(Received for publication, May 19, 1995; and in revised form, June 12, 1995)
From the
Polyhistidine-tagged horseradish peroxidase (hHRP) and its F41A, H42A, and H42V mutants have been expressed in an insect cell system. Kinetic studies show that the rates of Compound I formation and peroxidative catalysis are greatly decreased by the His-42 mutation. Furthermore, Compound II is not detected during turnover of the His-42 mutants. Compounds I and II are the two- and one-electron oxidized intermediates, respectively, of hHRP. In peroxygenative catalysis, the F41A and H42A mutants catalyze thioanisole sulfoxidation 100 and 10 times faster, respectively, than hHRP. Styrene epoxidation is catalyzed by both the Phe-41 and His-42 mutants but not by wild-type hHRP. The higher peroxygenase activity of the mutants reflects increased accessibility of the ferryl species. This is indicated by the finding that, contrary to the reaction with wild-type hHRP, reaction of phenyldiazene with the F41A mutant yields a new and unidentified product, and the same reaction with the His-42 mutants yields phenyl-iron complexes. Phe-41 and His-42 thus shield the iron-centered catalytic species, and His-42 plays a key catalytic role in the formation of Compound I. The peroxygenase activities of the Phe-41 and His-42 mutants approach those of cytochrome P450.
Catalytic turnover of classical peroxidases with a histidine as
the ligand to the heme ()iron involves reaction with
H
O
to give a two-electron oxidized species
known as Compound I(1, 2) . The iron atom in Compound
I is present as a ferryl (Fe
=O) complex and is
therefore 1 oxidation equivalent above the resting ferric state. The
2nd oxidation equivalent provided by the peroxide is stored either as a
porphyrin or protein radical cation. HRP is the prototype for
peroxidases in which the 2nd oxidation equivalent is located on the
porphyrin(3) , and cytochrome c peroxidase for
peroxidases in which the 2nd oxidation equivalent resides on the
protein(4) . Two single-electron transfers from substrate
molecules reduce Compound I, first to Compound II and then to the
resting state.
A high resolution crystal structure is not available for HRP.
However, the similarities among the known primary sequences of
peroxidases (5, 6, 7) and among the four
available peroxidase crystal structures (8, 9, 10, 11) indicate that the
catalytic machinery required to activate HO
is
highly conserved. The key catalytic residues are a histidine and an
adjacent arginine in the distal heme site. The histidine is thought to
facilitate formation of the initial iron-peroxide complex by
deprotonating the peroxide and subsequently to promote cleavage of the
oxygen-oxygen bond by protonating the distal oxygen (Fig. 1)(12) . The arginine is proposed to facilitate
oxygen-oxygen bond scission by electrostatic stabilization of dioxygen
bond cleavage(12) . Experimental support for these catalytic
roles comes from site-specific mutagenesis of cytochrome c peroxidase, which demonstrates that replacement of the catalytic
histidine (His-52) or arginine (Arg-48) by a leucine decreases the rate
of formation of Compound I by a factor of 10
(13) or 10
(14) , respectively.
Initial data on the corresponding HRP site-specific mutant suggest that
the distal arginine (Arg-38) is also important for
catalysis(15) , but the function of the distal histidine in HRP
has not yet been directly tested.
Figure 1: Proposed roles of the distal histidine and arginine residues in peroxidase catalysis.
In addition to the catalytic
histidine and arginine, a phenylalanine or tryptophan is found adjacent
to the histidine in the active sites of the four peroxidases for which
crystal structures are available. Sequence alignments suggest that an
aromatic residue is adjacent to the catalytic histidine in all plant
and fungal peroxidases (Fig. 2)(5, 6, 7, 8, 9, 10, 11) .
The reason for conservation of the aromatic residue is unclear, but
mutation of the tryptophan in cytochrome c peroxidase (16, 17) and replacement of the phenylalanine (Phe-41)
in HRP by a valine(18) , leucine(19, 20) , or
threonine (19, 20) indicate that the aromatic residue
is not essential for catalysis. We have proposed in earlier studies
that the ferryl species of Compounds I and II are partially or fully
shielded from direct interaction with
substrates(2, 21, 22) . We have furthermore
advanced the hypothesis that sequestration of the ferryl species is
responsible, at least in part, for the fact that peroxidases catalyze
electron abstraction (peroxidase) rather than ferryl oxygen transfer
(peroxygenase) reactions(2, 23) . The evidence
marshaled in support of this argument includes the finding that
reaction of HRP with phenylhydrazine (PhNHNH), via its
oxidation product phenyldiazene (PhN=NH), results in either
addition of the phenyl moiety to the
-meso-carbon of the
heme group or abstraction of a hydrogen atom from the 8-methyl group to
give, eventually, the 8-hydroxymethyl heme derivative(21) .
