Catalytic Cycle of Multicopper Oxidases Studied by Combined Quantum- and Molecular-Mechanical Free-Energy Perturbation Methods

Abstract

We have used combined quantum mechanical and molecular mechanical free-energy perturbation methods in combination with explicit solvent simulations to study the reaction mechanism of the multicopper oxidases, in particular the regeneration of the reduced state from the native intermediate. For 52 putative states of the trinuclear copper cluster, differing in the oxidation states of the copper ions and the protonation states of water-and O2-derived ligands, we have studied redox potentials, acidity constants, isomerisation reactions, as well as water-and O2 binding reactions. Thereby, we can propose a full reaction mechanism of the multicopper oxidases with atomic detail. We also show that the two copper sites in the protein communicate so that redox potentials and acidity constants of one site are affected by up to 0.2 V or 3 pK a units by a change in the oxidation state of the other site.

Laccase Redox Potentials: pH Dependence and Mutants, a QM/MM Study

“…8 Li and co-workers provide redox potentials of the T1 site depending on the TNC charge state; they state that the TNC state can affect the RP of the T1 site by up to 0.2 V (B3LYP-D3/def2-TZVPD). 9 In the specific case of NI vs. Red studied here, with both TNCs displaying a net charge of +2, they find however only a limited effect, 0.43 V (NI) compared to 0.41 to 0.47 V (Red) resulting only in a -0.02 to +0.04 V change in RP. Our results indicate the effect to be 0.22 V in the M06 case and 0.24 V for BP86; the difference between the two papers is however close to our standard deviations of the individual absolute RPs.…”

“…Note that we assigned the "NI" and "Red" labels in analogy to work presented by Li and coworkers. 9 From the optimised structure of NI low-pH , we created an additional model that contained a D206N change, "NI low-pH/D206N ", for which we explored the effect of a asparagine residue instead of a protonated aspartate close to the T1 copper centre. After steepest descent optimisation of the hydrogen atoms, the system was solvated in a water sphere of 35 Å radius, centred on the T1 Cu site.…”

“…It has been shown recently that the TNC can affect the T1 centre redox potential, 9 but it is unclear to what extent this effect can be reproduced in a model explicitly containing only the T1 site. (iI) How sensitive is laccase towards the environmental pH?…”

Mono- and binuclear non-heme iron chemistry from a theoretical perspective

“…Despite their importance in NHFe(2) chemistry, their calculations remain challenging due to large environmental effects that have to be properly described. This can be achieved by the inclusion of explicit solvent molecules [182] and/or remote parts of a protein through the QM/MM(+FEP) scheme [183], but this also usually requires an extensive sampling of the conformational space. On the other hand, the implicit solvent models are simplistic and do not account sufficiently for solvation-energy differences between protonated/deprotonated or oxidized/reduced species.…”

Protein effects in non-heme iron enzyme catalysis: insights from multiscale models

“…[21,83] There can of course be huge effects on calculated pKa and redox potentials, [84] where there is a change in the total charge of the system, but the difficulty in modeling these properties are discussed in more detail elsewhere, and are not considered further here. [85,86] The difficulty to predict the protonation state in the active site adds another important dimension to the modeling A strategy for modeling reactions where the protonation state is unknown is to include both alternatives and compare with available X-ray structures, spectroscopic data and/or reaction barrier heights. [65,87] These examples highlight the considerable challenges in correctly calculating and evaluating electrostatic contributions, which makes it difficult to determine their role in catalysis.…”