The mechanism of formation of N -formylkynurenine by heme dioxygenases

Article abstract of DOI:10.1021/ja207066z

Heme dioxygenases catalyze the oxidation of l-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O₂-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with ¹⁸O₂ confirm the origin of the oxygen atom as arising from O₂-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed.

Full text of DOI:10.1021/ja207066z

ARTICLE
pubs.acs.org/JACS
The Mechanism of Formation of N-Formylkynurenine by
Heme Dioxygenases
Jaswir Basran,‡ Igor Efimov,§ Nishma Chauhan,§ Sarah J. Thackray,|| James L. Krupa,§ Graham Eaton,§
Gerry A. Griffith,§ Christopher G. Mowat,|| Sandeep Handa,§ and Emma Lloyd Raven*,§
‡Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom
§Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom
EaStCHEM, School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, United Kingdom
S Supporting Information
b
ABSTRACT: Heme dioxygenases catalyze the oxidation of L-trypto-
phan to N-formylkynurenine (NFK), the first and rate-limiting step in
tryptophan catabolism. Although recent progress has been made on
early stages in the mechanism, there is currently no experimental data on
the mechanism of product (NFK) formation. In this work, we have used
mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data
identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O2-driven
turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent
chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling
experiments with 18O2 confirm the origin of the oxygen atom as arising from O2-dependent turnover. These data suggest that the
dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously
proposed.
’ INTRODUCTION
(presumed to be a histidine). And, in cases where histidine is
present, mutagenesis studies8,9 do not support an essential
role for it. This led to an alternative proposal7,10 that did not
require an obligate base (Scheme 1B). However, both proposals
(Scheme 1B) are problematic because neither is consistent with
the chemistry of indoles (which do not react by loss of the indole
proton11). That deprotonation of the indole proton might be a
red herring was first suggested12 from computational work but
the experimental evidence has so far been from mass
spectrometry13 which has shown that Me-Trp is not an inhibitor
(as previously suggested6) but a slow substrate. Since the indole
proton is missing in Me-Trp (Scheme 1A), the logical conclusion
is that the mechanism cannot involve its loss. Recent ENDOR
data14 and computational work15,16 support this idea.
The later parts of the mechanism leading to formation of NFK
are, as yet, unclarified. The literature1 describes either Criegee or
dioxetane rearrangements (Scheme 1C), but there is no experi-
mental evidence for either mechanism. Recent resonance Raman
work16 has suggested that the mechanism may proceed by
sequential insertion of oxygen. In the present paper, we have
examined product formation in a number of different heme
dioxygenases under turnover conditions. We find evidence, from
mass spectrometry data, for insertion of a single oxygen atom
into the substrate. This is not consistent with widely cited1
proposals for the concerted addition of both oxygen atoms.
Tryptophan is essential for all mammalian systems, and
tryptophan catabolism is regulated through the L-kynurenine
pathway. The first, rate-limiting step of the kynurenine pathway
is the oxidation of L-tryptophan to N-formylkynurenine (NFK,
Scheme 1A). This is an O2-dependent process and is catalyzed by
one of two heme enzymes: tryptophan 2,3-dioxygenase (TDO)
or indoleamine 2,3 dioxygenase (IDO).1 The mechanism of
tryptophan oxidation is of essential interest from a clinical
perspective because tryptophan catabolism generates a number
of secondary metabolites—such as 3-hydroxyanthranilic acid,
anthranilic acid, 3-hydroxykynurenine, and quinolic acid—that
are implicated in a wide range of neurological disorders, cataract
formation, and suppression of T cell proliferation. In this sense
IDO and TDO are emerging as important drug targets because
they have the power to regulate the supply of tryptophan (Trp)
and other kynurenine pathway metabolites.2 In consequence, the
development of dioxygenase inhibitors is ongoing3 (with some com-
pounds, such as 1-methyl-tryptophan (Me-Trp)), in clinical trials.4
Despite years of investigation going back as far as the 1930s,5
the reaction mechanism of the dioxygenases is still not clarified.
