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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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UV/Visible absorption spectra of a) holoCotA crystal and of b) apoCu(I) crystal.
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Figure 3: UV/Visible absorption spectra of a) holoCotA crystal and of b) apoCu(I) crystal.

Mentions: UV/Visible absorption spectra of holoCotA and apoCu(I) crystals (Figures 3a and 3b) were acquired as described above. Both spectra show the characteristics observed for the holoCotA solution [24], namely an intense peak at 610 nm, which is due to the charge transfer between the copper atom (in the +2 state) and the cysteine residue at the mononuclear T1 copper centre, and a shoulder at around 330 nm. This shoulder is usually attributed to coupling of the T3 copper ions through a hydroxyl moiety in between them. The biochemical and spectroscopic characterization of holoCotA and apoCu(I) showed that they have different catalytic and biophysical properties [24]. Indeed, the reconstituted apoCu(I) is less efficient towards the oxidation of non-phenolic substrates, shows a lower redox potential and is less thermostable than the holoCotA enzyme [24]. Since there are no significant changes in their overall three dimensional structures, such differences can be attributed to changes at the copper centres, namely the copper content and the nature of the bridging ligand in between the two T3 coppers. An enzyme that does not contain its full copper content, such as observed in the crystal structure of apoCu(I), will behave differently. The question as to why the enzyme was able to incorporate all the copper content in vivo and did not succeed in doing the same when incorporation was done in vitro, remains to be solved. One hypothesis is that, in vivo, the incorporation of copper occurs during the folding process, in a transition state that facilitates the incorporation of four positive charges (considering that the copper is incorporated in the Cu(I) state), whereas in vitro, when the final fold is already acquired, this incorporation process becomes less favourable and, therefore, less efficient. Work in our laboratories is currently being pursuit to address this question.


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)

UV/Visible absorption spectra of a) holoCotA crystal and of b) apoCu(I) crystal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: UV/Visible absorption spectra of a) holoCotA crystal and of b) apoCu(I) crystal.
Mentions: UV/Visible absorption spectra of holoCotA and apoCu(I) crystals (Figures 3a and 3b) were acquired as described above. Both spectra show the characteristics observed for the holoCotA solution [24], namely an intense peak at 610 nm, which is due to the charge transfer between the copper atom (in the +2 state) and the cysteine residue at the mononuclear T1 copper centre, and a shoulder at around 330 nm. This shoulder is usually attributed to coupling of the T3 copper ions through a hydroxyl moiety in between them. The biochemical and spectroscopic characterization of holoCotA and apoCu(I) showed that they have different catalytic and biophysical properties [24]. Indeed, the reconstituted apoCu(I) is less efficient towards the oxidation of non-phenolic substrates, shows a lower redox potential and is less thermostable than the holoCotA enzyme [24]. Since there are no significant changes in their overall three dimensional structures, such differences can be attributed to changes at the copper centres, namely the copper content and the nature of the bridging ligand in between the two T3 coppers. An enzyme that does not contain its full copper content, such as observed in the crystal structure of apoCu(I), will behave differently. The question as to why the enzyme was able to incorporate all the copper content in vivo and did not succeed in doing the same when incorporation was done in vitro, remains to be solved. One hypothesis is that, in vivo, the incorporation of copper occurs during the folding process, in a transition state that facilitates the incorporation of four positive charges (considering that the copper is incorporated in the Cu(I) state), whereas in vitro, when the final fold is already acquired, this incorporation process becomes less favourable and, therefore, less efficient. Work in our laboratories is currently being pursuit to address this question.

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