Identification of the rate-limiting step of the peroxygenase reactions catalyzed by the thermophilic cytochrome P450 from Sulfolobus tokodaii strain 7

Cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobustokodaii strain7 (P450st) is a thermophilic cytochrome P450 that shows high tolerance of harsh conditions and is capable of catalyzing some peroxygenase reactions. Here, we investigated the pH dependence of the peroxygenase reactions catalyzed by wild-type P450st and a mutant in which the residues located close to the proximal heme ligand are mutated. Both hydrogen peroxide-driven ethylbenzene hydroxylation and styrene epoxidation by wild-type P450st were found to be activated in weak acidic and weak basic solutions. Under these conditions, the Michaelis constant for hydrogen peroxide (K-m(H2O2)) was decreased. The turnover rate (k(cat)) of ethylbenzene hydroxylation was increased and followed an S-shaped curve, with an increase in the pH value. The apparent acid dissociation constant (pK(a)(app)) of the k(cat) was 7.0, which suggests that the rate-limiting step of this reaction is deprotonation of the Fe-III-H2O2 complex. By introducing a double mutation around the proximal heme ligand, the peroxygenase activity was increased over a wide pH range, and was dramatically increased at pH 5. The spectroscopic properties of this F310A/A320Q mutant indicated that the Lewis acidity of the heme was increased by this mutation. Kinetic investigations showed that the increase in the Lewis acidity of the heme facilitates the reaction rate of the rate-limiting step of peroxygenase reactions and decreases the K-m(H2O2) value. Differences in the pH dependence of the k(cat) value between wild-type P450st and the mutant suggest that the rate-limiting step switches to protonation of the ferric-hydroperoxo species (compound 0) under alkaline conditions.