The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

A synthesis route for puromycin caged with the photo-responsive 7-diethylaminocoumarinyl protecting group carbamate was developed. The inactivation and recovery of the cytotoxic effect of puromycin was tested with a XTT cell viability assay. The uncaging mechanism was studied by ultrafast transient absorption spectroscopy and by time-correlated single photon counting. The combination of these results with quantum-chemical calculations provided detailed insights in dynamics upon excitation. Interestingly, a change of the dipole moment due to structural rearrangements of the amino moiety led to an intermolecular charge transfer on the picosecond time-scale. IR measurements marked the successful uncaging via the release of CO, resulting from the carbamate linker. This decarboxylation constituted the rate-limiting step of the uncaging reaction and occurred on the subsecond timescale. DEACM-puromycin, thus, represents an efficient photo-activatable antibiotic for in-cell applications.

Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7‐N,N‐(diethylaminocumarin‐4‐yl)‐methoxycarbonyl‐puromycin (DEACM‐puromycin) was synthesized and compared with the previously developed 6‐nitroveratryloxycarbonyl puromycin (NVOC‐puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two‐photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light‐controlled puromycylation to visualize new translated proteins in neurons.

Photocleavable protecting groups (PPGs) play a pivotal role in numerous studies. They enable controlled release of small effector molecules to induce biochemical function. The number of PPGs attached to a variety of effector molecules has grown rapidly in recent years satisfying the high demand for new applications. However, until now molecules carrying PPGs have been designed to activate function only in a single direction, namely the release of the effector molecule. Herein, we present the new approach Two-PPGs-One-Molecule (TPOM) that exploits the orthogonal photolysis of two photoprotecting groups to first release the effector molecule and then to modify it to suppress its induced effect. The moiety resembling the tyrosyl side chain of the translation inhibitor puromycin was synthetically modified to the photosensitive ortho-nitrophenyl­alanine that cyclizes upon near UV-irradiation to an inactive puromycin cinnoline derivative. Additionally, the modified puromycin analog was protected by the thio-coumarylmethyl group as the second PPG. This TPOM strategy allows an initial wavelength-selective activation followed by a second light-induced deactivation. Both photolysis processes were spectroscopically studied in the UV/vis- and IR-region. In combination with quantum-chemical calculations and time-resolved NMR spectroscopy, the photoproducts of both activation and deactivation steps upon illumination were characterized. We further probed the translation inhibition effect of the new synthesized puromycin analog upon light activation/deactivation in a cell-free GFP translation assay. TPOM as a new method for precise triggering activation/deactivation of effector molecules represents a valuable addition for the control of biological processes with light.

Cytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure–activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.