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

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.

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