A histidine residue acting as a controlling sitefor dioxygen reduction and proton pumpingby cytochrome c oxidaseKazumasa Muramoto*, Kunio Hirata†, Kyoko Shinzawa-Itoh*, Shinji Yoko-o*, Eiki Yamashita†, Hiroshi Aoyama‡,Tomitake Tsukihara†, and Shinya Yoshikawa*§

*Department of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-1297, Japan; †Institute for Protein Research, OsakaUniversity, 3-2 Yamada-oka, Suita, Osaka 565-0871, Japan; and ‡RIKEN Harima Institute, Mikazuki Sayo, Hyogo 679-5148, Japan

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved March 6, 2007 (received for review November 11, 2006)

Cytochrome c oxidase transfers electrons and protons for dioxygenreduction coupled with proton pumping. These electron and pro-ton transfers are tightly coupled with each other for the effectiveenergy transduction by various unknown mechanisms. Here, wereport a coupling mechanism by a histidine (His-503) at the en-trance of a proton transfer pathway to the dioxygen reduction site(D-pathway) of bovine heart cytochrome c oxidase. In the reducedstate, a water molecule is fixed by hydrogen bonds betweenHis-503 and Asp-91 of the D-pathway and is linked via two waterarrays extending to the molecular surface. The microenvironmentof Asp-91 appears in the x-ray structure to have a proton affinityas high as that of His-503. Thus, Asp-91 and His-503 cooperativelytrap, on the fixed water molecule, the proton that is transferredthrough the water arrays from the molecular surface. On oxida-tion, the His-503 imidazole plane rotates by 180° to break thehydrogen bond to the protonated water and releases the protonto Asp-91. On reduction, Asp-91 donates the proton to the dioxy-gen reduction site through the D-pathway. The proton collectioncontrolled by His-503 was confirmed by partial electron transferinhibition by binding of Zn2� and Cd2� to His-503 in the x-raystructures. The estimated Kd for Zn2� binding to His-503 in the x-raystructure 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 suggestthat His-503 couples the proton transfer for dioxygen reductionwith the proton pumping.

proton pump � x-ray structure � proton collection �mitochondrial energy transduction

Cytochrome c oxidase (CcO), the terminal oxidase of cellularrespiration, reduces dioxygen (O2) to water coupled with

proton pumping. Thus, this enzyme transfers protons for waterformation and for proton pumping. The proton transfer must betightly coupled with the O2 reduction, which includes electrontransfer, for effective energy transduction. It is likely that each stepof the proton transfer is an actively controlled step driven by aredox-coupled conformational change. These redox-coupled con-formational changes can be directly identified in high-resolutionx-ray structures determined for different oxidation states of theenzyme. We have identified redox-coupled conformational changesof Asp-51 and the OH group of the long alkyl side chain of hemea. Both of these functional groups are located within a proposedproton 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 enzymesurface exposed to the inside and outside of the mitochondrial innermembrane 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 ofproton transfer of some proteins (3–6). Recent extensive studies onthe effect of Zn2� on the reaction of CcO have suggested that thisenzyme has Zn2�-binding sites on both its positive and negative

sides to slow down or abolish proton release and uptake (7–12).These experimental results would clarify the proton transfer mech-anism of CcO if the Zn2�-binding structure could be determined byx-ray structural investigations.

Here, we report observation of a redox-coupled conforma-tional change in a well-conserved histidine, His-503, which islocated near Asp-91 in the D-pathway. The conformationalchange suggests effective proton collection and a controlledsupply of protons to the D-pathway. X-ray structures of Cd2� andZn2� derivatives indicate that these metal ions bind to His-503.The estimated Zn2�-binding affinity to His-503 in the x-raystructures is consistent with previous reports of the Zn2� con-centration required for 50% inhibition of proton pumping (7).This result suggests that His-503 is directly involved in thecoupling of the proton-pumping process with the proton transferprocess used in the O2 reduction reaction.

