Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7167-7171, August 1995 Chemistry

The CUA center of cytochrome-c oxidase: Electronic structure and spectra of models compared to the properties of CUA domains (molecular model/exciton splitting/charge transfer/reorganization energy) SVEN LARSSONtt, BRUNO KALLEBRING§, PERNILLA WiTrUNGt, AND Bo G. MALMSTROM§ tDepartment of Physical Chemistry, Chalmers University of Technology, S-412 96 G6teborg, Sweden; and §Department of Biochemistry and Biophysics, Goteborg University, Medicinaregatan 9C, S-413 90 Goteborg, Sweden Communicated by Harry B. Gray, California Institute of Technology, Pasadena, CA, April 24, 1995

ABSTRACT The electronic structure and spectrum of copper proteins, and this was attributed to a second conserved several models of the binuclear metal site in soluble CuA cysteine in a stretch of the sequence containing the other domains of cytochrome-c oxidase have been calculated by the ligand cysteine and one of the ligand histidines (10). Recently, use of an extended version of the complete neglect of differ- however, one of the sites of nitrous oxide reductase has been ential overlap/spectroscopic method. The experimental spec- shown to have almost identical EPR parameters (13, 14). In tra have two strong transitions of nearly equal intensity addition, with the reductase the EPR spectrum gives strong around 500 nm and a near-IR transition close to 800 nm. The indications of a mixed-valence binuclear site (14, 15), suggest- model that best reproduces these features consists of a dimer ing that also CUA is binuclear (15, 16). This concept has, in fact, of two blue (type 1) copper centers, in which each Cu atom received support from work done with the soluble CUA do- replaces the missing imidazole on the other Cu atom. Thus, mains (9, 17, 18). both Cu atoms have one cysteine sulfur atom and one imida- Early structural models (19, 20) for CUA were mononuclear, zole nitrogen atom as ligands, and there are no bridging with two cysteine and two histidine residues as metal ligands. ligands but a direct Cu-Cu bond. According to the calcula- With the advent of the idea that CUA is binuclear, models tions, the two strong bands in the visible region originate from involving one (17) or two (18) cysteine sulfur atoms as a exciton coupling of the dipoles of the two copper monomers, bridging ligand have been formulated. An alternative structure and the near-IR band is a charge-transfer transition between has recently been suggested by Blackburn et al. (21) on the the two Cu atoms. The known amino acid sequence has been basis of extended x-ray absorption fine structure measure- used to construct a molecular model of the CuA site by the use ments. In their model, CUA is a dimeric copper site with a direct of a template and energy minimization. In this model, the two Cu-Cu bond, 2.5 A in length. ligand cysteine residues are in one turn of an a-helix, whereas In this paper we use the complete neglect of differential one ligand histidine is in a loop following this helix and the overlap/spectroscopic (CNDO/S) method to calculate the other one is in a a-strand. optical spectra of different CUA models. Models with bridging S atoms (17, 18) do not give a strong band in the near-IR C'ytochrome-c oxidase (ferrocytochrome-c:oxygen oxidoreduc- region. The model of Blackburn et al. (21), with the modifi- tase, EC 1.9.3.1), the terminal enzyme of aerobic respiration in cation that the two cysteine residues should be in a cis position, eukaryotic organisms and manybacteria, is a redox-linked proton on the other hand, can be used to reproduce the main features pump (1, 2). It couples the oxidation of cytochrome c by molec- of the experimental spectrum (a near-UV transition, two ular oxygen to the translocation of protons across a membrane. strong visible bands with a splitting close to 2000 cm-', and a The electrochemical gradient so created is used to drive the strong near-IR band). synthesis of ATP, in accordance with Mitchell's chemiosmotic Starting from the amino acid sequences around the metal theory (3). ligands and the fact that the binding domain has the secondary Cytochrome oxidase contains three redox centers, cyto- structure of blue copper proteins, the cupredoxin fold (22), we chrome a and CUA, which are the primary acceptors of have used molecular modeling and energy minimization to electrons from cytochrome c, and the binuclear cytochrome construct a CUA site. The restrictions of the sequence and the a3-CuB unit, which