Ion translocation by the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

2005 ◽  
Vol 33 (4) ◽  
pp. 836-839 ◽  
Author(s):  
T. Friedrich ◽  
S. Stolpe ◽  
D. Schneider ◽  
B. Barquera ◽  
P. Hellwig
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Fourier Transform ◽  
Complex I ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
E Coli ◽  
Ion Translocation ◽  
Activity Generation ◽  
Respiratory Complex I ◽  
Energy Converting

The energy-converting NADH:ubiquinone oxidoreductase, also known as respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of ions across the membrane. It was assumed that the complex exclusively works as a proton pump. Recently, it has been proposed that complex I from Klebsiella pneumoniae and Escherichia coli work as Na+ pumps. We have used an E. coli complex I preparation to determine the type of ion(s) translocated by means of enzyme activity, generation of a membrane potential and redox-induced Fourier-transform infrared spectroscopy. We did not find any indications for Na+ translocation by the E. coli complex I.

Nucleotide-induced conformational changes in the Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

2008 ◽  
Vol 36 (5) ◽  
pp. 971-975 ◽  
Author(s):  
Thomas Pohl ◽  
Daniel Schneider ◽  
Ruth Hielscher ◽  
Stefan Stolpe ◽  
Katerina Dörner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Conformational Changes ◽  
Complex I ◽  
Proton Translocation ◽  
Attenuated Total Reflection ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Respiratory Complex I ◽  
Part Structure ◽  
Energy Converting

The energy-converting NADH:ubiquinone oxidoreductase, also known as 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 consisting of a peripheral and a membrane arm. The peripheral arm contains all known cofactors and the NADH-binding site, whereas the membrane arm has to be involved in proton translocation. Owing to this, a conformation-linked mechanism for redox-driven proton translocation is discussed. By means of electron microscopy, we show that both arms of the Escherichia coli complex I are widened after the addition of NADH but not of NADPH. NADH-induced conformational changes were also detected in solution: ATR-FTIR (attenuated total reflection Fourier-transform infrared) of the soluble NADH dehydrogenase fragment of the complex indicates protein re-arrangements induced by the addition of NADH. EPR spectroscopy of surface mutants of the complex containing a covalently bound spin label at distinct positions demonstrates NADH-dependent conformational changes in both arms of the complex.

scholarly journals Semiquinone and Cluster N6 Signals in His-tagged Proton-translocating NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

2013 ◽  
Vol 288 (20) ◽  
pp. 14310-14319 ◽  
Author(s):  
Madhavan Narayanan ◽  
David J. Gabrieli ◽  
Steven A. Leung ◽  
Mahmoud M. Elguindy ◽  
Carl A. Glaser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Complex I ◽  
Genetic Manipulation ◽  
Affinity Purification ◽  
Power Level ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Saturation Power

NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ∼15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mm−1 cm−1 at 274 nm and 50.3 mm−1 cm−1 at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ∼12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.

scholarly journals Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Franziska Nuber ◽  
Johannes Schimpf ◽  
Jean-Paul di Rago ◽  
Déborah Tribouillard-Tanvier ◽  
Vincent Procaccio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Complex I ◽  
Proton Translocation ◽  
Gene Mutations ◽  
Stable Complex ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Common Source ◽  
Respiratory Complex ◽  
Quinone Reduction ◽  
Respiratory Complex I

AbstractNADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients’ tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253AMT-ND5 mutation completely disturbed the assembly of complex I, while the mutation D199GMT-ND1 led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199MT-ND1 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199MT-ND1 for energy conservation in complex I is discussed.

