Geometric and Electronic Structure Differences Between the Type 3 Copper Sites of the Multicopper Oxidases and Hemocyanin/Tyrosinase

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Geometric and electronic structure differences between the type 3 copper sites of the multicopper oxidases and hemocyanin/tyrosinase

Jungjoo Yoon, Satoshi Fujii1, and Edward I. Solomon2

Department of Chemistry, Stanford University, Stanford, CA 94305-5080

Contributed by Edward I. Solomon, February 25, 2009 (sent for review January 31, 2009)

The coupled binuclear ‘‘type 3’’ Cu sites are found in hemocyanin (Hc), tyrosinase (Tyr), and the multicopper oxidases (MCOs), such as laccase (Lc), and play vital roles in O2 respiration. Although all type 3 Cu sites share the same ground state features, those of Hc/Tyr have very different ligand-binding properties relative to those of the MCOs. In particular, the type 3 Cu site in the MCOs (LcT3)isa part of the trinuclear Cu cluster, and if the third (i.e., type 2) Cu is T3 removed, the Lc site does not react with O2. Density functional ؊1 theory calculations indicate that O2 binding in Hc is Ϸ9 kcal mol more favorable than for LcT3. The difference is mostly found in the Ϸ ؊1 total energy difference of the deoxy states ( 7 kcal mol ), where Fig. 1. O2 binding in the type 3 Cu sites of Hc/Tyr and MCOs. the stabilization of deoxy LcT3 derives from its long equilibrium Cu–Cu distance of Ϸ5.5–6.5 Å, relative to Ϸ4.2 Å in deoxy Hc/Tyr. The O2 binding in Hc is driven by the electrostatic destabilization Cu–Cu distance of Ϸ3.6 Å (Fig. 1A) (7, 8, 10). The side-on 2Ϫ ␲

of the deoxy Hc site, in which the two Cu(I) centers are kept close geometry promotes a large overlap between the O2 * and the BIOCHEMISTRY together by the protein for facile 2-electron reduction of O2. twoCudx2Ϫy2 orbitals and provides a direct pathway for 2-elec- T3 Alternatively, the lack of O2 reactivity in Lc reflects the flexibility tron reduction of O2 to peroxide (26). Detailed spectroscopic of the active site, capable of minimizing the electrostatic repulsion and theoretical studies have shown that the side-on bridged of the 2 Cu(I)s. Thus, the O2 reactivity of the MCOs is intrinsic to the geometry in oxy Hc/Tyr leads to a large energy splitting between trinuclear Cu cluster, leading to different O2 intermediates as the lowest-unoccupied molecular orbital (LUMO) and the high- required by its function of irreversible reduction of O2 to H2O. est-occupied molecular orbital (HOMO), which results in the strong antiferromagnetic coupling between the two Cu(II) cen- CHEMISTRY density functional theory ͉ laccase ͉ binuclear copper proteins ͉ ters (2, 3). Evaluation of the O2-binding reaction coordinate by oxygen binding ͉ oxygen reduction using density functional theory (DFT) calculations has shown that the spin-forbidden transition in the formation of the anti- number of important biological systems contain coupled ferromagnetically coupled (i.e., singlet) oxy Hc from triplet O2 involves a simultaneous 2-electron transfer, where the initial binuclear copper active sites that play important roles in O2 A ferromagnetically coupled coordination modes change along the binding, activation, and reduction to H2O. The term ‘‘coupled’’ refers to the exchange interaction between two Cu(II) (S ϭ 1/2) O2-binding coordinate with increasing metal–ligand overlap to centers caused by bridging ligation, leading to antiferromagnetic turn on antiferromagnetic coupling to generate the final planar coupled, diamagnetic, and thus, electron paramagnetic reso- singlet Cu2O2 structure (Fig. 1A). nance (EPR) silent ground states. They have been historically The type 3 Cu sites in the MCOs are part of the trinuclear Cu categorized as ‘‘type 3’’ Cu sites in biology (1) and consistently cluster where the 4-electron reduction of O2 to water occurs. referred to as one class of related sites. The simplest of the Reaction of the fully reduced form of MCO with O2 proceeds via metalloproteins containing type 3 sites are the binuclear Cu two sequential 2-electron steps generating, first, the peroxy proteins, hemocyanins (Hc), catechol oxidase (CatO), and ty- intermediate and then, the native intermediate. The spectral rosinase (Tyr) that reversibly bind O2; CatO and Tyr further features of the peroxy intermediate are very different from those activate O2 for substrate hydroxylation or oxidation (2, 3). of oxy Hc/Tyr, indicating that the peroxy intermediate acquires Hc/CatO/Tyr have been shown to have structurally equivalent a very different geometry (27). In our recent DFT study, we have active sites. The type 3 sites are also found in the multicopper generated a spectroscopically relevant structure, where the ␮ oxidases (MCOs), which include tree and fungal laccase (Lc), peroxide is bridged internally in a 3-1,1,2 geometry with the ceruloplasmin, Fet3p (the MCO found in yeast with ferroxidase type 2 and one of the type 3 coppers oxidized (Fig. 1B) (28). activity), and ascorbate oxidase. MCOs use a minimum of four Importantly, this structure allows for the irreversible binding of O Cu centers: a ‘‘blue,’’ type 1 Cu site and a trinuclear Cu cluster O2 at the trinuclear Cu site that leads to efficient