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

Area of Science:

• Biochemistry
• Computational Chemistry
• Enzyme Kinetics

Background:

• Laccases are copper-containing enzymes that catalyze oxidation reactions.
• The T1 site redox potential (RP) is crucial for laccase activity.
• Understanding factors influencing RP is key to enzyme engineering.

Purpose of the Study:

• To investigate the impact of oxidation states and protonation on the T. versicolor laccase T1 site RP.
• To computationally model the laccase system and predict RP changes in mutants.

Main Methods:

• Quantum mechanics/molecular mechanics (QM/MM) calculations with M06/6-311++G** and RI-BP86 levels of theory.
• Optimization of whole protein geometries and electronic embedding.
• Analysis of protonation state effects and single-point energy calculations.

Main Results:

• The trinuclear cluster oxidation state affects T1 site RP by 0.2-0.3 V.
• Neutral environments can lower computed laccase RP by up to -1.37 V.
• Mutations F463M and D206N were predicted to decrease RP by ~0.1 V and slightly, respectively.

Conclusions:

• Protein conformation favors a high redox potential, counteracting surface charge effects.
• Protonation state is a critical determinant of laccase RP.
• Computational predictions for mutants align with experimental observations, validating the approach.