Similar reactions have been observed with other
peroxidases(24, 25) . In contrast, the reaction of
phenyldiazene with hemoproteins in which the iron is known to be within
an open active site produce
-bonded phenyl-iron (Fe-Ph)
complexes. This has been observed with myoglobin(26) ,
hemoglobin, (27) catalase (28) , and cytochrome P450 (29, 30) and is unambiguously confirmed by crystal
structures of the myoglobin and cytochrome P450
complexes(26, 29) . Denaturation of the
phenyl-iron complexes under acidic, aerobic conditions results in
migration of the phenyl group from the iron to the nitrogens of the
porphyrin to yield N-phenylheme. This is followed by loss of
the iron to give N-phenylprotoporphyrin
IX(26, 27, 28, 29, 30, 31) .
Figure 2:
Superimposed images of active site
residues in the crystal structures of cytochrome c peroxidase
and lignin peroxidase. Panel a, side view with the
-meso edge of the heme facing the viewer. The residues
shown, in addition to the heme and the proximal histidine iron ligand,
are the catalytic histidine (His-52 in cytochrome c peroxidase, His-47 in lignin peroxidase), catalytic arginine
(Arg-48 in cytochrome c peroxidase, Arg-43 in lignin
peroxidase), and the aromatic residue (Trp-51 in cytochrome c peroxidase, Phe-46 in lignin peroxidase). These residues
correspond to Phe-41, His-42, and Arg-38 in HRP. Panel b, top
view with the
-meso edge at the bottom showing the same
distal residues as the side view.
The aromatic residue adjacent to the catalytic histidine, and the histidine itself, may be part of the proposed barrier that restricts access of substrates to the ferryl species. Evidence that the aromatic residue plays such a role is provided by our finding that mutation of Phe-41 to a leucine or threonine enhances the peroxygenase activity of HRP(19, 20) . These two mutants oxidize aryl alkyl thioethers to their sulfoxides at higher rates and, in the case of the F41L mutant, with higher enantioselectivity than native HRP. Furthermore, these mutations convey on HRP the ability to epoxidize styrene(20) . These results agree with our finding that replacement of the distal tryptophan in cytochrome c peroxidase by an alanine enhances both the accessibility of the iron and the peroxygenase activity of that enzyme (32) .
To investigate the catalytic role of the distal histidine and to determine if the histidine and the adjacent phenylalanine shield the ferryl species we have developed a new system for the heterologous expression and purification of HRP mutants and have expressed the F41A, H42A, and H42V mutants of HRP. We have examined the activities of these enzymes in the peroxidation of guaiacol, the sulfoxidation of thioanisole, and the epoxidation of styrene and have identified major differences in the catalytic activities of the mutants. The reactions of the three mutants with phenyldiazene have then been used to rationalize the differences in their peroxygenase activities in terms of differences in both the rate of Compound I formation and the accessibility of the heme iron.
The decay in absorption was fit to the equation A = A
e
+ C. The second-order rate constant was determined by taking
the slope of the plot, k
versus the
H
O
concentration. Increased
H
O
concentrations (0.025-1.5 M)
were required with H42A hHRP to see the 411 nm decay.
Figure 3: Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the purified hHRP, F41A hHRP, H42A hHRP, and H42V hHRP proteins. Lane A, molecular mass standards; lane B, native HRP; lane C, wild-type hHRP; lane D, F41A hHRP; lane E, H42A hHRP; and lane F, H42V hHRP.
Figure 4: Formation of Compound I of H42A hHRP and reversion to the ferric state on standing. A, resting state; B, 4 min after adding 5 equivalents of mCPBA; and C, 11 min, D, 18 min, E, 30 min, and F, 60 min after initiation of the reaction. A Compound II spectrum is not detected as Compound I reverts to the ferric state.