ResultsRedox-Coupled Conformational Change of His-503 at the Entrance ofD-Pathway. Previous reports based on 2.3-Å (or lower) x-raystructures indicated that Asp-91 is located at the entrance of theD-pathway (1, 13, 14). The water molecules near the entrance tothe D-pathway are not well resolved in these structures. Incontrast, our 1.8/1.9-Å x-ray structures (Fig. 1) indicate thatAsp-91 is located within the interior of the protein and con-nected with the negative side surface via a fixed water molecule(W207), which is linked, via two water arrays extending to twowater molecules (W4 and W201) at the molecular surface in thereduced state of the enzyme (Fig. 1a). Fixed water molecules aredenoted by W and arbitrary numbers in the text and by only anarbitrary number in the figures. In the reduced state, theimidazole group of His-503 is located between these two watermolecules at the surface.

In the present study, the 1.8/1.9-Å resolution x-ray structureswere used to examine the orientation of the imidazole group ofHis-503 for the oxidized and reduced states by inspectinghydrogen-bonding interactions with water molecules. In thereduced state, the imidazole group forms two hydrogen bonds to

Author contributions: K.M., T.T., and S.Y. designed research; K.M., K.H., K.S.-I., S.Y.-o, E.Y.,H.A., T.T., and S.Y. performed research; K.M., K.H., E.Y., H.A., T.T., and S.Y. analyzed data;and K.M., T.T., and S.Y. wrote the paper.

This article is a PNAS Direct Submission.

The authors declare no conflict of interest.

Abbreviations: CcO, cytochrome c oxidase; heme a, a low-spin heme A of cytochrome coxidase; f�atomic symbol, the imaginary part of the anomalous dispersion of the atom repre-sented by symbol.

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).

§To whom correspondence should be addressed. E-mail: yoshi@sci.u-hyogo.ac.jp.

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

© 2007 by The National Academy of Sciences of the USA

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W4 and W207. The location of W4 in the reduced state isstabilized by W28 and W490. The latter is fixed by the amidegroup of Asn-12 of subunit III and the peptide CO group ofMet-10 of subunit III as shown in Fig. 1a (in the figures and text,amino acid residues labeled without the subunit name refer toresidues of subunit I, the largest of the 13 different subunits inbovine heart CcO). The carboxyl group of Asp-91 is hydrogenbonded with two water molecules and two peptide NH groups(Fig. 1 a and b). This arrangement suggests that the pKa of thecarboxyl group is significantly higher than it is when exposed tobulk water phase (15). Thus, the pKa value of Asp-91 is similarto the pKa of His-503. These observations suggest that His-503and Asp-91 stabilize the hydronium ion state at W207.

Furthermore, four additional hydrogen bond-forming groupsare arranged such that they form an approximately rectangularplane, with W207 at the geometrical center. In the reduced state

of the enzyme, the two hydrogen bonds to Asp-91 and to His-503are roughly perpendicular to this plane (Fig. 1a). The sixfunctional groups detectable in the x-ray structure cannot si-multaneously participate in hydrogen-bonding interactions withW207, because a single water molecule may only form a maxi-mum of four hydrogen bonds with tetrahedral geometry. Thus,at least two of the six functional groups do not form hydrogenbonds with W207. These groups could be exchangeable. Four ofthe six groups can participate in hydrogen bond formation aseither hydrogen donors or acceptors without inducing a largeconformational change. This structural feature indicates thatW207 could form three ‘‘hydrogen-donating’’ hydrogen bondsand one ‘‘hydrogen-accepting’’ hydrogen bond. The hydroniumion state of Wat207 would also be stabilized by four suchhydrogen bonds. Furthermore, the exchangeability of the hy-drogen bond-forming pair without significant conformationalchange would increase the stability of the hydronium ion state,once formed between Asp-91 and His-503 with essentially thesame proton affinity as described above.

