother

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A family of benzimidazole derivatives (BI) was shown to possess potent and selective activity against Helicobacter pylori, although the precise cellular target of the BIs is unknown. Spontaneous H. pylori mutants were isolated as resistant to a representative BI (compound A). Genomic DNA was isolated from a BI-resistant mutant, transformed into a BI-sensitive strain, and found to be sufficient to confer BI resistance. The resistance determinant was localized to a 17-kb clone after screening a lambda-based genomic library constructed from the BI-resistant strain. Upon sequencing and mapping onto the H. pylori strain J99 genome, the 17-kb clone was shown to contain the entire nuo operon (NADH:ubiquinone oxidoreductase). Further subcloning and DNA sequencing revealed that a single point mutation in nuoD was responsible for BI resistance. The mutation resulted in a G398S amino acid change at the C terminus of NuoD. Thirty-three additional spontaneous BI-resistant mutants were characterized. Sequencing of nuoD from 32 isolated mutants revealed three classes of missense mutation resulting in amino acid changes in NuoD: G398S, F404S, and V407M. One BI-resistant isolate did not have a mutation in nuoD. Instead, a T27A amino acid change was identified in NuoB. MIC testing of the wild-type H. pylori strain and four classes of nuo mutants revealed that all NuoD mutant classes were hypersensitive to rotenone, a known inhibitor of complex I (NADH:ubiquinone oxidoreductase) suggested to bind to NuoD. Further, a nuoD knockout verified that it is essential in H. pylori and may be the target of the BI compounds.

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 95%

Molecular Characterization of Benzimidazole Resistance in Helicobacter pylori

Mills

Yang

MacCormack

2004

Antimicrob Agents Chemother

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“…Ubiquinone-analogous complex I inhibitors like 2-decyl-4-quinazolinyl amine (DQA), 1 rotenone, and pyridaben are thought to prevent the terminal electron transfer step from cluster N2 to ubiquinone (7). Using a trifluoromethyl derivative of pyridaben for photoaffinity labeling, the PSST subunit was identified as high-affinity target (11), suggesting that this subunit is in close vicinity to the ubiquinone binding site of complex I.…”

mentioning

confidence: 99%

Two Aspartic Acid Residues in the PSST-Homologous NUKM Subunit of Complex I from Yarrowia lipolytica Are Essential for Catalytic Activity

Garofano

Zwicker

Kerscher

et al. 2003

Journal of Biological Chemistry

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Mitochondrial proton-translocating NADH:ubiquinone oxidoreductase (complex I) couples the transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the mitochondrial inner membrane. Subunit PSST is the most likely carrier of iron-sulfur cluster N2, which has been proposed to play a crucial role in ubiquinone reduction and proton pumping. To explore the function of this subunit we have generated site-directed mutants of all eight highly conserved acidic residues in the Yarrowia lipolytica homologue, the NUKM protein. Mutants D99N and D115N had only 5 and 8% of the wild type catalytic activity, respectively. In both cases complex I was stably assembled but electron paramagnetic resonance spectra of the purified enzyme showed a reduced N2 signal (about 50%). In terms of complex I catalytic activity, almost identical results were obtained when the aspartates were individually changed to glutamates or to glycines. Mutations of other conserved acidic residues had less dramatic effects on catalytic activity and did not prevent assembly of iron-sulfur cluster N2. This excludes all conserved acidic residues in the PSST subunit as fourth ligands of this redox center. The results are discussed in the light of the structural similarities to the homologous small subunit of watersoluble [NiFe] hydrogenases.Proton-translocating NADH:ubiquinone oxidoreductase (complex I) is the first of five complexes of oxidative phosphorylation in the mitochondrial inner membrane. In complex I, two electrons are transported from NADH to ubiquinone via FMN and eight ironsulfur clusters (1). In this process four protons are translocated across the membrane (2). The mechanism of this process and the location and nature of the ubiquinone binding sites are still unclear (3). Evidence obtained with bovine heart submitochondrial particles strongly suggests that iron-sulfur cluster N2 is directly involved in ubiquinone reduction: based on paramagnetic interaction, the distance between an ubisemiquinone radical and cluster N2 was estimated to be 8-11 Å (4). Subunit PSST is the most likely carrier of iron-sulfur cluster N2. Site-directed mutagenesis of the homologue subunits in Escherichia coli (5) and Neurospora crassa (6) indicated that three cysteines of subunit PSST are ligands for cluster N2. These cysteines correspond to positions 86, 150, and 180 in the PSST homologous NUKM subunit of Yarrowia lipolytica. A glutamate residue (Glu-89 in Y. lipolytica) had been proposed as the fourth ligand (7), but this could be excluded by site-directed mutagenesis (8). Thus, the fourth ligand of cluster N2 is still not known. Albracht and co-workers (9) have proposed that complex I contains two iron-sulfur clusters N2 that have identical electron paramagnetic resonance (EPR) spectra and are bound by the two ferredoxin-like Fe 4 S 4 binding motifs in the TYKY subunit. However, these two clusters in the TYKY subunit have been shown to be EPR-silent and designated N6a and N6b by Friedrich and coworkers (10). Ubiquinone-analogous...

