Abstract
BackgroundLaccases 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.ResultsIn 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.ConclusionsThese 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.
Highlights
Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water
The pair of type 3 (T3) copper ions are localised in the trinuclear centre and are EPR silent, a fact attributed to their antiferromagnetic coupling in the presence of a bridging ligand, normally assumed to be hydroxyl
The proteins comprise three cupredoxin domains with a mononuclear copper centre localized in domain 3 and a trinuclear centre in between domains 1 and 3
Summary
Laccases are enzymes that couple the oxidation of substrates with the reduction of dioxygen to water. 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 Laccases, which are the simplest members of the MCOs family, show a characteristic fold that comprises three cupredoxin domains, with a mononuclear copper centre localised in the third domain, and a trinuclear copper centre located in between the first and the third domains (Figure 1a). The T3 site shows an absorption band at ca. 330 nm that has been attributed to the charge transfer between an hydroxyl bridging group and the copper atoms (OH- ® Cu2+) [10]
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