The cryoEM structure of cytochrome bd from C. glutamicum provides novel insights into structural properties of actinobacterial terminal oxidases

Abstract

Cytochromes bd are essential for microaerobic respiration of many prokaryotes including a number of human pathogens. These enzymes catalyze the reduction of molecular oxygen to water using quinols as electron donors. Their importance for prokaryotic survival and the absence of eukaryotic homologs make these enzyme ideal targets for antimicrobial drugs. Here, we determined the cryoEM structure of the menaquinol-oxidizing cytochrome bd-type oxygen reductase of the facultative anaerobic Actinobacterium Corynebacterium glutamicum at a resolution of 2.7 Å. The obtained structure adopts the signature pseudosymmetrical heterodimeric architecture of canonical cytochrome bd oxidases formed by the core subunits CydA and CydB. No accessory subunits were identified for this cytochrome bd homolog. The two b-type hemes and the oxygen binding heme d are organized in a triangular geometry with a protein environment around these redox cofactors similar to that of the closely related cytochrome bd from M. tuberculosis. We identified oxygen and a proton conducting channels emerging from the membrane space and the cytoplasm, respectively. Compared to the prototypical enzyme homolog from the E. coli, the most apparent difference is found in the location and size of the proton channel entry site. In canonical cytochrome bd oxidases quinol oxidation occurs at the highly flexible periplasmic Q-loop located in the loop region between TMHs six and seven. An alternative quinol-binding site near heme b ₅₉₅ was previously identified for cytochrome bd from M. tuberculosis. We discuss the relevance of the two quinol oxidation sites in actinobacterial bd-type oxidases and highlight important differences that may explain functional and electrochemical differences between C. glutamicum and M. tuberculosis. This study expands our current understanding of the structural diversity of actinobacterial and proteobacterial cytochrome bd oxygen reductases and provides deeper insights into the unique structural and functional properties of various cytochrome bd variants from different phylae.

Keywords: CryoEM; electrochemistry; microbiology; oxidases; proton channel.

FIGURE 1

CryoEM structure of C. glutamicum cytochrome bd at 2.7 Å resolution. (A and C) Side view representations of the cryoEM map density and its corresponding ribbon model of the heterodimeric C. glutamicum cytochrome bd structure. The substrate quinol binding site 1 at the Q-loop in proximity to heme b ₅₅₈ is highlighted in orange. The second putative quinol binding site next to heme b ₅₉₅ is shown in purple. (B and D) Top view representation of the cryoEM map density and the corresponding tube model of the Cgbd structure highlighting the pseudosymmetrical arrangement of the two four helix bundles and the additional peripheral helix per subunit. Color code: CydA, green; CydB, pink; b-type hemes, cyan; heme d, dark blue.

FIGURE 2

Cofactor arrangement in Cgbd. (A) Triangular heme arrangement in CydA. (B) Axial ligands and proximal side chains of the prosthetic heme groups in Cgbd. Color code: CydA, green; b-type hemes, cyan; heme d, dark blue.

FIGURE 3

Oxygen and proton pathways in Cgbd. (A) Oxygen (O) and proton (H) channels predicted by MOLE2. The O-channel starts at the membrane interface between TMHs one, nine and two of CydB and TMH three of CydA and continues towards heme d. The predicted H-channel allows proton transfer from the cytoplasm to the reduction site. (B) Hydrophilic residues lining the H-channel. (C) Variation in location and size of the cytoplasmic proton channel entry sites in cytochromes bd. Depicted are the cryoEM maps of Mtbd (EMD-12451) (Safarian et al., 2021), Cgbd and Ecbd-I (EMD-4908) (Safarian et al., 2019) viewed from the cytoplasmic side of the membrane. The cavity elongating into the H-channel is highlighted by a red ellipse.

FIGURE 4

Comparison of the Qh3-PL8 region in (A) Mtbd and (B) Cgbd. The stabilizing residues E320, Y321 and R324 of Qh3 in Mtbd (7NKZ) (Safarian et al., 2021) are replaced by alanines in Cgbd, presumably leading to a weaker interaction between Qh3 and PL8.

FIGURE 5

Second quinol binding site. (A) In Mtbd, site 2 harbors a bound MK-9 molecule stabilized by W9, R8 and M397. (B) Site 2 appears unoccupied in Cgbd, however the residues interacting with MK-9 in Mtbd are conserved in Cgbd.