Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature

Abstract

Cytochrome c oxidase catalyses the reduction of molecular oxygen to water while the energy released in this process is used to pump protons across a biological membrane. Although an extremely well-studied biological system, the molecular mechanism of proton pumping by cytochrome c oxidase is still not understood. Here we report a method to produce large quantities of highly diffracting microcrystals of ba ₃-type cytochrome c oxidase from Thermus thermophilus suitable for serial femtosecond crystallography. The room-temperature structure of cytochrome c oxidase is solved to 2.3 Å resolution from data collected at an X-ray Free Electron Laser. We find overall agreement with earlier X-ray structures solved from diffraction data collected at cryogenic temperature. Previous structures solved from synchrotron radiation data, however, have shown conflicting results regarding the identity of the active-site ligand. Our room-temperature structure, which is free from the effects of radiation damage, reveals that a single-oxygen species in the form of a water molecule or hydroxide ion is bound in the active site. Structural differences between the ba ₃-type and aa ₃-type cytochrome c oxidases around the proton-loading site are also described.

Figure 1

Microcrystals of ba ₃ CcO. (a) Microcrystals in an LCP drop. (b) Microcrystals in a string of LCP.

Figure 2

Room-temperature SFX structure of ba ₃ CcO. (a) Overall structure with subunit I in green, subunit II in blue, subunit IIa in pink, heme groups in black, copper ions in purple and lipids in orange. (b) Close-up view of the proton pathway where the SFX structure is shown in green/blue/pink and the cryo-LCP structure (PDB code 3S8G) is shown in grey. Water molecules are shown in red (SFX) and grey (cryo-LCP). (c) Close-up view of the area around the presumed proton-loading site. The SFX structure is shown in green/blue and the bovine heart aa ₃-type CcO (PDB code 3WG7) in grey. The magnesium ion of the bovine heart structure is shown in brown. Water molecules are shown in red (SFX) and grey (bovine heart). Heme a ₃ is shown in black (SFX) and grey (bovine heart). The amino acid numbering refers to the ba ₃ CcO.

Figure 3

Active site structure of the ba ₃ CcO from SFX data. (a) View of the heme a ₃ – Cu_B active site with a water molecule or hydroxide ion bound. The 2F_o-F_c density (blue) is contoured at 1.5 σ and the F_o-F_c difference density (green) at 4.0 σ. (b) The F_o-F_c omit map density, calculated without the water molecule, is contoured at 4.5 σ. Approximate distances between the heme a ₃ – Cu_B, the heme a ₃ – ligand and Cu_B – ligand are indicated.

Figure 4

Comparison of active site electron densities. (a–c) F_o-F_c omit map densities (green) contoured at 4.5 σ, calculated without any active site ligands. (a) The omit map density of ba ₃ CcO from SFX data. The bound water molecule/hydroxide ion is shown in red. (b) The omit map density of ba ₃ CcO from cryo-LCP data (PDB code 3S8G). The bound peroxide molecule is shown in red. (c) The omit map density of bovine heart aa ₃-type CcO from XFEL data of large crystals at cryo temperature (PDB code 3WG7). Two bound peroxide molecules with partial occupancies are shown in red. (d–f) F_o-F_c difference densities calculated with a water molecule bound in the active site are shown in green (positive) and red (negative), contoured at +4.0/−4.0 σ. The bound water molecules are shown in red. (d) The F_o-F_c density of ba ₃ CcO from SFX data. (e) The F_o-F_c density of ba ₃ CcO from cryo-LCP data (PDB code 3S8G). (f) The F_o-F_c density of bovine heart aa ₃-type CcO from XFEL data of large crystals at cryo temperature (PDB code 3WG7).