O O bond composed of a ‘‘normal,’’ type 2 Cu and a binuclear type 3 Cu site that together catalyze the 4-electron reduction of O2 to water with concomitant oxidation of substrates (2, 3). Crystal struc- Author contributions: J.Y. and E.I.S. designed research; J.Y. and S.F. performed research; E.I.S. contributed new reagents/analytic tools; J.Y. and E.I.S. analyzed data; and J.Y. wrote tures indicate that both the type 3 Cu active sites in Hc/CatO/Tyr the paper. (4–10) and the MCOs (11–25) are similarly held in the protein The authors declare no conflict of interest. by three His ligands on each Cu center. No additional ligands are 1Present address: Department of Chemistry, Faculty of Science and Engineering and Fron- present in the deoxy forms, whereas oxygen-derived ligands tier Institute for Biomolecular Engineering Research (FIBER) Konan University, 8-9-1 bridge and exchange couple the two Cu(II)s in the oxidized Okamoto, Higashinada-ku, Kobe 658-8501, Japan. forms. 2To whom correspondence should be addressed. E-mail: [email protected]. O2 binding to the type 3 Cu sites of deoxy Hc/Tyr generates This article contains supporting information online at www.pnas.org/cgi/content/full/ 2 2 a side-on ␮-␩ :␩ peroxo-bridged Cu2O2 oxy structure with 0902127106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902127106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 30, 2021 bond and therefore, does not contribute in orienting the Cu␣ atom out of the N3 plane. The pyramidal distortion in Hc and the in-plane geometry in the Cu␣ site of Lc is consistent with Cu K-edge X-ray absorption spectroscopy (XAS) data (29, 30, 32, 33) (see Discussion). The most conspicuous difference between the two deoxy structures is found in the equilibrium Cu–Cu distances, in which deoxy Hc is 4.23 Å and deoxy LcT3 is 6.48 Å (Fig. 2 A and B). For deoxy Hc, the calculated R(Cu–Cu) is in good agreement with those of known crystallographic data of Hc and Tyr, which range from 3.5 to approximately 4.6 Å (5–7, 10). For LcT3, the calculated R(Cu–Cu) is Ϸ1.2 to Ϸ1.5 Å larger than those of the known crystal structures of reduced ascorbate oxidase (13), CotA (the MCO found in bacteria Bacillus subtilis) (11) and Fet3p (24), which range from 5.0 to Ϸ5.3 Å. The deviation in the calculated Cu–Cu distance is reduced when two hydrogen bonds involving His ligands and backbone carbonyl groups† are included in the calculation [supporting information (SI) Fig. S1]. The resulting Cu–Cu distance is 5.80 Å, which is in reasonable agreement with those of the crystal structures. However, the additional hydrogen bonds have virtually no effect on the overall Cu geometries in deoxy LcT3 aside from the Cu–Cu distance. Moreover, the energy difference between deoxy LcT3 at R(Cu– Fig. 2. Optimized structures of deoxy and oxy forms of Hc and LcT3.(A) Deoxy Cu) of 6.48 Å and 5.4 Å is only Ϸ2 kcal molϪ1 (Ϸ1.4 kcal molϪ1 T3 ϭ T3 ϭ T3 ϭ Hc. (B) Deoxy Lc .(C) Oxy Hc (MS 0). (D) Oxy Lc (S 1). (E) Oxy Lc (MS 0). if a dielectric with ␧ ϭ 4.0 is included using the polarizable Relevant bond lengths are shown in Å. continuum model) caused by the shallow potential energy sur- face (PES) along R(Cu–Cu), implicating the flexible nature of T3 cleavage in the subsequent 2-electron reduction step to generate the Lc site. Thus, despite the long-calculated Cu–Cu distance, T3 the native intermediate. the deoxy Lc structure shown in Fig. 2B is a reasonable The different roles of type 3 Cu sites in Hc/Tyr and MCOs are representation of the type 3 site in the MCOs, in particular, in closely related to their distinctive ligand-binding properties. In providing energetic descriptions of O2 binding. particular, by using a derivative of tree Lc where the type 2 Cu is reversibly removed [i.e., type 2-depleted (T2D) Lc], we have Reaction Coordinate for O2 Binding. As a second step in evaluating T3 O2 binding in the type 3 sites of Hc and Lc ,anO2 molecule was demonstrated that the reduced type 3 Cu center alone cannot T3 bind O in the MCOs, (29, 30). However, no detailed account of introduced to the deoxy Hc and deoxy Lc structures. Geometry 2 ϩ T3ϩ the origin of this O reactivity difference between the two type optimizations of deoxy Hc O2 and deoxy Lc O2 complexes 2 ␣ 3 Cu sites has been available. In this work, DFT calculations are were performed with frozen C positions, in both the spin- ϭ ϭ systematically performed to compare the two classes of type 3 Cu unrestricted triplet (S 1) and broken-symmetry (MS 0) states; the energies of the pure-singlet (S ϭ 0) states were then sites in biology. Focus is on evaluating the different O2-binding properties of Hc/Tyr and the type 3 site in the MCOs and obtained from the spin-projection method (34–36) by using the elucidating their origins. This work provides molecular insight broken-symmetry state energies (see Computational Methods). into the critical role of the protein environment in directing the Calculations were performed both with unconstrained Cu–Cu distances and with these distances systemically varied. O2 reactivity of these catalytic active sites. The O2-binding mechanism in Hc has been studied in detail, Results and Analysis and the structure of HcϩO2 (i.e., oxy Hc) is well