Mechanisms of Electron Transfer Rate Modulations in Cytochrome P450 BM3

Bacterial cytochromes P450 BM3 (CYP450 BM3) catalyze reactions of industrial importance. Despite many successful biotransformations, robust (re)design for novel applications remains challenging. Rational design and evolutionary approaches are not always successful, highlighting a lack of complete understanding of the mechanisms of electron transfer (ET) modulations. Thus, the full potential of CYP450 reactions remains under-exploited. In this work, we report the first molecular dynamics (MD)-based explicit prediction of BM3 ET parameters (reorganization energies; λ and ET free energies; ΔG°), and log ET rates (log k_ET) using the Marcus theory. Overall, the calculated ET rates for the BM3 wild-type (WT), mutants (F393 and L86), ligand-bound state, and ion concentrations agree well with experimental data. In ligand-free (LF) BM3, mutations modulate k_ET via ET ΔG°. Simulations show that the experimental ET rate enhancement is due to increased driving force (more negative ΔG°) upon ligation. This increase is related to the protein reorganization required to accommodate the ligand in the binding pocket rather than binding interactions with the ligand. Our methodology (CYPWare 1.0) automates all the stages of the MD simulation step-up, energy calculations, and estimation of ET parameters. CYPWare 1.0 and this work thus represent an important advancement in the CYP450 ET rate predictions, which has the potential to guide the redesign of ET enzymes. This program and a Web tool are available on GitHub for academic research.