QM/MM study of cytochrome P450 TxtE catalysis: Substrate reorganization enables selective aromatic nitration

Cytochrome P450 enzymes are heme-dependent catalysts found in all forms of life (plants, bacteria, and mammals), playing pivotal roles in the transformation of both endogenous and exogenous molecules. Recently, a new subfamily of P450 enzymes has been identified that catalyzes the direct and selective nitration of aromatic amino acids, such as L-tryptophan, in the presence of oxygen and nitric oxide. To investigate the catalytic mechanism, we applied quantum mechanical (QM)/Molecular Mechanics (MM) computational methods to explore potential nitration pathways. Our study confirms that the initial substrate binding conformation from the crystal structures is inactive for the following nitration reactions. Instead, the substrate undergoes a conformational change, leading to the alternative conformations that are essential for the following nitration reaction at the C4 position. Moreover, comparative QM/MM scanning and QM/MM-based free energy calculations showed that the conformational reorganization of the intermediate indole/^·NO₂, which is coupled to the reorganization of enzyme pocket, greatly enhances the subsequent nitration reaction and H-atom transfer reaction. This study underscores the key roles of enzyme-mediated conformational changes of the substrate and intermediates in facilitating the selective biotransformation. Our study shows that aromatic nitration at C4 site is energetically favored, in agreement with experimental findings.