established (26). Reduced Structures. As a starting point for the evaluation of O Therefore, the optimized oxy Hc structure, in the broken- 2 ϭ binding to the type 3 sites in Hc/Tyr and MCOs, optimized symmetry (MS 0) state, is first obtained to validate our method structures of Hc and Lc [the type 3 Cu center in the absence of and model systems (Fig. 2C). Our calculations indicate that O2 Ϫ Ϫ1 the type 2 Cu (LcT3)] with reduced (i.e., deoxy) binuclear Cu binding in Hc is exothermic by 2.9 kcal mol , which is in ⌬ ϭϪ Ϫ A B reasonable agreement with experiment ( H 11.5 to 6.0 centers were obtained (Fig. 2 and ). Copper atoms are Ϫ1 Ϫ1 T3 kcal mol and ⌬G ϭϪ5.0 kcal mol at 25 °C) (37–41). Key labeled as CuA and CuB for Hc and Cu␣ and Cu␤ for Lc for clarity in further descriptions of these sites. Both type 3 sites were structural features are consistent with experimental data, yield- ␮ ␩2 ␩2 2Ϫ ing the side-on - : O2 core structure with R(Cu–Cu) of modeled with three His ligands on each Cu center; Cu centers Ϸ ␧ 3.55 Å (experimental 3.6 Å). The Cu2O2 structure is slightly were coordinated to N atoms except for one His ligand of the 2 2 2Ϫ T3 butterflied, and the ␮-␩ :␩ O bridge provides a strong Cu␣ center in Lc , which was coordinated to the ␦N atom as 2 found in all available crystal structures of MCOs. Each model superexchange pathway for a large antiferromagnetic coupling of Ϫ ˆ ϭϪ ˆ ⅐ˆ was geometry optimized with frozen ␣C positions to reflect the the Cu(II) centers with a calculated 2 J (H 2 J S1 S2)of Ϸ Ϫ1 Ͼ Ϫ1 different protein structures at the type 3 sites in Hc and LcT3. 1,280 cm , also consistent with experiment ( 600 cm ). A The geometry-optimized structures of deoxy Hc and deoxy reaction coordinate was further generated by varying the Cu–Cu LcT3 are shown in Fig. 2 A and B. In general, the optimized Cu distance from the starting R(Cu–Cu) of 4.2 Å (i.e., that of 10 reduced Hc) (Fig. 3 Lower Left) and optimizing the rest of the centers have trigonal planar geometry, typical for d -ML3 units ␣ ϭ (31). However, the Cu atoms in the Cu and Cu centers in Hc structure with frozen C positions (note that the S 0 energies A B shown in Fig. 3 are obtained from spin-projection method by and Cu␤ center in Lc are Ϸ0.2–0.3 Å out of the plane formed by the N3 ligands, whereas the Cu atom in the Cu␣ site in Lc remains in the plane. The planar geometry of the Cu␣ site is largely ␦ †The hydrogen-bonding interactions are present in the two HXH loop motifs in the LcT3 site. caused by the N coordination of one of its His ligands because In each HXH loop, the ␦NH of the ﬁrst His ligand is in position to hydrogen bond with the the ␣C constraint of the ␦N-His ligand is not along the CuON CAO group of the X residue.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902127106 Yoon et al. Downloaded by guest on September 30, 2021 Å, the broken-symmetry optimized structures (representing singlet states) are characterized by the side-on ␩2 superoxo structures formed between O2 and the oxidized Cu␤ center via internal 1-electron transfer from Cu␤ to O2 (see Fig. S3), in contrast to the end-on structure of the triplet state (Fig. 2D). When R(Cu–Cu) decreases below Ϸ4.5 Å in the broken- symmetry state, a bridged ␮-␩1-␩2 geometry is initially formed, then the final ␮-␩2-␩2 peroxo structure, with transfer of the second electron from the Cu␣ center to the bridging O2 moiety (spin density at the Cu␣ atom changes from 0.0 to Ϫ0.33 to Ϫ0.45 with change in R(Cu–Cu) from 6.13 Å to 4.20 Å to 3.66 Å in the broken-symmetry state). Notably, the spin state transition from S ϭ 1toSϭ 0 occurs at R(Cu–Cu) Ϸ4.0 Å, where an energy barrier is found to be Ϸ9 kcal molϪ1 relative to that of the triplet T3 oxy Lc . The presence of this energy barrier makes the O2 T3 T3 binding to deoxy Lc kinetically difficult, in contrast to Hc Fig. 3. Reaction coordinates for O2 binding in Hc and Lc along Cu–Cu. The where the O2 binding to the deoxy Hc is uniformly downhill (Fig. O2-binding energies were obtained by subtracting the sum of the energies of triplet O2 molecule and the most stable deoxy forms (i.e., deoxy forms in Fig. 3 Lower Left). Thus, our calculations indicate the O2 binding in T3 2) from that of the oxy forms at each R(Cu–Cu). The S ϭ 0 energies are obtained Lc is inhibited thermodynamically, as well as kinetically, and from the spin-projection method by using the broken-symmetry state ener- therefore the long Cu-Cu distance in deoxy LcT3 creates a large gies and geometries. energy barrier for the singlet oxy LcT3 formation. At this point, we evaluate the origin of different O2 reactivities T3 of Hc and Lc . As mentioned above, the O2-binding energies of using the broken-symmetry state energies and geometries). As a Hc and LcT3 are different by Ϸ8.5 kcal molϪ1 (Ϫ2.9 kcal molϪ1 ϭ ϭ Ϫ result, a spin-state transition from S 1toS 0 occurs with for Hc and ϩ5.6 kcal mol 1 for Lc). However, the total energy Ϸ Ϫ decreasing Cu–Cu distance (at 3.9 Å). Associated with this of the oxy LcT3 structure itself is only Ϸ1.6 kcal mol 1 higher ␮ ␩1 ␩2 spin-state transition is a structural change from a - : peroxy than that of the oxy Hc structure, which does not account for the ␮ ␩2 ␩2 bridge at large distances (Fig. S2) to the final - : Cu2O2 difference in O2-binding energies. Rather, the remaining 6.9 kcal BIOCHEMISTRY structure. These results are consistent with our previous study on molϪ1 difference is found in the energy difference of the deoxy O2 binding to deoxy Hc, where a reaction coordinate was structures of Hc and LcT3. As shown below, the relative desta- generated by systematically varying the distance of the peroxide bilization of the deoxy Hc structure leads to the thermodynam- above the molecular plane (26). It was shown that the charges on ically favorable O2 binding in Hc, whereas the relative stabili- T3 both copper centers change at the same rate even in the zation in the deoxy Lc structure results in unfavorable O2 asymmetric bridged structure, indicating that O2 binding in- binding in LcT3. volves the simultaneous transfer of 2 electrons. Likewise, it is CHEMISTRY found here that 2 electrons from both Cu centers have already Potential Energy Surfaces of Deoxy Sites. To gain insight into the Ϸ been transferred to O2 to form peroxide at an R(Cu–Cu) of 4.0 origin of the difference in deoxy Hc and deoxy LcT3, PESs along Å, as indicated by spin-density distribution among Cu and O Cu–Cu were generated, where geometry optimizations of the atoms in the broken-symmetry wave functions: At R(Cu–Cu) ϭ deoxy Hc and deoxy LcT3 were performed with frozen ␣C ϩ 4.0 Å, spin densities on CuA,CuB,O1,O2 atoms are 0.46, positions (Fig. 4A, filled symbols), as well as without the ␣C Ϫ0.46, ϩ0.09, and Ϫ0.11, whereas at the equilibrium R(Cu– constraints (Fig. 4A, open symbols, Hc* and LcT3*). As de- Cu) ϭ 3.55 Å, these are ϩ0.48, Ϫ0.48, Ϫ0.03, and ϩ0.04, scribed above, the deoxy LcT3 (with ␣C constraints) energy respectively. Thus, the Cu–Cu distance in deoxy Hc is preposi- minimum is 6.9 kcal molϪ1 lower in energy than that for deoxy tioned for facile 2-electron transfer upon O2 binding. Hc and has an equilibrium Cu–Cu distance of 6.48 Å, in contrast In contrast to oxy Hc, the most stable structure for oxy LcT3 to the 4.23 Å optimized distance of deoxy Hc. Alternatively, is found in the triplet (S ϭ 1) state. As shown in Fig. 2D, the unconstrained PESs for deoxy Hc and deoxy LcT3 (Hc* and triplet oxy LcT3 structure has R(Cu–Cu) of 6.30 Å. Consequently, LcT3* in Fig. 4A) are both effectively linear, and the slopes are Ϫ1 Ϫ1 O2 is unable to bridge the two Cu centers but is only very weakly very similar (approximately Ϫ2.5 kcal mol Å ), indicating that bound to the Cu␤ center with an end-on geometry and a low the Cu(I)–Cu(I) electrostatic repulsion of the two deoxy forms Ϫ1 O2-binding energy of Ϫ0.7 kcal mol (note that we were not are the same (note that the difference between the two uncon- able to generate a structure with O2 bound to the Cu␣ atom). strained PESs are from the small additional contribution caused Bond lengths, R(Cu␤OO) ϭ 2.96 Å and R(OOO) ϭ 1.23 Å, are by a decrease in steric interactions of the three His ligands also indicative of a very weak Cu␤OO2 bond that lacks any between Cu centers, which are eclipsed in Lc and staggered in electron transfer between Cu and O2 (i.e., no superoxide or Hc). Notably, the energy difference of the unconstrained deoxy peroxide formation). At shorter Cu–Cu distances, the side-on LcT3 at R(Cu–Cu) ϭ 6.5 Å and 4.2 Å is Ϸ5.8 kcal molϪ1 (i.e., the 2 2 ␮-␩ :␩ Cu2O2 structure in the broken-symmetry spin state is electrostatic energy difference), which accounts for most of the also obtained (Fig. 2E). The singlet oxy LcT3 structure has 6.9 kcal molϪ1 energy difference between deoxy Hc and deoxy R(Cu–Cu) of 3.66 Å and R(O–O) of 1.42 Å, indicative of a LcT3. Thus, the decrease in energy of the deoxy LcT3 site reflects 2-electron reduced peroxo-species. However, the singlet oxy the decrease in electrostatic repulsion of the two Cu(I) centers T3 ϩ Ϫ1 Lc is found to have O2-binding energy of 5.6 kcal mol (6.3 in the low dielectric of the protein. The lack of O2 reactivity of kcal molϪ1 higher than the triplet oxy LcT3), making its forma- the type 3 site in LcT3 reflects its electrostatic and structural tion thermodynamically unfavorable. stabilization at its long-calculated equilibrium Cu–Cu distance of We have also generated a reaction coordinate for the forma- 6.48 Å. tion of singlet oxy LcT3 by varying R(Cu–Cu), in both the triplet In Fig. 4B, the components of the PESs in Fig. 4A where the (S ϭ 1) and singlet (S ϭ 0) states (Fig. 3). The PES for the triplet electrostatic contributions (i.e., Hc* and LcT3* in Fig. 4A) are state is uphill from R(Cu–Cu) of 6.30 Å with decreasing Cu–Cu removed are given. The resultant PESs provide important insight distance, whereas that for the singlet state is downhill until the into the elasticity of the two type 3 sites imposed by the protein equilibrium R(Cu–Cu) of 3.66 Å is reached. At R(Cu–Cu) Ͼ 4.5 constraints (through the ␣C atoms in our models) in the absence

Yoon et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 30, 2021 Fig. 5. Features of Cu active sites Hc (A) and LcT3 (B) that affect the equilibrium Cu–Cu distances. Structures are adapted from crystal structure database 1JS8 (oxy Hc from O. doﬂeini Hc) (8) and 1GYC (resting oxidized fungal Lc from T. versicolor) (19).

