Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D

Abstract

Cytochrome bd quinol:O2 oxidoreductases are respiratory terminal oxidases so far only identified in prokaryotes, including several pathogenic bacteria. Escherichia coli contains two bd oxidases of which only the bd-I type is structurally characterized. Here, we report the structure of the Escherichia coli cytochrome bd-II type oxidase with the bound inhibitor aurachin D as obtained by electron cryo-microscopy at 3 Å resolution. The oxidase consists of subunits AppB, C and X that show an architecture similar to that of bd-I. The three heme cofactors are found in AppC, while AppB is stabilized by a structural ubiquinone-8 at the homologous positions. A fourth subunit present in bd-I is lacking in bd-II. Accordingly, heme b595 is exposed to the membrane but heme d embedded within the protein and showing an unexpectedly high redox potential is the catalytically active centre. The structure of the Q-loop is fully resolved, revealing the specific aurachin binding.

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 ]).…”

“…In that study, menaquinones were discussed as co-substrates of bd -II in aerobic respiration, when molecular oxygen is limited, thus extending the role of menaquinones in bd -II biosynthesis and enzyme catalysis. The observed divergence of the type of quinol found in AppB of E. coli bd -II, with binding of ubiquinol in one structure [ 14 ] and demethylmenaquinone in the other [ 15 ], is most likely due to different conditions of cell growth and protein production. Importantly, the bd oxidases of Mycobacteria lack these quinones bound to CydB [ 12 , 13 ].…”

“…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.…”