As already
noted, formation of Compound I from the H42A mutant requires a large
excess of HO
. Addition of the required excess
of peroxide to H42A, as shown by UV/visible spectroscopy, results in
rapid formation of Compound I followed within 30 s by formation of a
species with a Compound III-like spectrum and a species with a Soret
maximum at
680 nm (possibly verdoheme). It is therefore not
surprising that the secondary plot of the rate data for this mutant
shows a nonlinear dependence of k
on the
peroxide concentration (Fig. 5). Compound I, Compound III, and a
680 nm-absorbing species are also formed in the reactions of native HRP
with excess H
O
, hydroperoxides, or
peroxybenzoic acids (36, 37, 38) . One
consequence of multiple product formation in the reaction of H42A hHRP
is that the measurements of the change in absorbance at 411 nm used to
determine the rate constant for Compound I formation may not truly
reflect formation of that single species. It is nevertheless of
interest that, although the behavior of H42A hHRP differs greatly from
that of hHRP with regard to Compound I formation, the H42A mutant still
forms species with excess peroxide spectroscopically similar to those
obtained under comparable conditions with native HRP.
Figure 5:
Plot
of k[obs] versus the HO
concentration for the H42A hHRP mutant. The rate was determined
by measuring the rate of decrease of the absorbance at 411 nm as a
function of peroxide by stopped flow methods. The H
O
concentrations are 0.025, 0.05, 0.125, 0.250, 0.375, 0.5, 0.75,
1.0, 1.25, and 1.5 M.
The time-dependent increase in absorbance at 414 nm observed
during the oxidation of ABTS trails off in a nonlinear manner,
presumably because of enzyme inactivation similar to that seen with
native HRP at HO
concentrations above 1
mM(39) . Initial rates were therefore measured to
circumvent the apparent inactivation at higher peroxide concentrations.
The resulting data, plotted as 2[hHRP]
/v
versus 1/[ABTS], give a series of lines analogous to
those observed with the wild-type enzyme (Fig. 6a). The
lines at low H
O
concentrations are parallel but
deviate from this relationship at the higher H
O
concentrations. The deviation probably reflects competitive
formation of Compound III(39) . The rate of Compound I
formation (k
) was determined from the inverse of
the slope taken from a secondary plot of the y intercept
values from the primary plot versus 1/[H
O
] (Fig. 6b). The value thus obtained is k
= 19.4 M
s
for H42A hHRP and k
= 10.0 M
s
for H42V hHRP (Table 2). These values are
10
-fold lower than
the rate constant for wild-type hHRP.
Figure 6:
Steady-state kinetics for the oxidation of
ABTS by H42A hHRP. Panel a, the ABTS concentrations used for
the primary plot of the rate versus the inverse of the ABTS
concentration are 1.25, 1.67, 2.5, 5, and 10 mM. Panel
b, secondary plot of the rate constants determined from the y intercepts of the lines from the primary plot versus the
inverse of the peroxide concentration. The HO
concentrations are 65, 85, 125, and 250
mM.
In HRP and hHRP, the reduction
of Compound II is slower than that of Compound I, so that Compound II
is observed during steady-state catalytic turnover. A consequence of
the fact that k is larger than k
is that simplifies to and analysis of
HRP steady-state turnover yields a value for k
.
However, we do not spectrally detect Compound II under steady-state
turnover conditions with the distal histidine mutants. This suggests
that the situation is reversed for these mutants so that reduction of
Compound II is more rapid than reduction of Compound I (i.e.k
> k
). In this case simplifies to .
The average of the inverse of the slopes of the lines in Fig. 6a and of a parallel independent set of data gives
values of 1.9 ± 0.3 10
M
s
for H42A hHRP
and 1.6 ± 0.3
10
M
s
for H42V hHRP which, on the basis that
Compound II is not detected under steady-state conditions, can be
tentatively assigned to k
rather than k
. These values are smaller than the values of
both k
(
0.81
10
M
s
) and k
for the native enzyme (18) . Although
the value of k
for the reaction of ABTS with the
native enzyme is not known, it must be larger than k
because the reduction of Compound II limits the turnover rate of
the native enzyme under steady-state conditions. It appears, therefore,
that the distal histidine is important for both the formation and the
reduction of Compound I.
The sulfoxidation activities of the wild-type and native proteins are similar, although the commercially available enzyme is slightly more active (Table 3). A difference is observed in the sulfoxidation enantiospecificity of the native (83% ee) and wild-type recombinant (66% ee) enzymes. This minor difference presumably stems from the differences in glycosylation pattern or the presence of the N-terminal polyhistidine tag in hHRP. Asparagine 186, 198, and 214 of HRP are normally glycosylated. In the sequence alignment of lignin peroxidase with HRP, a gap is inserted into this region in lignin peroxidase. The residues that flank this gap, Val-184 and Asp-185, lie within the substrate channel in the crystal structure of lignin peroxidase. It is therefore probable that the unique HRP sequence that includes asparagine 186, 198, or 214 constitutes a portion of the HRP solvent accessible channel, and differences in glycosylation resulting from expression in insect cells might affect substrate docking in the active site.