Early studies6 concluded that the mechanism involved base-
catalyzed deprotonation of the indole NH group as the initiating
step (Scheme 1B), and were based on an assignment of Me-Trp
as an inhibitor. It has emerged that there are numerous difficulties
with this mechanism, however. Structural data are not consistent
with the proposal since not all dioxygenases—human indoleamine 2,
3-dioxygenase being one example7—contain an active site base
Received: July 28, 2011
Published: September 05, 2011
r
2011 American Chemical Society
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Scheme 1. Previous Proposals for Reaction Mechanism in the Heme Dioxygenasesa
a (A) The reaction catalyzed by the heme dioxygenases. X = H for tryptophan and X = Me for 1-methyl-tryptophan. (B) Left: Previous proposal1,6 for the
first step of the reaction mechanism, involving base-catalyzed abstraction. Right: A more recent proposal.7,10 (C) Proposed1 Criegee (top, solid line) and
dioxetane (bottom, dashed line) reaction mechanisms for formation of N-formylkynurenine.
An alternative mechanism is proposed for formation of
N-formylkynurenine.
dioxygenases, a peak eluting with m/z = 221 has been routinely
observed in addition to the expected peak at m/z = 237. A
representative example is shown in Figure 1 for hTDO, but
similar data have been obtained for other bacterial (xTDO) and
mammalian (hIDO) enzymes under a range of conditions. Here,
the products from the reaction of hTDO with O2 and L-Trp were
examined using LCÀMS (Figure 1A-D). Using selected ion
monitoring (Figure 1A), the elution profile revealed a peak
eluting with a mass at 221 (Figure 1B). This is consistent with
insertion of a single oxygen atom into the substrate (Mr = 204;
Table S1, SI). The observation of a peak at m/z = 221 did not
depend on the reaction time (varied between 10 and 120 min),
and is not a feature of the TCA precipitation treatment since the
equivalent peaks at m/z = 221 (and m/z = 237) are also observed
when TCA is not used. Nor is it a contaminant from the
adventitious reaction of Trp with O2 under aerobic conditions,
because no such peaks (either at m/z = 221 or m/z = 237) are
observed for steady state assays obtained in the absence of
enzyme. This is expected since Trp is only reactive with O2 in
the presence of a sensitizer; in fact, this is used for the chemical
synthesis of 3a-hydroxypyrroloindole-2-carboxylic acid below.
Samples of L-Trp alone (i.e., L-Trp in buffer under aerobic
conditions) similarly showed no peaks at m/z = 221 or m/z =
237 (and NMR analysis of L-Trp itself showed no peaks from
impurities, data not shown).
We speculated that this peak may originate from initial
conversion of the substrate to the corresponding 2,3-epoxide,
which had been initially identified12 by computational studies
(but considered energetically unfavorable in the gas phase) and
also implicated from resonance Raman work.16 The proposed 2,
3-epoxide is a known compound but is unstable and under-
goes facile conversion to the more stable cyclic aminoacetal
(Scheme 2). Mass spectrometry data for this derivative are
published,21 including the expected fragmentation patterns in
MS/MS experiments, which provides a useful control compound
for the enzymatic turnover experiments. In LCÀMS/MS ana-
lyses of product formation as above, a peak eluting at the same
position is observed (Figure 1C) and with m/z = 221, and
the fragmentation pattern, Figure 1D, matches that predicted for
’ MATERIALS AND METHODS
Human TDO (hTDO), human IDO (hIDO) and X. campestris TDO
(xTDO) were prepared as previously described.8,9,13,17À19 For LCÀMS
experiments, enzyme (1À5 μM), O2 and L-Trp (1 mM, Sigma) or
1-methyl-L-tryptophan (Me-Trp, 0.5 mM, Aldrich) were incubated in a