The 1.8-Å/1.9-Å resolution x-ray structures suggest that, onoxidation, the His-503 imidazole plane rotates by almost 180° atthe C�–C� 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 N� atom of the His-503imidazole, disappears on oxidation. Instead, the N� atom intro-duces W6 and the N� atom forms a hydrogen bond to W28. Inthe oxidized state, W28 is located at the molecular surfaceinstead of W4. The possible redox-coupled conformationalchanges are summarized in Fig. 1c. By removing the hydrogenbond from His-503, the proton on W207 would be destabilizedeven though the conformation of the rest of the five residuesinvolved in hydrogen bonds are not significantly influenced byloss of that hydrogen bond. Thus, the W207 proton would bereadily extracted by Asp-91, which has significant proton affinityas described above.

X-Ray Structures of Cd2� and Zn2� Derivatives of Bovine Heart CcO.X-ray diffraction data were collected from the crystals of theCd2� and Zn2� derivatives in the reduced and oxidized states.The metal ion treatments were performed by soaking crystalswith the metal ion-containing medium. Statistics of intensitydata are calculated for the best data set of both oxidation states[supporting information (SI) Text]. The anomalous differenceFourier maps revealed several anomalous difference electrondensities. In addition to those for the intrinsic metals (Fea, Fea3,and CuB in the subunit I; CuA in the subunit II; and Zn1 in thesubunit Vb), three Cd2�-binding sites or seven Zn2�-bindingsites appeared in cases where the crystals were soaked with Cd2�

or Zn2� ion (Fig. 2). The Cd1 and Cd2 sites are identical with theZn2 and Zn3 sites, respectively. The former and the latter sitesare denoted as Cd1/Zn2 site and Cd2/Zn3 site in this paper. Thedetailed x-ray structures of the metal-binding sites other than theCd2/Zn3 site are given in SI Text.

The enzyme exists in a dimer state in the crystal lattice. Oneof the monomers (denoted molecule A) has stronger specificprotein–protein interactions from the adjacent protein mole-cules in the crystal lattice relative to those of the other monomer(denoted molecule B). Because of the stronger specific interac-tions from the adjacent molecules in the lattice, the electrondensity of molecule A is better defined than that of molecule B.CcO molecule, as most other globular proteins, has variousconformations in equilibrium (conformers) (16). The unevenconstraints to the two monomers from the crystal lattice couldproduce different population of the conformers depending onthe location of the monomer in the dimer in the crystal (i.e.,molecule A or molecule B) to provide different reactivity to theexternal reactants. In fact, as described below, the reactivity ofthe Cd2/Zn3 site in the crystal is significantly dependent on the

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Fig. 1. Redox-coupled conformational changes in the His-503–Asp-91 regionnear the entrance of the D-pathway deduced from the x-ray structures ofbovine heart CcO from the 1.8-Å oxidized and 1.9-Å reduced x-ray structuresof bovine heart CcO. (a and b) The structures of the D-pathway entranceregions in the reduced and oxidized states shown in stereoviews. The reddotted lines indicate hydrogen bonds that are independent of oxidation state.The blue dotted lines indicate hydrogen bonds that are dependent on theoxidation state. The blue, green, and orange sticks represent the C� backbonesof subunits I, III, and VIIc, respectively. The red spheres represent fixed watermolecules. (c and d) Schematic representations of the redox-coupled confor-mational changes of His-503 in the Cd2�-free and bound CcO, respectively. Theblue and red drawings represent the structures in the reduced and oxidizedstates, respectively. The black dotted lines represent hydrogen bonds unin-fluenced by the oxidation state change. The blue and red dotted lines depicthydrogen bonds appearing in the reduced and oxidized states, respectively.The blue and red thick dotted lines in d represent the coordination bonds thatappear in the reduced and oxidized states, respectively.

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location in the dimer. Any conformer of the monomers thatappears in the crystal lattice exists as one of the conformers inthe physiological conditions (in the solution state) where anyconstraint in the crystal lattice is absent. In other words, it ispossible that the lattice constraints trap a conformer that is toounstable in the solution state to identify readily.

