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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 Glu 498 for the different CotA trinuclear centre states described in Figure 4. The state of T1 is set to oxidised for the dioxygen, hydroxyl and peroxide structures, and set to reduced for the reduced structure.
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Figure 5: Simulated pH titrations of Glu 498 for the different CotA trinuclear centre states described in Figure 4. The state of T1 is set to oxidised for the dioxygen, hydroxyl and peroxide structures, and set to reduced for the reduced structure.

Mentions: The simulation results reported above, and shown in figure 5, clearly suggest that, irrespectively of details, the protonation of Glu 498 is sensitive to the redox and liganted state of the trinuclear centre. Thus, being the only group in the vicinity of this centre to exhibit these characteristics, it is likely to play a proton transfer role for the reduction of oxygen to water. Additionally, its long redox titration curves, spanning almost 4 pH units in the cases of CotA-H2O2 and CotA-red (figure 5), clearly show that its binding fluctuations (that are maximum in the zone of active titration [44]) can be used for the deliverance of protons to the reaction, in a rather flexible way. Several reports describing mutants of other negatively charged residues in the oxygen access channel, namely in Fet3p (Glu 487) [16] and CueO (Glu 506 homologous to Glu 498 in CotA) [14], point to a role of these residues in the catalysis of oxygen reduction, a fact interpreted by the authors as their involvement in proton donation. DFT calculations [45] have also been used in trinuclear centre models to highlight the potential effect of this residue in proton transfer along the mechanism. Therefore, the data that is presented here further corroborates these findings.


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 Glu 498 for the different CotA trinuclear centre states described in Figure 4. The state of T1 is set to oxidised for the dioxygen, hydroxyl and peroxide structures, and set to reduced for the reduced structure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Simulated pH titrations of Glu 498 for the different CotA trinuclear centre states described in Figure 4. The state of T1 is set to oxidised for the dioxygen, hydroxyl and peroxide structures, and set to reduced for the reduced structure.
Mentions: The simulation results reported above, and shown in figure 5, clearly suggest that, irrespectively of details, the protonation of Glu 498 is sensitive to the redox and liganted state of the trinuclear centre. Thus, being the only group in the vicinity of this centre to exhibit these characteristics, it is likely to play a proton transfer role for the reduction of oxygen to water. Additionally, its long redox titration curves, spanning almost 4 pH units in the cases of CotA-H2O2 and CotA-red (figure 5), clearly show that its binding fluctuations (that are maximum in the zone of active titration [44]) can be used for the deliverance of protons to the reaction, in a rather flexible way. Several reports describing mutants of other negatively charged residues in the oxygen access channel, namely in Fet3p (Glu 487) [16] and CueO (Glu 506 homologous to Glu 498 in CotA) [14], point to a role of these residues in the catalysis of oxygen reduction, a fact interpreted by the authors as their involvement in proton donation. DFT calculations [45] have also been used in trinuclear centre models to highlight the potential effect of this residue in proton transfer along the mechanism. Therefore, the data that is presented here further corroborates these findings.

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