Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the P_R-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a₃ iron atom is in a ferryl (Fe⁴⁺ = O^2-) configuration, and heme a and Cu_B are oxidized while Cu_A is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Keywords: X-ray free electron laser; bioenergetics; catalytic intermediates; complex IV; crystallography.

Fig. 1.

Active site structure of bCcO (A) and the proposed dioxygen reduction reaction cycle (B). The structure shown in A illustrates the relative location of the four redox centers, Cu_A, heme a and the Cu_B-heme a₃ binuclear center, as well as the Mg site intervening the Cu_A center and the binuclear center. Helix-X (labeled in blue) contains the axial ligands of heme a₃ and heme a, H376 and H378, respectively. (Lower Right Inset) Expanded view of the binuclear center. One of the Cu_B ligands, H240, is posttranslationally linked to Y244. All these structural elements are in subunit I, except Cu_A and E198(II) reside in subunit II. (B) Electronic configuration of the heme a₃ iron, Cu_B, and Y244 in each intermediate is depicted. (See Introduction for the full description of the reaction sequence.) Each green arrow indicates the translocation of one pumped proton coupled to the designated reaction step (6).

Fig. 2.

Design and evaluation of the hydrodynamic focusing mixer (A and B) and time-resolved spectra associated with in crystallo dioxygen reduction reaction of bCcO (C and D). The schematic drawing in A illustrates the design of the mixer assembly. (B) Mixing behavior of the mixer simulated with COMSOL software, based on a convection-diffusion model. The two side streams, each flowing at a rate of 16 μL/min, squeeze the oxygen-containing central stream, flowing at a rate of 8 μL/min, such that complete mixing takes place before the mixed solution exits the mixer. The color bar indicates the normalized concentration of oxygen. (C) Microscopic image of typical bCcO microcrystals. (D) Optical absorption spectrum of the intermediate populated at 8 s after the mixing of a reduced bCcO microcrystal solution with an O₂-containing buffer. The spectra of the reduced and oxidized forms of the enzyme are shown as references.

Fig. 3.

Structural changes associated with the R → P_R transition. The structure of the P_R-intermediate (green) is superimposed on reduced bCcO (R, coral). (Insets) Expanded views of the structural regions of (i) E198(II), highlighting the resemblance of the C-O-Mg angle in P_R vs. R, demonstrating that Cu_A is in its reduced state; (ii) Loop-I-II, highlighting the large-scale movement of D51 and N55; and (iii) the [380-384] segment of Helix-X, showing the partial opening of the helical structure and the rotation in R438. The black curved arrows indicate R → P_R structural changes. The coral and green dashed lines represent H-bonds in reduced and intermediate structures, respectively.

Fig. 4.

Ligand density in the binuclear center of the P_R-intermediate of bCcO. A shows the 2F_o-F_c electron density map of the initial reduced bCcO (R), demonstrating that no ligand density is present in its binuclear center. (B) F_o-F_c difference density map of the P_R-intermediate, obtained without any ligand modeled, indicating the presence of exogenous ligand or ligands in the binuclear center. (C and D) Structure of the P_R-intermediate modeled with a ferryl oxygen coordinated to the heme a₃ and a hydroxide coordinated to Cu_B. (C) Polder OMIT map for each ligand. (D) 2F_o-F_c map. The Cu_B to heme a₃ iron distance (in Å) is indicated in each structure. The F_o-F_c density map (green) is contoured at σ = 3, the Polder map (black) is contoured at σ = 7, and the 2F_o-F_c density maps (blue) are contoured at σ = 2.

Fig. 5.

A water molecule detected in the binuclear center of the P_R-intermediate. The water molecule (W1) resides at the end of the K-channel, which may stabilize the tyrosinate configuration of Y244 and support proton transfer to the binuclear center. The numbers indicate the distances (in Å). The conserved water molecule, W2, is shown as a reference. The 2F_o-F_c difference map is contoured at σ = 1.

Fig. 6.

Postulated heme a gating mechanism. The mechanism is based on conformational changes induced by the change in the oxidation state of heme a and the associated rotation of the farnesyl side chain, as described in the text. PLS, proton loading site.