Oxygen Activation and Energy Conservation by Cytochrome c Oxidase

Abstract

This review focuses on the type A cytochrome c oxidases (C cO), which are found in all mitochondria and also in several aerobic bacteria. C cO catalyzes the respiratory reduction of dioxygen (O₂) to water by an intriguing mechanism, the details of which are fairly well understood today as a result of research for over four decades. Perhaps even more intriguingly, the membrane-bound C cO couples the O₂ reduction chemistry to translocation of protons across the membrane, thus contributing to generation of the electrochemical proton gradient that is used to drive the synthesis of ATP as catalyzed by the rotary ATP synthase in the same membrane. After reviewing the structure of the core subunits of C cO, the active site, and the transfer paths of electrons, protons, oxygen, and water, we describe the states of the catalytic cycle and point out the few remaining uncertainties. Finally, we discuss the mechanism of proton translocation and the controversies in that area that still prevail.

Figure 1

Thirteen-subunit A-type CcO is shown with subunits I, II, and III in blue, red, and green, respectively. Ten nuclear-coded accessory subunits are shown with transparent ribbon representation. Lipid bilayer boundaries (dotted lines) and electron, proton, and oxygen paths (arrows) are also marked. Low-spin heme (yellow), high-spin heme (orange), and copper atoms (purple) are displayed. Figure was prepared with VMD software.

Figure 2

(A) View of the catalytic subunit I from the P side of the membrane. Three helix clusters III–VI, VII–X, and XI, XII, I and II are shown in blue, green, and red, respectively. Two clusters (green and red) hold the redox-active centers. Three pore regions (filled circles) are found in each of the three clusters. (B) Side view of the cylindrically shaped catalytic subunit. TM helices are shown as ribbons, colored according to residue polarity (polar, green; acidic, red; basic, blue; hydrophobic, white). Hemes (yellow) and Cu_B (orange), buried in the catalytic subunit, are also displayed.

Figure 3

(A) Top view (from the P side of the membrane) showing histidine ligands of heme cofactors. Two TM helices in subunit I (II and X) that carry conserved histidine residues are marked in red. (B) Side view showing the histidine ligands of the Cu_B center. Two tandem histidines (H290 and H291) originate from helix VII, whereas the His240-Tyr244 cross-link is from helix VI. As before, hemes and Cu_B are shown in yellow and orange.

Figure 4

(A) Structure of subunit II. (Inset) Cu_A center and its ligand sphere. (B) Structure of subunit III. α-Helices and β-sheets are shown in purple and yellow, respectively. Amino acid residues are marked with their one letter codes and numbers. Phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) lipid molecules bound to subunit III are displayed in licorice representation.

Figure 5

Proton-pumping elements in HCO of type A. The D channel of proton transfer is displayed, which comprises the proton-uptake site (D91), the asparagine gate (circled with purple dotted line), the serine zone, and a highly conserved acidic residue, Glu-242. Heme propionate region is highly polar and consists of a water cluster (circled with red dotted line), two arginines, and one acidic residue, Asp-364. Water molecules (yellow), hemes (blue, left, heme a; right, heme a₃), Cu_B (orange), Mg ion (brown), and subunit I (green transparent ribbons) are also shown. Nonpolar cavity where water produced at the active site may be released is shown as a pink “cloud”.

Figure 6

Proton transfer pathways in type A (A), B (B), and C (C) HCOs.

Figure 7

Catalytic cycle. Square encompasses the binuclear site with the heme a₃, Cu_B, and the covalently linked tyrosine (HO-tyr). Distal histidine ligand of heme a₃ and the three histidine ligands of Cu_B are not shown for simplicity. Uptake of protons to complete the chemistry of water formation is shown, but proton pumping is not shown. One proton is pumped across the membrane in each of the one-electron reactions, but for the A → F reaction the situation is more complicated: formation of state P_R is linked to loading the PLS from the N side of the membrane; its release to the P side is driven by uptake of the chemical proton in formation of state F (see text). Structures of intermediates R, A, P_M, P_R, and F are well established (see text), whereas those of states O_H and E_H are still more hypothetical.

Figure 8

Oxygen-splitting A → P_M transition. Density functional theory-based geometry optimizations (def2-SVP/TZVP/BP86/disp3/MARIJ)⁻ and energy calculations (def2-TZVP/B3LYP/disp3/eps4)^,,,,− were performed with Turbomole software on large model systems of the BNC. Spin density (α, green; β, pink) are plotted at an isosurface value of 0.01 e/ų.

Figure 9

Comparison of structures of the F_H (A) and O_H (B) states from studies by Sharma et al. (orange) and Blomberg (green).

Figure 10

Redox potentials. Redox potentials for the four one-electron reactions reducing O₂ to water are shown in blue for the reaction in aqueous solution and in red for the reactions catalyzed by cytochrome c oxidase (Table 3). Note that the ordinate may also be the standard change in Gibbs free energy (−ΔG₀′, in meV), which is obtained by subtracting 250 mV from the E_m,7 values plotted. Red point at 2.5 reaction equivalents is not an E_m,7 value but the combined −ΔG₀′ value for the reactions R → A and A → P_M to which 250 mV was added (cf. Table 3).

Figure 11

Proton pump mechanism. Mechanism is depicted as revealed from time-resolved electron injection experiments of the O_H → E_H transition^, but revised from ref (178) to account for the effect of mutating the K channel lysine, which blocks the 150 μs phase.^, The 150 μs phase includes protonation and movement of lysine-319 closer to the BNC (blue circle below heme a₃), which is necessary to allow electron transfer from heme a to heme a₃ and the linked uptake to the PLS of the proton to be pumped (blue circle above heme a₃) in states marked III and IV, respectively. Red color indicates the position of the injected electron.