Single Proteoliposomes with E. coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks

2017 ◽  
Vol 57 (5) ◽  
pp. 437-445 ◽  
Author(s):  
Johan Berg ◽  
Stephan Block ◽  
Fredrik Höök ◽  
Peter Brzezinski
Keyword(s):  
Proton Pumping ◽  
Quinol Oxidase ◽  
E Coli

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered

Microbiology ◽  
1998 ◽  
Vol 144 (12) ◽  
pp. 3297-3308 ◽  
Author(s):  
G. M. Cook ◽  
C. Loder ◽  
B. Soballe ◽  
G. P. Stafford ◽  
J. Membrillo-Hernandez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Quinol Oxidase ◽  
E Coli ◽  
Cytochrome Bd ◽  
K 12

scholarly journals Rotational catalysis in proton pumping ATPases: From E. coli F-ATPase to mammalian V-ATPase

2012 ◽  
Vol 1817 (10) ◽  
pp. 1711-1721 ◽  
Author(s):  
Masamitsu Futai ◽  
Mayumi Nakanishi-Matsui ◽  
Haruko Okamoto ◽  
Mizuki Sekiya ◽  
Robert K. Nakamoto
Keyword(s):  
Proton Pumping ◽  
E Coli ◽  
Rotational Catalysis

scholarly journals The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship

2009 ◽  
Vol 284 (24) ◽  
pp. 16531-16540 ◽  
Author(s):  
Shane B. Claggett ◽  
Mac O'Neil Plancher ◽  
Stanley D. Dunn ◽  
Brian D. Cain
Keyword(s):  
Atp Synthase ◽  
Spatial Arrangement ◽  
Proton Pumping ◽  
Detectable Effect ◽  
Cross Link ◽  
Reaction Conditions ◽  
E Coli ◽  
Peripheral Stalk ◽  
Photosynthetic Organisms ◽  
Two Sectors

The peripheral stalk of F1F0 ATP synthase is essential for the binding of F1 to FO and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b′ subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463–5471). The spatial arrangement of the chimeric b and b′ subunits, abbreviated Tb and Tb′, has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between TbA83C and Tb′A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist.

scholarly journals Redox analysis of the cytochrome o-type quinol oxidase complex of Escherichia coli reveals three redox components

1991 ◽  
Vol 274 (3) ◽  
pp. 723-730 ◽  
Author(s):  
B Bolgiano ◽  
I Salmon ◽  
W J Ingledew ◽  
R K Poole
Keyword(s):  
Escherichia Coli ◽  
Soret Band ◽  
Oxygen Binding ◽  
Quinol Oxidase ◽  
E Coli ◽  
Cytochrome Bo ◽  
Twin Peaks ◽  
Cytochrome O ◽  
Potential Component ◽  
Maximal Peak

Potentiometric analyses of the cytochrome o-type oxidase of Escherichia coli, using membranes from a strain containing amplified levels of the cytochrome bo complex, were conducted to resolve the redox centres of the oxidase. The cytochrome o-type oxidase of E. coli, a quinol oxidase, contains 2 mol of b-type haem per mol of complex and copper. Detailed analysis of potentiometric titrations, based on the absorbance of the Soret band, suggests that there are three contributions with midpoint potentials (Em,7) around +55 mV, +211 mV and +408 mV, all with maxima at 426-430 nm in the reduced state. In the alpha region of the spectra, a component with Em,6.85 = +58 mV has a maximal peak at 557 nm, and twin peaks at 556 and 564 nm nitrate with Em,6.85 = +227 mV. A feature corresponding to the highest potential Soret contribution was not observed. These data can be explained either by a model incorporating haem-haem interaction or by attributing the shorter-wavelength band (557 nm) to haem b and a split alpha-band (556, 564 nm) to the haem o (oxygen-binding haem b). Absolute spectra of oxidized membranes show continuous absorbance from 460 to 530 nm and suggest the presence of a high-spin haem component in the membranes. Monitoring absorbance at 635 minus 672 nm, contributions with midpoints (Em,7) around +52 mV, +234 mV and +371 mV are observed. This latter contribution is possibly the highest-potential component which titrates with Em greater than +400 mV in the Soret region and may represent copper-haem coupling in the cytochrome o complex.

