Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli

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 bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II 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 bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I. Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target

“…Despite its structural homology to CydA/AppC, the second large subunit CydB/AppB does not carry any metal-containing cofactors. Instead, for CydB of E. coli bd -I and AppB of E. coli bd -II, it has been found that a ubiquinone-8 or demethylmenaqinone-8 occupies a position equivalent to the heme binding sites in CydA/AppC ( Figure 2 C) [ 10 , 11 , 14 , 15 ], perfectly tracing the positions of the hemes in the opposing subunit. Hence, it has been postulated that this quinone has mainly stabilizing properties.…”

“…Structural analyses of bacterial cytochrome bd oxidases by means of X-ray crystallography ( Geobacillus thermodenitrificans , pdb IDs 5DOQ & 5IR6, [ 9 ]) and cryo-electron microscopy (cryo-EM) ( Escherichia coli bd -I, pdb IDs 6RKO [ 11 ] and 6RX4 [ 10 ]; Mycobacterium smegmatis , 7D5I [ 12 ]; Mycobacterium tuberculosis , 7NKZ [ 13 ] and E. coli bd -II, 7OSE [ 14 ] and 7OY2 [ 15 ]) have shed light on the architecture of this class of oxidases.With the exception of the Geobacillus structures 5DOQ and 5IR6 that were obtained by X-ray crystallography, all structural data are based on cryo-EM. Resolutions range from 3.8 Å (5IR6, X-ray crystallography) to 2.5 Å (7NKZ, cryo-EM), providing near-atomic details (for a detailed discussion of optical resolutions in X-ray crystallography and cryo-EM, we suggest the work of Dubach and Guskov [ 85 ]).…”

“…This assumption is supported by the fact that mutations of amino acid residues in the ubiquinol-binding groove of E. coli CydB lead to a loss of activity [ 10 ]. The quinol must be very tightly bound to CydB because ubiquinone is not displaced by a high excess of aurachin D, a specific quinol-site inhibitor [ 10 ], and demethylmenaquinone is not replaced from AppB by a high excess of menaquinone-8 [ 15 ]. However, the presence of demethylmenaquinone-8 in one of the published E. coli bd -II structures (pdb ID 7OY2) was additionally suggested to indicate a close association of bd -II biosynthesis with menaquinone synthesis pathways [ 15 ].…”

“…The catalytically active subunit bears the three heme prosthetic groups, b 558 , b 595 , d , and the Q-loop. [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. The latter is a quinol-binding domain located in the hydrophilic loop region between the transmembrane helices 6 and 7.…”

“…It took more than 20 years to resolve the first three-dimensional structure of the bd oxidase from Geobacillus thermodenitrificans [ 9 ]. The single-particle cryoelectron microscopy (cryo-EM) structures of cytochromes bd from Escherichia coli , Mycobacterium smegmatis , and Mycobacterium tuberculosis have been reported over the past three years, with four structural papers published in 2021 [ 10 , 11 , 12 , 13 , 14 , 15 ]. The emergence of cytochrome bd structures has spurred the search for effective and selective inhibitors of this type of enzyme which could become new antibacterial agents.…”