Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

Abstract

Background: Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O2 reduction to H2O.

Results: The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 A. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, micro-eta 1:eta 1 peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (micro3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled.

Conclusion: This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.

Figure 1

Alignment of the LtL blue laccase amino acid sequence with other laccase sequences. Lt – Lentinus (Panus) tigrinus laccase [AAX07469 and Pdb code: 2QT6]; Tve – Trametes versicolor [gi:21730581, pdb:1KYA]; Tvi – Trametes villosa [AAC41686]; Pc – Pycnoporus cinnabarinus [AAF13052]; Ft – Funalia trogii [CAC13040]; So – Steccherinum ochraceum laccase [unpublished gene-xray]; Cc – Coprinus Cinereus [pdb:1A65]; Rl – Rigidoporus Lignosus [pdb:1V10]; Ts – Thizoctonia solani [CAA91042]; Ma – Melanocarpus albomyces [CAE00180]; Tov – Toxicodendron vernicifluum [BAB63411] ; Mt – Myceliophthora thermophila [AAC93841]; Bs – Bacillus subtilis Cota [pdb:1HKZ]. Positions identical in all sequences are marked with a black background. Regions, in which the sequences are similar are marked with light grey.

Figure 2

(A) Schematic representation of the structure of LtL; the copper ions are depicted as magenta spheres. (B) T2/T3 trinuclear cluster active site as observed in molecule A of LtL. (C) Substrate active T1 pocket residues. (D) Stereoview of the schematic representation of the four copper sites in LtL.

Figure 3

(A) and (B) Representations of the Fo-Fc difference Fourier omit map for the T2/T3 active site of molecules A and B of LtL respectively, each flanked by the corresponding schematic pictures. The electron density is contoured at 2.2 σ. (C) Schematic representation of the catalytic mechanism of multicopper oxidases including the intermediates observed in the present structural study and previous spectroscopic, kinetic, and structural investigations.

Figure 4

Schematic representations of possible adducts of the trinuclear T2/T3 copper cluster with 2-electrons reduced dioxygen (peroxide).

Figure 5

Schematic representations of different possible adducts of the trinuclear T2/T3 copper cluster. A and B: 4-electrons reduced dioxygen adducts, C: enzyme resting state.