sealed vessel for varying amounts of time (10À120 min), with condi-
tions as previously described.13 The reducing system in all cases was
ascorbate/methylene blue, as for normal dioxygenase assays,8 or in some
cases with dithionite-reduced hTDO with O2 and L-Trp (1 mM) in
50 mM Tris-HCl buffer, pH 8.0 (25.0 °C). Steady-state formation of
NFK was monitored at 321 nm using a Varian Cary 50 Probe
UVÀvisible spectrophotometer with a 1 cm light path. Reactions were
terminated by the addition of 30% (v/v) trichloroacetic acid although
control reactions were also carried out in which TCA was not used. In all
cases samples were centrifuged (13,000 rpm for 5 min in a micro-
centrifuge) to collect degraded protein and the supernatant was then
filtered using a Millipore 0.45 μM filter at 25 °C. For 18O2 experiments,
all buffers and solutions were purged with nitrogen and then left to
equilibrate in the glovebox for at least 2 h. Samples for the 18O2-
experiments were prepared by bubbling 18O2 (98%, CK Gas Products
Ltd.) into the assay mixture and the reaction initiated by the addition of
enzyme. For reactions carried out in 18O-labeled water, all solutions were
prepared using H218O (98%, CK Gas Products Ltd.) and the assays
carried out as outlined above. Reactions were terminated by the addition
of 30% (v/v) trichloroacetic acid as described above. LCÀMS analyses
used a Micromass Quatro LC spectrometer and samples were run with
acetonitrile/H2O (0.1% formic acid) as the solvent (in a ratio of 10:90 or
20:80) on a C18 column. The 3a-hydroxypyrroloindole-2-carboxylic
acid compound, referred to as the cyclic amino acetal in this paper (see
Scheme 2, species Y), was synthesized (>95% purity, assessed by NMR)
via dye-sensitized photooxygenation of L-tryptophan.20 NMR and mass
spectrometry data are given in the Supporting Information (SI).
’ RESULTS
Mass Spectrometry Analyses. In our LCÀMS analyses
looking for NFK (m/z = 237) formation in a number of
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Scheme 2. Proposed Mechanism for Formation of the Cyclic
Amino Acetal (3a-Hydroxypyrroloindole-2-carboxylic acid,
Species Y), Showing the Proposed Ring-Opening and Cycli-
zation Mechanisma
a The species labeled X and Y have also been suggested40 as intermedi-
ates in the PrnB mechanism (see Discussion).
the cyclic aminoacetal shown in Scheme 2 (peak at 175: loss of
COOH; peak at 203: loss of H2O; peak at 157: loss of H2O and
HCOOH).21 We thus assign the 221 peak detected in our mass
spectrometry experiments as arising from initial formation of an
epoxide intermediate, in which one atom of oxygen is inserted
into the substrate during turnover, followed by conversion to the
more stable amino acetal compound under the conditions of our
experiments.22 In these experiments, we can also isolate an ion
with m/z = 237 (Figure 1E), which is assigned as arising from
NFK formation (Mr = 236, Table S1, SI) by comparison with
previous work.13
Synthesis of the Cyclic Aminoacetal Intermediate. To
compare the mass spectrometry data above with the authentic
3a-hydroxypyrroloindole-2-carboxylic acid compound (Scheme 2),
we synthesized the compound (>95% purity, assessed by NMR)
via dye-sensitized photooxygenation of L-tryptophan.20 The
compound was isolated as a mixture of two diastereoisomers in
a ratio of 2:1, and the structure confirmed by NMR analysis
(see SI). Both isomers have a cis-5,5-ring junction with the major
isomer having a cis-relationship between the hydroxyl and
carboxylic acid groups (these assignments were confirmed by
NOE experiments). LCÀMS analysis of this synthesized sample
indicated that it had the same retention time and the fragmenta-
tion pattern (see Supporting Information) as the 221 peak
observed in the hTDO reactions.
