not interact ideally with any neighboring proton
donors. This lack of ideal hydrogen bonding
structure in the ferric form may favor formation
of the peroxide complex and the deprotonated
compound 0 (28), as structures of the closely
related compound III (oxy) complexes all suggest
that more favorable hydrogen bonding interac-
tions with the bound peroxide are likely (fig. S6).
Our data suggest that His52 retains a proton upon
formation of compound I. In terms of proton
count, this presumably means that delivery of
an additional proton to compound 0 is needed
for compound I formation if water is released
as product (Fig. 3A), aligning the mechanism
more closely with the related P450s in which
delivery of a single proton to compound 0 is also
widely assumed (2, 29). Arg48, which is proto-
nated (Figs. 1 and 2), could act as the source of
the additional proton required for release of
water in both formation and reduction of com-
pound I, as it is thought to play a role in proton-
relay networks (30). Hydroxide (instead of water,
Fig. 3A) formation is also possible; if this is the
case, a role for Arg48 in charge stabilization of
OH– is easily envisaged and would be consistent
with early predictions (31). Because the proton on
His52 must be removed before a second turn-
over of the enzyme can occur, the acid-catalysis
role of His52 must occur after compound I for-
mation and not [as is often assumed (1)] before
(Fig. 3A). An alternative and intriguing possibility
(Fig. 3B), which could apply generally to any
heme protein with a distal histidine in the heme
pocket, is that O–O bond cleavage initially forms
a transient Fe(IV)-OH species [as in the P450s
(32)], but the presence of the distal histidine
provides an escape route for the proton, thus dis-
favoring formation of Fe(IV)-OH. Proton transfer
in this direction, to form Fe(IV)=O, would be con-
sistent with published pKas (Ka, acid dissociation
constant) for the distal histidine across the perox-
idase family [estimated pKa ≈ 4 to 5 (1, 33)] if the
pKa of the ferryl heme, which is not known reli-
ably for a heme peroxidase, was lower than that.
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196 11 JULY 2014 • VOL 345 ISSUE 6193 sciencemag.org SCIENCE
Fig. 3. Possible movement of protons during O–O bond activation. (A)
Formation of a peroxide-bound complex and then compound 0 is followed
by O–O bond cleavage. The distal histidine retains a proton upon formation
of compound I, which means that an additional proton is required for release
of water in both formation and reduction of compound I. (See text for de-
tailed discussion.) The orientation of deuterium atoms on active site water
molecules (Figs. 2 and 3) seems to preclude a water-mediated mechanism
for O–O bond cleavage as suggested in horseradish peroxidase (34) be-
cause the deuterium atoms on W2 are oriented away from the ferryl oxygen
atom and a hydrogen bond would not be possible (Fig. 2E). (B) An alternative
mechanism from the same peroxide-bound complex. Cleavage of the peroxide
bond may lead initially to formation of a transient iron(IV)-hydroxide species
[Fe(IV)-OH], which, via proton transfer, leads to compound I, with Fe(IV)=O
and a protonated distal histidine.