A long-lived, substrate-hydroxylating peroxodiiron(III/III) intermediate in the amine oxygenase, AurF, from Streptomyces thioluteus

Article abstract of DOI:10.1021/ja9064969

(Chemical Equation Presented) The amine oxygenase AurF from Streptomyces thioluteus catalyzes the six-electron oxidation of p-aminobenzoate (pABA) to p-nitrobenzoate (pNBA). In this work, we have studied the reaction of its reduced Fe₂(II/II) c

Full text of DOI:10.1021/ja9064969

Published on Web 09/04/2009
A Long-Lived, Substrate-Hydroxylating Peroxodiiron(III/III) Intermediate in the
Amine Oxygenase, AurF, from Streptomyces thioluteus
†,‡
‡
‡
,†,‡
Victoria Korneeva Korboukh, Ning Li, Eric W. Barr, J. Martin Bollinger, Jr.,*
and
,
†,‡
Carsten Krebs*
Departments of Chemistry and Biochemistry and Molecular Biology, The PennsylVania State UniVersity,
UniVersity Park, PennsylVania 16802
Received August 1, 2009; E-mail: ckrebs@psu.edu; jmb21@psu.edu
2
AurF from Streptomyces thioluteus utilizes O as oxidant to effect
the six-electron oxidation of p-aminobenzoate (pABA) to p-nitroben-
1
zoate (pNBA), a building block of the antibiotic aureothin. This
conversion entails three successive two-electron oxidations via p-
2,3
hydroxylaminobenzoate(pHABA)andp-nitrosobenzoateintermediates.
Sequence analysis and the crystal structure of AurF showed that it
has a carboxylate-bridged dinuclear metal center, which was presumed
to activate O
as to the identities of the metal ions in the functional form(s),
and co-workers recently established that the diiron form is competent
to convert pABA to pNBA in the presence of O and a reducing
cluster in AurF reacts
in the absence of substrate to form a remarkably stable (t1/2
min at 20 °C) adduct with spectroscopic properties characteristic of
2
for the oxidation reactions. Following initial controversy
2-7
Zhao
Figure 1. Absorption spectra of a sample of AurF subjected to DT reduction
and O reoxidation: as-isolated protein (0.40 mM in 50 mM HEPES pH 7.5
and 10% glycerol buffer; blue line); after treatment with 1 equiv of DT for 10
2
2
8
II/II
min at 20 °C (red line); after addition of 0.2 volume of cold oxygenated buffer
system. Here we demonstrate that the Fe
with O
2
(
2
[O ]final ) 0.38 mM, green line), and after addition of pABA (0.92 mM final;
2
≈
black line). Inset: Kinetic traces after mixing DT-reduced AurF (0.64 mM) at
-saturated buffer, allowing the reaction to
7
2
1 °C with an equal volume of O
2
III/III
a peroxo-Fe
2
complex. The intermediate complex decays rapidly
proceed for 0.1 s, and then mixing the resultant solution with an equal volume
of anaerobic buffer either lacking pABA (9) or containing 1 equiv pABA (b).
The solid lines are simulations with parameters quoted in the text.
(
t
1/2 ≈ 0.005 s at 20 °C) when mixed with stoichiometric pABA,
implying that it is competent for at least the first oxidation in the three-
step sequence. The nearly quantitative (>80%) conversion of pABA
to pNBA upon addition of <0.3 equiv of the amine substrate to a
solution of the intermediate suggests that the complex might effect all
three steps in the sequence. The results establish a new reactivity of
nonheme diiron-peroxide intermediates: arylamine oxygenation.
III/III
2
B). These observations indicate that the Fe
2
protein is reduced to
II/II
the Fe
2
form by DT treatment.