The difference in the reactivity to the metal ions on thelocation of the monomer would not be detected if the metalderivatives prepared in the solution state were crystallized. Thelattice constraints influence the kinetics of the metal ion bind-ings. Because of the destabilizing effects of these metal ions tothe crystalline state of CcO, the conditions for soaking of theCcO crystals with these metal ion solutions are very limited.Therefore, any metal ion binding, found in the x-ray structure asshown in Fig. 2, is likely to occur in the solution state or underthe physiological conditions.

Structures of the Cd2/Zn3 site near the entrance to theD-pathway are influenced by the oxidation state of CcO as wellas by the location of the monomer in the crystal lattice. In thereduced state of the enzyme, Cd2� is coordinated to the N� atomof His-503 imidazole group in a conformation that is essentiallyidentical to that of the native enzyme in molecule A (Fig. 3a).The water molecules W28 and W490, and the imidazole ofHis-2VIIc are coordinated to the bound Cd2�. As a result,His-2VIIc moves closer to Cd2� relative to its location in CcObefore addition of Cd2�. At Cd2� concentration of 500 �M, theCd2� occupancy was estimated to be almost full by comparingthe peak height of the anomalous difference electron density ofCd2� ( f �Cd � 1.84 at the wavelength of 0.9 Å) with that of theintrinsic Zn2� ( f �Zn � 2.15 at the wavelength of 0.9 Å), whichfully occupied Zn1 site. In molecule B, another Cd2� forms abridge between Glu-506 and His-3III, in addition to the Cd2�

coordinated to His-503 (Fig. 3b).In the oxidized state, Cd2� in the Cd2/Zn3 site is coordinated to

the N� 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-coupledstructural change of the imidazole group of His-503 is essentially thesame as that of the Cd2�-free enzyme, although the imidazole ring

of the oxidized state moves further away from the position itoccupies before binding of Cd2� (Fig. 1d).

In the reduced state of the enzyme, the two Zn2�-bindingstructures at the Cd2/Zn3 site are similar to the Cd2�-bindingstructures at Cd2� site shown in Fig. 4 a and b. The metal-binding site between Glu-506 and His-3III seems to have weakeraffinity to both Cd2� and Zn2� ions than that of the site betweenHis-503 and His-2VIIc, because some crystals do not have themetal ions at the former site (three data sets/eight data sets). Inthe oxidized state, two Zn2� atoms occupy the positions of W5and W6 and are separated by 2.8 Å in molecule B. The atomicdistance is too short for coexistence of two Zn2� atoms at thesite. This x-ray structure indicates that two structures are inequilibrium, with a Zn2� replacing either W6 (Fig. 4c) or W5(Fig. 4d). The electron density assignments of the anomalousdifference map are consistent with the electron density peaks ofthe two Zn2� ions, each of which is �50% of that of the intrinsicZn2� electron density at the Zn1 site. The Zn2� replacing W6coordinates His-503, His-2VIIc, W5, and W500 in essentially thesame fashion (Fig. 4c) as the Cd2� ion coordinates the sameresidues in the oxidized state. The Zn2� replacing W5 coordi-nates W6, Glu-506, and His-3III (Fig. 4d). In molecule A, Zn2�

binding to oxidized CcO induces significant conformationalchanges in the coordinating residues His-503, His-2VIIc, andGlu-5VIIc (Fig. 4e). In this structure, the His-503 imidazole iscoordinated to the Zn2� at the N� atom without the need forrotation of the imidazole plane.

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�-bindingsites (Fig. 3 a–c), respectively. These structures confirm theredox-coupled conformational change in His-503, that is, therotation of the imidazole plane. The other two structures shown