F41A hHRP is a much better sulfoxidation catalyst than the native or wild-type enzymes, but the enantiospecificities of the three enzymes are comparable (Table 3). The distal histidine mutants also exhibit improved thioanisole sulfoxidation activity relative to the wild-type protein (Table 3), but the improvement is less than that found with the F41A mutant. Decreasing the size of the aliquot of peroxide added every 2 min to the reaction 5-fold causes the rate of thioanisole sulfoxidation catalyzed by His-42 mutants to decrease 2-3-fold but does not alter the sulfoxidation rates for hHRP and F41A hHRP. This suggests that Compound I formation is partially rate-limiting for the distal histidine mutants. The enantioselectivities of the distal histidine mutants are similar and relatively high compared with the enantioselectivities of the wild-type and F41A proteins (Table 3).
Figure 7: Changes in the absorption spectra caused by reaction hHRP and its mutants in sodium phosphate buffer, pH 7.0, with phenyldiazene. Panel a, hHRP (10 nmol) before (A) and after the addition of 57 (B), 113 (C), and 170 (D) nmol of phenyldiazene. Panel b, the F41A mutant before (A) and after the addition of 57 (B) and 113 nmol (C) of phenyldiazene. Panel c, the H42A mutant before (A) and after the addition of 57 (B), 113 (C), 227 (D), and 340 (E) nmol of phenyldiazene.
HPLC
analysis of the prosthetic group extracted from phenyldiazene-treated
HRP and hHRP, as reported for native HRP (21) , reveals the
presence of three heme-derived products (Fig. 8). Comparison of
the retention times and absorption spectra of these products with
authentic standards show that the substances eluting at 5.5 and 7 min
are, respectively, the 8-hydroxymethyl derivative of heme and heme
itself. The third peak with a retention time of 12 min is
-meso-phenylheme. As found before for the native
protein(21) , no trace is detected of the N-phenylprotoporphyrin IX isomers expected from rearrangement
of a
-bonded phenyl-iron complex.
Figure 8: HPLC analysis of the heme groups extracted after reaction with phenyldiazene from wild-type hHRP, F41A hHRP, and H42A hHRP. The details of the extraction and chromatographic methods are given under ``Experimental Procedures.''
In contrast to the native
and wild-type proteins, HPLC analysis of the prosthetic group from
phenyldiazene-treated F41A hHRP does not show peaks for heme or either
the 8-hydroxymethyl or meso-phenyl derivatives of heme (Fig. 8). Peaks indicative of the formation of the N-phenylprotoporphyrin IX isomers are also not observed. The
absence of a heme peak and the relative intensities of the product
peaks suggest that substantial heme degradation has occurred. The major
peak observed in the chromatogram corresponds to a new product with a
retention time of 9 min and = 400 nm (Fig. 8). The identity of this material has not yet been
established.
HPLC analyses of the prosthetic groups of phenyldiazene-treated H42A hHRP and H42V hHRP give similar chromatograms with a major peak attributable to intact heme (Fig. 8). The amplitude of the heme peak is double that of the heme peaks found in the analyses of the wild-type and native proteins. Moreover, for each of the two His-42 mutants, denaturation of the protein complex under acidic conditions gives small amounts of N-phenylprotoporphyrin IX, as confirmed by direct comparison of the retention time and absorption spectra of the product with that of an authentic standard (not shown).
Fusion of a polyhistidine tail to the N terminus of HRP greatly simplifies purification of the protein without causing significant alterations in the observable spectroscopic and catalytic properties of the enzyme. Not only are the ferric spectra of HRP and hHRP identical but there are only small differences between these two proteins in terms of their rates of Compound I formation (Table 2), guaiacol peroxidation (Table 3), and thioanisole sulfoxidation (Table 3), or their lack of styrene epoxidizing activity (Table 3). These results establish that the N-terminal polyhistidine extension does not significantly alter the catalytic function of HRP and therefore need not be removed from the protein for mechanistic studies.
