Escherichia coli cytochrome bd-II: an energy converting quinol peroxidase

• Applicant: Professor Dr. Thorsten Friedrich
• Subject Area: Biochemistry ; Metabolism, Biochemistry and Genetics of Microorganisms ; Structural Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 577813180

Cytochrome bd enzyme complexes are part of bacterial and archaeal respiratory chains. Members of this enzyme family are not present in eukaryotes making them a promising therapeutic target for next-generation antimicrobials. It is known that cytochrome bd catalyzes the reduction of molecular oxygen to water coupled with the oxidation of quinols. In doing so, it contributes to the generation of the protonmotive force (pmf) by releasing protons from quinol oxidation to the outside and taking protons for dioxygen reduction from the inside of the cell. Furthermore, cytochrome bd features several other functions as it protects dioxygen-labile protein, protects cells from heat stress and other environmental stressors and is part of bacterial defense mechanisms against antibiotic-induced stress. Surprisingly, Escherichia coli contains two highly similar isoforms called cytochrome bd-I and cytochrome bd-II. Due to structural and functional similarity it is thought that they have similar functions and could be interchangeable. However, the corresponding genes are differentially expressed and there are minor structural and functional differences between the two enzymes. Thus, the physiological role of cytochrome bd-II remains unclear as does its suitability as a target for antimicrobials. Initial experiments show that cytochrome bd-II has a low quinol oxidase but a high quinol peroxidase activity in agreement with its proposed role as detoxifying enzyme. According to its architecture, the complex will catalyze a novel energy converting quinol peroxidase activity contributing to the generation of the pmf by transmembrane charge separation. In this project we will determine the steady-state kinetics of the quinol oxidase and the quinol peroxidase reaction of cytochrome bd-II under anoxic conditions. The specificity constant of both reactions will reveal whether the native enzyme is an oxidase or a peroxidase. We will use a combination of He-temperature EPR-, stopped-flow UV/vis-, FTIR- and Raman-spectroscopy to identify intermediates of the oxidase and the peroxidase reaction. Spectra will be compared to those obtained with cytochrome bd-I under the identical experimental conditions. The structure of both complexes will be determined by cryogenic electron microscopy. Grids will be prepared under anoxic conditions and samples will be incubated with various quinols, hydrogen peroxide and various mixtures thereof. From the data, a mechanism of the peroxidase reaction will be derived. We will reconstitute cytochrome bd in liposomes and measure a proton gradient across the proteoliposome membranes by using optical dyes. The restricted permeability of hydrogen peroxide across the membrane will help to detect a transient gradient in stopped-flow measurements. All experiments will strongly benefit from the use of approximately 50 different site-directed cytochrome bd-I and bd-II mutants generated by our group.

• DFG Programme: Research Grants