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Fig. 2. Anomalous difference Fourier maps of CcO crystals treated with 0.5mM 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). C� backbone traces and porphyrin A (red)structures are shown. Anomalous difference electron densities of Cd2� andZn2� derivatives are calculated at 2.1 and 2.7 Å resolutions, respectively. Theelectron density cages are drawn at 6� and 4� for Cd2� and Zn2� derivatives,respectively, where one � for Cd2� derivative is 0.0125 e/Å3 and that for Zn2�

derivative is 0.0099 e/Å3. Zn1 represents the anomalous difference electrondensity of the intrinsic Zn2� in subunit Vb. Anomalous difference electrondensities appearing as a result of metal ion treatment are indicated witharrows. The treatment by 0.5 mM ZnSO4 showed the same anomalous differ-ence electron densities as shown in the right image. The peak heights of theCd2� derivative are 0.22 and 0.25 for CuA, 0.25 for CuB, 0.17 for Fea, 0.19 forFea3, 0.25 for Zn1, 0.19 for Cd1, 0.20 for Cd2, and 0.09 for Cd3 in e/Å3 unit.Those of Zn2� derivative are 0.13 and 0.12 for CuA, 0.12 for CuB, 0.06 for Fea,0.08 for Fea3, 0.11 for Zn1, 0.12 for Zn2, 0.08 for Zn3, 0.04 for Zn4, 0.05 for Zn5,0.06 for Zn6, and 0.06 for Zn7 in e/Å3 unit

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Fig. 3. His-503 site structures of Cd2� derivatives in stereoviews. The red andblue spheres represent fixed water molecules and Cd2�, respectively. The bluedotted lines indicate coordination bonds to the Cd2�. The blue, green, andbrown sticks denote the C� backbones of subunits I, III, and VIIc, respectively.The locations of the fixed water molecules not coordinated to Cd2� ions areidentical with those of the Cd2�-free structures shown in Fig. 1. The followingCd2�-bound structures have been identified: (a) a structure with a single Cd2�

bound in the reduced state; (b) a structure with two Cd2� ions bound in thereduced state; and (c) a structure with a single Cd2� bound in the oxidizedstate. Asp-91 is not shown in these atomic models. The resolution of theseatomic models is 2.1 Å

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in Fig. 4 d and e were not induced by Cd2� ion, perhaps becausethe size of the Cd2� ion is larger than that of Zn2�.

In the oxidized state, the lowest Zn2� concentration requiredto observe the anomalous difference electron density at theCd2/Zn3 site was found to be 100 �M. At this Zn2� concentra-tion, Zn2� binding was not detected in molecule B. The Zn2�

binding was detectable in both molecule A and molecule B at aZn2� concentration of 500 �M. The Cd2/Zn3 site occupancy wasestimated to be 70–90% (that is, essentially fully occupied) in thereduced state at Zn2� concentration of 500 �M. (Zn2� bindingfor the reduced form was not examined at lower Zn2� concen-tration.) These results suggest that Zn2� has similar affinity tothe Cd2/Zn3 site in both oxidation states. Because of the

significant effects of the lattice constraints on the metal ionbindings as described above, it seems impossible to determinethe Zn2� affinity in the solution state accurately only from theexperimental results obtained from CcO in the crystal lattice.Nevertheless, the above results suggest that the Cd2/Zn3 sites ofCcO in the solution state has Kd value of 100 �M or lower in bothoxidation states.

Recently, Cd2� binding at the entrance of K-pathway ofRhodobacter sphaeroides CcO between Glu-101 and His-96 ofsubunit II has been reported (17). However, this Cd2� bindingis unlikely to occur in the bovine CcO because the His-96 is notconserved in the bovine CcO.