ligand-binding properties compared with Hc/Tyr, in which exogenous ligands bridge the two-Cu center (42). We have calculated that O2 binding to Hc is exothermic by 2.9 Ϫ1 T3 kcal mol . This is in contrast to O2 binding to the deoxy Lc in the formation of singlet oxy LcT3, which is found to be endothermic by 5.6 kcal molϪ1 (Fig. 3), consistent with the fact that T2D Lc does ‡ Ϫ1 not bind O2. The origin of this 8.5 kcal mol destabilization of O2 binding in LcT3 relative to Hc reflects the relative stabilization of the Fig. 4. PESs of deoxy Hc and deoxy LcT3 along Cu–Cu. (A) PESs of deoxy Hc and deoxy LcT3 structure in the protein environment of the MCOs. The deoxy LcT3 with ␣C constraints are shown as Hc and LcT3 in ﬁlled symbols, 6.9 kcal molϪ1 stabilization of deoxy LcT3 relative to deoxy Hc whereas PESs of deoxy Hc and deoxy LcT3 without ␣C constraints are shown as T3 T3 derives from the decrease in electrostatic repulsion of the two Hc* and Lc * in open symbols. (B) PESs of deoxy Hc and deoxy Lc , where the Cu(I)s at the long Cu–Cu distance in deoxy LcT3 in the low dielectric energies of Hc* and LcT3*inA are subtracted from those of Hc and LcT3 in A. Symbols are the results of the subtraction, whereas the solid lines are the ﬁt of the protein. This increase in Cu–Cu distance is allowed by the curves using a second-order parabolic function. For comparison of the two PES combined effects of the protein constraints and the flexibility in the components, the minima are set as zero in relative energies. active-site environment. From Fig. 5, the large structural differences between the coupled binuclear site in Hc and the type 3 site in MCOs relate to the very of electrostatic contributions. The PES components (Fig. 4B, different structural constraints in the two protein environments. filled symbols) were fitted to second-order parabolic functions The binuclear Cu(I) site of deoxy Hc is kept at its 4.2 Å Cu–Cu (Fig. 4B, solid lines) to obtain spring force constants, k.Itis distance because of the constraints of the protein. The most T3 Ϫ1 Ϫ2 conspicuous constraint is associated with two His ligands, one on found that k for deoxy Lc is 2.3 kcal mol Å , whereas that ␣ ␣ Ϸ for deoxy Hc is 14.1 kcal molϪ1ÅϪ2, which is Ϸ7 times larger than each Cu, for which the C– C distance is only 5 Å compared with Ͼ9 Å for other ␣C–␣C distances between His ligands on the two that of deoxy LcT3. Notably, in Fig. 4B, the minimum in the deoxy Cu centers. The origin of the close ␣C–␣C distance is attributed to LcT3 PES component is Ϸ1 Å shorter than that of the original an Arg(Lys)–Asp salt bridge between two helices that is conserved PES in Fig. 4A (from 6.48 Å to 5.51 Å), reflecting the low value in Hc/CatO/Tyr (see sequence alignment in Fig. S4). Alternatively, of k and thus, the flexible nature of the Cu geometry in deoxy T3 T3 in the Lc Cu site, the two Cu(I)s are kept at an electrostatically Lc . In contrast, the minimum in the deoxy Hc PES is shifted stable distance of Ϸ6.5 Å (calculated) by two sets of two His ligands by only 0.08 Å (from 4.23 Å to 4.15 Å). Thus, the large k in deoxy (␣C–␣C distances of the two pairs of His ligands are Ϸ7 Å), where Hc demonstrates that the Cu–Cu distance is kept short by the each set is from an H–X–H motif that is on a loop extending from T3 protein environment, whereas it is not in deoxy Lc . a ␤-sheet, leaving the Cu(I)s relatively unconstrained. Importantly, this shows that the different ligand architecture in Hc and LcT3 Discussion account for the large difference in equilibrium Cu–Cu distances in Generally, the binuclear Cu sites in Hc/Tyr and MCOs (i.e., T3 deoxy Hc and deoxy Lc . This is a critical factor in the O2-binding T3 Lc ) have been classified into the same type 3 group because properties of Hc and LcT3, where the close Cu–Cu distance in deoxy both have antiferromagnetically coupled diamagnetic ground Hc leads to simultaneous 2-electron transfer from both Cus to O2, states and thus, no EPR feature. Despite the same active-site whereas the long Cu–Cu distance in deoxy LcT3 leads to stabiliza- components of [Cu(His)3]2, however, the two type 3 sites have tion of this state and energetically unfavorable O2 binding. very different ligand-binding properties. In particular, the re- In addition, the protein constraints further contribute to the T3 duced type 3 Cu site in T2D Lc is stable to O2 binding, in strong very different PESs for deoxy Hc and Lc along Cu–Cu (Fig. T3 contrast to Hc/Tyr, which reversibly bind O2 to form oxy Hc/Tyr 4B), where the binuclear force constant k in deoxy Lc is Ϸ7 (29, 30). Moreover, exogenous ligands (carbon monoxide and azide) were shown to bind to the type 3 site of T2D Lc, demonstrating that small molecules are not blocked from having ‡We have also calculated deoxy LcT3 structures with a fourth His ligand, as found in some of the crystal structures of T2D Lc (14, 15). Our results show that the fourth His ligand does access to this active site. However, these ligands bind only to one not coordinate with the Cu center, whereas instead, a H2O molecule can coordinate very Cu center in T2D Lc, further demonstrating the different weakly.

