The
cytochromes P450 are heme-dependent enzymes that catalyze many
vital reaction processes in the human body related to biodegradation
and biosynthesis. They typically act as mono-oxygenases; however,
the recently discovered P450 subfamily TxtE utilizes O
2
and NO to nitrate aromatic substrates such as
L
-tryptophan.
A direct and selective aromatic nitration reaction may be useful in
biotechnology for the synthesis of drugs or small molecules. Details
of the catalytic mechanism are unknown, and it has been suggested
that the reaction should proceed through either an iron(III)-superoxo
or an iron(II)-nitrosyl intermediate. To resolve this controversy,
we used stopped-flow kinetics to provide evidence for a catalytic
cycle where dioxygen binds prior to NO to generate an active iron(III)-peroxynitrite
species that is able to nitrate
l
-Trp efficiently. We show
that the rate of binding of O
2
is faster than that of NO
and also leads to
l
-Trp nitration, while little evidence
of product formation is observed from the iron(II)-nitrosyl complex.
To support the experimental studies, we performed density functional
theory studies on large active site cluster models. The studies suggest
a mechanism involving an iron(III)-peroxynitrite that splits homolytically
to form an iron(IV)-oxo heme (Compound II) and a free NO
2
radical via a small free energy of activation. The latter activates
the substrate on the aromatic ring, while compound II picks up the
ipso
-hydrogen to form the product. The calculations give
small reaction barriers for most steps in the catalytic cycle and,
therefore, predict fast product formation from the iron(III)-peroxynitrite
complex. These findings provide the first detailed insight into the
mechanism of nitration by a member of the TxtE subfamily and highlight
how the enzyme facilitates this novel reaction chemistry.