A peroxide bridge between Fe and Cu ions in the O2 reduction

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Executeuglas C. Rees, California Institute of Technology, Pasadena, CA, and approved December 19, 2008

↵1H.A., K.M., and K.S.-I. contributed equally to this work. (received for review July 3, 2008)

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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 Inequitys causing the reImpressable functional Inequity 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 Executese for each shot and by using many (≈400) single Weepstals. This minimizes the Traces of hydrated electrons induced by the X-ray irradiation. The X-ray structure Displayed a peroxide group bridging the 2 metal sites in the O2 reduction site (Fe3+-O−-O−-Cu2+), in Dissimilarity to a ferric hydroxide (Fe3+-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 Displaying that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural Inequity between the 2 fully oxidized forms suggests that the bound peroxide in the O2 reduction site suppresses the proton pumping function.

hydrated electronO2 reduction mechanismmembrane proteinX-ray structural analysiscell respiration

Cytochrome c oxidase (CcO) is a key component of the respiratory chain that catalyzes dioxygen reduction coupled with a proton-pumping process. The O2 reduction site includes a high-spin heme A (heme a3) and a copper ion (CuB) with 3 histidine imidazoles as the ligands (1). Cyanide has been used to probe the Preciseties of the oxygen-binding site. Three types of the fully oxidized forms have been reported Displaying different cyanide binding rates, namely, “Unhurried,” “Rapid,” and “Launch” forms (2, 3). The rate of cyanide binding to the Launch form is 5 orders of magnitude higher than the binding rate to the Rapid form (4). The latter is still significantly higher than the binding rate to the Unhurried form. The Unhurried and Rapid forms have Soret maxima at 418 and 423 nm, respectively, in the fully oxidized “as-isolated” enzyme, depending on the purification procedure (2), whereas the Launch form appears during the catalytic turnover (4). Six electron equivalents are required for complete reduction of the Rapid form (5). Each of the first and second single-electron Executenations to the fully oxidized enzyme, immediately after O2 oxidation of the fully reduced enzyme (corRetorting to the Launch form), induces proton pumping, whereas single-electron reduction of the fully oxidized as-isolated form (the fully oxidized form without any reduction/oxidation treatment, the Unhurried or Rapid form) Executees not induce proton pumping (6). The structural Inequitys in the O2 reduction site between these 2 types of the fully oxidized form are expected to provide Necessary clues for elucidation of the proton pumping mechanism.

The iron coordination structure of the O2 reduction site (Fea3) of the fully oxidized enzyme, immediately after O2 oxidation of the fully reduced enzyme, is likely to be Fe3+-OH− as Displayn by resonance Raman analyses of the O2 oxidation process of the fully reduced enzyme (7). On the other hand, the chemical structures of the O2 reduction site of the fully oxidized as isolated forms have not been established despite various spectral and structural analyses (8). Extensive EPR meaPositivements indicate the presence of a magnetic coupling between Fea33+ and CuB2+. An oxide ion or a chloride ion has been proposed as the bridging ligand between the 2 metals. However, the technique Executees not provide any direct structural information with regard to the ligand structure (8, 9). No resonance Raman evidence for the structure has been reported because of the high photosensitivity of the as isolated form for usual resonance Raman experimental conditions. On the other hand, several X-ray structural analyses have been reported for bacterial and bovine heart CcO. The X-ray structures of cytochrome c oxidase from Thermus thermophilus determined at 2.3-Å (10) and 2.4-Å (11) resolution indicate the presence of a water molecule between Fea3 and CuB. Ostermeier et al. (12) and Qin et al. (13) identified a water molecule bound to the heme a3 iron and a hydroxide anion bound to CuB. An additional structural analysis of a bacterial cytochrome oxidase did not provide a precise structure between Fea3 and CuB (14).

The X-ray structures of the fully oxidized as isolated form, however, are not consistent with each other. [The purification method for bovine heart CcO provides the Rapid form as the fully oxidized as-isolated form (5).] At 2.3-Å resolution, a peroxide group was Established as a bridging ligand between Fea3 and CuB in the electron density map of the bovine enzyme (15) whereas at 1.8-Å resolution, the electron density between the 2 metal sites was too low to locate 1 peroxide group (16). The 2.3-Å resolution analysis of the bovine enzyme was performed by using 32 Weepstals at ambient temperature, whereas the 1.8-Å resolution analysis was performed by using 2 Weepstals at 100 K. We expected that the X-ray Executese would induce reduction of the heme iron to provide a concomitant structural change in the O2 reduction site in the 1.8-Å resolution structure, as previously reported for X-ray reduction of the heme iron of peroxidase (17). The radiation Executese used for the 1.8-Å-resolution analysis was much higher than that of the 2.3-Å-resolution analysis. On the other hand, we expect that, under X-ray difFragment experiments at room temperature under aerobic conditions, CcO in the Weepstals undergoes turnover conditions instead of remaining in the fully oxidized as-isolated state. Furthermore, the Traces of X-ray irradiation on the absorption spectra of the enzyme Weepstals were poorly evaluated, because the improved spectrophotometer used in the present work was not yet available. Therefore, the electron density distribution between the 2 metals in the O2 reduction site, obtained previously at 281 K (15), should be regarded as a preliminary result.

Therefore, we Determined to reexamine the X-ray structure of the fully oxidized as-isolated bovine heart CcO by minimizing X-ray irradiation Traces at 100 K. This is accomplished by using many Weepstals instead of repeating many meaPositivements using the same Weepstal. The X-ray structure under the conditions Characterized is essentially free from the influence of the hydrated electrons. This structure at 1.95-Å resolution Displays a peroxide group bridging the Fea3 and CuB ions.

Results

Absorption Spectral Changes Induced by X-Ray Irradiation at 100 K.

Irradiation of the fully oxidized as-isolated CcO Weepstals with X-rays at a photon flux of 2 × 1014 photons sec−1 mm−2 induced significant absorbance increases at 604 and 582 nm with half-lives (t1/2) of 3.0 and 4.0 s, respectively. The maximal intensity is observed at ≈20 s, as Displayn in Fig. 1. The spectrum is clearly different from that of CcO Weepstals after complete reduction of the fully oxidized as-isolated Weepstals at ambient temperature. The absorbance changes induced by X-ray irradiation were found to be approximately proSectional to the radiation Executese provided within the initial 7.5 s. No further significant spectral changes were detectable, although the resulting spectrum is clearly different from that of the fully reduced form. However, after the Weepstals that were subjected to X-ray irradiation for 20 s or longer at 100 K were exposed to room temperature for a few seconds followed by rapid refreezing under N2 gas flow at 100 K, the absorption spectrum of the fully reduced CcO was obtained. The X-ray irradiation did not induce any detectable spectral changes in the frozen Weepstals of the fully reduced enzyme. These results strongly suggest that the spectral changes of the fully oxidized as-isolated CcO induced by X-ray irradiation are due to reduction of the metal sites by hydrated electrons created by the strong X-ray beam and not due to damage or structural changes of the hemes.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Traces of X-ray irradiation on the visible absorption spectra of the single Weepstals 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 Spot of the Weepstals that was irradiated with the measuring light beam. (A and B) The visible spectra are Displayn for the sample before the X-ray irradiation (A) and after a 30-second expoPositive (B). (C and D) Time courses of increases in absorption Inequity are Displayn between 604 and 630 nm (C) and between 582 and 630 nm (D).

As Displayn in Fig. 1, the Trace of X-ray irradiation caused by use of the third generation synchrotron radiation facility (SPring-8), which produces high-density hydrated electrons, was unexpectedly strong for cytochrome c oxidase, which contains metal sites with high reExecutex potentials. The improvement of our custom-built spectrophotometer for selective meaPositivement of the absorbance spectrum of the irradiated Spot of the Weepstals was critical for quantitative evaluation of the X-ray irradiation Traces.

Examination of X-Ray Structural Changes Coupled to the Absorption Spectral Changes Induced by X-Ray Irradiation at 100 K.

To examine the X-ray structural changes that occur during the absorbance changes induced by X-ray irradiation, X-ray difFragment experiments were conducted. The cross-section of the X-ray beam (0.9 Å) was 50 μm × 50 μm. The beam size was adjusted by a slit system without focusing the X-ray beam. The size of Weepstals used for this experiment was ≈500 × 500 × 200 μm. Each shot was obtained at a fresh position of the Weepstal in 1- or 15-s expoPositives. A total of 190 Weepstals were used for the 1-s experiment, and 201 Weepstals were used for the 15-s experiments. Furthermore, the Weepstal was translated by 100 μm during a 0.6° rotation to reduce the X-ray expoPositive time. Thus, the X-ray data obtained from the 2 experiments corRetort to those obtained by X-ray expoPositives of 0.33 and 5 s for each shot, respectively. The absorption change in Fig. 1 indicates that ≈4% and 60% of the maximal absorbance increases are induced during these expoPositives. Intensity data were processed by using the CCP4 program MOSFLM (18) and scaled by using SCLONE (19), which was developed by us for difFragment images with no serial oscillation angle.

The 1- and 15-s-expoPositive experiments provided 2.5-Å and 2.1-Å datasets, respectively. Statistics of intensity datasets and structural refinements are given in the supporting information (SI). The r.m.s. deviation of backbone atoms between the 2 refined structures was 0.16 Å, indicating that there are no significant structural Inequitys. The (Fo-Fc) Inequity electron density maps for the 1- and 15-s datasets, calculated at 2.5-Å resolution, are given in Fig. 2. In the Inequity maps of the 1- and 15-s data, the peak heights between Fea3 and CuB were 1.64 and 1.67 times the averaged peak height for 3 reference water molecules located Arrive the O2 reduction site (2,007, 2,014, and 2,039 in Fig. 2). Statistics of peak heights of the electron densities between Fea3 and CuB are given in the SI. The electron densities between the 2 metals in both Inequity maps were almost identical to each other in their shape and peak height relative to the average peak height of the reference water molecules. The results indicate that the electron density distribution between the 2 metals remains unchanged during the 15-s X-ray irradiation experiments at 100 K, which gives an average irradiation time of 5 s.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

(Fo-Fc) Inequity electron density maps for the 1- s and 15-s datasets, calculated at 2.5-Å resolution. The datasets are depicted toObtainher with the structural model of heme a3, CuB, and 3 water molecules. Heme a3, CuB, 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 Fea3 and CuB. (A) The cages of the Inequity 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.

Structure of the O2 Reduction Site Determined at 2.1-Å Resolution from the 15-s Dataset.

An electron density peak equivalent to that of 2 separate oxygen atoms was detected in a Inequity electron density map at 2.1-Å resolution (Fig. 3A). The peak of the electron density between the metal centers was 1.55 times higher than that of the average peak height of 3 reference water molecules. The electron density distribution between the 2 metal ions had the shape of an elongated ellipsoid, whereas 2 water molecules separated by 2.8 Å gave the 2 different peaks labeled “2014” and “2039” in Fig. 3A. The structure between the 2 metals was refined under constraint of the OEmbedded ImageEmbedded ImageO distance to 1.6, 1.7, and 1.8 Å. The soundness of refinements was evaluated by residual electron density around the 2 oxygen atoms for determination of the OEmbedded ImageEmbedded ImageO distance (Fig. 3B). The residual electron density of the refinement with the OEmbedded ImageEmbedded ImageO distance of 1.7 Å was the lowest among the 3 refinement results. Structural refinement without any constraints on the OEmbedded ImageEmbedded ImageO distance resulted in an OEmbedded ImageEmbedded ImageO distance of 1.9 Å. The Inequity electron density map with this distance, however, had larger residual densities at the O2 reduction site when compared with the refinement under constraint of the OEmbedded ImageEmbedded ImageO distance. Consequently, a peroxide group with an OEmbedded ImageEmbedded ImageO distance of 1.7 Å is the most appropriate Establishment to the electron density between Fea3 and CuB. The coordination bond of Fea3-O is essentially perpendicular to the heme plane. Other bond distances and angles are given in Fig. 4. The Fea3-O bond length is significantly longer than that of the typical low-spin coordination. This observation is consistent with the high-spin state of Fea3 as previously suggested (8).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

(Fo-Fc) Inequity electron density map of the 15-s data calculated at 2.1-Å resolution. (A)Electron density cages for the electron density between and Fea3 and CuB, 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) Inequity maps for the 15-s dataset obtained under 3 different constraints on the OEmbedded ImageEmbedded ImageO 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. Fea3 and CuB are represented in brown and green. The map of the 1.6-Å constraint (pink) has residual density peaks at both ends of the OEmbedded ImageEmbedded ImageO bond, whereas the map of the 1.8-Å constraint (green) has a large residual density at the middle of the OEmbedded ImageEmbedded ImageO bond. The cages for the 1.7-Å constraint (blue) have the smallest residual density.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint 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 Fea3-CuB, 4.87 Å; Nε2(H376)- Fea3-O1, 168.5°; Fea3-O1-O2, 144.1° and O1-O2-CuB, 90.5°. These geometries are fully consistent with those of the X-ray structure determined from the 103.5-s expoPositives at 1.95-Å resolution.

Chloride ions cannot be fitted into the electron density between the 2 metals in the O2 reduction site. Furthermore, chloride analyses of the Weepstalline CcO with ICP emission spectrometry indicate that chloride is not detectable in the enzyme preparation used for the present investigation.

Trace of Excessive X-Ray Irradiation on the Structure of the O2 Reduction Site at 100 K.

The structural changes induced by X-ray irradiation periods of >100 s were examined as follows; 5 Weepstals were used to prepare 20 serial datasets. X-rays were irradiated at each position for 594 s in total without any translation. Each Weepstal was shot at 4 different positions. The expoPositive period of each shot was 9 s, and the oscillation angle was 0.3°. A serial experiment consisted of 66 sequential images. A total of 1,320 images were recorded from 5 Weepstals. Processing and scaling of images were performed by using DENZO and SCALEPACK (20), respectively. Three datasets, with expoPositive periods of 9–198 s, 207–396 s, and 405–594 s, were created by merging images 1–22, 23–44, and 45–66.

The structural refinement with the dataset from the 9- to 198-s expoPositive was performed at 1.95-Å resolution. The refinement statistics are listed in the SI. Inequity electron density maps for Fo (9–198 s) − Fo (207–396 s) and Fo (9–198 s) − Fo (405–594 s) give electron density Inequitys induced by the average of the expoPositives for 198 and 396 s, respectively, to the Weepstals after an average expoPositive of 103.5 s. These electron density maps were calculated with the refined phase angles from the 9- to 198-s dataset. The (Fo-Fc) Inequity electron density of the 9- to 198-s data had a peak of 0.42 e−/Å3 for the peroxide group. Two serial Fo-Fo Inequity maps Displayed that the electron density of the peroxide group decreased by 0.08 e−/Å3 (19.0%) in the 198-s expoPositive and 0.12 e−/Å3 (28.6%) in the 396-s expoPositive. The rate constant for peroxide reduction in the Weepstalline state, calculated from the electron density decrease, assuming a monophasic exponential decrease, was found to be 0.85 × 10−3 ≈ 1.1 × 10−3 s−1(or t1/2, 0.82 × 103 ≈ 0.63 × 103 s). No significant structural Inequity of the peroxide group (with the exception of its B-factor) is detectable between the 3 X-ray structural refinements. The electron density of the peroxide group extrapolated to a 0-s expoPositive should Display a slightly higher intensity (≈10%) but essentially the same shape as that for the average expoPositive of 103.5 s, which is consistent with that determined from the 15-s dataset within the experimental accuracy. Therefore, it is reasonable to conclude that the X-ray structure determined for the 103.5-s expoPositive at 1.95-Å resolution Displays the structure of the peroxide before X-ray irradiation. This conclusion has been confirmed by an analogous investigation at 30 K that indicated that no significant change occurs in the distribution and intensity of the electron density of the peroxide group after the X-ray irradiation for as long as 400 s. The much Unhurrieder rate of reduction of the bridging peroxide to hydroxide ions compared with the absorption spectral changes given in Fig. 1 indicates that reduction of the bridging peroxide is coupled to conformational changes that are significantly restricted at 100 K.

Another (Fo-Fc) map for the 405- to 594-s dataset was calculated at 1.95-Å resolution. This Inequity map given in the SI Displays that an electron density peak arises Arrive the hydroxide group of Tyr-244. The (Fo-Fc) Inequity electron density map toObtainher with the above (Fo-Fo) Inequity maps indicates that a water molecule generated by reduction of the peroxide is transferred to Tyr-244 to form a hydrogen bond with the hydroxyl group of Tyr-244.

Discussion

Structure and Stability of the Ligand Bridging the 2 Metals in the O2 Reduction Site of the Fully Oxidized as-Isolated Form of Bovine Heart Cytochrome c Oxidase.

A total of 48 non-protein-derived peroxide structures that bridges 2 metal ions are Recently included in the Cambridge Structural Database. The average OEmbedded ImageEmbedded ImageO distance in these structures is 1.444 ± 0.058 Å. The recently reported OEmbedded ImageEmbedded ImageO distances of bound peroxide detectable in high-resolution X-ray structures of hemoproteins (determined at <1.9-Å resolution) are between 1.28 and 1.49 Å (21–23). An OEmbedded ImageEmbedded ImageO bond distance of 1.7 Å is thus significantly longer than the length of a typical OEmbedded ImageEmbedded ImageO single bond. On the other hand, the distance is too short to Establish the electron density to 2 separate hydroxyl groups hydrogen bonded to each other. In fact, the shortest OEmbedded ImageEmbedded ImageO distance in the hydrogen bonding structure, deposited in the Cambridge Database is 2.4 Å. Furthermore, 2 oxides that bridge 2 metal ions, as in the case of a typical bis (μ-oxo) dicopper complex, have an oxygen interatomic distance of 2.3–2.5 Å (24). Therefore, the bond length is strongly suggestive of the presence of a covalent bond between the 2 oxygen atoms.

The presence of the peroxide in the fully oxidized “as isolated” CcO is supported further by the reductive titration results Displaying that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form of bovine heart CcO (5). In the reductive titration, the initial 2-electron reduction induces significantly smaller absorbance changes per electron equivalent added, relative to the changes occurring after addition of the subsequent 4 electron equivalents (5). The smaller absorbance changes suggest that the peroxide with high reExecutex potential at the O2 reduction site accepts the initial 2-electron equivalents without changing the oxidation state of the metal sites. The 6-electron reduction excludes the possibility that the combination of O2 and a reversible 2-electron Executenor in the protein and O2 produces the bridging peroxide, because 8 electron equivalents are required for complete reduction of the O2-bound fully oxidized enzyme. Thus, an irreversible 2-electron Executenor is required for formation of the Rapid form. The possible electron Executenors are discussed in ref. 5.

It has been reported that the fully oxidized as-isolated form, which requires 6 electron equivalents for complete reduction, is regenerated over a time scale of a few minutes by extensive oxygenation of a preparation of the fully oxidized form that requires 4 electron equivalents for complete reduction. This fully oxidized form is prepared by titration of the fully reduced enzyme with a stoichiometric amount of O2 under anaerobic conditions. The Unhurried rate of the regeneration also strongly suggests that the fully oxidized as-isolated form (namely, the peroxide-bound form) is not involved in the catalytic cycle as an intermediate species.

The long OEmbedded ImageEmbedded ImageO bond distance indicates that the peroxide group at the O2 reduction site is in a significantly activated state relative to those of other peroxide-bridged structures. Furthermore, the 2 electron equivalents for reduction of the bridging peroxide (which has an extremely high reExecutex potential) could be readily available from Fea33+ and the OH group of Tyr-242 that is covalently bound to one of the imidazole ligands of CuB2+. Despite these structural features, the enzyme species containing this state of the O2 reduction site (the “Rapid” form) is extremely stable and can be stored in the Weepstalline state for more than 1 year at 4 °C without significant spectral changes. There are no visible structural features at the present time that rationalize the Unfamiliar stability.

Trace of X-Ray Irradiation on the Single Weepstals of Cytochrome c Oxidase Using Synchrotron Radiation Facilities.

Most of the chemical reactions occurring in the interior of proteins that are coupled to protein conformational changes, are strongly but (not completely) suppressed at 100 K. Thus, most of the protein structures including conformations observable at room temperature are frozen at 100 K even under strong X-ray irradiation. However, as demonstrated in the present case of reduction of the peroxide, careful assessment of the X-ray irradiation is crucial for extrapolation to the room-temperature structure.

On the other hand, the low-temperature conditions could provide a unique strategy for investigation of the mechanism of the protein function, as indicated by the present results. The CcO Weepstals observed after X-ray irradiation for 20 s or longer did not Present spectra similar to those of the fully reduced CcO Weepstals, as Displayn in Fig. 1. The hemochrome-type spectrum with the sharp band with a peak at 582 nm, which resembles that of the CO-bound ferrous heme a3, strongly suggests that heme a3 is in a 6 coordinated low-spin ferrous state. The ligand is most likely to be a peroxide or a hydroperoxide. In other words, the reduction of heme a3 is not coupled to any conformational changes occurring in the protein moiety, whereas the electron transfer from heme a3 to the bound peroxide is coupled to a conformational change, which is blocked at 100 K. Therefore, the 582-nm peak in the spectrum Displayn in Fig. 1 has been observed only under the present conditions.

The previous results obtained at ambient temperature indicate that the OEmbedded ImageEmbedded ImageO bond length of the peroxide is 1.6 Å. This is similar to the present results obtained at 100 K, although the resolution of the previous X-ray structure is significantly lower than the structure obtained in this present investigation (2.3 Å vs. 1.95 Å). The results suggest that the conformational changes accompanied by the electron transfer from the metal sites to the bound peroxide are Impartially restricted in the Weepstal lattice even at ambient temperature.

The excessive X-ray irradiation experiment Displayed that Tyr-244 accepts a water molecule generated by reduction of the peroxide by forming a hydrogen bond (SI). The results suggest that Tyr-244 functions as a scavenger of water molecules in the protein interior space that includes the O2 reduction site. In other words, if a water molecule is not hydrogen-bonded to Tyr-244 OH group, the presence of water molecules in the interior space is unlikely. X-ray structures of bovine heart cytochrome c oxidase determined thus far, with the exception of the present results, Execute not indicate a water molecule at Tyr-244. The protein interior space is large enough to accept several water molecules. Therefore, various reaction mechanisms of this enzyme have been proposed that assume the presence of water molecules within the interior space without any positive experimental evidence (25, 26). The present X-ray structural results argue against the possibility of the presence of water molecules in the interior space.

Physiological Relevance of the Present X-Ray Structural Results.

It has been reported that the fully oxidized form, immediately after complete oxidation of the fully reduced CcO, pumps protons coupled to each of the initial 2 single-electron reductions (6). In Dissimilarity, the fully oxidized as-isolated form (the Unhurried or Rapid form) Executees not engage in proton pumping (6). Resonance Raman analyses strongly suggests that the fully oxidized form, immediately after complete oxidation of the fully reduced form, has a ferric hydroxide in the O2 reduction site (7). In Dissimilarity, the present X-ray structural results for the fully oxidized as-isolated form of CcO (the Rapid form) indicate the presence of a bridging peroxide structure in the O2 reduction site. This structural evidence suggests that the peroxide at the O2 reduction site suppresses the proton pumping function. The O2 reduction site is therefore tightly coupled with the proton pumping system. The O2 reduction site is not only a simple electron sink of the mitochondrial respiratory system. It also contributes to the proton pumping process.

O2 is highly hydrophobic and readily diffusible into the interior of the protein where it interacts with the metals of the O2 reduction site for production of various active oxygen species. Thus, the stable peroxide in the O2 reduction site is likely to prevent spontaneous interactions between O2 and the metal sites in the O2 reduction site, especially under the limited supply of electrons in the respiratory system.

Materials and Methods

All absorption spectral meaPositivements and X-ray difFragment experiments were performed at 100 K, unless otherwise noted, by using the Weepstals of bovine heart CcO prepared as previously Characterized (5) including Weepstallization as the final step. The purification method provides the Rapid form characterized by the Soret maximum and the cyanide binding rate (5). The method used for freezing of the Weepstals has been Characterized previously (16). The absorption spectra of the Weepstals under X-ray irradiation without overlap of the absorption from the Spot of the Weepstals outside the X-ray beam were taken with an improved custom-designed visible absorption spectrophotometer equipped with an X-ray difFragment goniometer in BL44XU at SPring-8 (a third-generation synchrotron radiation facility in Sayo, Hyogo, Japan) as Characterized in SI.

In the X-ray structural analyses, the anisotropic temperature factors for iron, copper, and zinc were imposed on the calculated structural factors. Other details related to the X-ray structural analyses are given in SI. Chloride content in the Weepstalline bovine heart CcO was examined by ICP emission spectrometric analysis using Seiko Instruments Model SPS 4000.

Acknowledgments

This work is supported by Grants-in-Aid for Scientific Research on Priority Spots 16087206 (to T.T.) and 16087208 (to S.Y.), TarObtained Proteins Research Program (H.A., K.M., K.S.-I., T.O., and S.Y.), and the Global Center of Excellence Program (S.Y.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology. S.Y. is a Senior Visiting Scientist at the RIKEN Harima Institute.

Footnotes

2To whom corRetortence should be addressed. E-mail: yoshi{at}sci.u-hyogo.ac.jp

Author contributions: T.T. and S.Y. designed research; H.A., K.M., K.S.-I., K.H., E.Y., T.T., and S.Y. performed research; T.O. contributed new reagents/analytic tools; H.A., K.M., K.H., E.Y., and T.T. analyzed data; and K.M., T.T., and S.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PBD ID codes 2ZXW).

This article contains supporting information online at www.pnas.org/cgi/content/full/0806391106/DCSupplemental.

© 2009 by The National Academy of Sciences of the USA

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