• Cytochrome P450;
  • enzyme catalysis;
  • substrate dynamics;
  • substrate activation barriers;
  • essential dynamics;
  • protein dynamics


Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants—F87A, L188Q/A74G, and F87V/L188Q/A74G—were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (ω−1, ω−2, ω−3) but not the terminal (ω) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen–atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.