The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

Abstract

Multicopper oxidases (MCOs) catalyze the 4e(-) reduction of O(2) to H(2)O. The reaction of the fully reduced enzyme with O(2) generates the native intermediate (NI), which undergoes a slow decay to the resting enzyme in the absence of substrate. NI is a fully oxidized form, but its spectral features are very different from those of the resting form (also fully oxidized), because the type 2 and the coupled-binuclear type 3 Cu centers in the O(2)-reducing trinuclear Cu cluster site are isolated in the resting enzyme, whereas these are all bridged by a micro(3)-oxo ligand in NI. Notably, the one azide-bound NI (NI(Az)) exhibits spectral features very similar to those of NI, in which the micro(3)-oxo ligand in NI has been replaced by a micro(3)-bridged azide. Comparison of the spectral features of NI and NI(Az), combined with density functional theory (DFT) calculations, allows refinement of the NI structure. The decay of NI to the resting enzyme proceeds via successive proton-assisted steps, whereas the rate-limiting step involves structural rearrangement of the micro(3)-oxo-bridge from inside to outside the cluster. This phenomenon is consistent with the slow rate of NI decay that uncouples the resting enzyme from the catalytic cycle, leaving NI as the catalytically relevant fully oxidized form of the MCO active site. The all-bridged structure of NI would facilitate electron transfer to all three Cu centers of the trinuclear cluster for rapid proton-coupled reduction of NI to the fully reduced form for catalytic turnover.

Fig. 1.

Optimized structures of the R_Az and NI_Az. Geometric parameters are listed in SI Table 2.

Fig. 2.

Contour plots of αLUMOs of R_Az (A) and NI_Az (B) in the broken symmetry states |Cu^T2Cu^T3αCu^T3β〉 = |βαα〉, |αβα〉, and |ααβ〉, based on the d_x²−y² ground states of the Cu^T2, Cu^T3α, and Cu^T3β centers, respectively. The N₃⁻ π_σ^nb or N₃⁻ π_v^nb characters in these MOs are indicated in parentheses, where N₃⁻ π_σ^nb and N₃⁻ π_v^nb are components of N₃⁻ π^nb HOMO with P orbitals in and vertical to the Cu₃ plane, respectively. Note that higher energy N₃⁻ π* orbital character is also mixed in these MOs.

Fig. 3.

EPR of NI_AZ and the origin of the low g_eff = 1.86. (A) Low-temperature EPR spectrum of NI_Az of Lc. T1 Cu contribution is subtracted using the EPR spectrum of T2-depleted Lc ([NI_Az] = 0.5 mM, 10 eq of NaN₃; T = 9.0 K; power = 200 mW, pH 7.5). (B) Zeeman splitting (B ⊥ z) of the ground and low-lying excited doublet states of NI_Az with and without SOC. (C) Depiction of the ground-to-ground and ground-to-excited superexchange pathways in NI_Az.

Fig. 4.

Absorption [(Upper) 277 K, [NI_Az] = 45 μM] and MCD [(Lower) 5K, 7T, [NI_Az] = 0.25 mM] spectra of NI_Az (10 protein equivalents of NaN₃, pH 7.5) with Gaussian fits obtained by simultaneous fits. Result of the simultaneous Gaussian fit of the absorption and MCD spectra are given in SI Table 6. The contributions from the aromatic residues have been subtracted in the absorption spectrum by using that of the fully reduced Lc (the fully reduced Lc has no MCD feature). Note that bands 4 and 5 are the S → T1 CT transitions, indicative of the oxidized T1 site in NI_Az.

Fig. 5.

Nonphase-shift-corrected FT data of NI (red) and NI_Az (black) forms of Lc at pH 7.5. (Inset) The corresponding EXAFS data. EXAFS least-squares fitting results are given in SI Table 3.

Fig. 6.

Optimized structures of NI with H₂O and OH⁻ on the Cu^T2 center (NI^H₂O and NI^OH, respectively). Geometric parameters are listed in SI Table 4.

Fig. 7.

Proposed decay mechanism of NI to the resting enzyme. The O atoms in red indicate that these are from O₂ (Upper) and the energy profiles of the rotation of the internal O atom to the external side of the T2 site in doubly protonated NI^H₂O (in red) and NI^OH (in blue) (Lower). A water molecule near the T3 site found in all MCO crystal structures is also included in these calculations.

Fig. 8.

Possible mechanisms for the reduction of NI to the fully reduced enzyme.