Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

Abstract

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a₃ iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu_B, which, in turn, moves closer to the heme a₃ iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a₃ iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a₃, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Keywords: X-ray free electron laser; bioenergetics; crystallography; cytochrome c oxidase; serial femtosecond crystallography.

Fig. 1.

The heme a and heme a₃-Cu_B binuclear center of bCcO. The heme a₃ and heme a axial ligands, H376 and H378, respectively, are both located in helix-X, which passes between the two hemes. Cu_B is coordinated by three histidine ligands, H240, H290, and H291. Small molecule ligands, such as O₂, CO, CN, and NO, bind to the heme a₃ iron atom, which is only ∼5 Å from Cu_B.

Fig. 2.

Active site structures of bCcO-CO and bCcO-CO* obtained with (A–C) XFEL and (D–F) synchrotron radiation X-rays. (A) Unbiased F_o–F_c difference map (green; contoured at 10 σ), in the absence of the CO ligand showing positive electron density in the binuclear center. (B) The 2F_o–F_c electron density map (blue; contoured at 3.0 σ) of the binuclear center modeled in the presence of the CO. (C) Expanded view of the binuclear center. (D) Unbiased F_o–F_c difference map (green; contoured at 8 σ), in the absence of CO showing positive electron density near Cu_B. (E) The 2F_o–F_c electron density (blue; contoured at 2 σ) of the binuclear center showing that the CO was photodissociated and located near Cu_B. (F) Expanded view of the binuclear center.

Fig. 3.

Allosteric structural transition in the Helix-X fragment induced by ligand binding to heme a₃. (A) Superimposed structures of helix-X in bCcO-CO and bCcO-CO* obtained by SFX (green) and by synchrotron radiation (coral), respectively. The arrows designate the [380–384] peptide backbone movements (in Å) induced by ligand binding to heme a₃. In the comparison of the two structures, the Cα carbon atoms of S382 and M383 differ by 1.5 Å, and the backbone carbonyl groups differ by 2.8 Å. Comparison of the alpha-helix hydrogen bonding in (B) the bCcO-CO structure obtained by SFX and (C) the bCcO-CO* structure obtained by synchrotron radiation. In C, in which the CO ligand was dissociated from the heme a₃ iron atom, the normal α-helical H-bonding is established, whereas in B, the SFX structure, the α-helical H-bonding patterns of the carbonyl groups of Y379, S382, and M383 are disrupted.

Fig. 4.

Helix-X mediated allosteric structural transition induced by ligand binding to heme a₃. Superimposed structures of bCcO-CO and bCcO-CO* obtained by SFX (green) and synchrotron radiation (coral), respectively, showing the ligand binding induced conformational change to the heme a₃, in particular the C-pyrrole ring, and its propagation to heme a via the 380–384 residue segment of the helix-X. The farnesyl OH group of heme a is highlighted by the blue background. The expanded view shows the interaction region between V380 and the C-pyrrole ring of heme a₃. The red arrows identify the distance change of the C2 atom (0.5 Å) due to the increased heme distortion in the photodissociated state.

Fig. 5.

Cascade of structural changes induced by ligand binding to heme a₃. The letters a–e indicate the sequential structural changes initiated by ligand binding to heme a₃ (red arrows). The letters f and g indicate structural changes induced by the change in the redox state of heme a (gray arrows) (31).

Fig. 6.

Schematic illustration of the reaction coordinate for the ligand binding reaction of bCcO. The heme ligand, CO or O₂, first enters the protein matrix by overcoming the Outer Barrier to form the encounter complex (FeCuB··L), in which the ligand is loosely associated with the protein. It then binds to Cu_B via passing over inner barrier 1 and subsequently to heme a₃ by passing over inner barrier 2. Inset shows the equation for the reversible ligand-binding and photolysis reactions.