The Inhibitory Effects of Cd2� and Zn2�. The inhibitory effects ofCd2� and Zn2� on ferrocytochrome c oxidation activity bybovine heart CcO were examined at pH 7.4 at 20°C by usingsulfate salts of the metal ions. Inclusion of Cd2� or Zn2� in thereaction mixture did not result in complete enzyme inhibition. Asingle phase inhibition by Zn2� was observed with a Kd of 76 �Mand 30% residual activity, which is the enzyme activity asymp-totically approaching with increase in the Cd2� concentration.The Cd2� inhibited the reaction in a biphasic manner with a Kdof 0.3 �M for 17% inhibition and 450 �M for 70% inhibition. Theresidual activity was 13%. The observation of partial enzymeinhibition seems to be consistent with the occurrence of redox-coupled conformational changes in the metal derivatives, whichare essentially similar to the conformational changes occurringin 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 SIFig. 6 a and c) showed detectable anomalous difference electrondensity, at the wavelength of 0.9 Å ( f �Zn � 2.15) with the peakheight depending on the individual crystals used for x-raydiffraction experiments. In some cases, the peak height wasfound to be as high as that of the intrinsic Zn2� at the Zn1 site.The average peak height of Zn2� determined for 20 independentdata collections is �30% of that of Zn2� at the Zn1 site. Theanomalous dispersion of the Cd1/Zn2 site was examined atvarious x-ray wavelengths for identification of the metal ion atthe Cd1/Zn2 site in the absence of exogenous Zn2�. Theanomalous difference electron density at the Cd1/Zn2 site wasdetected at the wavelength of 1.20 Å ( f �Zn � 3.44), together withthe intrinsic Zn2� site (Zn1), the two copper ( f �Cu � 3.07) sites,and the two iron ( f �Fe � 2.12) sites. X-rays with longer wave-length, 1.76 Å ( f �Zn � 0.86), 1.50 Å ( f �Zn � 0.65), and 1.35 Å( f �Zn � 0.53), did not show the anomalous difference electrondensity at the Cd1/Zn2 site. Addition of 5 mM EDTA resultedin disappearance of the anomalous difference electron density atthe Cd1/Zn2 site. These anomalous difference maps are given inSI Text. Metal analysis indicated the presence of �25% addi-tional Zn2� relative to the Zn2� content of the sample washedwith 5 mM EDTA. These results indicate that the Cd1/Zn2 sitebinds Zn2� at various saturation levels in a manner dependenton individual crystals. On the other hand, the enzyme activity isquite consistent regardless of the enzyme preparation. There-fore, the binding of Zn2� to subunit III is unlikely to influenceelectron and proton transfer through the enzyme.

The peak height of anomalous difference electron density atthe Cd1/Zn2 site increases significantly on exposure of thecrystals to Zn2� at concentrations of 1 �M or higher. Therefore,the Kd of the Cd1/Zn2 site should be �1 �M, which representsa higher affinity than that of the Cd2/Zn3 site at His-503.

DiscussionThe Redox-Coupled Conformational Change of His-503. The redox-coupled positional changes in water molecules and Cd2� and Zn2�

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Fig. 4. His-503 site structures of Zn2� derivatives in stereoviews. The red andblue spheres denote fixed water molecules and Zn2� ions, respectively. The bluedotted lines represent coordination bonds. The blue, green, and brown sticksdenote C� backbones of subunits I, III, and VIIc, respectively. The locations of fixedwater molecules that are not coordinated to Zn2� ions are identical with those ofthe Zn2�-free structures shown in Fig. 1. The five different species identified areas follows: (a) a structure with a single Zn2� bound in the reduced state; (b) astructure with two Zn2�ions bound in the reduced state; (c) a structure in whichthe W6 site is occupied by a single Zn2� in the oxidized state; (d) a structure inwhich the W5 site is occupied by a single Zn2� in the oxidized state; and (e) astructure in which Zn2� is coordinated by His-503 and His-2 and Glu-5 of subunitVIIc in the oxidized state. The structures shown in c and d are in 1:1 equilibrium.Asp-91 is not shown in these atomic models. The resolution of the atomic modelsis 2.6 Å for a, 2.8 Å for b, and 2.7 Å for c–e

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ions bound to His-503 (Figs. 1, 3, and 4) are consistent with therotation 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 metalsites. No significant x-ray structural change in the protein moietybetween His-503 and the redox-active metal sites has been detectedat the present resolutions. However, structural changes in theprotein moiety much smaller than those detectable at the presentresolutions of the x-ray structure could significantly influenceinteractions between amino acid residues inside the protein. Thus,the present x-ray structural results do not provide the conclusiveevidence against the presence of communication systems betweenHis-503 and the redox-active sites. On the other hand, no change inthe crystal packing induced by the oxidation state change of CcOhas been detected. At present, no experimental result positivelysuggests that the conformational change in His-503 is induced by acrystallization effect.

