Publications by authors named "Jo Hoeser"

7 Publications

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Stabilization of the Highly Hydrophobic Membrane Protein, Cytochrome Oxidase, on Metallic Surfaces for Direct Electrochemical Studies.

Molecules 2020 Jul 16;25(14). Epub 2020 Jul 16.

Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg, CNRS, 67081 Strasbourg, France.

The cytochrome oxidase catalyzes the reduction of oxygen to water in bacteria and it is thus an interesting target for electrocatalytic studies and biosensor applications. The oxidase is completely embedded in the phospholipid membrane. In this study, the variation of the surface charge of thiol-modified gold nanoparticles, the length of the thiols and the other crucial parameters including optimal phospholipid content and type, have been performed, giving insight into the role of these factors for the optimal interaction and direct electron transfer of an integral membrane protein. Importantly, all three tested factors, the lipid type, the electrode surface charge and the thiol length mutually influenced the stability of films of the cytochrome oxidase. The best electrocatalytic responses were obtained on the neutral gold surface when the negatively charged phosphatidylglycerol (PG) was used and on the charged gold surface when the zwitterionic phosphatidylethanolamine (PE) was used. The advantages of the covalent binding of the membrane protein to the electrode surface over the non-covalent binding are also discussed.
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http://dx.doi.org/10.3390/molecules25143240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397230PMC
July 2020

Low cost, microcontroller based heating device for multi-wavelength differential scanning fluorimetry.

Sci Rep 2018 01 23;8(1):1457. Epub 2018 Jan 23.

Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstr. 21, 79104, Freiburg i. Br., Germany.

Differential scanning fluorimetry is a popular method to estimate the stability of a protein in distinct buffer conditions by determining its 'melting point'. The method requires a temperature controlled fluorescence spectrometer or a RT-PCR machine. Here, we introduce a low-budget version of a microcontroller based heating device implemented into a 96-well plate reader that is connected to a standard fluorescence spectrometer. We demonstrate its potential to determine the 'melting point' of soluble and membranous proteins at various buffer conditions.
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http://dx.doi.org/10.1038/s41598-018-19702-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5780519PMC
January 2018

Reduction of the off-pathway iron-sulphur cluster N1a of Escherichia coli respiratory complex I restrains NAD dissociation.

Sci Rep 2017 08 18;7(1):8754. Epub 2017 Aug 18.

Albert-Ludwigs-Universität, Institut für Biochemie, Albertstr. 21, Chemie-Hochhaus, 79104, Freiburg i. Br., Germany.

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The reaction starts with NADH oxidation by a flavin cofactor followed by transferring the electrons through a chain of seven iron-sulphur clusters to quinone. An eighth cluster called N1a is located proximally to flavin, but on the opposite side of the chain of clusters. N1a is strictly conserved although not involved in the direct electron transfer to quinone. Here, we show that the NADH:ferricyanide oxidoreductase activity of E. coli complex I is strongly diminished when the reaction is initiated by an addition of ferricyanide instead of NADH. This effect is significantly less pronounced in a variant containing N1a with a 100 mV more negative redox potential. Detailed kinetic analysis revealed that the reduced activity is due to a lower dissociation constant of bound NAD. Thus, reduction of N1a induces local structural rearrangements of the protein that stabilise binding of NAD. The variant features a considerably enhanced production of reactive oxygen species indicating that bound NAD represses this process.
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http://dx.doi.org/10.1038/s41598-017-09345-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562879PMC
August 2017

Significance of [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I.

Biochemistry 2017 06 25;56(22):2770-2778. Epub 2017 May 25.

Spemann Graduate School of Biology and Medicine (SGBM), Albert-Ludwigs-University , Freiburg, Germany.

NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with proton translocation across the membrane. NADH reduces a noncovalently bound FMN, and the electrons are transported further to the quinone reduction site by a 95 Å long chain of seven iron-sulfur (Fe-S) clusters. Binuclear Fe-S cluster N1a is not part of this long chain but is located within electron transfer distance on the opposite site of FMN. The relevance of N1a to the mechanism of complex I is not known. To elucidate its role, we individually substituted the cysteine residues coordinating N1a of Escherichia coli complex I by alanine and serine residues. The mutations led to a significant loss of the NADH oxidase activity of the mutant membranes, while the amount of the complex was only slightly diminished. N1a could not be detected by electron paramagnetic resonance spectroscopy, and unexpectedly, the content of binuclear cluster N1b located on a neighboring subunit was significantly decreased. Because of the lack of N1a and the partial loss of N1b, the variants did not survive detergent extraction from the mutant membranes. Only the C97A variant retained N1a and was purified by chromatographic steps. The preparation showed a slightly diminished NADH/ferricyanide oxidoreductase activity, while the NADH:decyl-ubiquinone oxidoreductase activity was not affected. N1a of this preparation showed unusual spectroscopic properties indicating a different ligation. We discuss whether N1a is involved in the physiological electron transfer reaction.
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http://dx.doi.org/10.1021/acs.biochem.6b01058DOI Listing
June 2017

Creation of a gold nanoparticle based electrochemical assay for the detection of inhibitors of bacterial cytochrome bd oxidases.

Bioelectrochemistry 2016 Oct 7;111:109-14. Epub 2016 Jun 7.

Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg, CNRS, Strasbourg, France. Electronic address:

Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections.
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http://dx.doi.org/10.1016/j.bioelechem.2016.06.001DOI Listing
October 2016

Cytochrome bd Displays Significant Quinol Peroxidase Activity.

Sci Rep 2016 06 9;6:27631. Epub 2016 Jun 9.

Department of Biotechnology, Delft University of Technology, The Netherlands.

Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme.
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http://dx.doi.org/10.1038/srep27631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4899803PMC
June 2016

Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site.

FEBS Lett 2014 May 26;588(9):1537-41. Epub 2014 Mar 26.

Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Albertstr. 21, 79104 Freiburg i. Br., Germany. Electronic address:

Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.
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http://dx.doi.org/10.1016/j.febslet.2014.03.036DOI Listing
May 2014
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