Detecting antimicrobial resistance in Escherichia coli using benchtop attenuated total reflectance-Fourier transform infrared spectroscopy and machine learning

The Analyst ◽  
2021 ◽  
Vol 146 (20) ◽  
pp. 6211-6219
Author(s):  
Hewa G. S. Wijesinghe ◽  
Dominic J. Hare ◽  
Ahmed Mohamed ◽  
Alok K. Shah ◽  
Patrick N. A. Harris ◽  
...  
Keyword(s):  
Machine Learning ◽  
Escherichia Coli ◽  
Fourier Transform ◽  
Infrared Spectroscopy ◽  
Antimicrobial Resistance ◽  
Fourier Transform Infrared Spectroscopy ◽  
Attenuated Total Reflectance ◽  
Total Reflectance ◽  
E Coli ◽  
Machine Learning Model

ATR–FTIR with a machine learning model predicts ESBL genotype of unknown E. coli strains with 86.5% AUC.

scholarly journals Structure of the deactive state of mammalian respiratory complex I

2017 ◽  
Author(s):  
James N. Blaza ◽  
Kutti R. Vinothkumar ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Bos Taurus ◽  
Atp Synthesis ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Structural Elements ◽  
Highly Active ◽  
Mammalian Mitochondria ◽  
Energy Conserving ◽  
Respiratory Complex I ◽  
Active Preparation

AbstractComplex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, ‘active’ complex I gradually enters a pronounced resting or ‘deactive’ state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly-active preparation of Bos taurus complex I into the biochemically-defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. The deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding templates their reordering. Our structure both rationalizes biochemical data on the deactive state, and offers new insights into its physiological and cellular roles.

scholarly journals Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation

2019 ◽  
Vol 201 (9) ◽  
Author(s):  
Birgit Schilling ◽  
Nathan Basisty ◽  
David G. Christensen ◽  
Dylan Sorensen ◽  
James S. Orr ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Regulatory Mechanism ◽  
Growth Conditions ◽  
Lysine Acetylation ◽  
Label Free ◽  
Content Type ◽  
E Coli ◽  
Regulatory Mode ◽  
Specific Sugar

ABSTRACT Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli. IMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.

scholarly journals Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms andEscherichia coli

1998 ◽  
Vol 64 (3) ◽  
pp. 1018-1023 ◽  
Author(s):  
I. Tryland ◽  
L. Fiksdal
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Environmental Water ◽  
Coliform Bacteria ◽  
Galactosidase Activity ◽  
Fluorogenic Substrates ◽  
Rapid Methods ◽  
E Coli ◽  
Aerococcus Viridans

ABSTRACT Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities ofBacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified asAeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

scholarly journals Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I

2017 ◽  
Vol 114 (48) ◽  
pp. 12737-12742 ◽  
Author(s):  
Justin G. Fedor ◽  
Andrew J. Y. Jones ◽  
Andrea Di Luca ◽  
Ville R. I. Kaila ◽  
Judy Hirst
Keyword(s):  
Electron Transfer ◽  
Complex I ◽  
Mammalian Cells ◽  
Membrane Surface ◽  
Proton Translocation ◽  
Structural Data ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Respiratory Complex ◽  
Respiratory Complex I ◽  
Ubiquinone 10

Respiratory complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in mammalian cells, powers ATP synthesis by using the energy from electron transfer from NADH to ubiquinone-10 to drive protons across the energy-transducing mitochondrial inner membrane. Ubiquinone-10 is extremely hydrophobic, but in complex I the binding site for its redox-active quinone headgroup is ∼20 Å above the membrane surface. Structural data suggest it accesses the site by a narrow channel, long enough to accommodate almost all of its ∼50-Å isoprenoid chain. However, how ubiquinone/ubiquinol exchange occurs on catalytically relevant timescales, and whether binding/dissociation events are involved in coupling electron transfer to proton translocation, are unknown. Here, we use proteoliposomes containing complex I, together with a quinol oxidase, to determine the kinetics of complex I catalysis with ubiquinones of varying isoprenoid chain length, from 1 to 10 units. We interpret our results using structural data, which show the hydrophobic channel is interrupted by a highly charged region at isoprenoids 4–7. We demonstrate that ubiquinol-10 dissociation is not rate determining and deduce that ubiquinone-10 has both the highest binding affinity and the fastest binding rate. We propose that the charged region and chain directionality assist product dissociation, and that isoprenoid stepping ensures short transit times. These properties of the channel do not benefit the exhange of short-chain quinones, for which product dissociation may become rate limiting. Thus, we discuss how the long channel does not hinder catalysis under physiological conditions and the possible roles of ubiquinone/ubiquinol binding/dissociation in energy conversion.

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