Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography

Abstract

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu _B ) and a heme a ₃ iron, where O ₂ binds and is reduced to water. CO is a versatile O ₂ surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a ₃ iron leads to a metastable intermediate (Cu _B -CO), where CO is bound to Cu _B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a ₃ iron and moved to a temporary binding site midway between the Cu _B and the heme a ₃ iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a ₃ iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

Fig. 1.. Proposed CO dissociation mechanism of bCcO (A) and the associated structural transition (B).

The structure of the CO complex (PDB ID: 5W97) (grey) is superimposed with that of the ligand-free R species (PDB ID: 7THU) (green) in (B) to illustrate the CO dissociation induced structural transition (as indicated by the arrows), in particular (i) the out-of-plane movement of the heme a₃ iron-H376 moiety, (ii) the displacement of the Cu_B-H240 moiety towards the heme a₃, and (iii) the open to closed conformational change in Helix-X. The inset shows the expanded view of the [380–383] fragment of Helix-X. The arrows indicate the rotation of the backbone carbonyl groups. The sidechains, except that of S382, are not shown for clarity.

Fig. 2.. Structure of the geminate photoproduct derived from this work (PDB ID: 8GBT) (A) and that of the reported Cu_B-CO intermediate (PDB ID: 5X1B) (B).

The structures are superimposed with that of the bCcO-CO complex (PDB ID 5W97) (grey) to highlight the structural differences between the two photoproducts. In the geminate photoproduct reported here, the CO is photolyzed and moved to a new position midway between heme a₃ and Cu_B, while the BNC and the Helix-X remain in the CO-complex state; in contrast, in the Cu_B-CO intermediate, CO coordinates to Cu_B, the heme a₃ iron-H376 moiety moves out of the heme a₃ plane and the Cu_B-H240 moiety displaces towards the heme a₃ (as indicated by the arrows), while the Helix-X remains in the CO-complex (open) state. The inset (i) in (A) displays the F_O-F_C difference map of the BNC (contoured at 6σ), showing the clear electron density associated with CO between heme a₃ and Cu_B in the BNC. The inset (ii) shows the 2F_O-F_C map of the BNC (contoured at 2σ) with the ligand electron density modeled with CO.

Fig. 3.. CO dissociation reaction trajectory in bCcO.

(A) The three elementary steps of the CO dissociation reaction and the interatomic distances in each species involved in the reaction. (B) The bCcO-CO complex structure (PDB ID 5W97) (grey) superimposed with the CO molecules taken from the geminate photoproduct (this work) (green) and the Cu_B-CO complex (PDB ID: 5X1B) (cyan). The green, blue and red arrows indicate the conformational transition associated with the step (1), (2) and (3) of the reaction, respectively.