Mechanistic and structural diversity between cytochrome <i>bd</i> isoforms of <i>Escherichia coli</i>

Abstract

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome <i>bd</i>-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. <i>Escherichia coli</i> encodes for two cytochrome <i>bd</i> isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between <i>cydABX</i> (<i>Ecbd-I</i>) and <i>appCBX</i> (<i>Ecbd-II</i>) operon encoded cytochrome <i>bd</i> variants have remained elusive in the past. Here, we demonstrate that cytochrome <i>bd</i>-<i>II</i> catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome <i>bd-II</i> from <i>E. coli</i>, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome <i>bd</i>-<i>II</i> to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two <i>bd</i> oxidase isoforms, but two notable differences are apparent upon inspection: (i) <i>Ecbd-II</i> does not contain a CydH-like subunit, thereby exposing heme <i>b</i>₅₉₅ to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of <i>Ecbd-I</i> Our work completes the structural landscape of terminal respiratory oxygen reductases of <i>E. coli</i> and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in <i>E. coli</i>.