II/II
Reaction of Fe
2
AurF with O
2
produces a new broad absorption
-1
-1
band centered at ∼500 nm (ε500 ≈ 0.5 mM cm ; Figure 1, green
trace). Formation of the 500-nm-absorbing species is complete within
Diiron AurF was isolated from overproducing Escherichia coli cells
as described in the Supporting Information. The UV/visible absorption
0
.01 s at ∼0.6 mM O
2
and 20 °C (Figure S1). The species does not
decay noticeably on the 100-s time scale of the SF absorption
spectrum of the protein (Figure 1, blue trace) has a band at ∼360 nm
experiment. The 4.2-K/53-mT M o¨ ssbauer spectrum of the protein
III/III
similar to those exhibited by other proteins with µ-oxo-Fe
2
clusters,
II/II
following vigorous stirring of the Fe
2 atm of O
quantitatively oxidized. In addition to small amounts of the subspectra
2
complex for ∼2 min under
wherein it has been assigned to an oxo-to-iron charge transfer
∼
2
(Figure 2C) shows that the diiron cluster is almost
9
transition. The 4.2-K/53-mT M o¨ ssbauer spectrum of AurF isolated
5
7
57
from cultures supplemented with FeSO
4
(>95% Fe) can be
II/II
III/III
of Fe
2
(5%, purple line) and µ-oxo-Fe
2
(18%, orange line)
simulated as two quadrupole doublets with parameters typical of high-
complexes, the spectrum reveals several new sharp peaks, which can
be simulated as two quadrupole doublets with similar parameters. The
III
spin Fe [δ
Q,2 ) 0.80 mm/s] and relative contributions to the experimental
spectrum of 78% (orange line) and 22% (green line), respectively
1
) 0.54 mm/s, ∆EQ,1 ) -1.86 mm/s; δ
2
) 0.48 mm/s,
∆E
parameters [δ
and δ ) 0.61 mm/s, ∆EQ,2 ) 0.35 mm/s (33%, red line)] are typical
of high-spin Fe(III). Spectra collected in external fields of 5 T and
1
) 0.54 mm/s, ∆EQ,1 ) -0.66 mm/s (49%, blue line)
2
III/III
(
Figure 2A). The large |∆EQ,1| of 1.86 mm/s is typical of µ-oxo-Fe
2
10
clusters, consistent with the absorption spectrum and the crystal
11
8
T (Figure 2G and 2H) confirm that the new state has S ) 0 ground
8
structure. The complex with smaller ∆E
Q
could have one or more
III
state(s), due to antiferromagnetic coupling of two high-spin Fe sites.
µ-hydroxo bridge(s). M o¨ ssbauer spectra collected in externally applied
fields of 5 T and 8 T (Figure 2E and 2F) can be simulated well with
the same parameters and the assumption of a diamagnetic ground state.
The S ) 0 ground state arises from antiferromagnetic coupling between
III/III
II/II
Because it is an Fe
2
cluster derived from the Fe
2
enzyme by
III/III
treatment with O , we assign this state as peroxo-Fe
2
2
. The ∼1.5:1
intensity ratio of the two quadrupole doublets suggests the presence
of two or more isomeric complexes, perhaps in equilibrium (as has
been observed in the reactive intermediate states of other iron
III
the two high-spin Fe sites.
12-14
Treatment of the protein with 1 equiv of sodium dithionite (DT)
bleaches the band at 360 nm in the absorption spectrum (Figure 1,
enzymes).
M o¨ ssbauer spectra of samples of the peroxide state
frozen after subsequent incubation at 20 °C show that it is stable for
red trace) and produces a new quadrupole doublet with parameters (δ
minutes in the absence of substrate (t1/2 ≈ 7 min) and decays to the
II
III/III
)
1.24 mm/s, ∆E
Q
) 3.06 mm/s) typical of high-spin Fe (Figure
µ-oxo-Fe
2
species (Figure S2).
III/III
The reactivity of the peroxo-Fe
2
state was examined by stopped-
II/II
†
flow experiments in which the Fe
2
protein was allowed to react with
Department of Chemistry.
Department of Biochemistry and Molecular Biology.