In control experiments, we found that the purified cyclic
aminoacetal (as a mixture of isomers) was not turned over to
product by any of the dioxygenases examined in this work
(no identifiable product peaks in mass spectrometry). This
would be consistent with observations from mass spectrometry
that the 221 species is a minor product (with most of the sub-
strate turned over to NFK, as judged by comparing intensities of
peaks eluting from LCÀMS). Since the epoxide itself is not an
isolatable compound, it was not possible to spike the reaction
with epoxide in the same way.
Figure 1. Formation of NFK formed during O2-dependent turnover by
hTDO. LCÀMS and LCÀMS/MS analyses of the products obtained
from the O2-dependent reaction of hTDO with L-Trp in the steady state.
(A) Elution profile for selected ion chromatogram with m/z = 221. The
peak eluting at t = 8.58 min is kynurenine. L-Trp elutes at t = 13 min. (B)
Corresponding positive ESI mass spectrum for the product eluted at
5.72 min, showing a peak at m/z = 221. (C) Elution profile for the same
peak (at 5.60 min in this elution profile) and (D) corresponding MS/MS
spectrum, showing the peak at 221 and a fragmentation pattern
corresponding to the structure proposed in Scheme 2. (E) Positive
ESI mass spectrum for the product eluted at 7.02 min, showing a peak at
m/z = 237 corresponding to NFK formation. For all enzymes examined,
the intensity of the 221 peak is invariably lower than that for the 237
peak, but we have been unable to extract quantitative time profiles from
the mass spectrometric analyses.
Labeling Experiments Using 18O2. To confirm that the 221
peak was generated from O2-dependent turnover, the reactions
with L-Trp were repeated under the same conditions but using
18O2 to assess whether the oxygen atom in the 221 species
originated from O2 (or 18O2). The results of these LCÀMS/MS
experiments are shown in Figure 2 for hTDO with L-Trp and for
hIDO with Me-Trp, but similar data have been observed for
xTDO with L-Trp. For hTDO, a peak is observed with m/z = 223
(Figure 2A), which corresponds to insertion of a single atom of
18O into L-Trp (Table S1, (SI)). In this spectrum there is also a
peak at m/z = 177, which corresponds to the 18O-equivalent of
the m/z = 175 fragmentation peak reported above (loss of
COOH, as above). An ion with m/z = 241 was also detected,
Figure 2B, which corresponds to the mass of the NFK product
carrying two atoms of 18O (m/z = 241, Table S1 (SI)). In the
corresponding reactions of hIDO with Me-Trp and 18O2, we
also observe peaks eluting with m/z = 237 and m/z = 255
(Figures 2C,D), which are as expected for the 18O-labeled mono-
oxygenated intermediate species and 18O-labeled N-formyl-
methylkynurenine product (Mr = 254, Table S1(SI)).23
For the reaction of hTDO and 18O2, we also observed a
product peak with m/z = 239 for the reaction with L-Trp
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Figure 2. Formation of NFK during dioxygenase-catalyzed turnover using 18O2. LCÀMS/MS analyses of the products obtained on reaction of hTDO
with L-Trp and 18O2 ((A) fragmentation pattern for ion with m/z = 223, (B) fragmentation pattern for ion with m/z = 241) and on reaction of hIDO with
Me-Trp and 18O2 ((C) fragmentation pattern for ion with m/z = 237, (D) fragmentation pattern for ion with m/z = 255).
Figure 3. Evidence for mixed incorporation of 18O and 16O in NFK in reactions with 18O2. LCÀMS/MS analyses of NFK (m/z = 239) carrying
one atom of 16O and one atom of 18O from reaction of (A) hTDO with L-Trp and 18O2 (16O18O-NFK) and (B) hIDO with Me-Trp and 18O2 (16O18O-
Me-NFK).
