The Mycobacterium smegmatis bd -II terminal oxidase employs a carboxylate shift mechanism

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here we characterise the first qOR-2 type bd oxidase, the cyt bd -II from Mycobacterium smegmatis , by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. By over-expressing the appCB operon in its native host, we produce a highly active bd -II ( k _cat =30 e ^- s ^-1 ) that together with a high-resolution (2.8 Å) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that O ₂ -scavenging activates a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues that regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.