Limits...
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.

Show MeSH
Structural detail of the trinuclear Copper centre. In each picture the electron density for the moiety is derived from omit Fourier syntheses computed with SigmaA weighted coefficients /Fo/ - /Fc/; the moieties were not included in the structure factor calculations and five cycles of maximum likelihood refinement were computed using REFMAC prior to Fourier synthesis to minimise phase bias. Contour levels are 5 rms for both electron density maps. (a) In the holoCotA structure a dioxygen molecule is bound into the trinuclear centre. (b) In the apoCu(I) a hydroxyl group is bound to the trinuclear centre. Both pictures were made with PyMol [69].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2944330&req=5

Figure 2: Structural detail of the trinuclear Copper centre. In each picture the electron density for the moiety is derived from omit Fourier syntheses computed with SigmaA weighted coefficients /Fo/ - /Fc/; the moieties were not included in the structure factor calculations and five cycles of maximum likelihood refinement were computed using REFMAC prior to Fourier synthesis to minimise phase bias. Contour levels are 5 rms for both electron density maps. (a) In the holoCotA structure a dioxygen molecule is bound into the trinuclear centre. (b) In the apoCu(I) a hydroxyl group is bound to the trinuclear centre. Both pictures were made with PyMol [69].

Mentions: The overall comparison with holoCu(II) [20] of the three-dimensional structures obtained for both holoCotA and apoCu(I), showed no significant differences. 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. The rms deviation of the Cα trace of the holoCotA and apoCu(I) structures from the structure of holoCu(II) is about 0.1 Å. The most significant differences found between the structures were localized at the trinuclear centre and in the copper content. The holoCotA structure, in a similar manner as holoCu(II), shows full occupancy for all copper atoms in both copper centres and has a dioxygen moiety modelled almost symmetrically between the two T3 coppers (Figure 2a). However, in the holoCotA structure, a fifth copper atom was modelled with half occupancy at the surface of the molecule, located ca. 20 Å away from the other two copper centres (Figure 1a). This copper atom, coordinated by two histidine residues and by two water molecules, is localised close to a disordered coil region that has been poorly defined in the calculated electron density maps for all other CotA structures determined until now. As the crystallisation conditions are different from the ones used previously, we cannot rule out the possibility that this may be an artefact of the crystallization procedure. However, we did not add any extra copper to the crystallization solutions. Therefore, this extra copper may be a consequence of the new protocol used to produce the protein. The overall structure of holoCotA determined here, with the exception of this extra copper, which was never been observed before, is basically the same as holoCu(II) determined previously [20], and should correspond to the same chemical species.


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)

Structural detail of the trinuclear Copper centre. In each picture the electron density for the moiety is derived from omit Fourier syntheses computed with SigmaA weighted coefficients /Fo/ - /Fc/; the moieties were not included in the structure factor calculations and five cycles of maximum likelihood refinement were computed using REFMAC prior to Fourier synthesis to minimise phase bias. Contour levels are 5 rms for both electron density maps. (a) In the holoCotA structure a dioxygen molecule is bound into the trinuclear centre. (b) In the apoCu(I) a hydroxyl group is bound to the trinuclear centre. Both pictures were made with PyMol [69].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Structural detail of the trinuclear Copper centre. In each picture the electron density for the moiety is derived from omit Fourier syntheses computed with SigmaA weighted coefficients /Fo/ - /Fc/; the moieties were not included in the structure factor calculations and five cycles of maximum likelihood refinement were computed using REFMAC prior to Fourier synthesis to minimise phase bias. Contour levels are 5 rms for both electron density maps. (a) In the holoCotA structure a dioxygen molecule is bound into the trinuclear centre. (b) In the apoCu(I) a hydroxyl group is bound to the trinuclear centre. Both pictures were made with PyMol [69].
Mentions: The overall comparison with holoCu(II) [20] of the three-dimensional structures obtained for both holoCotA and apoCu(I), showed no significant differences. 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. The rms deviation of the Cα trace of the holoCotA and apoCu(I) structures from the structure of holoCu(II) is about 0.1 Å. The most significant differences found between the structures were localized at the trinuclear centre and in the copper content. The holoCotA structure, in a similar manner as holoCu(II), shows full occupancy for all copper atoms in both copper centres and has a dioxygen moiety modelled almost symmetrically between the two T3 coppers (Figure 2a). However, in the holoCotA structure, a fifth copper atom was modelled with half occupancy at the surface of the molecule, located ca. 20 Å away from the other two copper centres (Figure 1a). This copper atom, coordinated by two histidine residues and by two water molecules, is localised close to a disordered coil region that has been poorly defined in the calculated electron density maps for all other CotA structures determined until now. As the crystallisation conditions are different from the ones used previously, we cannot rule out the possibility that this may be an artefact of the crystallization procedure. However, we did not add any extra copper to the crystallization solutions. Therefore, this extra copper may be a consequence of the new protocol used to produce the protein. The overall structure of holoCotA determined here, with the exception of this extra copper, which was never been observed before, is basically the same as holoCu(II) determined previously [20], and should correspond to the same chemical species.

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