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

Zickermann, Volker; Bostina, Mihnea; Hunte, Carola; Ruíz, Teresa; Radermacher, Michael; Brandt, Ulrich

doi:10.1074/jbc.m302713200

Cited by 80 publications

(66 citation statements)

References 43 publications

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“…Therefore, it is not clear how the membrane-integral quinones can oxidize the cytoplasmic Cys180 and Cys241. In a similar case, it has been shown that reduced quinones reduce periplasmic Cys residues of the membrane-bound protein disulfide reductase DsbB (17), and the quinone site of the NADH:ubiquinone oxidoreductase seems to be distant from the membrane, too (290). Presumably, the membrane-integral quinones are able to react at sites outside the membrane, when hydrophobic parts of the protein enable emerging of the reactive head group from the membrane.…”

Section: Membrane-anchored Hks With C-terminal Cytoplasmic Sensing Domentioning

confidence: 97%

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

Mascher

Helmann

Unden

2006

Microbiol Mol Biol Rev

615

652

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SUMMARY Two-component signal-transducing systems are ubiquitously distributed communication interfaces in bacteria. They consist of a histidine kinase that senses a specific environmental stimulus and a cognate response regulator that mediates the cellular response, mostly through differential expression of target genes. Histidine kinases are typically transmembrane proteins harboring at least two domains: an input (or sensor) domain and a cytoplasmic transmitter (or kinase) domain. They can be identified and classified by virtue of their conserved cytoplasmic kinase domains. In contrast, the sensor domains are highly variable, reflecting the plethora of different signals and modes of sensing. In order to gain insight into the mechanisms of stimulus perception by bacterial histidine kinases, we here survey sensor domain architecture and topology within the bacterial membrane, functional aspects related to this topology, and sequence and phylogenetic conservation. Based on these criteria, three groups of histidine kinases can be differentiated. (i) Periplasmic-sensing histidine kinases detect their stimuli (often small solutes) through an extracellular input domain. (ii) Histidine kinases with sensing mechanisms linked to the transmembrane regions detect stimuli (usually membrane-associated stimuli, such as ionic strength, osmolarity, turgor, or functional state of the cell envelope) via their membrane-spanning segments and sometimes via additional short extracellular loops. (iii) Cytoplasmic-sensing histidine kinases (either membrane anchored or soluble) detect cellular or diffusible signals reporting the metabolic or developmental state of the cell. This review provides an overview of mechanisms of stimulus perception for members of all three groups of bacterial signal-transducing histidine kinases.

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Section: Membrane-anchored Hks With C-terminal Cytoplasmic Sensing Domentioning

confidence: 97%

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

Mascher

Helmann

Unden

2006

Microbiol Mol Biol Rev

615

652

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“…Purification of Complex I-Unsealed mitochondrial membranes were prepared from parental and mutant haploid strains (nukm::LEU2, ura3, leu2, lys11, pUB4-nukm mut /Hyg) as described previously (17,19). Complex I was purified by extraction of mitochondrial membranes with dodecyl maltoside followed by Ni 2ϩ -agarose and size exclusion chromatography (17).…”

Section: Methodsmentioning

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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“…The location shown for NuoB takes into account the direct interaction demonstrated between subunits NuoB and NuoA (47). The location of NuoCD is according to recent antibody labeling studies (48). The fact that NuoCD was readily dissociated from the otherwise intact complex under some conditions (Figs.…”

Section: Subunits Nuol and Nuom Are Removed From Complex I In Diheptamentioning

confidence: 99%

The Location of NuoL and NuoM Subunits in the Membrane Domain of the Escherichia coli Complex I

Holt

Morgan

Sazanov

2003

Journal of Biological Chemistry

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The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,Ndimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.NADH:ubiquinone oxidoreductase (complex I, EC 1.6.5.3) is the first enzyme of the respiratory chains of most mitochondria and many bacteria. It catalyzes the transfer of two electrons from NADH to quinone, coupled to the translocation of about 4 protons across the membrane (for reviews, see Refs. 1-4). Complex I is one of the largest known membrane protein complexes. The bovine enzyme has a mass of ϳ980 kDa and is composed of about 46 subunits, including the seven hydrophobic ND subunits encoded in the mitochondrial genome (5). The simplest version of the complex in terms of protein content is the prokaryotic enzyme that has 13-14 subunits and a combined molecular mass of about 550 kDa (6). All of the subunits of bacterial complex I (also referred to as NDH-1) have analogues in the mitochondrial enzyme (4). Because of its relatively simple subunit composition, NDH-1 represents a useful "minimal" model to study the structure and function of complex I. It is currently the least understood component of the respiratory chain. In contrast to other enzymes of the respiratory chain, the atomic structure of complex I is not known and the mechanisms of proton pumping and electron transfer are not established.Electron microscopy has shown that both the mitochondrial and the bacterial enzyme have a characteristic L-shaped structure. One arm is embedded in the membrane and the other, the peripheral arm, protrudes into the mitochondrial matrix or bacterial cytoplasm. This was demonstrated at about 20 to 30 Å resolution for the enzyme from fungus Neurospora crassa (7, 8), yeast Yarrowia lipolytica (9), beef heart mitochondria (10), Escherichia coli (11,12), and the thermophile Aqu...

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

Cited by 80 publications

References 43 publications

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

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

The Location of NuoL and NuoM Subunits in the Membrane Domain of the Escherichia coli Complex I

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