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902127106 Yoon et al. Downloaded by guest on September 30, 2021 and B); in deoxy Hc, both Cu centers show a pyramidal distortion (Cu centers Ϸ0.2–0.3 Å out of the N3 plane) whereas in deoxy LcT3, only one Cu is distorted, and the other, Cu␣ in Fig. 2B, retains a trigonal planar geometry, where the planarity derives largely from the ␦N ligation of one of its His ligands. These different Cu geometries are closely related to the different force constants; in the trigonal planar geometry of the Cu␣ center in T3 deoxy Lc , the vibrational mode normal to the N3 plane involves only bends, whereas for the trigonal pyramidal geometries of the Cu centers in deoxy Hc (and Cu␤ in deoxy LcT3), both bends and bond stretches are involved. Thus, the larger force constant along Cu–Cu in deoxy Hc largely reflects the pyramidal distortion of the Cu center that is imposed by the protein environment, whereas the low force constant in deoxy LcT3 derives from its relatively unconstrained environment, resulting in the trigonal 10 planar structure typical of Cu(I) d –ML3 complexes. Fig. 6. Normalized Cu K-edge XAS spectra of deoxy Hc and T2D Lc. The two In summary, deoxy Hc is electrostatically destabilized to react Hc spectra were obtained from Hcs from an arthropod Panulirus interruptus (spiny lobster) (32) and a mollusk Busycon canaliculatum (sea snail) (32), and with O2 to form oxy Hc, and this can be cooperatively regulated the T2D Lc spectrum was obtained from Lc from Rhus vernicifera (lacquer tree) by changing the Cu(I)–Cu(I) distance (26) in the protein qua- (29, 30). The features of the type 1 Cu (T1 Cu) was subtracted from the T2D Lc ternary structure (i.e., tense and relaxed conformations) (44). spectrum by using the XAS spectrum of the blue Cu protein plastocyanin. Alternatively, the deoxy LcT3 Cu site in the multicopper oxidases is electrostatically stable and does not react with O2 when the T2 Cu is not present. When the type 2 Cu is also present, the times smaller than that of deoxy Hc. This large force constant trinuclear Cu cluster does react with O2 to form a 2-electron difference results in the shift in the equilibrium Cu–Cu distance reduced peroxide, bridging between the type 2 and type 3 Cu by Ϸ0.1 Å in deoxy Hc and Ϸ1 Å in deoxy LcT3 because of the centers (27, 28). The trinuclear Cu cluster further promotes linear distorting force from the electrostatic repulsion (Fig. 4). cleavage of the OOO bond by stabilizing the products of Ϸ T3 Note that the 1 Å shift in deoxy Lc results in an energy peroxide reduction (oxo and hydroxo moieties) through multiple BIOCHEMISTRY stabilization of Ϸ1.4 kcal molϪ1, whereas the energy of deoxy bridging interactions requiring a large change in Cu–Cu distance. Hc is lowered by only Ϸ0.2 kcal molϪ1 by the Ϸ0.1 Å shift.§ Thus, the difference in force constants between deoxy LcT3 and deoxy Computational Methods Hc contributes Ϸ20% of the total energy difference (i.e., Ϸ7 kcal Spin-unrestricted DFT calculations were performed on Gaussian 03 (45). All ge- molϪ1) between these sites. Importantly, the large force constant ometry optimizations were performed with B3LYP hybrid functional (46) by using in deoxy Hc indicates that the active site is tightly controlled to TZVP basis sets for Cu and coordinated N/O atoms and TZV basis sets for the rest keep the Cu–Cu distance short for facile reversible O binding. (47, 48). The initial geometries of the type 3 sites of Hc and Lc were adapted from 2 CHEMISTRY T3 the crystal structures of the oxy form of Octopus doﬂeini Hc [Protein Data Bank Alternatively in deoxy Lc , the low force constant allows a large (PDB) ID code 1JS8] (8) and the resting oxidized form of Trametes versicolor change in Cu–Cu distance with little change in energy, which is fungal Lc (PDB ID code 1GYC) (19), where the bridging O atoms were removed. required in the reaction coordinate for OOO bond cleavage at Three His ligands on each Cu center were replaced with methyl-imidazolyl li- the trinuclear Cu cluster in the MCOs (43). gands, where Cu centers were coordinated to ␧N atoms, except for one imidazolyl Insight into the origin of the difference in force constants in ligand of the Cu␣ center in Lc, which was coordinated to ␦N atom, as shown deoxy Hc and deoxy LcT3 can be gained from Cu K-edge XAS consistently in all available crystal structures of MCOs. All geometry optimizations were performed with frozen methyl C atoms (i.e., ␣C atoms; depicted by gray data. As shown in Fig. 6, the preedge features of deoxy Hc and T3 T3 spheres in Fig. 2, except for the deoxy Hc and deoxy Lc structures in generating deoxy T2D Lc (i.e., deoxy Lc ) are very different (29, 30, 32, 33). ␣ Ϸ the Hc* and Lc* potential energy surfaces shown in Fig. 4A, in which all of the C In deoxy T2D Lc, the sharp intense peak at 8,983 eV is the Cu constraints were removed). For oxy Hc and oxy LcT3 structures, geometry optimi- 1s 3 Cu 4pz transition in a trigonal planar geometry, with the Cu zations were performed in both the triplet (S ϭ 1) and the broken-symmetry 1s 3 Cu 4px,y transitions shifted up in energy with lower (MS ϭ 0) states. The energies of the pure singlet (S ϭ 0) states were then computed intensities because of the equatorial ligands. In deoxy Hc, the Cu by using the spin-projection method (34–36): 3 3 1s Cu 4pz transition has lower intensity whereas the Cu 1s 2 2E Ϫ ͗S ͘E ϭ Cu 4px,y transitions shift to lower energy and higher intensity, ϭ BS BS S 1 ESϭ0 Ϫ ͗ 2 ͘ , [1] reflecting the pyramidal distortion of the Cu centers out of the 2 SBS ligand N3 plane. These Cu K-edge XAS data are consistent with the calculated structures of deoxy Hc and deoxy LcT3 (Fig. 2 A where the triplet energy, ES ϭ 1, is computed for the single-determining high- ͗ 2 ͘ spin conﬁguration at the broken-symmetry geometry and SBS is the spin- expectation value of the broken-symmetry calculation. The O2-binding ener-

§ 2 2 gies were obtained by subtracting the sum of the energies of triplet O2 If the PES is described by E0 ϭ 1/2 k(Q Ϫ Q0) ϭ 1/2 k(⌬Q) and the linear distortion by ␭(Q Ϫ 2 molecule and the most stable deoxy forms from that of the oxy forms. Q0) ϭ ␭(⌬Q), the total energy becomes E ϭ 1/2 k(⌬Q) ϩ ␭(⌬Q). Therefore, the energy will be lowered Ϫ␭2/2k by the distortion, with a shift in Q by Ϫ␭/k. With k of 14.1 and 2.3 kcal molϪ1ÅϪ2 and the slope of the distorting electrostatic force ␭ of Ϫ2.5 kcal molϪ1ÅϪ1, Q will ACKNOWLEDGMENTS. We thank Dr. Ritimukta Sarangi for assistance with the be shifted by ϩ0.2 Å and ϩ1.2 Å (within 10% of those in Fig. 4B) with energies lowered by Cu K-edge XAS data analysis. This work was supported by National Institutes 0.2 kcal molϪ1 and 1.4 kcal molϪ1 for deoxy Hc and deoxy LcT3, respectively. of Health Grant DK-31450.