The Role of the His-503 in Supplying Protons to the D-Pathway. Inaddition to the rotation of the imidazole plane of His-503, the higheffective pKa value of Asp-91 suggested by the hydrogen-bondingstructures shown in Fig. 1 is also critical for redox-controlled protoncollection. This structural feature is consistent with the abolishmentof the enzyme activity induced by mutation of Asp-91 to Asn (18).Asn-91, which has extremely weak basicity, cannot stabilize thehydronium 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-91system occurs as shown in Fig. 5. In this scheme, for the sake ofsimplicity, only one of the two water arrays connecting themolecular surface with W207 is shown and W6 is not shown inthe oxidized state. In the reduced state (a), W207 is stabilized inthe hydronium ion state by His-503 and Asp-91 via the twohydrogen bonds. The x-ray structure of the reduced state cor-responds to this structure. On oxidation, the imidazole planerotates by 180° and the hydrogen bond between His-503 andW207 is disrupted. As a result, the proton at W207 is transferredto Asp-91 (b) and W4 is released (c). In this state, the enzymeis stable in the absence of oxidation state changes at theredox-active metal sites. The x-ray structure of the oxidized statewas 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 thehydrogen bond with W207 is formed (e), followed by introduc-tion of a hydronium ion as W4 (f). The proton at W4 is thentransferred to W207 giving the original state (a).

This scheme includes introduction of protons to the system viaa hydronium ion at step e3f. The system could also accept aproton after receiving W4 or through other water arrays notshown in this scheme. One of the critical points of this schemeis that, on reduction, the Asp-91 proton is released (c3d) to theO2 reduction site before the rotation of the imidazole planeoccurs (d3e). Otherwise, protons at Asp-91 would be returnedto W207 on reduction. This conformational change in His-503could be driven by, at most, four sites because CcO has fourredox-active metal sites. Thus, a single equivalent of electronequivalent from cytochrome c could collect more than oneproton equivalents during the electron transfer to the oxygen atheme a3. In other words, this redox-controlled proton supplymechanism does not include any proposal for the H�/e� ratio.The redox site (or sites) driving this conformational change ofthe His-503 imidazole has yet to be identified.

Proton collecting functions of carboxyl groups and imidazolegroups on the negative side surface of CcO have been proposedwithout assuming any redox-coupled conformational change (19,20). The x-ray structural changes in His-503, determined by thepresent work, indicate that His-503–Asp-91 system not onlycollects protons from the negative side surface but also facilitatesthe redox-controlled proton supply to the D-pathway.

Effects of Binding of Zn2� on the Function of Bovine CcO. The presentx-ray structural analyses show six Zn2�-binding sites (Fig. 2). TheCd1/Zn2 site seems to have the Zn2� affinity stronger than thatof the Cd2/Zn3 site (including His-503) as described above.Therefore, the subunit III site could scavenge Zn2� in themitochondrial negative side space to prevent the decouplingeffect of Zn2� at the His-503 site as described below. The x-raystructures (Fig. 4) strongly suggest that the Cd2/Zn3 site is theZn2� inhibition site. The remaining four Zn2�sites locatedwithin nuclear-coded subunits are unlikely to influence theenzymatic function of CcO, because the sites are far apart fromthe active sites of CcO.

