Investigation of the Proton-Assisted Pathway to Formation of the Catalytically Active, Ferryl Species of P450s by Molecular Dynamics Studies of P450eryF

The recently determined crystal structure of cytochrome P450eryF
(6-deoxyerythronolide B hydroxylase;
CYP107A1) in its ferric heme substrate-bound form has been used to
address one of the most fundamental unresolved
aspects of the mechanism of oxidation common to this ubiquitous family
of metabolizing heme proteins, the pathway
from the twice reduced dioxygen species to the putative catalytically
active ferryl oxygen species. Both of these
species are too transient to have been characterized experimentally,
and the transformation from one to the other has
been only partially characterized. The observed requirement of two
protons and the formation of water in this
transformation suggests a proton-assisted dioxygen bond cleavage as a
plausible pathway. However, this pathway
is difficult to establish by experiment alone, and the source of the
protons in the largely hydrophobic binding pocket
of the P450s remains unclear. In this work we have performed
molecular dynamics simulations of the twice reduced
dioxygen substrate-bound form of this isozyme in order to
(i)determine the plausibility of the proposed pathway
to
compound I formation, a proton-assisted cleavage of the dioxygen bond,
and (ii) investigate the possible source of
these protons. The analysis of the molecular dynamics trajectories
of this species does indeed provide further evidence
for this pathway and points to a source of protons. Specifically,
two dynamically stable hydrogen bonds to the
distal oxygen atom of the dioxygen ligand, one by the substrate and the
other by a bound water, are found, consistent
with the proposed proton-assisted cleavage of the bond and formation of
water. In addition, an extensive dynamically
stable hydrogen bond network is formed that connects the distal oxygen
to Glu 360, a well-conserved residue in a
channel accessible to solvent that could be the ultimate source of
protons. The simulations were done for both a
protonated and unprotonated Glu and led to a proposed mechanism of
proton transfer by it to the distal oxygen
atom. In order to validate the procedures used for the simulation
of this transient twice-reduced species, we have
used these same procedures to perform molecular dynamics simulations of
two other forms of P450eryF, the ferric
and ferryl substrate-bound species, and compared the results with
experiment. The results for the ferric substrate-bound species were assessed by comparisons to the experimentally
determined X-ray structure and fluctuations, and
good agreement was found. The simulations performed for the ferryl
substrate-bound species led to the correct
prediction of the observed regio- and stereospecific hydroxylation of
its natural substrate, 6-deoxyerythronolide B
(6-DEB) at the 6S position. The results of these two additional
studies lend credibility to the important mechanistic
inferences from the simulations of the transient twice reduced dioxygen
species:  further evidence for a proton-assisted pathway from it to the catalytically active ferryl species and
a possible source of the protons.