(Figure 3A), and in parallel experiments a peak with m/z = 253
for the reaction with Me-Trp (Figure 3B). In both instances, this
corresponds to the mass of NFK carrying one atom each of 16O
and 18O (i.e., 16O18O-NFK and 16O18O-Me-NFK, instead of the
expected product carrying two atoms of 18O2). It is known from
other work24À26 that both carbonyl oxygen atoms in NFK are
exchangeable with solvent. In recent analyses, Ronsein and co-
workers26 have observed exactly the same ‘mixed’ species
(16O18O-NFK, m/z = 239) for NFK formed using singlet O2
and have used the fragmentation patterns to show that the two
carbonyl oxygen atoms in NFK have different rates of exchange
with solvent, with the carbonyl oxygen group of NFK (blue in
Figure 4) exchanging faster than the carbonyl oxygen of the
amide group (red in Figure 4).26 In our experiments for hTDO,
xTDO, and hIDO with 18O2 (Figures 4AÀC), the m/z = 239
peak for the original mixed 16O18O species fragments in all cases
to give m/z = 222 (loss of the NH3 group of the side chain), 204
(loss of NH3 then H2O), and m/z = 192 (loss of NH3 then C18O
from the carbonyl of the amide group26). This is consistent with
initial formation of 18O18O-NFK, with subsequent exchange of
the carbonyl oxygen to give 16O18O-NFK (m/z = 239) followed
by fragmentation leading to loss of 18O (from loss of C18O, red)
and leaving 16O in the product (blue).26 To verify this, we carried
out a control reaction with 16O2 but with the protein exchanged
into H218O-containing buffer. In these experiments, 16O16O-NFK
(m/z = 237) formed initially, followed by exchange (as evidenced
by a peak at m/z = 239, Figure 4D), but the fragmentation
pattern is different (peaks at m/z = 222 as above, 202 (loss
of NH3 then H218O), and 194 (loss of NH3 then C16O),
Figure 4D)). This is again consistent with faster exchange of
the carbonyl oxygen with 18O (from H218O water), because the
fragmentation (Figure 4D) now leads to loss of C16O (red)
from the carbonyl of the amide group (not 18O, as above in
Figure 4AÀC). Together, these experiments provide assurance
that the peaks for the mixed 16O18O-NFK species are secondary
products derived from subsequent exchange reactions with water
(during handling).
’ DISCUSSION
The activation of atmospheric oxygen is a fundamental
requirement in biology that all aerobic organisms have to
overcome. The difficulty is that oxygen is intrinsically unreactive,
both thermodynamically and kinetically. Heme iron is often
used to overcome these difficulties, and Nature uses heme
enzymes for a large number of quite different, and sometimes
difficult, biological oxidations. The mechanism of O2 activation
and NFK formation in the heme dioxygenases has yet to be
clarified.
Unraveling the Dioxygenase Mechanism. The mass spec-
trometry data presented in this paper reveal the presence of a
stable amino acetal species, assigned as originating from initial
formation of an unstable 2,3-epoxide, and consistent with inser-
tion of a single atom of oxygen into the substrate during O2-
driven reactions. This is in contrast to the originally proposed
mechanisms1 for conversion of L-Trp to NFK, Scheme 1C, but
would agree with more recent evidence16 from resonance Raman
work in which sequential insertion of oxygen into the substrate
has been suggested. We envisage two possible mechanisms
(Scheme 3, steps 1a and 1b27), both of which fit with our
data and with the supposed formation of ferryl heme.16,28,29
One route is through electrophilic addition (steps 1b and
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2b12,13), which is well-known in indole chemistry. However, in
this mechanism the bound O2 is denoted as an electrophile which
could be problematic since O2 bound to ferrous heme is often
formulated as a ferric-superoxide species.30 An alternative sug-
gested route12,16,31 is through a radical-mediated reaction and
formation of ferryl heme (Scheme 3, steps 1a and 2a). This would
be attractive if the ferrous oxy species exists as ferric-superoxide30,32
(for which there is evidence16) and would at the same time
overcome the thermodynamic barrier to the required activation
of O2 (by one-electron reduction). Recent computational work
favors the radical addition mechanism.33 It is perhaps also worth
remembering that heterolytic cleavage typically requires consid-