The Zn2� effects on the reaction of CcO reported thus far(7–12) indicate that CcO has Zn2�-binding sites on both thepositive and negative side surfaces to inhibit the enzymaticfunction (7, 10, 12). It should be noted that the Zn2�-binding siteon the positive side surface that could inhibit the enzymaticfunction has not been identified in the present x-ray structuralanalyses. The Zn2� effects depend strongly on the membranepotential of the reconstituted vesicles (10). Furthermore, Zn2�

inhibition depends on the stage of O2 reduction process as wellas the assay conditions. In the reaction of the fully reduced CcOwith O2, the intermediate species P, F, and O appear after theformation of the oxygenated species. The 50% decreases in theP3F and F3O transition rates are caused by �120 and 2 �MZn2� to the inside surface, exhibiting �30% and 40% of theresidual activities, respectively (8). The steady-state turnoverrate of CcO determined by using the soluble O2 consumptionassay system provided a Zn2� inhibition constant (KI) of 9 �M(10, 11), whereas the KI value of the steady-state turnovermeasured with ferrocytochrome c oxidation is 76 �M Zn2�

(present work). Both inhibitions showed residual activities.These differences in the Zn2� effect are likely to be caused bydifference in the rate-limiting step that provides different overallZn2� effects. (In a partial inhibition system, the affinity of therate-limiting intermediate species to the inhibitor determines theoverall inhibition level.) X-ray structural analyses for the Zn2/Cd3 site as described above show five Zn2�-binding structures intotal (Fig. 4). The structural diversity suggests that different

H3O+ H+

(reduction)

H2O

N

NH

H2OH2O

H2O

HO

OC

H2O(oxidation)H2O

H3O+

HN N

H2OH2O

O

O- C

D91

260

207

28

4H503

de

f

a b

cH3O

+

H2O

HN N

H2OH2O

O

O- C

H2O

HN N

H2OH2O

O

O- C

N

NH

H2OH2O

H2O

HO

OC

N

NH

H2OH2O

H2O

O

O- C

Fig. 5. Redox-controlled proton collection and supply by His-503. Only oneof the water arrays connecting W207 and W4 and two other possiblehydrogen-bonding groups to W207 is shown for the sake of simplicity. The redand blue structures represent the oxidized and reduced (or electron-releasedand electron-accepted) states of the redox site (or sites) controlling theconformation of the imidazole of His-503. The structures before the oxidationstate change are the stable oxidized and reduced states. The circles witharrowheads indicate the rotation of the imidazole group during the transition

.

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intermediates show different Zn2� affinity to provide thesevarieties in the Zn2� effect as described above.

The KI for complete inhibition of proton pumping is �70 �MZn2� (7), whereas the partial inhibition of ferrocytochrome coxidation with 30% residual activity showed KI of 76 �M (presentwork). The Zn2�-binding site for these inhibitions is likely to beHis-503, because these KI values are essentially consistent with thatto the His-503 site estimated by x-ray structural analyses (the levelof 100 �M). This consistency suggests that the Zn2�-binding sitecontrols both the proton transfer through D-pathway and theproton pumping. The proton transfer through D-pathway is tightlycoupled with the electron transfer for the O2 reduction that drivesthe proton pumping. However, it is unlikely that the inhibition ofproton pumping by the Zn2� binding is induced indirectly byblocking the proton transfer through D-pathway, because thepartial inhibition of the electron transfer by Zn2� cannot induce thecomplete abolishment of proton pumping. In other words, Zn2�

binding at His-503 is likely to influence directly the proton-pumpingsite 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 wereidentified. Two proton-pumping mechanisms have been pro-posed: one that includes the D-pathway (18) and another thatincludes the H-pathway, a proton transfer pathway located nearheme a (2). Both proposals include proton-pumping sites locatedfar away from His-503. Improvement of the resolution of theZn2� derivative of CcO would provide important clues forelucidation of the mechanism of proton pumping.

Materials and MethodsCrystals of Cd2� and Zn2� complexes of bovine heart CcO wereprepared by soaking the CcO crystals obtained by the methoddescribed previously (21) in a medium containing 0.5 mM CdSO4or 0.5–5 mM ZnSO4. Other experimental conditions for crys-tallizations, x-ray structural analyses, and the measurements ofthe inhibitory effects of Cd2� and Zn2� on CcO are given in SIText. The values of f �Cu, f �Fe, f �Zn, and f �Cd are cited from ref. 22.

This work was supported in part by grants from the Ministry ofEducation, Culture, Sports, Science, and Technology, and the CoreResearch for Evolutional Science and Technology.

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