Origin of Catalysis by Nitroalkane Oxidase

Area of Science:

• Enzyme catalysis
• Biochemistry
• Computational chemistry

Background:

• Nitroalkane oxidase (NAO) catalyzes nitroalkane oxidation with a 10⁸-fold rate enhancement over acetate in water.
• Nitroalkanes exhibit an unusual linear free energy relationship between acidity and deprotonation rates in solution.
• The enzyme utilizes the flavin adenine dinucleotide (FAD) cofactor for the oxidation step.

Purpose of the Study:

• To elucidate the catalytic mechanism of NAO, specifically the origin of rate enhancement in proton transfer.
• To investigate the role of the FAD cofactor in the initial proton abstraction step.
• To understand how NAO restores the normal behavior of the Bronsted acid linear free energy relationship.

Main Methods:

• Combined quantum mechanics/molecular mechanics (QM/MM) simulations were employed.
• Calculated free energy reaction profiles (potentials of mean force) for enzymatic and model reactions.
• Free energy perturbation and QM calculations were used to analyze cofactor interactions.

Main Results:

• Enzyme catalysis primarily arises from transition-state stabilization, accounting for the observed rate enhancement.
• The FAD cofactor increases Cα proton acidity through hydrogen bonding and π-stacking interactions.
• π-stacking between the FAD isoalloxazine ring and nitroethane stabilizes the anionic form, contributing 17.3 kcal/mol.

Conclusions:

• NAO catalysis is driven by transition-state stabilization, re-establishing normal Bronsted acid behavior.
• The FAD cofactor significantly enhances proton transfer by increasing substrate acidity via noncovalent interactions.
• FAD's π-stacking ability contributes substantially to the catalytic efficiency beyond its oxidative role.