A peroxide bridge between Fe and Cu ions in the O ₂ reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump

Abstract

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-Å resolution by limiting the X-ray dose for each shot and by using many (Ϸ400) 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.hydrated electron ͉ O2 reduction mechanism ͉ membrane protein ͉ X-ray structural analysis ͉ cell respiration

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

“…5 A and B). The results are consistent with the maintenance of the oxidized protein conformation (36), even though the metal centers are reduced, before annealing at warmer temperature allows the protein to change its structure. The strained configuration shows an interesting spectral feature at 589 nm, also observed in the bovine and thermus CcO irradiated crystals (34,37).…”

X-ray structure of cyanide-bound bovine heart cytochromecoxidase in the fully oxidized state at 2.0 Å resolution

“…The (F o À F c ) electron density of the fully oxidized form calculated at 2.1 Å resolution reveals an elongated peak distinct from that of the CN À ion at the dioxygen-reduction site, which is 1.55 times higher than the average peak height of the reference water molecules ( Fig. 3a; Aoyama et al, 2009). The peak in (F o À F c ) electron density between the metal centres was 1.56 times higher than the average peak height of the same reference water molecules as those in the fully oxidized form.…”

“…The peak electron density in the vicinity of the hydroxide group of Tyr244 in the oxidized enzyme increases in proportion to the period of X-ray exposure, whereas the density of the peroxide group between Fe a3 and Cu B decreases (Aoyama et al, 2009). This is because haem irons are reduced to the ferrous state to activate the oxygen-reduction centre and generate a water molecule from the peroxide anion.…”

“…Thus, the occupancy of the water O atom was estimated to be 0.37. The electron density at this site in the fully oxidized form increases in proportion to the X-ray exposure period (Aoyama et al, 2009). Because CN À completely removes the bound peroxide, X-ray reduction of heme a 3 of the CN À -bound enzyme is unlikely to generate any water molecules.…”

“…Previously, we determined the crystal structure of fully oxidized CcO at 1.8 Å resolution (Aoyama et al, 2009). The peak electron density in the vicinity of the hydroxide group of Tyr244 in the oxidized enzyme increases in proportion to the period of X-ray exposure, whereas the density of the peroxide group between Fe a3 and Cu B decreases (Aoyama et al, 2009).…”