functions as the dioxygen-reducing unit. secondary structure still allow the geometry of the site pre- The CUA site is the point of entry of the electron from dicted by our theoretical calculations, and it is consistent with cytochrome c (4-6), and it is in rapid redox equilibrium with the Blackburn model, including the Cu-Cu bond (21). cytochrome a (7). It is located in subunit II, which is anchored to the membrane by two transmembrane helices, but the metal site is situated in a domain outside the membrane. Recently, METHODS the gene for this domain has been cloned, and the protein has The CNDO/S Method. This model is based on Hartree- been expressed in soluble form (8, 9). This allowed for the Fock and configuration interaction (CI) methods, and it was recording of the complete optical spectrum of CUA, most of originally designed to provide theoretical spectra of organic which is hidden by the heme absorption in the intact oxidase; molecules, including the valence electrons of oa or X type (23). the only part of the optical spectrum that can be observed in The parameterization scheme has been generalized, so that the the whole oxidase is a near-IR band close to 800 nm. method may now be applied to molecules containing not only The amino acid sequences of the soluble domains show great H, C, N, and 0 atoms but any nonrelativistic atom (24). homology to small blue copper proteins (10), such as azurin The molecular orbitals (MOs) are expressed as linear com- and plastocyanin, but still its EPR spectrum (11) is quite binations of atomic orbitals. A self-consistent field calculation distinct from the spectra of type 1 sites (12). Until a few years is first carried out for the ground state. Singly substituted Slater ago, the CUA EPR spectrum was considered unique among determinants are subsequently constructed by replacing an

The publication costs of this article were defrayed in part by page charge Abbreviations: CNDO/S, complete neglect of differential overlap/ payment. This article must therefore be hereby marked "advertisement" in spectroscopic; CI, configuration interaction; MO, molecular orbital. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

7167 Downloaded by guest on September 27, 2021 7168 Chemistry: Larsson et al. Proc. Natl. Acad. Sci. USA 92 (1995) occupied spin orbital by an unoccupied one. Linear combina- respectively. The geometry is either cis or trans, as shown in tions of spin doublet projected Slater determinants are Fig. 1. formed, and the matrix elements are calculated as given in ref. The MO energy level scheme for the cis form is given in Fig. 25. The electronic states are calculated by solving the eigen- 2. MOs that are fr bonding and antibonding between Cu and value problem for these projected determinants. S are of particular interest. The one with the highest energy is We have included 100 CI configurations; a calculation using singly occupied and denoted 4m (3b2 in Fig. 2). All transitions 300 configurations did not change the energy levels to any seen in the spectrum are due to excitations into this MO. MOs significant extent. For this type of system, there is a large with a local symmetry different from ( close to the Cu atoms, mixing between the atomic orbitals from metal ion and ligand such as 2b2 in Fig. 2, give a small intensity to the transition (for atoms and also a strong mixing between CI configurations. example, 2b2 -* 3b2). In the mononuclear blue copper center, Screened electron repulsion is introduced in our extension (24) it is the Cu-S wi bonding to fT antibonding transition that gives in such a way that the method is consistent with local exchange rise to the strong absorption (26-28). In the binuclear center, methods. The result is that the CNDO/S method, as well as the there are three possible transitions of this type: la1 -> 3b2, lb2 multiple scattering Xa or other local exchange methods, -> 3b2, and 2a1 -* 3b2- predicts ionization energies and electron affinities well. The The calculation shows that the bonding-antibonding energy method has also been tested on simple Cu2+ complexes, with splitting is smaller between the two Cu atoms than between Cu good agreement with local exchange methods (26, 27) and with and S. It follows that the two upper MOs in Fig. 2 are both experiments (28). Cu-S antibonding and that the lowest energy transition is In a blue copper protein site, the hole state is an antibonding Cu-Cu bonding to antibonding. The calculated energy of the r orbital between Cu and S, which is at the same time