An Altered Heme Environment in an Engineered Cytochrome P450 Enzyme Enables the Switch from Monooxygenase to Peroxygenase Activity

Cytochrome
P450 heme-thiolate monooxygenases are exceptionally
versatile enzymes which insert an oxygen atom into the unreactive
C–H bonds of organic molecules. They source O2 from
the atmosphere and usually derive electrons from nicotinamide cofactors
via electron transfer proteins. The requirement for an expensive nicotinamide
adenine dinucleotide (phosphate) cofactor and the redox protein partners
can be bypassed by driving the catalysis using hydrogen peroxide (H2O2). We demonstrate that the mutation of a highly
conserved threonine residue, involved in dioxygen activation, to a
glutamate shuts down monooxygenase activity in a P450 enzyme and converts
it into a peroxygenase. The reason for this switch in the threonine
to glutamate (T252E) mutant of CYP199A4 from Rhodopseudomonas
palustris HaA2 was linked to the lack of a spin state
change upon the addition of the substrate. The crystal structure of
the substrate-bound form of this mutant highlighted a modified oxygen-binding
groove in the I-helix and the retention of the iron-bound aqua ligand.
This ligand interacts with the glutamate residue, which favors its
retention. Electron paramagnetic resonance confirmed that the ferric
heme aqua ligand of the mutant substrate-bound complex had altered
characteristics compared to a standard ferric heme aqua complex. Significant
improvements in peroxygenase activity were demonstrated for the oxidative
demethylation of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and
veratric acid to vanillic acid (up to 6-fold). The detailed characterization
of this engineered heme peroxygenase will facilitate the development
of new methods for driving the biocatalytic generation of oxygenated
organic molecules via selective C–H bond activation using heme
enzymes.