Investigating the Correlation between Product Releaseand Solvation in Cytochrome P450 Enzymes

Cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyze oxidation reactions. The binding of substrates and inhibitors can be assessed using a set of standard techniques, but less is known about how the products bind within the active site. Although substrate binding and product removal from the active site are generally not rate-determining steps, they are important components of the multistep catalytic cycle and the selectivity of the enzyme. The bacterial P450 enzyme CYP199A4, from Rhodopseudomonas palustris HaA2, catalyzes highly selective oxidation reactions on <i>para</i>-substituted benzoic acids such as the oxidative <i>O</i>-demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and the hydroxylation of 4-methylbenzoic acid to 4-(hydroxymethyl)benzoic acid. Here, we examine the binding of the products of these reactions to this enzyme using UV–visible absorbance spectroscopy, biochemical assays, X-ray crystallography, and molecular dynamics (MD) simulations. Experimental results show that the sixth aqua ligand is not displaced on addition of either product ligand and they bind less tightly than their respective substrates. Structural changes included an increase in the number of active site water molecules present, and changes in the position of several hydrophobic amino acid residues were observed. These experimental findings were compared with computational studies simulating both the 4-methoxybenzoic acid substrate and 4-hydroxybenzoic acid product bound to CYP199A4. Combining experimental and theoretical analyses, this study provides a detailed molecular rationale on how this enzyme can bind its substrates tightly yet effectively release the products, facilitating efficient catalysis with solvent molecules playing an important role in the process of product release.