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

Abstract

We have studied the T. versicolor laccase T1 site redox potential (RP) at the M06/6-311++G**/SDD(Cu) level of theory, employing QM/MM optimised geometries (RI-BP86/def2-SVP/def2-TZVP(Cu):CHARMM) of the whole protein system with electronic embedding. The oxidation state of the trinuclear cluster was found to affect the T1 site RP by about 0.2-0.3 V, depending on the protein protonation state. The computed laccase RP can be drastically lowered upon introduction of a protonation state corresponding to a neutral environment, by up to -1.37 V, which is likely an overestimation of the effect in vivo. The gradual change of the protonation state by single points without optimisation or equilibration results in a change that is even larger, namely up to about -2.6 V. Thus, the preferred protein conformation supports a high redox potential, compensating for the RP-lowering effect of surface charges. The predicted change in RP on going to the F463M mutant, ca. -0.1 V, is consistent with observations for a related laccase. Based on our results, we also propose and test a D206N mutant, but find it to be locked in a conformation with slightly lower RP.

Computational Modeling Methods for Understanding the Interaction of Lignin and Its Derivatives with Oxidoreductases as Biocatalysts

“…Moreover, they also calculated the RP of a F463M laccase mutant protein to probe if their adopted methodology could describe the change upon mutation of this residue. Notably, they were able to reproduce the experimental change in the RP of a related enzyme (0.09-0.1 V) very closely (0.08 V) [48]. Other relevant research works have been published with the aim to understand the differential affinity of several substrates against laccase T1 site and to explain the electron transfer pathways and the enzyme residues involved in that process.…”

Loss of Catalytic Activity in the E134D, H67A, and H349A Mutants of DapE: Mechanistic Analysis with QM/MM Investigation

“…However, the multireference methods are computationally demanding for systems with a large number of atoms in the QM region, further compounded by the lack of a straightforward choice of active space for electron correlation. In such cases, DFT methods with metahybrid functionals have been shown to explain experimental results very well and hence are widely used. , …”

Open-Source, Python-Based Redevelopment of the ChemShell Multiscale QM/MM Environment

“…Previously, ChemShell has been used to model a wide range of chemical systems using QM/MM methods, particularly in the fields of biomolecular and materials modeling. In biochemistry, QM/MM methods are well-suited to simulating enzymatic reaction mechanisms and ChemShell has been used to study many important enzymatic processes, with recent investigations targeting oxidoreductases such as flavin-containing amine oxidases, lignin peroxidase, multicopper oxidases, [Fe] hydrogenase, Ni–Fe carbon monoxide dehydrogenases, iron-containing dioxygenases, − and iron-containing halogenases; , transferases such as protein kinases and glycosyltransferases; hydrolases such as the lipase family and matrix metalloproteinases; , lyases such as the radical SAM superfamily; and ligases such as carbapenem synthase . ChemShell continues to be particularly heavily used for studies of the cytochrome P450 superfamily of enzymes owing to their importance in metabolic processes, with recent studies investigating reactivity networks, active site chemoselectivity, regio- and enantioselectivity of fatty acid hydroxylation, , and redox-catalyzed drug metabolism mechanisms .…”