Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer

Abstract

Background: Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. They are the simplest members of the multi-copper oxidases and contain at least two types of copper centres; a mononuclear T1 and a trinuclear that includes two T3 and one T2 copper ions. Substrate oxidation takes place at the mononuclear centre whereas reduction of oxygen to water occurs at the trinuclear centre.

Results: In this study, the CotA laccase from Bacillus subtilis was used as a model to understand the mechanisms taking place at the molecular level, with a focus in the trinuclear centre. The structures of the holo-protein and of the oxidised form of the apo-protein, which has previously been reconstituted in vitro with Cu(I), have been determined. The former has a dioxygen moiety between the T3 coppers, while the latter has a monoatomic oxygen, here interpreted as a hydroxyl ion. The UV/visible spectra of these two forms have been analysed in the crystals and compared with the data obtained in solution. Theoretical calculations on these and other structures of CotA were used to identify groups that may be responsible for channelling the protons that are needed for reduction of dioxygen to water.

Conclusions: These results present evidence that Glu 498 is the only proton-active group in the vicinity of the trinuclear centre. This strongly suggests that this residue may be responsible for channelling the protons needed for the reduction. These results are compared with other data available for these enzymes, highlighting similarities and differences within laccases and multicopper oxidases.

Figure 1

Three-dimensional structure of CotA. (a) Three-dimensional structure of CotA with each cupredoxin domain coloured in a different colour; domain I coloured in blue, domain II coloured in gray and domain III coloured in violet. Copper atoms are represented by spheres coloured in yellow. These correspond to the mononuclear coppers 1 and 5, and the trinuclear centre, comprising coppers 2, 3 and 4. (b) Structural detail of the catalytic copper centres, the mononuclear type 1 copper centre (T1) where the copper atom is coordinated by a cysteine and two histidines, and the trinuclear centre which comprises a type 2 copper atoms (T2) and two type 3 (T3) copper atoms. The cysteine residue (C492) that coordinates the T1 copper atom is bound to two of the histidine residues (H491 and H493) that coordinate the two T3 coppers in the trinuclear centre. This motif has been proposed to constitute the path for transfer electrons from the T1 copper centre to the trinuclear centre. (c) Close view of the CotA trinuclear centre - Important acidic groups are labelled (E498 and D116) as well as the histidine ligands to the copper that establish hydrogen bonds with D116.

Figure 2

Structural detail of the trinuclear Copper centre. In each picture the electron density for the moiety is derived from omit Fourier syntheses computed with SigmaA weighted coefficients |Fo| - |Fc|; the moieties were not included in the structure factor calculations and five cycles of maximum likelihood refinement were computed using REFMAC prior to Fourier synthesis to minimise phase bias. Contour levels are 5 rms for both electron density maps. (a) In the holoCotA structure a dioxygen molecule is bound into the trinuclear centre. (b) In the apoCu(I) a hydroxyl group is bound to the trinuclear centre. Both pictures were made with PyMol [69].

Figure 3

UV/Visible absorption spectra of a) holoCotA crystal and of b) apoCu(I) crystal.

Figure 4

Dioxygen reduction to water by multicopper oxidases: crystal structures of several potential intermediates in the dioxygen reduction to water by multicopper oxidases: a) HoloCotA. b) CotA-H₂O₂ (1W8E) [20]. c) Laccase from Trametes hirsuta [43] (3FPX). d) Ascorbate oxidase from Zucchini (1ASO) [21]. e) ApoCu(I) f) reduced CotA (2BHF) [20].

Figure 5

Simulated pH titrations of Glu 498 for the different CotA trinuclear centre states described in Figure 4. The state of T1 is set to oxidised for the dioxygen, hydroxyl and peroxide structures, and set to reduced for the reduced structure.

Figure 6

Simulated pH titrations of Asp 116 for the different CotA trinuclear centre states described in Figure 4. The state of T1 is set to oxidised for the dioxygen, hydroxyl, double hydroxyl and peroxide structures, and set to reduced for the reduced structure. Note that the maximum of the scale is one tenth of the maximum used for the other plots.

Figure 7

Simulated pH titrations of Glu 498 for the CotA-H₂O₂ state upon changes on the redox state of centre T1.

Figure 8

Simulated redox titrations at pH 7 of the T1 site for the different trinuclear CotA trinuclear centre states described in Figure 4. The redox potentials for each of the situations are 525 mV for the structure with dioxygen (which was set, see text), 473 mV for the structure with hydroxyl, 457 mV for the structure with peroxide, and 424 mV for the reduced structure.