A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump

Abstract

The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529-533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-A resolution by limiting the X-ray dose for each shot and by using many (approximately 400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O(2) reduction site (Fe(3+)-O(-)-O(-)-Cu(2+)), in contrast to a ferric hydroxide (Fe(3+)-OH(-)) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O(2) reduction site suppresses the proton pumping function.

Fig. 1.

Effects of X-ray irradiation on the visible absorption spectra of the single crystals of the fully oxidized “as isolated” bovine heart cytochrome c oxidase. The measuring light beam focused to ≈50 μm in diameter was injected perpendicularly to the most extended plane of (010). The X-ray beam injected along the plane was thick enough to irradiate the entire area of the crystals that was irradiated with the measuring light beam. (A and B) The visible spectra are shown for the sample before the X-ray irradiation (A) and after a 30-second exposure (B). (C and D) Time courses of increases in absorption difference are shown between 604 and 630 nm (C) and between 582 and 630 nm (D).

Fig. 2.

(Fo-Fc) difference electron density maps for the 1- s and 15-s datasets, calculated at 2.5-Å resolution. The datasets are depicted together with the structural model of heme a₃, Cu_B, and 3 water molecules. Heme a₃, Cu_B, and the oxygen atom of each of the water molecules are colored in red, green, and light blue, respectively. Three water molecules, 2,007, 2,014 and 2,039, were not included in the structural refinements performed to compare their electron densities with the electron density between Fe_a3 and Cu_B. (A) The cages of the difference map for the 1-s data are drawn at the 4.5σ level. (B) The cages for the 15-s data are drawn at the 5σ level.

Fig. 3.

(Fo-Fc) difference electron density map of the 15-s data calculated at 2.1-Å resolution. (A)Electron density cages for the electron density between and Fe_a3 and Cu_B, and for 3 water molecules depicted at the 5.2σ level in a stick model around the O₂ reduction site. Carbon, nitrogen, and oxygen atoms are colored in yellow, blue, and red, respectively. (B) (Fo-Fc) difference maps for the 15-s dataset obtained under 3 different constraints on the OO distance. Cages are depicted at the 3.8σ level. Heme a₃ is depicted by a ball-and-stick model. Carbon, nitrogen, and oxygen atoms are yellow, blue, and red, respectively. The peroxide anion is illustrated by a stick model. Fe_a3 and Cu_B are represented in brown and green. The map of the 1.6-Å constraint (pink) has residual density peaks at both ends of the OO bond, whereas the map of the 1.8-Å constraint (green) has a large residual density at the middle of the OO bond. The cages for the 1.7-Å constraint (blue) have the smallest residual density.

Fig. 4.

Coordination geometries of the peroxide anion obtained from structural refinement of the 15-s dataset calculated at 2.1-Å resolution. The interatomic distances are given in angstroms. Other distances and angles are Fe_a3-Cu_B, 4.87 Å; N_ε2(H376)- Fe_a3-O₁, 168.5°; Fe_a3-O₁-O₂, 144.1° and O₁-O₂-Cu_B, 90.5°. These geometries are fully consistent with those of the X-ray structure determined from the 103.5-s exposures at 1.95-Å resolution.