Electron transfer and reaction mechanism of laccases

Abstract

Laccases are part of the family of multicopper oxidases (MCOs), which couple the oxidation of substrates to the four electron reduction of O2 to H2O. MCOs contain a minimum of four Cu's divided into Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3) Cu sites that are distinguished based on unique spectroscopic features. Substrate oxidation occurs near the T1, and electrons are transferred approximately 13 Å through the protein via the Cys-His pathway to the T2/T3 trinuclear copper cluster (TNC), where dioxygen reduction occurs. This review outlines the electron transfer (ET) process in laccases, and the mechanism of O2 reduction as elucidated through spectroscopic, kinetic, and computational data. Marcus theory is used to describe the relevant factors which impact ET rates including the driving force, reorganization energy, and electronic coupling matrix element. Then, the mechanism of O2 reaction is detailed with particular focus on the intermediates formed during the two 2e(-) reduction steps. The first 2e(-) step forms the peroxide intermediate, followed by the second 2e(-) step to form the native intermediate, which has been shown to be the catalytically relevant fully oxidized form of the enzyme.

Fig. 1

UV-Vis absorbance (a) and EPR spectra (b) of resting laccase. Reprinted with permission from [3]. Copyright 2014 American Chemical Society

Fig. 2

T1 sites of CotA from B. subtilis with axial methionine (a) and of laccase from T. versicolor without the axial methionine (b). PDB accession numbers: 2X88 and 1GYC

Fig. 3

T2 and T3 Cu sites in MCOs. Labeled histidines H454 and H452 connect T3α and T3β to the T1 Cu through the Cys-His bridge. Also shown are conserved carboxylates D77 and D456. All residues are numbered according to PDB:1GYC

Fig. 4

β-LUMO coupled into Cys-His pathway (a). Calculated spin density of ground state wavefunction with H-bond pathway shown in blue (b). π to σ superexchange pathway from T1 Cu to TNC (c). Reprinted with permission from [21]. Copyright 2006 American Chemical Society

Fig. 5

Mechanism of O₂ reduction by MCOs. Red arrows show steps in the catalytic cycle. Black arrows show reduction of resting enzyme to enter catalytic cycle and decay of the native intermediate which terminates catalysis. Adapted with permission from [80]. Copyright 2010 American Chemical Society

Fig. 6

a Absorbance spectra of the peroxy intermediate at different times during its decay. Inset shows the decay at 340 nm. b Absorbance difference spectra of the spectra in (a) relative to fully oxidized T1Hg laccase. Reprinted with permission from [90]. Copyright 1996 American Chemical Society

Fig. 7

Optimized structures of the peroxy intermediate without (a) and with (b) the D94 residue. Reprinted with permission from [94]. Copyright 2007 American Chemical Society

Fig. 8

Schematic showing the roles of carboxylates E487 and D94 in O–O bond cleavage during PI decay. Reprinted with permission from [94]. Copyright 2007 American Chemical Society

Fig. 9

2D potential energy surface of the decay of the peroxy intermediate. Reprinted with permission from [94]. Copyright 2007 American Chemical Society

Fig. 10

Schematic of the overlap between the HOMOs on the T2 and T3α sites and the ${\text{O}}_2^{2-}$ σ* orbital. Reprinted with permission from [94]. Copyright 2007 American Chemical Society

Fig. 11

Origin of the low g values observed in NI. Reprinted from [104] with permission of The Royal Society of Chemistry

Fig. 12

Optimized structure of the NI. Reprinted from [103]. Copyright 2007 National Academy of Science, USA