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

Abstract

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 O(2) 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 (Lc(T3)) is a part of the trinuclear Cu cluster, and if the third (i.e., type 2) Cu is removed, the Lc(T3) site does not react with O(2). Density functional theory calculations indicate that O(2) binding in Hc is approximately 9 kcal mol(-1) more favorable than for Lc(T3). The difference is mostly found in the total energy difference of the deoxy states (approximately 7 kcal mol(-1)), where the stabilization of deoxy Lc(T3) derives from its long equilibrium Cu-Cu distance of approximately 5.5-6.5 A, relative to approximately 4.2 A in deoxy Hc/Tyr. The O(2) binding in Hc is driven by the electrostatic destabilization of the deoxy Hc site, in which the two Cu(I) centers are kept close together by the protein for facile 2-electron reduction of O(2). Alternatively, the lack of O(2) reactivity in Lc(T3) reflects the flexibility of the active site, capable of minimizing the electrostatic repulsion of the 2 Cu(I)s. Thus, the O(2) reactivity of the MCOs is intrinsic to the trinuclear Cu cluster, leading to different O(2) intermediates as required by its function of irreversible reduction of O(2) to H(2)O.

Fig. 1.

O₂ binding in the type 3 Cu sites of Hc/Tyr and MCOs.

Fig. 2.

Optimized structures of deoxy and oxy forms of Hc and Lc^T3. (A) Deoxy Hc. (B) Deoxy Lc^T3. (C) Oxy Hc (M_S = 0). (D) Oxy Lc^T3 (S = 1). (E) Oxy Lc^T3 (M_S = 0). Relevant bond lengths are shown in Å.

Fig. 3.

Reaction coordinates for O₂ binding in Hc and Lc^T3 along Cu–Cu. The 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. 2) from that of the oxy forms at each R(Cu–Cu). The S = 0 energies are obtained from the spin-projection method by using the broken-symmetry state energies and geometries.

Fig. 4.

PESs of deoxy Hc and deoxy Lc^T3 along Cu–Cu. (A) PESs of deoxy Hc and deoxy Lc^T3 with αC constraints are shown as Hc and Lc^T3 in filled symbols, whereas PESs of deoxy Hc and deoxy Lc^T3 without αC constraints are shown as Hc* and Lc^T3* in open symbols. (B) PESs of deoxy Hc and deoxy Lc^T3, where the energies of Hc* and Lc^T3* in A are subtracted from those of Hc and Lc^T3 in A. Symbols are the results of the subtraction, whereas the solid lines are the fit curves using a second-order parabolic function. For comparison of the two PES components, the minima are set as zero in relative energies.

Fig. 5.

Features of Cu active sites Hc (A) and Lc^T3 (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).

Fig. 6.

Normalized Cu K-edge XAS spectra of deoxy Hc and T2D Lc. The two Hc spectra were obtained from Hcs from an arthropod Panulirus interruptus (spiny lobster) (32) and a mollusk Busycon canaliculatum (sea snail) (32), and the T2D Lc spectrum was obtained from Lc from Rhus vernicifera (lacquer tree) (29, 30). The features of the type 1 Cu (T1 Cu) was subtracted from the T2D Lc spectrum by using the XAS spectrum of the blue Cu protein plastocyanin.