1. Malkin R, Malmstro¨m BG (1970) State and function of copper in biological systems. Adv 5. Volbeda A, Hol WGJ (1989) Crystal structure of hexameric haemocyanin from Panulirus Enzymol 33:177–244. interruptus reﬁned at 3.2 Å resolution. J Mol Biol 209:249–279. 2. Solomon EI, Chen P, Metz M, Lee SK, Palmer AE (2001) Oxygen binding, acti- 6. Hazes B, et al. (1993) Crystal structure of deoxygenated Limulus polyphemus subunit vation, and reduction to water by copper proteins. Angew Chem Int Ed 40:4570– II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation. 4590. Protein Sci 2:597–619. 3. Solomon EI, Sundaram UM, Machonkin TE (1996) Multicopper oxidases and oxygen- 7. Magnus KA, et al. (1994) Crystallographic analysis of oxygenated and deoxygenated ases. Chem Rev 96:2563–2605. states of arthropod hemocyanin shows unusual differences. Proteins 19:302–309. 4. Gaykema WPJ, et al. (1984) 3.2 Å structure of the copper-containing, oxygen-carrying 8. Cuff ME, Miller KI, van Holde KE, Hendrickson WA (1998) Crystal structure of a protein Panulirus interruptus haemocyanin. Nature 309:23–29. functional unit from octopus hemocyanin. J Mol Biol 278:855–870.

Yoon et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 30, 2021 9. Klabunde T, Eicken C, Sacchettini JC, Krebs B (1998) Crystal structure of a plant catechol 29. Lubien CD, et al. (1981) Chemical and spectroscopic properties of the binuclear copper oxidase containing a dicopper center. Nat Struct Biol 5:1084–1090. active site in Rhus laccase: Direct confirmation of a reduced binuclear type 3 copper site 10. Matoba Y, Kumagai T, Yamamoto A, Yoshitsu H, Sugiyama M (2006) Crystallographic in type 2-depleted laccase and intramolecular coupling of the type 3 to the type 1 and evidence that dinuclear copper center of tyrosinase is flexible during catalysis. J Biol type 2 copper sites. J Am Chem Soc 103:7014–7016. Chem 281:8981–8990. 30. Kau LS, Spira-Solomon DJ, Penner-Hahn JE, Hodgson KO, Solomon EI (1987) X-ray 11. Bento I, Martins LO, Lopes GG, Carrondo MA, Lindley PF (2005) Dioxygen reduction by absorption-edge determination of the oxidation state and coordination number of multicopper oxidases: A structural perspective. Dalton Trans 3507–3513. copper: Application to the type 3 site in Rhus vernicifera laccase and its reaction with 12. Messerschmidt A, et al. (1992) Refined crystal structure of ascorbate oxidase at 1.9 Å oxygen. J Am Chem Soc 109:6433–6442. resolution. J Mol Biol 224:179–205. 31. Albright TA, Burdett JK, Whangbo M-H (1985) Orbital Interactions in Chemistry (Wiley, 13. Messerschmidt A, Luecke H, Huber R (1993) X-ray structures and mechanistic implica- New York). tions of three functional derivatives of ascorbate oxidase from zucchini: Reduced, 32. Kau LS (1988) Chemical and spectroscopic studies on metal active sites in metallopro- peroxide and azide forms. J Mol Biol 230:997–1014. teins and heterogeneous catalysts. PhD dissertation, (Stanford Univ, Stanford, CA). 14. Ducros V, et al. (1998) Crystal structure of the type 2 Cu depleted laccase from Coprinus 33. Brown JM, Powers L, Kincaid B, Larrabee JA, Spiro TG (1980) Structural studies of the cinereus at 2.2 Å resolution. Nat Struct Biol 5:310–316. hemocyanin active site. 1. Extended X-ray absorption fine structure (EXAFS) analysis. 15. Ducros V, et al. (2001) Structure of the laccase from Coprinus cinereus at 1.68 Å J Am Chem Soc 102:4210–4216. resolution: Evidence for different ‘‘type 2 Cu-depleted’’ isoforms. Acta Crystallogr D 34. Noodleman L (1981) Valence bond description of antiferromagnetic coupling in tran- 57:333–336. 16. Zaitsev VN, Zaitseva I, Papiz M, Lindley PF (1999) An X-ray crystallographic study of the sition metal dimers. J Chem Phys 74:5737–5743. binding sites of the azide inhibitor and organic substrates to ceruloplasmin, a multi- 35. Yamaguchi K, Jensen F, Dorigo A, Houk KN (1988) A spin correction procedure for copper oxidase in the plasma. J Biol Inorg Chem 4:579–587. unrestricted Hartree–Fock and Moller–Plesset wavefunctions for singlet diradicals and 17. Zaitseva I, et al. (1996) The X-ray structure of human serum ceruloplasmin at 3.1 Å: polyradicals. Chem Phys Lett 149:537–542. Nature of the copper centers. J Biol Inorg Chem 1:15–23. 