The bacterial cytochromes P450 BM3 (CYP450 BM3) catalyzes reactions of industrial importance. Despite many successful biotransformations, robust re(design) for novel applications remains challenging. Rational design and evolution-ary approaches are not always successful highlighting a lack of complete understanding of the mechanisms of electron trans-fer (ET) modulations. Thus, the full potential of CYP450 reactions remains under-exploited. In this work, we report the first MD-based explicit prediction of BM3 ET parameters (reorganization energies; λ and ET free energies; ΔG°), and log ET rates (log kET) using 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 kET 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 interaction with the ligand. Our methodology (CYPWare 1.0) automates all the stages of 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.
|Journal||The Journal of Physical Chemistry Part B|
|Publication status||Accepted/In press - 7 Nov 2022|
- Electron Transfer
- Marcus Theory
- CYP450 BM3
- Molecular Dynamics