Experimental and Theoretical Examination of the Kinetic Isotope Effect in Cytochrome P450 Decarboxylase OleT

Using
a combination of experimental studies, theory, simulation,
and modeling, we investigate the hydrogen atom transfer (HAT) reaction
by the high-valent ferryl cytochrome P450 (CYP) intermediate known
as Compound I, a species that is central to innumerable and important
detoxification and biosynthetic reactions. The P450 decarboxylase
known as OleT converts fatty acids, a sustainable biological feedstock,
into terminal alkenes and thus is of high interest as a potential
means to produce fungible biofuels. Previous experimental work has
established the intermediacy of Compound I in the CC scission
reaction catalyzed by OleT and an unprecedented ability to monitor
the HAT process in the presence of bound fatty acid substrates. Here,
we leverage the kinetic simplicity of the OleT system to measure the
activation barriers for CYP HAT and the temperature dependence of
the substrate 2H kinetic isotope effect. Notably, neither
measurement has been previously accessible for a CYP to date. Theoretical
analysis alludes to the significance of substrate fatty acid coordination
for generating the hydrogen donor/acceptor configurations that are
most conducive for HAT to occur. The analysis of the two-dimensional
potential energy surface, based on multireference electronic wave
functions, illustrates the uncoupled character of the hydrogen motion.
Quantum dynamics calculations along the hydrogen reaction path demonstrate
that hydrogen tunneling is essential to qualitatively capture the
experimental isotope effect, its temperature dependence, and appropriate
activation energies. Overall, a more fundamental understanding of
the OleT reaction coordinate contributes to the development of biomimetic
catalysts for controlled CH bond activation, an outstanding
current challenge for (bio)­synthetic chemistry.