36. Isobe H, et al. (2002) Extended Hartree–Fock (EHF) theory of chemical reactions. VI. 18. Bertrand T, et al. (2002) Crystal structure of a four-copper laccase complexed with an Hybrid DFT and post-Hartree–Fock approaches for concerted and nonconcerted tran- arylamine: Insights into substrate recognition and correlation with kinetics. Biochem- sition structures of the Diels–Alder reaction. Mol Phys 100:717–727. istry 41:7325–7333. 37. Brunori M, Kuiper HA, Zolla L (1983) in Life Chemistry Reports, ed Woods EJ (Harwood, 19. Piontek K, Antorini M, Choinowski T (2002) Crystal structure of a laccase from the New York), pp 239–250. fungus Trametes versicolor at 1.90 Å resolution containing a full complement of 38. Zolla L, Kuiper HA, Brunori M, Antonini E (1979) Thermodynamics of Oxygen Binding coppers. J Biol Chem 277:37663–37669. to Helix pomatia ␤-Hemocyanin (Dekker, New York). 20. Hakulinen N, et al. (2002) Crystal structure of a laccase from Melanocarpus albomyces 39. Klarman A, Daniel E (1980) Oxygen binding properties of stripped (calcium ion and with an intact trinuclear copper site. Nat Struct Biol 9:601–605. magnesium ion free) hemocyanin from the scorpion Leirus quinquestriatus. Biochem- 21. Roberts SA, et al. (2002) Crystal structure and electron transfer kinetics of CueO, a istry 19:5176–5180. multicopper oxidase required for copper homeostasis in Escherichia coli. Proc Natl 40. Er-El Z, Shaklai N, Daniel E (1972) Oxygen binding properties of hemocyanin from Acad Sci USA 99:2766–2771. Levantina hierosolima. J Mol Biol 64:341–352. 22. Enguita FJ, et al. (2004) Substrate and dioxygen binding to the endospore coat laccase 41. Antonini E, Brunori M, Colosimo A, Kuiper HA, Zolla L (1983) Kinetic and thermody- from Bacillus subtilis. J Biol Chem 279:23472–23476. namic parameters for oxygen binding to the allosteric states of Panulirus interruptus 23. Smith AW, Camara-Artigas A, Wang MT, Allen JP, Francisco WA (2006) Structure of hemocyanin. Biophys Chem 18:117–124. phenoxazinone synthase from Streptomyces antibioticus reveals a new type 2 copper 42. Spira-Solomon DJ, Solomon EI (1987) Chemical and spectroscopic studies of the cou- center. Biochemistry 45:4378–4387. pled binuclear copper site in type 2 depleted Rhus laccase: Comparison to the hemo- 24. Taylor AB, Stoj CS, Ziegler L, Kosman DJ, Hart PJ (2005) The copper–iron connection in cyanins and tyrosinase. J Am Chem Soc 109:6421–6432. biology: Structure of the metallo-oxidase Fet3p. Proc Natl Acad Sci USA 102:15459–15464. 43. Solomon EI, Augustine AJ, Yoon J (2008) O reduction to H O by the multicopper 25. Garavaglia S, et al. (2004) The structure of Rigidoporus lignosus laccase containing a 2 2 oxidases. Dalton Trans 3921–3932. full complement of copper ions, reveals an asymmetrical arrangement for the type 3 44. Brouwer M, Bonaventura C, Bonaventura J (1978) Analysis of effect of three different copper pair. J Mol Biol 342:1519–1531. 26. Metz M, Solomon EI (2001) Dioxygen binding to deoxyhemocyanin: Electronic struc- allosteric ligands on oxygen binding by hemocyanin of shrimp, Penaeus setiferus. Biochemistry 17:2148–2154. ture and mechanism of the spin-forbidden two-electron reduction of O2. J Am Chem Soc 123:4938–4950. 45. Frisch MJ, et al. (2004) Gaussian 03, Revision C 01 (Gaussian, Pittsburgh). 27. Shin W, et al. (1996) Chemical and spectroscopic definition of the peroxide-level 46. Becke AD (1993) Density functional thermochemistry. 3. The role of exact exchange. intermediate in the multicopper oxidases: Relevance to the catalytic mechanism of J Chem Phys 98:5648–5652. dioxygen reduction to water. J Am Chem Soc 118:3202–3215. 47. Scha¨fer A, Horn H, Ahlrichs R (1992) Fully optimized contracted Gaussian basis sets for 28. Yoon J, Solomon EI (2007) Electronic structure of the peroxy intermediate and its atoms Li to Kr. J Chem Phys 97:2571–2577. correlation to the native intermediate in the multicopper oxidases: Insights into the 48. Scha¨fer A, Huber C, Ahlrichs R (1994) Fully optimized contracted Gaussian basis sets of reductive cleavage of the OOO bond. J Am Chem Soc 129:13127–13136. triple zeta valence quality for atoms Li to Kr. J Chem Phys 100:5829–5835.

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