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

Abstract

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O₂ with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.

Keywords: cytochrome oxidase; electron transport chain; enzyme structure; inhibition; membrane protein; molecular bioenergetics; terminal oxidase.

Figure 1

Structures of bacterial cytochrome bd oxidases. (A) bd oxidase of G. thermodenitrificans (pdb ID 5DOQ) is composed of subunits CydA (light beige), CydB (red), and CydS (dark red). The heme groups are located in subunit CydA. (B) E. coli bd-I (pdb ID 6RKO in grey tones, pdb ID 6RXO in green colours) comprises four subunits, termed CydA, CydB, CydX, and CydY. While CydA, CydB, and CydX have homologues in G. thermodenitrificans, CydY is exclusive for E. coli bd-I. (C) bd-II oxidase from E. coli (pdb ID 7OSE in blue colours, pdb ID 7OY2 in grey tones) is built by subunits AppC (homologue to CydA), AppB (homologue to CydB), and AppX (homologous to CydS/X). 7OSE has been solved with the inhibitor aurachin D (AurD) bound to the Q-loop. (D) The mycobacterial bd oxidase (M. smegmatis, pdb ID 7D5I, in yellow colours, M. tuberculosis, pdb ID 7NKZ, in orange and salmon) consists of only two subunits, CydA and CydB.

Figure 2

Triangular heme arrangement in CydA/AppC and equivalent positions in CydB/AppB. (A) In G. thermodenitrificans, hemes b₅₅₈ and b₅₉₅ (light beige sticks) are found in a plane, while heme d sits orthogonally on top (grey sticks). Here, the orthogonal heme is the active site. Axial heme ligands of CydA are shown as thick lines. Thr14 and Leu18 form a hydrophobic roof above heme d. (B) The heme arrangement in E. coli bd-I (green sticks), E. coli bd-II (blue sticks), as well as the mycobacterial bds (M. smegmatis in yellow sticks, M. tuberculosis in red sticks) is conserved and differs from G. thermodenitrificans in the relative position of hemes b₅₉₅ and d. Heme b₅₉₅ is now orthogonally placed with respect to the plane and replaces heme d (bold colours), which in turn resides in the plane with b₅₅₈ (light colours). Nonetheless, heme d (bold colours) remains the active site with molecular oxygen (red spheres) being found as axial ligand in pdb ID 7NKZ (M. tuberculosis). (C) Superposition of E. coli bd-I subunit CydA with subunits CydB/AppB of E. coli bd-I and bd-II. The Q-loop was removed from CydA prior to superposition. In CydB/AppB, either ubiquinone-8 or menaquinone-8 occupies the position corresponding to the heme groups in CydA. (D) Mycobacterial bds do not employ a quinone in subunit CydB to fill the equivalent positions of the heme groups, but instead utilise aromatic side chains (shown as thick lines, M. smegmatis in orange, M. tuberculosis in yellow) to seamlessly fill the available space. Ubiquinone-8, as found in E. coli bd-I (6RX4), is given as reference in olive-green sticks.

Figure 3

Quinol binding sites in bacterial bd oxidases. (A) Quinol binding site in E. coli bd-II (pdb ID 7OSE). The specific inhibitor aurachin D is shown as sticks, the interacting surface of CydA is shown in green, the residual protein surface in light blue. The Q-loop is involved in binding and provides the top half of the binding site. (B) Aurachin D (modelled from 7OSE after superimposing 5DOQ and 7OSE) perfectly fits to the putative quinol binding site in G. thermodenitrificans (5DOQ, green surface, residual protein surface given in red). (C) The corresponding cleft below the Q-loop in mycobacterial bd oxidases (shown for M. tuberculosis) is too narrow for aurachin D (putative clashes shown in hotpink). Instead, a quinol binding site was identified close to heme b₅₉₅, where menaquinone-9 was found to interact with CydA (green surface, residual protein surface in light salmon).

Figure 4

Interaction interfaces of additional subunits with the bd core subunit CydA/AppC. (A–C) Subunit CydX/AppX (bold colours) binds to subunit CydA/AppC (light colours) in a largely conserved position close to heme b₅₅₈, lateral to the Q binding site (not shown). Interactions are mainly driven by hydrophobic contacts. (A) Interaction patterns between CydX and CydA in G. thermodenitrificans bd. (B,C) CydX/AppX of E. coli bd-I and bd-II bind in a nearly identical manner to CydA/AppC, underlining the close homology of both bd oxidases. As compared to G. thermodenitrificans bd, the N-terminus (top) of CydX/AppX is tilted further away from heme b₅₅₈. (D) Subunit CydH (limon), exclusive to E. coli bd-I, is found at the opposite side of CydA (light green) and appears to be further stabilised by a glycerophospholipid (shown as sticks). Again, mainly hydrophobic interactions contribute to binding.

Figure 5

Oxygen channels to the active site in bacterial bd oxidases. (A) Oxygen channel in E. coli bd-I (cyan) leading from Trp11 in CydB through the protein core to heme d. The channel features a constriction that is thought to serve as selectivity filter for linear molecules such as dioxygen. (B) Other bd oxidases feature an equivalent oxygen channel (E. coli bd-II in marine blue, M. smegmatis bd in yellow, M. tuberculosis bd in salmon) with a similar constriction. Only G. thermodenitrificans bd, as a consequence of the altered arrangement of the heme groups, features a very short channel (red) from the opposing side of the enzyme and leading directly to the active site heme d. (C–E) A conserved isoleucine residue (shown as thick lines) blocks diffusion of dioxygen between hemes b₅₉₅ and d by perfect surface complementarity. Hemes are given as sticks, protein surfaces (smooth surfaces) and surfaces of hemes (meshes) are provided to illustrate the excellent surface match. (C) G. thermodenitrificans bd, (D) E. coli bd-I (pale green) and E. coli bd-II (slate blue), (E) M. smegmatis (pale yellow) and M. tuberculosis bd (salmon).

Figure 6

Proton pathways in bacterial bd oxidases to the heme triangle. (A) G. thermodenitrificans features a pathway along the CydA/CydB interface, termed the CydB pathway (red). A second pathway running through CydA has been proposed as well, termed CydA pathway (light beige). (B) A pathway in E. coli bd-I (olive green), largely equivalent to the CydB pathway in G. thermodenitrificans, is lined by several hydrophilic amino acid sidechains (shown as thick lines) and directs water molecules (i.e., protons) to the propionate of the active site heme d. Numerous water molecules (blue spheres) have been found in that channel, highlighting its full accessibility. (C) E. coli bd-II features a comparable channel. But due to the much wider cavity protruding deeply into the protein core, the actual channel requires a only a glutamate and an aspartate sidechain (shown as sticks) to coordinate water molecules and guide them to the active site. Here again, the presence of water molecules illustrates the accessibility of solvent molecules.

Figure 7

Simplified schematic representation of the effects of compounds targeting the bd oxidase on the level of the isolated enzyme, membrane vesicles or whole cells. Shown are the structures of aurachin D, 3-[[2-(4-chlorophenyl)ethylamino]methyl]-1-ethyl-indole-2-carboxylic acid (MQL-H₂), N-(4-(4-(trifluoromethyl)phenoxy)phenyl)quinazolin-4-amine (ND-011992), and N-(4-(tert-butyl)phenethyl)thieno [3,2-d]pyrimidin-4-amine (Compound 19). See the main text for details. Data collected from: [10,13,14,116,117,118,119,120,121,122,123,124].