Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels

Abstract

Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.

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

“…This could indicate that both enzymes use different substrates under physiological conditions in accordance with the fact that the corresponding genes are differently expressed under different growth conditions 14 – 17 . Recently, the structure of the Mycobacterium smegmatis bd -oxidase consisting only of the two major subunits was reported 43 . Two oxygen access channels were identified from the structure due to the lack of small subunits, one channel leading to heme d , the other to heme b 595 43 .…”

“…Recently, the structure of the Mycobacterium smegmatis bd -oxidase consisting only of the two major subunits was reported 43 . Two oxygen access channels were identified from the structure due to the lack of small subunits, one channel leading to heme d , the other to heme b 595 43 . Unfortunately, the redox potentials of the heme groups were not determined making it impossible to say whether both channels are used.…”

“…It was speculated that the N-terminal region of the Q-loop is intrinsically disordered or flexible and therefore cannot be resolved in structural analysis 43 . However, as shown here, this is not the case but it should be kept in mind that the incompletely resolved Q-loop belongs to a bd oxidase from the S-subfamily and shows in part a conformation different from that of other bd oxidases 43 . Due to its different length the Q-loop can be exchanged between E. coli bd -I and bd -II but not between E. coli bd -I and the G. thermodenitrificans enzyme 32 , 33 .…”

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

“…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.…”