scholarly journals A Single Point Mutation Converts a Proton-pumping Rhodopsin into a Turn-on Fluorescent Sensor for Chloride

2020 ◽  
Author(s):  
Jasmine Tutol ◽  
Jessica Lee ◽  
Hsichuan Chi ◽  
Farah Faizuddin ◽  
Sameera Abeyrathna ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Single Point ◽  
Proton Pumping ◽  
Single Point Mutation ◽  
E Coli ◽  
Turn On ◽  
Natural Function ◽  
Fluorescent State

<p>The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from <i>Gloeobacter violaceus</i> into GR1, a turn-on fluorescent sensor for chloride in detergent micelles and in live <i>Escherichia coli</i>. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The addition of chloride tunes the p<i>K</i><sub>a </sub>of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single cell fluorescence microscopy experiments with <i>E. coli</i>, expressing a GR1 fusion with cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of chloride. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin fluorescent chloride sensors for cellular applications and learn how proteins can evolve and adapt to bind anions in water.</p>

Resolving the mechanism of proton pumping coupled to hydride transfer in E. coli Transhydrogenase

2018 ◽  
Vol 1859 ◽  
pp. e119
Author(s):  
Simone Graf ◽  
Sangjin Hong ◽  
Robert Gennis ◽  
Christoph von Ballmoos
Keyword(s):  
Hydride Transfer ◽  
Proton Pumping ◽  
E Coli

scholarly journals Cytochrome bd promotes Escherichia coli biofilm antibiotic tolerance by regulating accumulation of noxious chemicals

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Connor J. Beebout ◽  
Levy A. Sominsky ◽  
Allison R. Eberly ◽  
Gerald T. Van Horn ◽  
Maria Hadjifrangiskou
Keyword(s):  
Escherichia Coli ◽  
Quinol Oxidase ◽  
E Coli ◽  
Cytochrome Bd ◽  
External Agents ◽  
Specific Increase ◽  
Matrix Production ◽  
Noxious Chemicals ◽  
Biofilm Development ◽  
Outer Membrane Permeability

AbstractNutrient gradients in biofilms cause bacteria to organize into metabolically versatile communities capable of withstanding threats from external agents including bacteriophages, phagocytes, and antibiotics. We previously determined that oxygen availability spatially organizes respiration in uropathogenic Escherichia coli biofilms, and that the high-affinity respiratory quinol oxidase cytochrome bd is necessary for extracellular matrix production and biofilm development. In this study we investigate the physiologic consequences of cytochrome bd deficiency in biofilms and determine that loss of cytochrome bd induces a biofilm-specific increase in expression of general diffusion porins, leading to elevated outer membrane permeability. In addition, loss of cytochrome bd impedes the proton mediated efflux of noxious chemicals by diminishing respiratory flux. As a result, loss of cytochrome bd enhances cellular accumulation of noxious chemicals and increases biofilm susceptibility to antibiotics. These results identify an undescribed link between E. coli biofilm respiration and stress tolerance, while suggesting the possibility of inhibiting cytochrome bd as an antibiofilm therapeutic approach.

scholarly journals A Single Point Mutation Converts a Proton-pumping Rhodopsin into a Turn-on Fluorescent Sensor for Chloride

2020 ◽  
Author(s):  
Jasmine Tutol ◽  
Jessica Lee ◽  
Hsichuan Chi ◽  
Farah Faizuddin ◽  
Sameera Abeyrathna ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Single Point ◽  
Proton Pumping ◽  
Single Point Mutation ◽  
E Coli ◽  
Turn On ◽  
Natural Function ◽  
Fluorescent State

<p>The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from <i>Gloeobacter violaceus</i> into GR1, a turn-on fluorescent sensor for chloride in detergent micelles and in live <i>Escherichia coli</i>. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The addition of chloride tunes the p<i>K</i><sub>a </sub>of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single cell fluorescence microscopy experiments with <i>E. coli</i>, expressing a GR1 fusion with cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of chloride. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin fluorescent chloride sensors for cellular applications and learn how proteins can evolve and adapt to bind anions in water.</p>

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