Coupling and uncoupling mechanisms in the methoxythreonine mutant of cytochrome P450cam: a quantum mechanical/molecular mechanical study

Abstract

The Thr252 residue plays a vital role in the catalytic cycle of cytochrome P450cam during the formation of the active species (Compound I) from its precursor (Compound 0). We investigate the effect of replacing Thr252 by methoxythreonine (MeO-Thr) on this protonation reaction (coupling) and on the competing formation of the ferric resting state and H2O2 (uncoupling) by combined quantum mechanical/molecular mechanical (QM/MM) methods. For each reaction, two possible mechanisms are studied, and for each of these the residues Asp251 and Glu366 are considered as proton sources. The computed QM/MM barriers indicate that uncoupling is unfavorable in the case of the Thr252MeO-Thr mutant, whereas there are two energetically feasible proton transfer pathways for coupling. The corresponding rate-limiting barriers for the formation of Compound I are higher in the mutant than in the wild-type enzyme. These findings are consistent with the experimental observations that the Thr252MeO-Thr mutant forms the alcohol product exclusively (via Compound I), but at lower reaction rates compared with the wild-type enzyme.

Scheme 1

a Two mechanisms for the conversion of Compound 0 (Cpd 0) to Compound I (Cpd I, coupling reaction). b Two mechanisms for ferric resting state (Fe RS) formation (uncoupling reaction)

Fig. 1

Quantum mechanical region for the Thr252MeO-Thr mutant in the Glu366 and Asp251 channels

Fig. 2

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism I (coupling reaction) in the Glu366 channel

Fig. 3

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism I (coupling reaction) in the Asp251 channel

Fig. 4

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism II (coupling reaction) in the Glu366 channel

Fig. 5

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism III (uncoupling reaction) in the Glu366 channel

Fig. 6

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism III (uncoupling reaction) in the Asp251 channel

Fig. 7

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism IV (uncoupling reaction) in the Glu366 channel

Fig. 8

Optimized geometries (UB3LYP/B1/CHARMM) for mechanism IV (uncoupling reaction) in the Asp251 channel