o- latter transition is 0.8 eV compared to the experimental value antibonding between Cu and the histidine imidazole nitrogens. of 1.6 eV. Since the CNDO/S method has not been parame- We also found that the other possible alternative-namely, trized to reproduce intermetal interactions, one cannot expect that the hole state is o- antibonding between Cu and S-cor- a higher accuracy. responds to an excited state about 0.8 eV above the ground The energy difference (A) between the Cu-Cu bonding and state. The strong absorption at about 600 nm is due to a antibonding MOs is of special interest in connection with an transition from a wi bonding S-Cu orbital to its antibonding electron-transfer theory of Hush (29). We consider a hypo- counterpart. We also found in our calculations that a blue thetical electron transfer between two Cu-S centers. When a copper center may be simplified to a minimum model in which blue copper center is reduced or oxidized, we have shown that the cysteine-CH=S- group is replaced by H-S-, the the reorganization energy (A) cannot exceed 1 eV (28). The methionine-CH2-S--CH3 group is replaced by H-S-H, main contribution to A comes from the Cu-S bond, and we and imidazole is replaced by NH3. consequently expect that A for the Cu-S bonds in a binuclear Molecular Modeling and Energy Minimization. On the basis site cannot exceed 1 eV. Hush (29) has shown that if A > A, of the primary structure of subunit II in cytochrome oxidase then the electron hole is delocalized, whereas if A < A, the hole from Paracoccus denitrificans (10), the following sequence of is localized. In the delocalized case, the spectral widths are 14 amino acids was chosen for the main structural element in narrow and the intensity is high. The transition at 800 nm (1.6 the building of a model of the binuclear CUA site: GQCSE- eV) is indeed narrow. Furthermore, our calculation suggests LCGINHAYM. First, 7 amino acids were used to form an that A is of the order of 1 eV. We believe, therefore, that the a-helix, and the remaining ones were allowed to make a turn. system is, in fact, delocalized in the same way as the well-known Then, two copper atoms were attached to the cysteine residues Creutz-Taube complex (30, 31). in positions 3 and 7. A model for the binuclear site, We now turn to the remaining two transitions, la, -* 3b2 and NH3(SH)Cu-Cu(SH)NH', which according to our calcula- lb2 -> 3b2. It is of interest to discuss these two transitions in tions has the right spectral properties (see Results), was used the light of a theory of Forster (32). In this theory the spectrum as template to minimize the degrees of freedom of the of the dimer is obtained from the transition dipole moments structure. By the introduction of a strong template force, the of the monomers in an excitonic interaction scheme. In the sequence was forced to align with the site model. The sulfur present case, when the spin state is a doublet (S = 1/2), the atoms, with their attached copper atoms, of cysteine residues Forster equation does not apply. We derive below the corre- 3 and 7 were forced toward the template. Likewise, the N3 sponding equation for the doublet case, using the matrix atom of H11 was forced toward the nitrogen in the NH3 group elements of Ishitani and Nagakura (25). The strong monomer of the template under energy minimization. After that, a /3 transitions can be assumed to be #m and h Om, where strand was built from a sequence of 5 amino acids, including 4pm is assumed to be delocalized, and 4i and 04h are localized the second conserved ligand histidine residue: MGHNW. The Cu-S bonding MOs of the monomers. They combine into two Na atom of this histidine was superimposed on the N atom in delocalized MOs [lb2 = (4i - la, = + 4h)/\/2], but, the second NH3 group of the template. Then, the distances and 4h)/1V2; ((P angles for the two copper atoms and their ligands were fixed while energy minimization was performed on the whole sys- y tem. For the molecular graphics work, the software INSIGHTII from Biosym Technologies (San Diego) was used together with the molecular dynamics module DISCOVER. > x