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“…This poses the problem of how the redox chemistry taking place in the peripheral arm can drive proton translocation across the membrane arm. Evidence from different laboratories (10,11) and the identification of several pathogenic mutations (12) suggested a key mechanistic role for the 49-kDa, PSST, and TYKY subunits. Based on these indications and a well established homology (13) between the 49-kDa and PSST subunits of complex I and the large and small subunits of [NiFe] hydrogenases, we proposed that at least part of the ubiquinone binding pocket and possibly the proton translocation machinery of complex I have evolved from the domains surrounding the [NiFe] site of the hydrogenase (14).…”

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confidence: 99%

Functional Implications from an Unexpected Position of the 49-kDa Subunit of NADH:Ubiquinone Oxidoreductase

Zickermann

Bostina

Hunte

et al. 2003

Journal of Biological Chemistry

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Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.Complex I (NADH:ubiquinone oxidoreductase) 1 is the major entry point for electrons into the respiratory chain. By linking redox chemistry to vectorial proton translocation, complex I converts up to 40% of the energy used in mitochondria to make ATP. The molecular mechanism of catalysis and its structural basis is not at all understood. Complex I from bovine heart mitochondria is composed of more than 40 different subunits adding to a molecular mass of almost 1000 kDa (1). The enzyme from the strictly aerobic yeast Yarrowia lipolytica is of similar size and has been established as a model system to study eucaryotic complex I employing yeast genetics (2, 3). A major obstacle to progress in understanding this extremely large and complicated enzyme complex has been the lack of detailed structural information. Resolutions in the 20 -30 Å range have been obtained by electron microscopy of single particles from different organisms that show an L-shaped structure with a hydrophobic arm residing in the membrane and a peripheral arm protruding into the mitochondrial matrix space (2, 4 -6). Recently, projection maps from two-dimensional crystals of the bovine enzyme have been obtained at 13 Å resolution (7). have reported that the enzyme from Escherichia coli may change from an L shape to a horseshoe-like shape at zero ionic strength.The minimal form of the enzyme consists of 14 subunits that are found in both bacterial and mitochondrial complex I (9). In higher eucaryotes seven highly hydrophobic polypeptides are encoded by the mitochondrial genome. All redox prosthetic groups that have been identified so far (eight iron-sulfur clusters and one FMN) are associated with a total of seven hydrophilic and nuclear-coded polypeptides. This poses the problem of how the redox chemistry taking place in the peripheral arm can drive proton translocation across the membrane arm. Evidence from different laboratories (10, 11) and the identification of several pathogenic mutations (12) suggested a key mechanistic role for the 49-kDa, PSST, and TYKY subunits. Based on these indications and a well established homology (13) between the 49-kDa and PSST subunits of complex I and the large and small subunits of [NiFe] hydrogenases, we proposed th...

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NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

Cited by 162 publications

References 38 publications

Molecular Characterization of Benzimidazole Resistance in Helicobacter pylori

Molecular Characterization of Benzimidazole Resistance in Helicobacter pylori

Two Aspartic Acid Residues in the PSST-Homologous NUKM Subunit of Complex I from Yarrowia lipolytica Are Essential for Catalytic Activity

Functional Implications from an Unexpected Position of the 49-kDa Subunit of NADH:Ubiquinone Oxidoreductase

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