Photoaffinity Capture Compounds to Profile the Magic Spot Nucleotide Interactomes.

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.

H2O2 selectively damages the binuclear iron-sulfur cluster N1b of respiratory complex I.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism by coupling electron transfer with proton translocation. Electron transfer is catalyzed by a flavin mononucleotide and a series of iron-sulfur (Fe/S) clusters. As a by-product of the reaction, the reduced flavin generates reactive oxygen species (ROS). It was suggested that the ROS generated by the respiratory chain in general could damage the Fe/S clusters of the complex. Here, we show that the binuclear Fe/S cluster N1b is specifically damaged by H2O2, however, only at high concentrations. But under the same conditions, the activity of the complex is hardly affected, since N1b can be easily bypassed during electron transfer.

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.