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

Bento I, Silva CS, Chen Z, Martins LO, Lindley PF, Soares CM - BMC Struct. Biol. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. bento@itqb.unl.pt

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.

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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].
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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].

Mentions: The mechanism of dioxygen reduction to water includes a state where a dioxygen moiety is located between the two T3 coppers and another state where a hydroxyl group is at this position (Figure 4). The latter is formed after the oxidation of four substrate molecules and the transfer of four electrons to the trinuclear centre [20]. For the majority of studies on the multi-copper oxidases, this state has been assumed to be the resting state. However, in CotA this state has only been observed in the structure of a semi-reduced crystal (1GSK) [33] and, in the present study, in the structure of the reconstituted apoCu(I). The apo protein was incubated with Cu(I) in anaerobic conditions [24], implying that when oxygen became available the enzyme was ready to reduce it to water (Figure 4). Indeed, in many studies where such a moiety is observed, the experimental starting point is the reduced state of the enzyme. Moreover, studies on the effect of the X-ray radiation on laccase crystals reported by Hakulinen et al. [34] showed that crystals exposed to high radiation doses present a hydroxyl moiety in the trinuclear site. This shows that reduction of the copper centres may occur during data collection, leading to an end-product that can be different from the starting state. Altogether, these data highlight that the observable state of the enzyme depends critically on the experimental conditions. Indeed, this has been clearly shown for bilirubin oxidase by Sakurai et. al. [12]. On the other hand, laccases usually show a characteristic absorption spectrum with a shoulder around 330 nm that has been attributed to the presence of the bridging hydroxyl in between the T3 coppers. The spectrum obtained for the holoCotA (Figure 3a) also shows this shoulder, but the crystal structure has a dioxygen moiety at the trinuclear cluster. Moreover, the same shoulder was observed in the spectrum acquired from crystals of the laccase from M. albomyces, before being exposed to the X-ray radiation [34], and this laccase has also a dioxygen moiety in the trinuclear centre. It could be argued that the dioxygen moiety was the well characterised peroxide intermediate (PI) [11,16,17,35,36]. However, in both cases, the enzymes were in the oxidised state and no electrons were available to reduce the dioxygen molecule. It is therefore clear that a shoulder around 330 nm in the absorption spectrum can also be attributed to a dioxygen moiety. What is the resting state of multi-copper oxidases is a question that still remains to be answered. It is possible that MCOs have more than one resting state depending on the residues that surround the trinuclear centre. In fact, the current work clearly shows that the final results are dependent on the process of obtaining the protein; therefore, it is still not possible to give a clear unbiased answer.


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

Bento I, Silva CS, Chen Z, Martins LO, Lindley PF, Soares CM - BMC Struct. Biol. (2010)

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].
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2944330&req=5

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].
Mentions: The mechanism of dioxygen reduction to water includes a state where a dioxygen moiety is located between the two T3 coppers and another state where a hydroxyl group is at this position (Figure 4). The latter is formed after the oxidation of four substrate molecules and the transfer of four electrons to the trinuclear centre [20]. For the majority of studies on the multi-copper oxidases, this state has been assumed to be the resting state. However, in CotA this state has only been observed in the structure of a semi-reduced crystal (1GSK) [33] and, in the present study, in the structure of the reconstituted apoCu(I). The apo protein was incubated with Cu(I) in anaerobic conditions [24], implying that when oxygen became available the enzyme was ready to reduce it to water (Figure 4). Indeed, in many studies where such a moiety is observed, the experimental starting point is the reduced state of the enzyme. Moreover, studies on the effect of the X-ray radiation on laccase crystals reported by Hakulinen et al. [34] showed that crystals exposed to high radiation doses present a hydroxyl moiety in the trinuclear site. This shows that reduction of the copper centres may occur during data collection, leading to an end-product that can be different from the starting state. Altogether, these data highlight that the observable state of the enzyme depends critically on the experimental conditions. Indeed, this has been clearly shown for bilirubin oxidase by Sakurai et. al. [12]. On the other hand, laccases usually show a characteristic absorption spectrum with a shoulder around 330 nm that has been attributed to the presence of the bridging hydroxyl in between the T3 coppers. The spectrum obtained for the holoCotA (Figure 3a) also shows this shoulder, but the crystal structure has a dioxygen moiety at the trinuclear cluster. Moreover, the same shoulder was observed in the spectrum acquired from crystals of the laccase from M. albomyces, before being exposed to the X-ray radiation [34], and this laccase has also a dioxygen moiety in the trinuclear centre. It could be argued that the dioxygen moiety was the well characterised peroxide intermediate (PI) [11,16,17,35,36]. However, in both cases, the enzymes were in the oxidised state and no electrons were available to reduce the dioxygen molecule. It is therefore clear that a shoulder around 330 nm in the absorption spectrum can also be attributed to a dioxygen moiety. What is the resting state of multi-copper oxidases is a question that still remains to be answered. It is possible that MCOs have more than one resting state depending on the residues that surround the trinuclear centre. In fact, the current work clearly shows that the final results are dependent on the process of obtaining the protein; therefore, it is still not possible to give a clear unbiased answer.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. bento@itqb.unl.pt

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.

Show MeSH