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
Simulated pH titrations of Glu 498 for the CotA-H2O2 state upon changes on the redox state of centre T1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Simulated pH titrations of Glu 498 for the CotA-H2O2 state upon changes on the redox state of centre T1.

Mentions: Electron-proton coupling is at the core of many redox enzyme mechanisms, and it is possible that multicopper oxidases are no exception. The methodology applied here was early developed to consider these effects in a complete way [44,47]. Despite the fact that the complete interplay between the two redox centres and protonatable groups cannot be studied, at this time, in an exact way, the coupling between the redox state of T1 and the protonation of any protonatable group is amenable to be analysed by our simulations. These clearly show that the titration of Glu 498 depends on the redox state of T1. As an example, figure 7 shows the effect of the reduction of this centre alone on the titration of Glu 498 in the peroxide adduct structure (CotA-H2O2). The pKa of this group increases by 1.4 pH units, from 5.3 to 6.7, evidencing the influence of the redox state of T1 on its protonation. On the other hand, the different redox and bridging moieties of the trinuclear centre (which can include protonation changes of Glu 498) also show an influence on the redox affinity of the T1 centre, as shown in the redox titrations (at pH 7) of the T1 centre performed in the different structurally characterised states of the trinuclear centre (figure 8). Although the simulated situations correspond to "pure" states, that may not have direct experimental counterparts (given that the reduction of the protein may occur in the two copper centres at the same time), these results are illustrative of the interplay between the redox states of the two centres. The redox potential of T1 has a span of 100 mV in the situations analysed, and it decreases with higher reduction of the trinuclear centre and bound entities. This data can be compared to experimental data obtained previously for CotA (comparing the holoCotA and apoCu(I)). Experimentally it was found [24] that the redox potential of apoCu(I) (found here with a hydroxyl ion at the trinuclear centre) was 498 mV versus 525 mV for the holoCotA (found here with a dioxygen moiety in the trinuclear centre). The present simulations also show a decrease, from 525 mV for holoCotA (value set) to 473 mV for apoCu(I), even if this decrease is larger than that experimentally observed. However, it must be stressed again that the experimental and simulated situations may not correspond to the same exact process. Nevertheless, and irrespectively of the actual value of the calculated redox potential, which is prone to considerable error in the present simulations (for instance, it is highly dependent on the value of the internal dielectric constant used [42]), the present simulations show the same trend, and this is the important conclusion to be retained here. These observations show how the coupling between the T1 and trinuclear centres is important in the mechanism of laccases.


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 CotA-H2O2 state upon changes on the redox state of centre T1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Simulated pH titrations of Glu 498 for the CotA-H2O2 state upon changes on the redox state of centre T1.
Mentions: Electron-proton coupling is at the core of many redox enzyme mechanisms, and it is possible that multicopper oxidases are no exception. The methodology applied here was early developed to consider these effects in a complete way [44,47]. Despite the fact that the complete interplay between the two redox centres and protonatable groups cannot be studied, at this time, in an exact way, the coupling between the redox state of T1 and the protonation of any protonatable group is amenable to be analysed by our simulations. These clearly show that the titration of Glu 498 depends on the redox state of T1. As an example, figure 7 shows the effect of the reduction of this centre alone on the titration of Glu 498 in the peroxide adduct structure (CotA-H2O2). The pKa of this group increases by 1.4 pH units, from 5.3 to 6.7, evidencing the influence of the redox state of T1 on its protonation. On the other hand, the different redox and bridging moieties of the trinuclear centre (which can include protonation changes of Glu 498) also show an influence on the redox affinity of the T1 centre, as shown in the redox titrations (at pH 7) of the T1 centre performed in the different structurally characterised states of the trinuclear centre (figure 8). Although the simulated situations correspond to "pure" states, that may not have direct experimental counterparts (given that the reduction of the protein may occur in the two copper centres at the same time), these results are illustrative of the interplay between the redox states of the two centres. The redox potential of T1 has a span of 100 mV in the situations analysed, and it decreases with higher reduction of the trinuclear centre and bound entities. This data can be compared to experimental data obtained previously for CotA (comparing the holoCotA and apoCu(I)). Experimentally it was found [24] that the redox potential of apoCu(I) (found here with a hydroxyl ion at the trinuclear centre) was 498 mV versus 525 mV for the holoCotA (found here with a dioxygen moiety in the trinuclear centre). The present simulations also show a decrease, from 525 mV for holoCotA (value set) to 473 mV for apoCu(I), even if this decrease is larger than that experimentally observed. However, it must be stressed again that the experimental and simulated situations may not correspond to the same exact process. Nevertheless, and irrespectively of the actual value of the calculated redox potential, which is prone to considerable error in the present simulations (for instance, it is highly dependent on the value of the internal dielectric constant used [42]), the present simulations show the same trend, and this is the important conclusion to be retained here. These observations show how the coupling between the T1 and trinuclear centres is important in the mechanism of laccases.

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