Visualizing the mechanism of quinol oxidation and inhibition of a bd-type oxidase using cryo-EM

Cytochrome bd is a prokaryotic terminal oxidase recognized as an antibiotic target against various pathogens. Despite its critical role in respiration, failure to capture the mechanism of enzyme catalysis and inhibition prohibits structure guided drug discovery. Here, we present cryo-electron microscopy structures of Escherichia coli cytochrome bd-I in monomeric and dimeric forms, along all stages of quinol turn-over and in an inhibitor-bound state. We identify a dynamic Q-loop lid that undergoes a disorder-to-order transition upon substrate binding to the dimer, completing the active site and enabling catalysis. Structure-guided mutagenesis confirms Tyr243 and Arg298 as essential catalytic residues unique to long Q-loop oxidases, highlighting evolutionary divergence from short Q-loop variants. Inhibition by Aurachin D triggers refolding of the active site, occluding substrate access via a conserved Asp239-mediated mechanism. The structural and mechanistic insights presented here establish a comprehensive framework, opening new ways for drug discovery.