RESULTS NH13 NH3 NH3 S-H Application of the CNDO/S Method to CUA Models. The I / first model to be considered has the formula Cu Cu Cu - Cu NH3(SH)Cu-Cu(SH)NH' and has the structure used by Blackburn et al. (21) to interpret their extended x-ray absorp- H-S S-H H-S NH3 tion fine structure results. The model may be regarded as obtained from two blue centers, if each Cu replaces the missing cis trans imidazole on the other Cu. In the blue center the N-Cu-N FIG. 1. Structure of two stereoisomers of the model compound angle should be close to 900, whereas the S-Cu-N angles are used in our calculation. The MO diagram in Fig. 2 is based on the cis close to 135°; in plastocyanin these angles are 970 and 123°, form with the use of the coordinate system indicated at the top. Downloaded by guest on September 27, 2021 Chemistry: Larsson et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7169

-10' --w + 4+ -1 ll. i" 3b2 c11 c -11 C+ + C+ 00 4~ I -12- 2a, -13 4+ + +--F 2b2 -14 4+ lb2

Kt . lal

FIG. 2. Schematic MO diagram for the cis form of the model compound in Fig. 1, with the symmetries indicated to the right of the orbital levels. The size of the orbitals has been scaled to the relative amplitude (coefficient) on that atomic orbital. In the right-hand part ofthe figure, the transition dipoles of the transitions corresponding to the three optical bands in the visible and near-IR regions are shown. since the latter are nearly degenerate, they may be replaced by 4i according to the C2, point group (4m = 3b2). The excitonic and oh. The energy difference between the odd and even states interaction corresponds to transitions lb2 -- 3b2 and 1a, -3 3b2- is then twice the matrix element between the two states 0, Om Since la1 and lb2 are nondegenerate, the excitonic interaction and oh -i 4, (25): is not the only interaction determining the energy splitting. The calculated splitting for the case of the perpendicular H(i m, h -> m) =2 (hmlim) - (hilmm), [1] Cu-S bonds is 1400 cm-1. The contributions have different signs. With reasonable S-Cu-Cu angles, the splitting ranges between 1000 and 2000 cm-1. where A S-Cu--Cu angle of 1350 in this model (Fig. 1) results in electronic states at 6000 (0.19), 16,100 (0.17), 17,300 (0.08), = I4 [2] and 22,900 cm-' (0.01) (with oscillator strengths given in (ijlkl) i(T1)Oj(T1) r- k(T2)44(T2)dT,dT2. parentheses), and similar results are obtained with other reasonable'angles. The oscillator strengths are a little smaller If 4i and oh do not overlap, (hilmm) is equal to zero. The term than for the strong absorption at 16,000 cm-1 in blue copper (hmlim) is a coulomb interaction between the two charge proteins, where it has been calculated to be 0.22 (28). This is distributions 44 Om and +4Om. Since the latter are also the consistent with the measured extinction coefficient for CUA (8, transition densities for the monomer, (hmlim) is approximately 9). The theoretical polarization is different in the two close an interaction between two transition dipole moments, mi and lines at about 500 nm. The rather strong absorption at 22,900 mh, directed from Cu to S; given by: cm-1 is due to excitation from orbitals with Cu 3d character, 1 ~~~3 Ir-bonding between the Cu ions, to Om. This also agrees with 4 mh X m ((Mh X Rhi)(Mi X Rhi)], [3] the experimental spectrum. Rhi i-42hi_ A model with two bridging cysteine- S atoms has been suggested in ref. 18. We have studied a number of models of where Rhi is the distance vector between the monomers. this type, permitting the Cu-NH3 groups to fold back around The transition dipole moment is obtained as the sum and the S-S axis. A model folded back 200 with a Cu-Cu distance difference, divided by \/2-, of the monomer transition mo- and same as ments. In the trans case, the two monomers have parallel of 2.5 A, and Cu-S Cu-N distances the transition moments. The dimer then has one transition (red- previously, has electronic states at 15,300 (0.05), 20,700 (0.05), shifted) with V times the transition moment of the monomer and 23,800 cm-1 (0.13). Except for the last one, the oscillator and another one (blue-shifted) with a zero transition moment. strengths in these states are considerably smaller than for the This is inconsistent with the experimental spectrum, which strong absorption in blue proteins. Increasing the Cu-Cu shows two strong absorptions with nearly equal intensity distance to 3 A leads to smaller energies, and it is possible to around 500 nm. In the cis case, however, the monomer obtain strong absorptions at about 500 nm with quite large transition moments are approximately perpendicular. There oscillator strengths. The theoretical polarization would then be are two transitions with perpendicular polarization and equal the same. The near-IR band has, however, too small an intensity. The wavenumber splitting is of the order of 1000 oscillator strength in all the models with bridging S atoms that cm-1. Thus we