A single-site mutation (F429H) converts the enzyme CYP 2B4 into a heme oxygenase: a QM/MM study

Abstract

The intriguing deactivation of the cytochrome P450 (CYP) 2B4 enzyme induced by mutation of a single residue, Phe429 to His, is explored by quantum mechanical/molecular mechanical calculations of the O-OH bond activation of the (Fe(3+)OOH)(-) intermediate. It is found that the F429H mutant of CYP 2B4 undergoes homolytic instead of heterolytic O-OH bond cleavage. Thus, the mutant acquires the following characteristics of a heme oxygenase enzyme: (a) donation by His429 of an additional NH---S H-bond to the cysteine ligand combined with the presence of the substrate retards the heterolytic cleavage and gives rise to homolytic O-OH cleavage, and (b) the Thr302/water cluster orients nascent OH(•) and ensures efficient meso hydroxylation.

Figure 1

B3LYP optimized geometries (Å and degrees), and doublet-quartet energy gap, ^,ΔE (kcal/mol) of Cpd 0 in the conformations LW1 (a) and LM1 (b). Note the NH---S H-bond in (b).

Figure 2

Key bond distances (Å) of the QM region in TS_O-O (het) of the WT conformations LW1 (a) and LW3 (b).

Figure 3

Key bond distances (Å) in TS_O-O (hom) and TS_O-O (het) of the MT conformations, LM1 (a) LM2 (b) LM3 (c), and the homolytic intermediate point I_H of LM3 (d).

Scheme 1

The conversion of Cpd 0 to Cpd I vs. the HO option of generating a hydroxylated heme

Scheme 2

Generic energy profiles starting from Cpd 0 (in its doublet ground state) and leading to Cpd I (to the right) and/or to meso-hydroxylation HO activity (to the left)

Scheme 3

Electronic shift diagrams during O-OH cleavage of ²Cpd 0 in (a), during PCET and OH expulsion past ²TS_O-O in (b), and the electronic configuration of ²Cpd I (c) and ²Heme-OH products (d).

Scheme 4

Qualitative molecular orbital diagrams of mixing of the sulfur hybrid into the a_2u orbital in WT (a) and MT (b)