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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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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.
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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.

Mentions: In laccases, the exit channel for water molecules also contains, at least, one acidic residue, Asp116 in CotA (Figure 1c), in close proximity to the T2 copper centre. The equivalent residue in Fet3p (Asp 94) [16] and in CueO (Asp 112) [13,14] has undergone mutation studies and been subject to DFT calculations [45]. These studies have demonstrated the importance of such a residue for the mechanism of oxygen reduction in multi-copper oxidases. In some of these reports [13,16,45], it is suggested that this residue is involved in proton transfer. However, the present calculations on CotA shows that this group is not proton active around pH 7 in any of the states analysed; in fact, it is mostly charged in all the pH range considered (see figure 6). Given its proximity, this residue could become protonated when a hydroxyl group is bound to the T2 copper ion (the situation evidenced in ascorbate oxidase, figure 4). We have not yet characterised this state in CotA, but we did perform test calculations on the apoCu(I) conformation by decreasing the overall charge of the T2 Cu bound water molecule by one charge unit (an exaggerated situation to simulate a hydroxyl group). These calculations (Figure 6, blue line), despite showing an increased protonation pattern of this acid, did not show an increase in the protonation of Asp116 to values that would make it proton-active around pH 7, further suggesting that this group is unlikely to be involved in proton transfer mechanisms. A possible explanation for its reported relevance in the activity of laccases, may be related with the role of this aspartate in maintaining a proper structural arrangement of the trinuclear centre, by being hydrogen bonded to His 424 and His 105, which coordinate one of the T3 and the T2 coppers, respectively (see figure 1c). The requirement for a charged residue at this position is suggested by the mutants made on Fet3p [16] and CueO [13,14], which show that its mutation to glutamate results in the smaller perturbation in activity, whereas its mutation to alanine or to asparagine results in a drastic decrease of the enzyme activity.


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)

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

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

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.
Mentions: In laccases, the exit channel for water molecules also contains, at least, one acidic residue, Asp116 in CotA (Figure 1c), in close proximity to the T2 copper centre. The equivalent residue in Fet3p (Asp 94) [16] and in CueO (Asp 112) [13,14] has undergone mutation studies and been subject to DFT calculations [45]. These studies have demonstrated the importance of such a residue for the mechanism of oxygen reduction in multi-copper oxidases. In some of these reports [13,16,45], it is suggested that this residue is involved in proton transfer. However, the present calculations on CotA shows that this group is not proton active around pH 7 in any of the states analysed; in fact, it is mostly charged in all the pH range considered (see figure 6). Given its proximity, this residue could become protonated when a hydroxyl group is bound to the T2 copper ion (the situation evidenced in ascorbate oxidase, figure 4). We have not yet characterised this state in CotA, but we did perform test calculations on the apoCu(I) conformation by decreasing the overall charge of the T2 Cu bound water molecule by one charge unit (an exaggerated situation to simulate a hydroxyl group). These calculations (Figure 6, blue line), despite showing an increased protonation pattern of this acid, did not show an increase in the protonation of Asp116 to values that would make it proton-active around pH 7, further suggesting that this group is unlikely to be involved in proton transfer mechanisms. A possible explanation for its reported relevance in the activity of laccases, may be related with the role of this aspartate in maintaining a proper structural arrangement of the trinuclear centre, by being hydrogen bonded to His 424 and His 105, which coordinate one of the T3 and the T2 coppers, respectively (see figure 1c). The requirement for a charged residue at this position is suggested by the mutants made on Fet3p [16] and CueO [13,14], which show that its mutation to glutamate results in the smaller perturbation in activity, whereas its mutation to alanine or to asparagine results in a drastic decrease of the enzyme activity.

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