conclude that the geometry is cis. we have tested. We, therefore, conclude that the model of ref. The excitonic interaction just discussed is automatically 18 is less likely than the cis model in Fig. 1. included in the CI part of the CNDO/S method. Calculated A linear S-Cu--Cu-S model with ir bonds is excluded MOs and transition moments are drawn in Fig. 2. The exper- because of steric problems with the nitrogen ligands (see Fig. imental value of the splitting between the two visible bands in 3). Parallel trigonal planes of the same type as in blue proteins a CUA center is 1960 cm-1 (8, 9). The orbital notation is are not possible, since there are not enough ligands. Downloaded by guest on September 27, 2021 7170 Chemistry: Larsson et aL Proc. Natl. Acad. Sci. USA 92 (1995) P.W. and B.G.M., unpublished results), indicating that the origin of the splitting is different compared to the CUA site. The absorption in the near-IR region is a so-called charge- transfer transition between the Cu atoms. The term is only appropriate when the hole is localized on a single center. If the hole is delocalized equally over both centers, there is no actual charge transfer, but the intensity of the transition is high and the absorption bands are narrow. This is clearly the case for the experimentally measured spectra. The same coupling between the Cu ions is relevant for the strong doublet absorption in the visible region. The width of the two bands is also remarkably small, lending further support to our suggested geometry. One may ask what would be the advantage of a binuclear structure of the CUA site? In small soluble electron-transfer proteins, such as cytochrome c or azurin, electrons generally enter and leave the redox site through the same path (34, 35). The CUA center is, however, located in a subunit, which is immobilized in the membrane structure by interactions with the other subunits of the oxidase, and consequently CUA must receive an electron from cytochrome c via a different path compared to that used to donate the electron to cytochrome a. Such a long-distance electron transfer would be facilitated by the highly delocalized electron hole of a binuclear site. FIG. 3. Molecular model of the binuclear CuA site in cytochrome The binuclear site with a delocalized hole has the additional oxidase. The picture has been prepared with the program MOLSCRIPr (33). advantage that it results in a reduction of the electron transfer reorganization energy, A. If the site is symmetric, the bond- Molecular Model. The molecular model constructed on the length changes on electron transfer are only half of those for basis of the known sequence and metal ligands is shown in Fig. a mononuclear complex. Consequently, the inner reorganiza- 3. As described in Methods, the degrees of freedom have been tion energy, Ain, is only 25% of A for a mononuclear site. The restricted by the introduction of a strong template force. In best estimate of Ain in a mononuclear blue site is 0.40 eV (36, addition, energy minimization was performed on the whole 37). There should also be a decrease in A0ut in a binuclear site system. due to its larger size. We conclude that A = Ain + Aout for the binuclear site should be between 0.15 and 0.25 eV. It is possible to construct a binuclear CUA site within the DISCUSSION restrictions of the amino acid sequence of the metal binding On the basis of CNDO/S calculations, we can account for the domain (10), as shown in Fig. 3. As in blue copper proteins, one main features of the optical spectrum of CuA, if we assume the cysteine and one histidine ligand are close together in the model of Blackburn et al. (21), modified so that the two primary structure as shown by the sequence comparison in Fig. cysteine sulfur atoms are in a cis position. The calculations 4. In the oxidase sequence, however, there is an additional predict four optical bands, as found experimentally. There are cysteine and also two more amino acids between the glycine relatively large errors in the calculated transition energies, residue and the ligand histidine. In blue copper proteins, the which is not unexpected in view of the simplified model. The two ligands are in a loop formed by this sequence, which is oscillator strengths are smaller than for the intense transitions consistent with it containing helix-breaking amino acids, par- in a blue copper site, consistent with the lower extinction ticularly a proline residue. In the oxidase, on the other hand, coefficient of a CUA center. The same calculational