How Metal Coordination in the Ca-, Ce- and Eu-Containing …
491
For Ce-MDH, further calculations with ε 24 and ε 78 have been also done
to test the influence of the more polar environments on its catalytic activity.
More accurate energies with the larger basis set 6-311+G(2d,2p) for all atoms
except for the metal ion described by ECP have been obtained by single-point calculations performed on the optimized structures.
The given energies presented are corrected for ZPE, solvation, and dispersion
effects. The same computational protocol has been successfully used in the mechanistic studies of other metal proteins [33, 34, 47, 51, 53, 54].
As far as the cerium-containing enzyme is concerned, the doublet spin multiplicity
(2S + 1 2 ) previously determined [53, 55] has been considered as the ground state
in all the calculations by using spin-unrestricted wavefunctions. Almost no spin
contaminations have been evidenced due to the Sˆ2 value equal to 0.77.
Natural bond orbital (NBO) analysis [56] was performed on all the intercepted
stationary points of the investigated PESs.
3 Results and Discussion
3.1 Ce-MDH PES
Different mechanisms have been proposed for MDH enzymes over the years [9, 18,
20, 21, 23, 27, 28, 30, 53, 57], but the exact oxidation mechanism of CH 3 OH by
MDH is still under debate.
In the investigated mechanism shown in Scheme 2, we propose the metal ion,
during the first step evidenced in the rectangular box, to facilitate the nucleophile
attack of OH group of the coordinated methanol that with its lone pair oriented toward
the PQQ is preparing to transfer the proton to the Asp299 base. As a result, the O5
of PQQ coordinated to the metal ion completes the activation role played by the
metal ion making C5 more suitable for the nucleophilic addition. Successively, the
reaction proceeds with the protonation of the O4 of PQQ with simultaneous CH 2 O
formation (TS2) and then the H
+ abstraction from Asp299 with the formation of
PQQH 2 (TS3). Summarizing during the oxidation of methanol to formaldehyde, the
PQQ cofactor suffers a reduction in two steps (Scheme 1): from PQQ (starting point)
to semiquinone PQQH (intermediate) and the fully reduced PQQH 2 form.
During the redox cycling of the PQQ cofactor, it was demonstrated the metal ion
to retain the same oxidation state equal to +3 [55].
As reported in our previous works on methanol dehydrogenase enzymes [23,
53], the PESs have been obtained considering the polarization effects caused by the
portion of the surrounding enzyme that is not explicitly included in the quantum
model by cavity techniques. Other than the environment simulated with ε 4, in the
present investigation, single-point calculations with other dielectric constant values
have been performed. In this way, a set of three different dielectric constants (ε 4,
ε 24 and ε 78) characterized by a trend of increasing polarity have been tested.
491
For Ce-MDH, further calculations with ε 24 and ε 78 have been also done
to test the influence of the more polar environments on its catalytic activity.
More accurate energies with the larger basis set 6-311+G(2d,2p) for all atoms
except for the metal ion described by ECP have been obtained by single-point calculations performed on the optimized structures.
The given energies presented are corrected for ZPE, solvation, and dispersion
effects. The same computational protocol has been successfully used in the mechanistic studies of other metal proteins [33, 34, 47, 51, 53, 54].
As far as the cerium-containing enzyme is concerned, the doublet spin multiplicity
(2S + 1 2 ) previously determined [53, 55] has been considered as the ground state
in all the calculations by using spin-unrestricted wavefunctions. Almost no spin
contaminations have been evidenced due to the Sˆ2 value equal to 0.77.
Natural bond orbital (NBO) analysis [56] was performed on all the intercepted
stationary points of the investigated PESs.
3 Results and Discussion
3.1 Ce-MDH PES
Different mechanisms have been proposed for MDH enzymes over the years [9, 18,
20, 21, 23, 27, 28, 30, 53, 57], but the exact oxidation mechanism of CH 3 OH by
MDH is still under debate.
In the investigated mechanism shown in Scheme 2, we propose the metal ion,
during the first step evidenced in the rectangular box, to facilitate the nucleophile
attack of OH group of the coordinated methanol that with its lone pair oriented toward
the PQQ is preparing to transfer the proton to the Asp299 base. As a result, the O5
of PQQ coordinated to the metal ion completes the activation role played by the
metal ion making C5 more suitable for the nucleophilic addition. Successively, the
reaction proceeds with the protonation of the O4 of PQQ with simultaneous CH 2 O
formation (TS2) and then the H
+ abstraction from Asp299 with the formation of
PQQH 2 (TS3). Summarizing during the oxidation of methanol to formaldehyde, the
PQQ cofactor suffers a reduction in two steps (Scheme 1): from PQQ (starting point)
to semiquinone PQQH (intermediate) and the fully reduced PQQH 2 form.
During the redox cycling of the PQQ cofactor, it was demonstrated the metal ion
to retain the same oxidation state equal to +3 [55].
As reported in our previous works on methanol dehydrogenase enzymes [23,
53], the PESs have been obtained considering the polarization effects caused by the
portion of the surrounding enzyme that is not explicitly included in the quantum
model by cavity techniques. Other than the environment simulated with ε 4, in the
present investigation, single-point calculations with other dielectric constant values
have been performed. In this way, a set of three different dielectric constants (ε 4,
ε 24 and ε 78) characterized by a trend of increasing polarity have been tested.