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

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.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.Multicopper oxidases (MCOs) are a group of enzymes that are able to couple the oxidation of a variety of different substrates concomitantly with dioxygen reduction to water [1-3]. This protein family comprises laccases, (p-diphenol: dioxygen oxidoreductase, EC 1.10.3.2), metalloxidases, ascorbate oxidase and ceruloplasmin [2,4,5]. They are found in prokaryotes, eukaryotes as well as in archea. In plants, they have been associated with lignin formation; in fungi, with pigment formation, lignin degradation and detoxification processes; in yeast and mammals with iron metabolism; in bacteria with copper homeostasis and manganese oxidation ([6,7] and references therein). Overall, they are capable of oxidise substrates that vary from organic compounds, such as ascorbic acid, phenolate siderophores and organic