The active sites of the four peroxidases for
which crystal structures are available suggest that the catalytic
histidine and the adjacent aromatic residue act as part of a barrier
that restricts interaction of the ferryl oxygen with the substrate. The
changes in the catalytic properties of the enzyme caused by the F41A
mutation support this inference. Thus, the F41A mutation causes a small
(4 nm) shift in the Soret maximum and small percentage increases in the
rate of Compound I formation (50%) and the ability of the mutant
to catalyze guaiacol peroxidation (
30%) ( Table 2and Table 3). Nevertheless, the F41A mutation increases the rate of
thioanisole sulfoxidation 10-fold (Table 3). Replacement of
Phe-41 by leucine or threonine, residues that are intermediate in size
between a phenylalanine and an alanine, increases the V
for thioanisole sulfoxidation 1.9- and
1.6-fold, respectively(40) . The 10-fold increase observed here
in the thioanisole sulfoxidation rate when Phe-41 is replaced by an
alanine is considerably higher, as might be expected, than that
obtained when Phe-41 is replaced by groups of intermediate size. The
large increase in sulfoxidation activity in the face of minor changes
in the rates of Compound I formation and guaiacol oxidation supports
the conclusion that the F41A mutation makes the ferryl oxygen more
accessible to thioanisole and thereby facilitates ferryl oxygen
transfer to the sulfur. The conclusion that the F41A mutation increases
access to the ferryl oxygen is confirmed by the observation that F41A
hHRP (Table 3), like the F41L and F41T mutants(40) ,
catalyzes the epoxidation of styrene whereas the wild-type and native
enzymes are virtually inactive in this regard (Table 3).
The
increase in substrate access to the ferryl oxygen provided by the F41A
mutation occurs without significantly enlarging the active site cavity
directly above the iron atom. The evidence for this is provided by the
finding that reaction of F41A hHRP with phenyldiazene results in
modification of the heme group without the detectable formation of a
phenyl-iron complex. The failure to form a phenyl-iron complex is
indicated by the absence of a shift of the Soret to 430 nm in the
reaction with phenyldiazene and by the absence of N-phenylprotoporphyrin IX from among the modified heme
products extracted from the protein. In fact, a modified heme is formed
in good yield which does not correspond to the normally observed
-meso-phenyl- or 8-hydroxymethyl derivatives of heme (Fig. 8)(21) . The absence of these two products and the
formation of a new heme product are consistent with a modification of
the active site topology in the vicinity of the heme.
Mutations of
His-42, the putative HRP distal histidine, have not been reported
previously. Replacement of His-42 by an alanine or valine compromises
the catalytic machinery required for activation of
HO
. This is clearly demonstrated by a decrease
of
10
in the rate (k
) of Compound
I formation (Table 2). This agrees well with the decrease of
10
observed in the rate of Compound I formation when the
distal histidine of cytochrome c peroxidase is replaced by a
leucine(13) . The rate constant for Compound I formation was
determined under steady-state conditions due to complications
introduced by the requirement for a large excess of
H
O
for catalytic turnover of the H42A and H42V
mutants. The
10
-fold decrease in k
confirms that His-42 is, indeed, the distal residue in HRP and
establishes that it plays a direct catalytic role in peroxide
activation (Fig. 1). The decrease is due, in part, to the fact
that the peroxide anion (HOO
) is probably required
for uncatalyzed binding to the iron of H42A and H42V hHRP in the first
step of Compound I formation, and this peroxide species is present in
10
-fold lower concentration than H
O
at pH 6.0 (the pK
of
H
O
is 11.6)(41) .
A further
consequence of replacing His-42 by an alanine or valine appears to be
destabilization of Compound II with respect to Compound I. Thus,
Compound I of the H42A and H42V mutants formed by reaction with mCPBA
is reduced by 1 equivalent of ferrocyanide or by spontaneous decay to
the ferric species without the detectable formation of Compound II. The
distal histidine residue in HRP apparently stabilizes Compound II,
probably by hydrogen bonding to the ferryl oxygen, and loss of this
interaction destabilizes the ferryl intermediate with respect to
reduction to the ferric state. Resonance Raman studies suggest the
existence in native HRP Compound II of a hydrogen bond between a
protein residue, presumably the distal histidine with a
pK = 8.5-8.8, and the ferryl
oxygen(42, 43) . The H42A and H42V mutants show some
resemblance to the Arthromyces rhamosus peroxidase. At neutral
pH, Compound I of the A. rhamosus peroxidase is reduced by
ascorbate or hydroquinone to the ferric state without the apparent
accumulation of Compound II, although spectroscopic analysis suggests
that Compound II is formed under steady-state conditions at neutral pH,
and Compound II formation is readily observed at pH 10(44) .