erable charge separation; hence, there is a requirement for
electrostatic stabilization by residues in the distal pocket (Arg
for example in the peroxidases), and the dioxygenase structures
solved so far do not reveal large numbers of charged residues
close to the heme iron (although there is an active-site Arg
involved in substrate binding18). Regardless of which mechanism
is used (Scheme 3, steps 1a or 1b), neither requires abstraction of
the indole proton and would thus be in agreement with most of
the recent data.9,12À16,33
Formation of NFK. The later stages of the mechanism leading
to formation of NFK have been proposed1,34 as occurring through
either Criegee or dioxetane pathways (Scheme 1C). A singularly
misleading aspect of the literature is that these proposals are
based on early speculation34 rather than on experimental fact
(formation of a peroxyindole species, usually generated photo-
lytically, is an implicit requirement,11,35 and we presume this
would be difficult to generate under enzymatic conditions). The
conditions of our turnover experiments and mass spectrometry
analyses allow us to identify a cyclic aminoacetal derivative, which
we assign as a byproduct of the reaction that originates from ring-
opening of the initially formed (and unstable) 2,3-epoxide
(Scheme 2). It is well-known that epoxides can undergo facile
C2ÀO bond cleavage (due to the adjacent nitrogen lone pair36),
and for this reason we propose that the amino acetal species
arises from ring-opening of the initial epoxide (Scheme 2).37 By
analogy, this indicates to us that a heterolytic (two-electron)
mechanism, again involving ring-opening of the initial epoxide,
may be the initiating step in the dioxygenase enzymes (Scheme 3,
step 3). This would allow for reactivity of both L-Trp and Me-
Trp, both of which are substrates.13 Direct attack of the ferryl
heme on the substrate can then be envisaged (step 4), after which
Figure 4. Analysis of oxygen exchange in NFK. LCÀMS/MS analyses of
the NFK product peak (m/z = 239) in reactions of (A) hTDO, (B) hIDO,
and (C) xTDO with L-Trp and 18O2 in H216O buffer; and LCÀMS/MS
analyses of the NFK product peak (m/z = 239) in reactions of hTDO (D)
with L-Trp and 16O2 in H218Obuffer. For clarity of discussion in the Results,
the two different carbonyl groups are differentiated using color (see text).
Scheme 3. Mechanistic Proposal for NFK Formation in the Heme Dioxygenasesa
a Epoxide formation is envisaged by two possible routes, an electrophilic mechanism (step 1a) or by radical addition (step 1b): as we highlight in the
Discussion, both routes can lead to formation of an epoxide, but neither mechanism has been definitely proven. Ring-opening is indicated in step 3
(leading to the species labeled as X in Scheme 2), but could be synchronous with step 4. Cleavage of the C2ÀC3 bond and formation of NFK is
+
subsequently straightforward. For further details see text. R = CH2CH(NH3 )CO2À. The amine group of the side chain could potentially form a
hydrogen bond to the OÀ of the singly oxygenated intermediate.
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Scheme 4. Comparisons of the Reactivity of the Dioxygenase
and PrnB Enzymesa
The proposed lack of protons in the dioxygenase active site14 might
be a controlling factor in discriminating one pathway against
the other.
Epoxidation of substrates is, of course, well-known in the
P450s41,42—in this case through direct reaction with the more
powerfully oxidizing Compound I intermediate—but for indole com-
pounds hydroxylation42 rather than epoxidation43,44 is more
common. Drawing on this, we considered other mechanisms for
NFK formation involving H atom abstraction from different
positions on the substrate. Direct H atom abstraction from the
C2ÀH group of the epoxide by Compound II was considered
(Scheme S1A, SI) but subsequent conversion to NFK is not
easily visualized. The most plausible alternative was considered
to involve abstraction of the indole NH (which has precedent in
P450 chemistry41,42) and is attractive because it leads to NFK
formation through a simple and chemically convincing radical
mechanism, Scheme S1B (SI). However, this mechanism has the
disadvantage that it does not accommodate the known reactivity
of Me-Trp,13 and we thus considered it less likely because Me-
Trp and L-Trp are, at this stage, presumed to react through
similar mechanisms.
