Cytochrome bd quinol:O2 oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b595 is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.

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The reduction of oxygen to water is crucial to life and a central metabolic process. To fulfil this task, prokaryotes use among other enzymes cytochrome bd oxidases (Cyt bds) that also play an important role in bacterial virulence and antibiotic resistance. To fight microbial infections by pathogens, an in‐depth understanding of the enzyme mechanism is required. Here, we combine bioinformatics, mutagenesis, enzyme kinetics and FTIR spectroscopy to demonstrate that proton delivery to the active site contributes to the rate limiting steps in Cyt bd‐I and involves Asp58 of subunit CydB. Our findings reveal a previously unknown catalytic function of subunit CydB in the reaction of Cyt bd‐I.

The cytochrome bd oxidase catalyzes the reduction of oxygen to water in bacteria and it is thus an interesting target for electrocatalytic studies and biosensor applications. The bd 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 bd 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.