Jo Hoeser

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

cydB , cydA , and cydX physically interacts by affinity technology (1, 2)

Cytochrome bd Displays Significant Quinol Peroxidase Activity

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.

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

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.

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

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 C97AE 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.

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

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.

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

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.