The <i>Mycobacterium smegmatis bd</i>-II terminal oxidase employs a carboxylate shift mechanism.

Abstract

Cytochrome <i>bd</i> is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here, we characterize the cyt <i>bd</i>-II from <i><i>Mycobacterium</i> smegmatis</i>, a member of a hitherto uncharacterized evolutionary group (qOR-2) of <i>bd</i> oxidases, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. Overexpressing the <i>appCB</i> operon in its native host led to production of a highly active <i>bd</i>-II (<i>k</i>_obs = 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 a pH-dependent molecular switch, involving coordination changes of heme <i>d</i> and surrounding bulky residues regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type <i>bd</i> oxidases, and a basis for understanding the evolution of the superfamily.