‡
III/III
O
2
for 100 ms and the resultant peroxo-Fe
2
state was then mixed
1
3608 9 J. AM. CHEM. SOC. 2009, 131, 13608–13609
10.1021/ja9064969 CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
with the substrate, pABA, or (as a control) buffer. The 500 nm band
the AurF reaction, involving nucleophilic attack of the lone pair of
the neutral arylamine on the peroxo electrophile (Scheme 1), seems
plausible. An attractive possibility is that the peroxo complexes in
ToMO and AurF differ structurally from the canonical µ-1,2-peroxides
in ways that activate them as electrophiles. Protonation of the peroxide
unit, perhaps with rearrangement to a µ-1,1-bridging geometry, seems
likely to have this effect.
III/III
of the peroxo-Fe
2
state, stable in the absence of pABA (inset to
-1
Figure 1, 9), decays rapidly (kobs ) 150 ( 20 s ) upon exposure to
1
equiv of the substrate (inset to Figure 1, b).
The 4.2-K/53-mT M o¨ ssbauer spectra of samples prepared by
II/II
oxygenation of Fe
2
AurF (Figure 2C, replotted in Figure 2D as solid
line for clarity) and subsequent treatment of the oxygenated sample
with 0.3 equiv of pABA for 1 min (Figure 2D, hashed marks) confirm
The remarkable stability of the AurF intermediate, which permits
its preparation in concentration, purity, and physical form (e.g., a
transmitting glass) appropriate for most spectroscopic methods, should
be a considerable asset in ongoing efforts to define the structure of
the complex by a combination of experiments and density functional
theory calculations.
III/III
the reactivity of the peroxo-Fe
2
state toward the substrate. The
features of the intermediate complex(es) decay, and those of the µ-oxo-
III/III
Fe
2
cluster develop. Analysis of the small-molecule components
of reactions performed similarly (described in Supporting Information)
showed that, at these low pABA/AurF ratios of e0.3, the substrate is
15
converted to pNBA with a yield of >80%.
Scheme 1. Proposed Mechanism for Hydroxylation of pABA by a
III/III
Putative µ-1,2 (left) or µ-1,1 (right) Hydroperoxo-Fe
2
Intermediate
Acknowledgment. This work was supported by the National
Institutes of Health (GM-55365 to J.M.B. and C.K.), the Dreyfus
Foundation (Teacher-Scholar Award to C.K.), and the Pennsylvania
State University.
Supporting Information Available: Procedures to produce AurF and
quantify pABA and pNBA in reaction samples; stopped-flow absorption
II/II
and M o¨ ssbauer spectra of samples from the reaction of Fe
2
-AurF with
2
O in the absence of substrate; M o¨ ssbauer analysis of the sample used for
III/III
high-field studies of the peroxo-Fe
2
state. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
Figure 2. 4.2-K M o¨ ssbauer spectra (hashed marks) of as-isolated AurF (A,
E, F); DT-reduced AurF (B); DT-reduced AurF exposed to 2 atm O for 2
min, and then either directly frozen for M o¨ ssbauer analysis (C) or further treated
with 0.3 equiv pABA prior to being frozen for analysis (D); and DT-reduced
(
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(
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II/II
(10) An alternative solution of similar quality yields: δ
.59 mm/s (49%) and δ
1
) 0.50 mm/s, ∆EQ,1
)
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0
2
) 0.66 mm/s, ∆EQ,2 ) 0.45 mm/s (33%); both
III/III
long-lived state that has properties consistent with a peroxo-Fe
2
solutions indicate that the iron sites are high-spin Fe(III).
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(
formulation. The stopped-flow data show that the intermediate state
reacts rapidly with pABA, implying that it is competent for at least
the first oxidation in the three step AurF sequence. The possibility
that it is competent for all three oxidations seems likely (on the basis
of the efficient conversion of limiting pABA to pNBA) but requires
more rigorous evaluation.
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(
(
(
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15) Experiments were performed both by addition of substrate to intermediate
and by oxygenation of reduced enzyme in the presence of pABA. The
results were not significantly different.
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III/III
those of peroxo-Fe
2
complexes detected in related diiron oxidases
(
16) Bollinger, J. M., