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Proc Natl Acad Sci U S A. May 8, 2007; 104(19): 7881–7886.
Published online Apr 30, 2007. doi:  10.1073/pnas.0610031104
PMCID: PMC1876541

A histidine residue acting as a controlling site for dioxygen reduction and proton pumping by cytochrome c oxidase


Cytochrome c oxidase transfers electrons and protons for dioxygen reduction coupled with proton pumping. These electron and proton transfers are tightly coupled with each other for the effective energy transduction by various unknown mechanisms. Here, we report a coupling mechanism by a histidine (His-503) at the entrance of a proton transfer pathway to the dioxygen reduction site (D-pathway) of bovine heart cytochrome c oxidase. In the reduced state, a water molecule is fixed by hydrogen bonds between His-503 and Asp-91 of the D-pathway and is linked via two water arrays extending to the molecular surface. The microenvironment of Asp-91 appears in the x-ray structure to have a proton affinity as high as that of His-503. Thus, Asp-91 and His-503 cooperatively trap, on the fixed water molecule, the proton that is transferred through the water arrays from the molecular surface. On oxidation, the His-503 imidazole plane rotates by 180° to break the hydrogen bond to the protonated water and releases the proton to Asp-91. On reduction, Asp-91 donates the proton to the dioxygen reduction site through the D-pathway. The proton collection controlled by His-503 was confirmed by partial electron transfer inhibition by binding of Zn2+ and Cd2+ to His-503 in the x-ray structures. The estimated Kd for Zn2+ binding to His-503 in the x-ray structure is consistent with the reported Kd for complete proton-pumping inhibition by Zn2+ [Kannt A, Ostermann T, Muller H, Ruitenberg M (2001) FEBS Lett 503:142–146]. These results suggest that His-503 couples the proton transfer for dioxygen reduction with the proton pumping.

Keywords: proton pump, x-ray structure, proton collection, mitochondrial energy transduction

Cytochrome c oxidase (CcO), the terminal oxidase of cellular respiration, reduces dioxygen (O2) to water coupled with proton pumping. Thus, this enzyme transfers protons for water formation and for proton pumping. The proton transfer must be tightly coupled with the O2 reduction, which includes electron transfer, for effective energy transduction. It is likely that each step of the proton transfer is an actively controlled step driven by a redox-coupled conformational change. These redox-coupled conformational changes can be directly identified in high-resolution x-ray structures determined for different oxidation states of the enzyme. We have identified redox-coupled conformational changes of Asp-51 and the OH group of the long alkyl side chain of heme a. Both of these functional groups are located within a proposed proton transfer pathway. The structural changes suggest a proton-pumping mechanism from the positive side to the negative side, driven by heme a (negative and positive sides refer to the enzyme surface exposed to the inside and outside of the mitochondrial inner membrane or the bacterial cytoplasmic membrane) (1, 2).

X-ray structural and functional analyses by using Zn2+ and Cd2+ as probes have provided various insights into the mechanisms of proton transfer of some proteins (36). Recent extensive studies on the effect of Zn2+ on the reaction of CcO have suggested that this enzyme has Zn2+-binding sites on both its positive and negative sides to slow down or abolish proton release and uptake (712). These experimental results would clarify the proton transfer mechanism of CcO if the Zn2+-binding structure could be determined by x-ray structural investigations.

Here, we report observation of a redox-coupled conformational change in a well-conserved histidine, His-503, which is located near Asp-91 in the D-pathway. The conformational change suggests effective proton collection and a controlled supply of protons to the D-pathway. X-ray structures of Cd2+ and Zn2+ derivatives indicate that these metal ions bind to His-503. The estimated Zn2+-binding affinity to His-503 in the x-ray structures is consistent with previous reports of the Zn2+ concentration required for 50% inhibition of proton pumping (7). This result suggests that His-503 is directly involved in the coupling of the proton-pumping process with the proton transfer process used in the O2 reduction reaction.


Redox-Coupled Conformational Change of His-503 at the Entrance of D-Pathway.

Previous reports based on 2.3-Å (or lower) x-ray structures indicated that Asp-91 is located at the entrance of the D-pathway (1, 13, 14). The water molecules near the entrance to the D-pathway are not well resolved in these structures. In contrast, our 1.8/1.9-Å x-ray structures (Fig. 1) indicate that Asp-91 is located within the interior of the protein and connected with the negative side surface via a fixed water molecule (W207), which is linked, via two water arrays extending to two water molecules (W4 and W201) at the molecular surface in the reduced state of the enzyme (Fig. 1a). Fixed water molecules are denoted by W and arbitrary numbers in the text and by only an arbitrary number in the figures. In the reduced state, the imidazole group of His-503 is located between these two water molecules at the surface.

Fig. 1.
Redox-coupled conformational changes in the His-503–Asp-91 region near the entrance of the D-pathway deduced from the x-ray structures of bovine heart CcO from the 1.8-Å oxidized and 1.9-Å reduced x-ray structures of bovine heart ...

In the present study, the 1.8/1.9-Å resolution x-ray structures were used to examine the orientation of the imidazole group of His-503 for the oxidized and reduced states by inspecting hydrogen-bonding interactions with water molecules. In the reduced state, the imidazole group forms two hydrogen bonds to W4 and W207. The location of W4 in the reduced state is stabilized by W28 and W490. The latter is fixed by the amide group of Asn-12 of subunit III and the peptide CO group of Met-10 of subunit III as shown in Fig. 1a (in the figures and text, amino acid residues labeled without the subunit name refer to residues of subunit I, the largest of the 13 different subunits in bovine heart CcO). The carboxyl group of Asp-91 is hydrogen bonded with two water molecules and two peptide NH groups (Fig. 1 a and b). This arrangement suggests that the pKa of the carboxyl group is significantly higher than it is when exposed to bulk water phase (15). Thus, the pKa value of Asp-91 is similar to the pKa of His-503. These observations suggest that His-503 and Asp-91 stabilize the hydronium ion state at W207.

Furthermore, four additional hydrogen bond-forming groups are arranged such that they form an approximately rectangular plane, with W207 at the geometrical center. In the reduced state of the enzyme, the two hydrogen bonds to Asp-91 and to His-503 are roughly perpendicular to this plane (Fig. 1a). The six functional groups detectable in the x-ray structure cannot simultaneously participate in hydrogen-bonding interactions with W207, because a single water molecule may only form a maximum of four hydrogen bonds with tetrahedral geometry. Thus, at least two of the six functional groups do not form hydrogen bonds with W207. These groups could be exchangeable. Four of the six groups can participate in hydrogen bond formation as either hydrogen donors or acceptors without inducing a large conformational change. This structural feature indicates that W207 could form three “hydrogen-donating” hydrogen bonds and one “hydrogen-accepting” hydrogen bond. The hydronium ion state of Wat207 would also be stabilized by four such hydrogen bonds. Furthermore, the exchangeability of the hydrogen bond-forming pair without significant conformational change would increase the stability of the hydronium ion state, once formed between Asp-91 and His-503 with essentially the same proton affinity as described above.

The 1.8-Å/1.9-Å resolution x-ray structures suggest that, on oxidation, the His-503 imidazole plane rotates by almost 180° at the Cβ–Cγ bond, causing loss of the hydrogen bonds to W207 (Fig. 1b). The W4 water molecule, which, in the reduced state, is visibly hydrogen bonded to the Nε atom of the His-503 imidazole, disappears on oxidation. Instead, the Nδ atom introduces W6 and the Nε atom forms a hydrogen bond to W28. In the oxidized state, W28 is located at the molecular surface instead of W4. The possible redox-coupled conformational changes are summarized in Fig. 1c. By removing the hydrogen bond from His-503, the proton on W207 would be destabilized even though the conformation of the rest of the five residues involved in hydrogen bonds are not significantly influenced by loss of that hydrogen bond. Thus, the W207 proton would be readily extracted by Asp-91, which has significant proton affinity as described above.

X-Ray Structures of Cd2+ and Zn2+ Derivatives of Bovine Heart CcO.

X-ray diffraction data were collected from the crystals of the Cd2+ and Zn2+ derivatives in the reduced and oxidized states. The metal ion treatments were performed by soaking crystals with the metal ion-containing medium. Statistics of intensity data are calculated for the best data set of both oxidation states [supporting information (SI) Text]. The anomalous difference Fourier maps revealed several anomalous difference electron densities. In addition to those for the intrinsic metals (Fea, Fea3, and CuB in the subunit I; CuA in the subunit II; and Zn1 in the subunit Vb), three Cd2+-binding sites or seven Zn2+-binding sites appeared in cases where the crystals were soaked with Cd2+ or Zn2+ ion (Fig. 2). The Cd1 and Cd2 sites are identical with the Zn2 and Zn3 sites, respectively. The former and the latter sites are denoted as Cd1/Zn2 site and Cd2/Zn3 site in this paper. The detailed x-ray structures of the metal-binding sites other than the Cd2/Zn3 site are given in SI Text.

Fig. 2.
Anomalous difference Fourier maps of CcO crystals treated with 0.5 mM CdSO4 (Left) or 5 mM ZnSO4 (Right). The wavelength is 0.9 Å (f″Cd = 1.84, f″Zn = 2.15, f″Cu = 1.91, f″Fe = 1.30). Cα backbone traces ...

The enzyme exists in a dimer state in the crystal lattice. One of the monomers (denoted molecule A) has stronger specific protein–protein interactions from the adjacent protein molecules in the crystal lattice relative to those of the other monomer (denoted molecule B). Because of the stronger specific interactions from the adjacent molecules in the lattice, the electron density of molecule A is better defined than that of molecule B. CcO molecule, as most other globular proteins, has various conformations in equilibrium (conformers) (16). The uneven constraints to the two monomers from the crystal lattice could produce different population of the conformers depending on the location of the monomer in the dimer in the crystal (i.e., molecule A or molecule B) to provide different reactivity to the external reactants. In fact, as described below, the reactivity of the Cd2/Zn3 site in the crystal is significantly dependent on the location in the dimer. Any conformer of the monomers that appears in the crystal lattice exists as one of the conformers in the physiological conditions (in the solution state) where any constraint in the crystal lattice is absent. In other words, it is possible that the lattice constraints trap a conformer that is too unstable in the solution state to identify readily.

The difference in the reactivity to the metal ions on the location of the monomer would not be detected if the metal derivatives prepared in the solution state were crystallized. The lattice constraints influence the kinetics of the metal ion bindings. Because of the destabilizing effects of these metal ions to the crystalline state of CcO, the conditions for soaking of the CcO crystals with these metal ion solutions are very limited. Therefore, any metal ion binding, found in the x-ray structure as shown in Fig. 2, is likely to occur in the solution state or under the physiological conditions.

Structures of the Cd2/Zn3 site near the entrance to the D-pathway are influenced by the oxidation state of CcO as well as by the location of the monomer in the crystal lattice. In the reduced state of the enzyme, Cd2+ is coordinated to the Nε atom of His-503 imidazole group in a conformation that is essentially identical to that of the native enzyme in molecule A (Fig. 3a). The water molecules W28 and W490, and the imidazole of His-2VIIc are coordinated to the bound Cd2+. As a result, His-2VIIc moves closer to Cd2+ relative to its location in CcO before addition of Cd2+. At Cd2+ concentration of 500 μM, the Cd2+ occupancy was estimated to be almost full by comparing the peak height of the anomalous difference electron density of Cd2+ (ƒ″Cd = 1.84 at the wavelength of 0.9 Å) with that of the intrinsic Zn2+ (ƒ″Zn = 2.15 at the wavelength of 0.9 Å), which fully occupied Zn1 site. In molecule B, another Cd2+ forms a bridge between Glu-506 and His-3III, in addition to the Cd2+ coordinated to His-503 (Fig. 3b).

Fig. 3.
His-503 site structures of Cd2+ derivatives in stereoviews. The red and blue spheres represent fixed water molecules and Cd2+, respectively. The blue dotted lines indicate coordination bonds to the Cd2+. The blue, green, and brown sticks denote the C ...

In the oxidized state, Cd2+ in the Cd2/Zn3 site is coordinated to the Nδ atom of the His-503 imidazole group and to His-2VIIc (Fig. 3c) in molecule A but not in molecule B. Two water molecules (W500 and W5) are coordinated to the Cd2+. The redox-coupled structural change of the imidazole group of His-503 is essentially the same as that of the Cd2+-free enzyme, although the imidazole ring of the oxidized state moves further away from the position it occupies before binding of Cd2+ (Fig. 1d).

In the reduced state of the enzyme, the two Zn2+-binding structures at the Cd2/Zn3 site are similar to the Cd2+-binding structures at Cd2+ site shown in Fig. 4 a and b. The metal-binding site between Glu-506 and His-3III seems to have weaker affinity to both Cd2+ and Zn2+ ions than that of the site between His-503 and His-2VIIc, because some crystals do not have the metal ions at the former site (three data sets/eight data sets). In the oxidized state, two Zn2+ atoms occupy the positions of W5 and W6 and are separated by 2.8 Å in molecule B. The atomic distance is too short for coexistence of two Zn2+ atoms at the site. This x-ray structure indicates that two structures are in equilibrium, with a Zn2+ replacing either W6 (Fig. 4c) or W5 (Fig. 4d). The electron density assignments of the anomalous difference map are consistent with the electron density peaks of the two Zn2+ ions, each of which is ≈50% of that of the intrinsic Zn2+ electron density at the Zn1 site. The Zn2+ replacing W6 coordinates His-503, His-2VIIc, W5, and W500 in essentially the same fashion (Fig. 4c) as the Cd2+ ion coordinates the same residues in the oxidized state. The Zn2+ replacing W5 coordinates W6, Glu-506, and His-3III (Fig. 4d). In molecule A, Zn2+ binding to oxidized CcO induces significant conformational changes in the coordinating residues His-503, His-2VIIc, and Glu-5VIIc (Fig. 4e). In this structure, the His-503 imidazole is coordinated to the Zn2+ at the Nε atom without the need for rotation of the imidazole plane.

Fig. 4.
His-503 site structures of Zn2+ derivatives in stereoviews. The red and blue spheres denote fixed water molecules and Zn2+ ions, respectively. The blue dotted lines represent coordination bonds. The blue, green, and brown sticks denote Cα backbones ...

The structures of the three Zn2+-binding sites of the five states (Fig. 4 a–c) are closely similar to those of the three Cd2+-binding sites (Fig. 3 a–c), respectively. These structures confirm the redox-coupled conformational change in His-503, that is, the rotation of the imidazole plane. The other two structures shown in Fig. 4 d and e were not induced by Cd2+ ion, perhaps because the size of the Cd2+ ion is larger than that of Zn2+.

In the oxidized state, the lowest Zn2+ concentration required to observe the anomalous difference electron density at the Cd2/Zn3 site was found to be 100 μM. At this Zn2+ concentration, Zn2+ binding was not detected in molecule B. The Zn2+ binding was detectable in both molecule A and molecule B at a Zn2+ concentration of 500 μM. The Cd2/Zn3 site occupancy was estimated to be 70–90% (that is, essentially fully occupied) in the reduced state at Zn2+ concentration of 500 μM. (Zn2+ binding for the reduced form was not examined at lower Zn2+ concentration.) These results suggest that Zn2+ has similar affinity to the Cd2/Zn3 site in both oxidation states. Because of the significant effects of the lattice constraints on the metal ion bindings as described above, it seems impossible to determine the Zn2+ affinity in the solution state accurately only from the experimental results obtained from CcO in the crystal lattice. Nevertheless, the above results suggest that the Cd2/Zn3 sites of CcO in the solution state has Kd value of 100 μM or lower in both oxidation states.

Recently, Cd2+ binding at the entrance of K-pathway of Rhodobacter sphaeroides CcO between Glu-101 and His-96 of subunit II has been reported (17). However, this Cd2+ binding is unlikely to occur in the bovine CcO because the His-96 is not conserved in the bovine CcO.

The Inhibitory Effects of Cd2+ and Zn2+.

The inhibitory effects of Cd2+ and Zn2+ on ferrocytochrome c oxidation activity by bovine heart CcO were examined at pH 7.4 at 20°C by using sulfate salts of the metal ions. Inclusion of Cd2+ or Zn2+ in the reaction mixture did not result in complete enzyme inhibition. A single phase inhibition by Zn2+ was observed with a Kd of 76 μM and 30% residual activity, which is the enzyme activity asymptotically approaching with increase in the Cd2+ concentration. The Cd2+ inhibited the reaction in a biphasic manner with a Kd of 0.3 μM for 17% inhibition and 450 μM for 70% inhibition. The residual activity was 13%. The observation of partial enzyme inhibition seems to be consistent with the occurrence of redox-coupled conformational changes in the metal derivatives, which are essentially similar to the conformational changes occurring in the enzyme when it is free of exogenous metal ions.

A Zn2+/Cd2+ Site in Subunit III.

In the absence of exogenous Zn2+ or Cd2+, the Cd1/Zn2 site of subunit III (where Zn2+ or Cd2+ is coordinated by His-148, His-232, and Glu-236 as shown in SI Fig. 6 a and c) showed detectable anomalous difference electron density, at the wavelength of 0.9 Å (ƒ″Zn = 2.15) with the peak height depending on the individual crystals used for x-ray diffraction experiments. In some cases, the peak height was found to be as high as that of the intrinsic Zn2+ at the Zn1 site. The average peak height of Zn2+ determined for 20 independent data collections is ≈30% of that of Zn2+ at the Zn1 site. The anomalous dispersion of the Cd1/Zn2 site was examined at various x-ray wavelengths for identification of the metal ion at the Cd1/Zn2 site in the absence of exogenous Zn2+. The anomalous difference electron density at the Cd1/Zn2 site was detected at the wavelength of 1.20 Å (ƒ″Zn = 3.44), together with the intrinsic Zn2+ site (Zn1), the two copper (ƒ″Cu = 3.07) sites, and the two iron (ƒ″Fe = 2.12) sites. X-rays with longer wavelength, 1.76 Å (ƒ″Zn = 0.86), 1.50 Å (ƒ″Zn = 0.65), and 1.35 Å (ƒ″Zn = 0.53), did not show the anomalous difference electron density at the Cd1/Zn2 site. Addition of 5 mM EDTA resulted in disappearance of the anomalous difference electron density at the Cd1/Zn2 site. These anomalous difference maps are given in SI Text. Metal analysis indicated the presence of ≈25% additional Zn2+ relative to the Zn2+ content of the sample washed with 5 mM EDTA. These results indicate that the Cd1/Zn2 site binds Zn2+ at various saturation levels in a manner dependent on individual crystals. On the other hand, the enzyme activity is quite consistent regardless of the enzyme preparation. Therefore, the binding of Zn2+ to subunit III is unlikely to influence electron and proton transfer through the enzyme.

The peak height of anomalous difference electron density at the Cd1/Zn2 site increases significantly on exposure of the crystals to Zn2+ at concentrations of 1 μM or higher. Therefore, the Kd of the Cd1/Zn2 site should be ≈1 μM, which represents a higher affinity than that of the Cd2/Zn3 site at His-503.


The Redox-Coupled Conformational Change of His-503.

The redox-coupled positional changes in water molecules and Cd2+ and Zn2+ ions bound to His-503 (Figs. 1, ,3,3, and and4)4) are consistent with the rotation of the imidazole ring of His-503 as summarized in Fig. 1c. The His-503 site is a long way from any of the redox-active metal sites. No significant x-ray structural change in the protein moiety between His-503 and the redox-active metal sites has been detected at the present resolutions. However, structural changes in the protein moiety much smaller than those detectable at the present resolutions of the x-ray structure could significantly influence interactions between amino acid residues inside the protein. Thus, the present x-ray structural results do not provide the conclusive evidence against the presence of communication systems between His-503 and the redox-active sites. On the other hand, no change in the crystal packing induced by the oxidation state change of CcO has been detected. At present, no experimental result positively suggests that the conformational change in His-503 is induced by a crystallization effect.

The Role of the His-503 in Supplying Protons to the D-Pathway.

In addition to the rotation of the imidazole plane of His-503, the high effective pKa value of Asp-91 suggested by the hydrogen-bonding structures shown in Fig. 1 is also critical for redox-controlled proton collection. This structural feature is consistent with the abolishment of the enzyme activity induced by mutation of Asp-91 to Asn (18). Asn-91, which has extremely weak basicity, cannot stabilize the hydronium ion state of W207 in the reduced state.

These redox-coupled structural changes suggest that redox-controlled proton collection and supply by the His-503–Asp-91 system occurs as shown in Fig. 5. In this scheme, for the sake of simplicity, only one of the two water arrays connecting the molecular surface with W207 is shown and W6 is not shown in the oxidized state. In the reduced state (a), W207 is stabilized in the hydronium ion state by His-503 and Asp-91 via the two hydrogen bonds. The x-ray structure of the reduced state corresponds to this structure. On oxidation, the imidazole plane rotates by 180° and the hydrogen bond between His-503 and W207 is disrupted. As a result, the proton at W207 is transferred to Asp-91 (b) and W4 is released (c). In this state, the enzyme is stable in the absence of oxidation state changes at the redox-active metal sites. The x-ray structure of the oxidized state was determined for this form. On reduction, Asp-91 releases protons that migrate to the O2 reduction site via the D-pathway (d). At this point, the His-503 imidazole plane rotates and the hydrogen bond with W207 is formed (e), followed by introduction of a hydronium ion as W4 (f). The proton at W4 is then transferred to W207 giving the original state (a).

Fig. 5.
Redox-controlled proton collection and supply by His-503. Only one of the water arrays connecting W207 and W4 and two other possible hydrogen-bonding groups to W207 is shown for the sake of simplicity. The red and blue structures represent the oxidized ...

This scheme includes introduction of protons to the system via a hydronium ion at step e→f. The system could also accept a proton after receiving W4 or through other water arrays not shown in this scheme. One of the critical points of this scheme is that, on reduction, the Asp-91 proton is released (c→d) to the O2 reduction site before the rotation of the imidazole plane occurs (d→e). Otherwise, protons at Asp-91 would be returned to W207 on reduction. This conformational change in His-503 could be driven by, at most, four sites because CcO has four redox-active metal sites. Thus, a single equivalent of electron equivalent from cytochrome c could collect more than one proton equivalents during the electron transfer to the oxygen at heme a3. In other words, this redox-controlled proton supply mechanism does not include any proposal for the H+/e ratio. The redox site (or sites) driving this conformational change of the His-503 imidazole has yet to be identified.

Proton collecting functions of carboxyl groups and imidazole groups on the negative side surface of CcO have been proposed without assuming any redox-coupled conformational change (19, 20). The x-ray structural changes in His-503, determined by the present work, indicate that His-503–Asp-91 system not only collects protons from the negative side surface but also facilitates the redox-controlled proton supply to the D-pathway.

Effects of Binding of Zn2+ on the Function of Bovine CcO.

The present x-ray structural analyses show six Zn2+-binding sites (Fig. 2). The Cd1/Zn2 site seems to have the Zn2+ affinity stronger than that of the Cd2/Zn3 site (including His-503) as described above. Therefore, the subunit III site could scavenge Zn2+ in the mitochondrial negative side space to prevent the decoupling effect of Zn2+ at the His-503 site as described below. The x-ray structures (Fig. 4) strongly suggest that the Cd2/Zn3 site is the Zn2+ inhibition site. The remaining four Zn2+sites located within nuclear-coded subunits are unlikely to influence the enzymatic function of CcO, because the sites are far apart from the active sites of CcO.

The Zn2+ effects on the reaction of CcO reported thus far (712) indicate that CcO has Zn2+-binding sites on both the positive and negative side surfaces to inhibit the enzymatic function (7, 10, 12). It should be noted that the Zn2+-binding site on the positive side surface that could inhibit the enzymatic function has not been identified in the present x-ray structural analyses. The Zn2+ effects depend strongly on the membrane potential of the reconstituted vesicles (10). Furthermore, Zn2+ inhibition depends on the stage of O2 reduction process as well as the assay conditions. In the reaction of the fully reduced CcO with O2, the intermediate species P, F, and O appear after the formation of the oxygenated species. The 50% decreases in the P→F and F→O transition rates are caused by ≈120 and 2 μM Zn2+ to the inside surface, exhibiting ≈30% and 40% of the residual activities, respectively (8). The steady-state turnover rate of CcO determined by using the soluble O2 consumption assay system provided a Zn2+ inhibition constant (KI) of 9 μM (10, 11), whereas the KI value of the steady-state turnover measured with ferrocytochrome c oxidation is 76 μM Zn2+ (present work). Both inhibitions showed residual activities. These differences in the Zn2+ effect are likely to be caused by difference in the rate-limiting step that provides different overall Zn2+ effects. (In a partial inhibition system, the affinity of the rate-limiting intermediate species to the inhibitor determines the overall inhibition level.) X-ray structural analyses for the Zn2/Cd3 site as described above show five Zn2+-binding structures in total (Fig. 4). The structural diversity suggests that different intermediates show different Zn2+ affinity to provide these varieties in the Zn2+ effect as described above.

The KI for complete inhibition of proton pumping is ≈70 μM Zn2+ (7), whereas the partial inhibition of ferrocytochrome c oxidation with 30% residual activity showed KI of 76 μM (present work). The Zn2+-binding site for these inhibitions is likely to be His-503, because these KI values are essentially consistent with that to the His-503 site estimated by x-ray structural analyses (the level of 100 μM). This consistency suggests that the Zn2+-binding site controls both the proton transfer through D-pathway and the proton pumping. The proton transfer through D-pathway is tightly coupled with the electron transfer for the O2 reduction that drives the proton pumping. However, it is unlikely that the inhibition of proton pumping by the Zn2+ binding is induced indirectly by blocking the proton transfer through D-pathway, because the partial inhibition of the electron transfer by Zn2+ cannot induce the complete abolishment of proton pumping. In other words, Zn2+ binding at His-503 is likely to influence directly the proton-pumping site to impair its function.

At the present resolution of the x-ray structures of the Zn2+ derivatives, no significant structural effects that block of the proton pump as a result of binding of Zn2+ at His-503 site were identified. Two proton-pumping mechanisms have been proposed: one that includes the D-pathway (18) and another that includes the H-pathway, a proton transfer pathway located near heme a (2). Both proposals include proton-pumping sites located far away from His-503. Improvement of the resolution of the Zn2+ derivative of CcO would provide important clues for elucidation of the mechanism of proton pumping.

Materials and Methods

Crystals of Cd2+ and Zn2+ complexes of bovine heart CcO were prepared by soaking the CcO crystals obtained by the method described previously (21) in a medium containing 0.5 mM CdSO4 or 0.5–5 mM ZnSO4. Other experimental conditions for crystallizations, x-ray structural analyses, and the measurements of the inhibitory effects of Cd2+ and Zn2+ on CcO are given in SI Text. The values of ƒ″Cu, ƒ″Fe, ƒ″Zn, and ƒ″Cd are cited from ref. 22.


This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology, and the Core Research for Evolutional Science and Technology.


cytochrome c oxidase
heme a
a low-spin heme A of cytochrome c oxidase
f″atomic symbol
the imaginary part of the anomalous dispersion of the atom represented by symbol.


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 (PDB ID codes 2DYR, 2EIJ, 2EIK, 2EIL, 2EIM, and 2EIN).

This article contains supporting information online at www.pnas.org/cgi/content/full/0610031104/DC1.


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