However, Compound II is not observed at pH 10 with the H42A and H42V
mutants.
The decreased rates of Compound I formation and changes in the reactivities of Compounds I and II of the H42A and H42V mutants are reflected in the overall rate of guaiacol oxidation, which decreases by approximately 5 orders of magnitude (Table 3). Comparison of the kinetic sensitivity of guaiacol oxidation with the H42A and H42V mutations with the insensitivity of this reaction to mutations of Phe-41 indicates that the rate effects of the His-42 mutations are not simply due to enlargement of the active site cavity or increased exposure to solvent water.
In contrast to the large decreases in the
rate of Compound I formation and the peroxidase activity caused by the
His-42 mutations, the peroxygenase activities of the H42A and H42V
mutants actually increase (Table 3). Thus, the thioanisole
sulfoxidation rate increases 13-fold for the H42A mutant and 9-fold for
the H42V mutant with respect to wild-type hHRP. The increase in the
rate of thioanisole sulfoxidation does not match that obtained with the
F41A mutation, but this is not surprising given that the rate of
Compound I formation is increased by the F41A mutation but drastically
decreased by the H42A mutation. It may be that Compound I formation
becomes partly rate-limiting in the thioanisole sulfoxidation reaction
catalyzed by the His-42 mutants. The same trend is not observed for
styrene epoxidation, a reaction that requires intimate contact of the
ferryl oxygen with both carbons of the -bond rather than simply
with a sulfur electron pair and is therefore more sterically demanding
than thioanisole sulfoxidation. If access to the ferryl oxygen is
particularly critical for epoxidation, the finding that the His-42
mutants are approximately three times more active than the F41A mutant (Table 3) suggests that the distal histidine mutations are more
effective than those of Phe-41 in providing this access.
The changes in reactivity observed with the mutants are consistent with the topological data obtained in their reactions with phenyldiazene. The reactions of phenyldiazene with the H42A and H42V mutants, in contrast to the reactions with native, hHRP, or F41A hHRP, produce a phenyl-iron complex. The formation of a phenyl-iron complex is signaled by the observation of an absorption maximum at 434 nm (Fig. 7) and is confirmed by the isolation of small amounts of N-phenylprotoporphyrin IX when the prosthetic group is extracted from the phenyldiazene-treated protein under aerobic, acidic conditions (not shown). The high recovery of unmodified heme in this reaction suggests that the H42A and H42V phenyl-iron complexes are unstable and revert to a considerable extent to unmodified heme during the workup procedure. The formation of an iron-phenyl complex is a hallmark of hemoproteins in which the iron atom is accessible to exogenous substrates. Analysis of the location of the histidine and the conserved adjacent aromatic residue in the four peroxidases for which crystal structures are available shows that the histidine sits directly above the iron, whereas the aromatic residue is positioned to one side (Fig. 2). The results obtained with phenyldiazene provide direct experimental evidence that the histidine and phenylalanine are similarly disposed in the active site of HRP.
The enantioselectivities of both thioanisole sulfoxidation and styrene epoxidation are higher for the distal histidine mutants than for F41A hHRP (Table 3). Preferred formation of the (R)-thioanisole sulfoxide and (S)-styrene oxide enantiomers by the F41A mutant requires the same spatial orientation of the substrate phenyl group and the side chain with respect to the ferryl oxygen, whereas the favored formation of the S-enantiomers of both products by the H42A and H42V mutants requires that the aryl substituents assume opposite orientations with respect to the ferryl oxygen. The basis for this difference in substrate orientation cannot be deduced from the available structural information.
The present studies provide direct experimental evidence that His-42 is a key catalytic residue, strengthen the evidence that both His-42 and Phe-41 sterically insulate the ferryl species of Compound I from interaction with the substrate, and demonstrate that reductions in the size of these residues greatly increase the peroxygenase activity of HRP. In fact, the F41A, H42A, and H42V mutants compare favorably with cytochrome P450 as thioanisole sulfoxidation catalysts even if they are still 1 order of magnitude weaker than cytochrome P450 as styrene epoxidation catalysts (Table 3). To improve the peroxygenase properties of HRP, it will be necessary to construct mutants that retain a high rate of Compound I formation but have active sites that allow substrates unhindered access to the ferryl oxygen.