Structural basis for functional properties of cytochrome c oxidase

Abstract

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis¹. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized O_H state, and a reductive phase, in which O_H is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes². However, if O_H is allowed to relax to the resting oxidized state (O), a redox equivalent to O_H, its subsequent reduction to R is incapable of driving proton translocation^2,3. How the O state structurally differs from O_H remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)⁴, we show that the heme a₃ iron and Cu_B in the active site of the O state, like those in the O_H state^5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three Cu_B ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O_H, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

Fig. 1.. Oxygen reduction reaction catalyzed by bCcO.

(A) Schematic illustration of the four redox active metal centers in bCcO and the electron and proton transfers associated with the O₂ reduction reaction. The entry of O₂ and four substrate protons into the BNC, as well as the release of the product water molecules out of it, are indicated by the blue arrows. The associated entry of four electrons into the BNC and the translocation of four pumped protons across the membrane are indicated by the green and red arrows, respectively. The putative PLS between heme a₃ and the Mg center is highlighted by the light blue background. (B) The overall O₂ reduction reaction and the associated mechanism. The P intermediate is a general term for the P_M and P_R intermediates. The entry of the electrons and substrate protons into the BNC and the release of the product water molecules are indicated in each step of the reaction as described in the main text. The coupled proton translocation reactions are indicated by the white arrows. If the O_H intermediate produced at the end of the oxidative phase is allowed to relax to the resting O state, its reduction to R does not support proton translocation.

Fig. 2.. Resonance Raman spectrum of the O state of bCcO in H216O (black) versus H218O (red).

The H₂¹⁶O-H₂¹⁸O difference spectrum (expanded by 5 fold) is shown at the bottom. The oxygen sensitive mode in the H₂¹⁶O sample centered at 451 cm⁻¹ that shifted to 428 cm⁻¹ in the H2¹⁸O sample is assigned to the νFe-OH mode.

Fig. 3.. Electron density maps of the BNC in the O state of bCcO.

(A) The F_O-F_C electron density map (contoured at 7.0 σ) showing clear 2-lobe electron density associated with the BNC ligands. (B) The 2F_O-F_C electron density map (contoured at 2.5 σ) obtained with the electron density modeled with a hydroxide ion coordinated to the heme a₃ iron and a water molecule coordinated to Cu_B. (C) The Polder map (contoured at 7.0 σ) associated with the BNC ligands.

Fig. 4.. Protonation state of Y244 in the P_R (A) and O (B) states.

The post translationally modified Y244 forms a H-bonding network with the OH group of the farnesyl side chain of heme a₃, a water molecule (W¹) and T316. In the P_R state, an additional water, W², is recruited into the BNC to stabilize the tyrosinate configuration of Y244. This water is absent in the O state reported here, as evident in the 2FO-FC electron density map (contoured at 1.0 σ) shown in the lower inset, signifying that Y244 is in the neutral protonated state. For clarity the BNC ligands are not shown. The oxygen atom and hydrogen atoms are shown as red and white spheres.

Fig. 5.. Hypothesized ligand transformation in the BNC during the CcO reaction cycle.

The oxidative and reductive phase of the reaction cycle are highlighted with green and blue backgrounds, respectively. The white arrows indicate the proton translocation reactions associated with the reaction cycle. The lifetimes associated with the P_R→F, F→O_H and O_H→O transitions are indicated in red.

Fig. 6.. Functional role of Y244 during the CcO reaction cycle.

(A) During the active turnover, Y244 cycles between the neutral form (YH), tyrosyl radical form (Y^•) and deprotonated tyrosinate form (Y⁻) as depicted by the blue cycle. In all the intermediate states active in proton translocation, Y244 is in the Y⁻ form (in red), which ensures the tight coupling of electron transfer (green arrows) and substrate proton transfer (blue arrows), via the D and K-channel into the BNC, to drive the proton translocation (red arrows) via the D or H channel from the N to P-side of the membrane. (B) The protonation of the tyrosinate to the YH form (red) in the O state disables the proton translocation upon its reduction to R.