There is logic in the idea that the shared heme structure used
across all heme enzymes, together with the similarity of their
reactions with O2 or its derivatives, means that there may be
common patterns of reactivity across the entire family. Typically,
other O2-dependent oxidative enzymes (e.g., P450s, NO synthase)
react with O2 or derivatives of it (H2O2 in the peroxidases) to
give an oxidized Compound I intermediate. Irreversible forma-
tion of Compound I is a reaction that the dioxygenases appar-
ently need to avoid because formation of Compound I and
recycling of the heme demands both protons (for evolution of
water) and a continuous supply of electrons, neither of which is
known to be required for dioxygenase turnover. Quite how such
discrimination is achieved in the dioxygenases has yet to be
established, but regulation of the reactivity of the ferrous-oxy
species by limiting the supply of protons/electrons to the heme
would be one solution to the problem. In this sense, the dioxy-
genases are unique because, rather than using the same oxidizing
intermediate, they appear to use different heme intermediates for
insertion of the two atoms of oxygen. This may be one of the key
controlling features that helps the dioxygenases to specialize in
NFK formation.
a (A) The reaction catalyzed by PrnB.40 (B) Comparison of possible
reaction mechanisms in PrnB (right) and the dioxygenases (left). In
both, formation of the species labeled X is implicated, but after that the
mechanisms branch (the species labeled X and Y are the same as also
shown in Scheme 2). In PrnB this leads to formation of the tricyclic
(amino acetal) intermediate, without insertion of oxygen, whereas in the
dioxygenases C2ÀC3 bond cleavage and further O atom insertion is the
preferred route.
’ ASSOCIATED CONTENT
S
Supporting Information. Calculated masses for species
b
observed in mass spectra (Table S1), alternative mechanisms
(Scheme S1), NMR and MS data for the cyclic amino acetal
compound, and a full citation for reference 18. This material is
available free of charge via the Internet at http://pubs.acs.org.
a heterocyclic (two-electron) mechanism for the required clea-
vage of the C2ÀC3 bond is feasible (step 5).39
Comparisons with Other Heme Enzymes. We have found
that the amino acetal is not, under the conditions of our experi-
ments, turned over by the enzyme to NFK, but we note that the
very same species (labeled X and Y in Scheme 2) have been
proposed40 as intermediates in the PrnB enzyme (species Y being
referred to as the ‘tricylic’ intermediate by Naismith et al.40),
which catalyzes cleavage of the C2ÀN bond of 7-Cl-Trp but
without insertion of O2 (Scheme 4A). In Scheme 4B, we suggest
how the two enzyme mechanisms may align: both proceed
through intermediate X but then branch, in the dioxygenases
leading to further oxygen insertion and NFK formation, while in
PrnB a different product emerges via cyclization and protonation.40
’ AUTHOR INFORMATION
Corresponding Author
emma.raven@le.ac.uk.
’ ACKNOWLEDGMENT
We thank Paul Ortiz de Montellano and James Naismith for
helpful discussions on the mechanism. This work was supported
by grants from The Wellcome Trust (Project Grant 083636 to
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Journal of the American Chemical Society
ARTICLE
E.R. and to C.G.M.), BBSRC/EaStChem (studentship to S.T.)
and EPSRC (studentship to N.C.).
amounts of 16O2 introduced during handling and/or exchange processes
described below.
(24) Hayaishi, O.; Hirata, F.; Ohnishi, T.; Henry, J. P.; Rosenthal, I.;
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(27) It is not known whether addition occurs at C2 or C3 of the
substrate; both options are feasible for both mechanisms in Scheme 3
(but we only show one in each case).12,13,16,26
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(37) The same tricylic compound is also known to be generated via
formation of a 3-peroxyindole species, and is well-known in indole
chemistry.11,34,36 However, this reaction requires photolytic conditions
and a sensitizer for formation of (the much more reactive) singlet oxygen,
and we assume that formation of singlet oxygen is not possible under the
steady state turnover conditions of our experiments.
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+
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