method has it is only possible to achieve the Cu-S geometry of the model earlier been shown to give approximately the correct spectrum compound (21) by forming a short helix with the sequence and electronic structure of blue copper proteins (28). The between the two cysteine residues (Fig. 3), and the three amino CNDO/S results reveal a very complex mixing of atomic acids in this stretch can all be found in helices (38). orbitals in the self-consistent field part as well as between In cytochrome oxidase and in blue proteins, the second in the CI The schematic picture shown in histidine is further removed in the primary structure (10). In configurations part. is the Fig. 2 appears to be essentially correct. The spectrum is both cases, however, the secondary structure cupredoxin sensitive to the distance between the two S atoms of the fold (22), a Greek key 3-barrel, and it is consequently likely histidine is in a in the oxidase, cysteine residues. This suggests a possible mechanism for that this second located 3-strand fine-tuning the properties of the site, explaining the small as it is in the crystal structure of azurin (39) (see Fig. 3). The differences in the spectra of CUA domains from different site geometry shown in Fig. 3, obtained by energy minimization of our molecular model, is in close agreement with that species (8, 9, 34). whole In the binuclear model, the strong absorption band of the suggested by Blackburn et al. (21). A model for the CuA domain can he obtained by attaching the structure in Fig. 3 to monomers are split into two because of dipole-dipole inter- bands the known structure of azurin (40). This is justified by the action. This is also consistent with the fact that the CD that this domain has the same secondary structure as corresponding to the optical transitions in the CuA domains finding have opposite signs (P.W. and B.G.M., unpublished results). I N H The exciton splitting explains why the CUA spectrum has two Paracoccus oxidase C S E L C G in the visible region, in bands of approximately equal intensity Pseudomonas azurin C T F P G H contrast to blue copper proteins, which have only one strong band. The blue copper site in plastocyanin has an absorption FIG. 4. A comparison of the amino acid sequence containing the band at a wavelength below the strong band close to 600 nm two ligand cysteine residues and one ligand histidine residue in (16,700 cm-1), at 18,700 cm-, but this is very weak, and both cytochrome oxidase with the corresponding sequence in azurin. The CD bands associated with these transitions are positive (ref. 27; ligand amino acid residues are in boldfaced type. Downloaded by guest on September 27, 2021 Chemistry: Larsson et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7171 small blue proteins (22), and this approach was used earlier for 15. Antholine, W. E., Kastrau, D. H. W., Steffens, G. C. M., Buse, constructing mononuclear models of CUA (10, 20). G., Zumft, W. G. & Kroneck, P. M. H. (1992) Eur. J. Biochem. In conclusion, we have shown that it is possible in theoretical 209, 875-881. terms to account for the experimental electronic spectrum of 16. Malmstrom, B. G. & Aasa, R. (1993) FEBS Lett. 325, 49-52. 17. Kelly, M., Lappalainen, P., Talbo, G., Haltia, T., van der Oost, J. CUA on the basis of an electronic structure for a binuclear site. & Saraste, M. (1993) J. Bio. Chem. 268, 16781-16787. We have, in addition, argued that such a site facilitates electron 18. Lappalainen, P. & Saraste, M. (1987) Biochim. Biophys. Acta transfer from cytochrome c to cytochrome a in at least two 1187, 222-225. ways: by allowing electrons to enter and leave the site by 19. Martin, C. T., Scholes, C. P. & Chan, S. I. (1988) J. Biol. Chem. different paths and by lowering the reorganization energy. 263, 8420-8429. Thus, the structure of the CUA site that we have presented here 20. Holm, L., Saraste, M. & Wikstrom, M. (1987) EMBO J. 6, is expected to facilitate the function of this center in the 2819-2823. operation of cytochrome oxidase as a redox-linked proton 21. Blackburn, N. J., Barr, M. E., Woodruff, W. H., van der Oost, J. & de Vries, S. (1994) Biochemistry 33, 10401-10407. pump-namely, to mediate rapid electron transfer from cyto- 22. Wittung, P., KYillebring, B. & Malmstrom, B. G. (1994) FEBS chrome c to cytochrome a. Lett. 349, 286-288. 23. Del Bene, J. & Jaffe, H. H. (1968) J. Chem. Phys. 48, 1807-1813. We acknowledge very helpful discussions with Prof. Harry B. Gray 24. 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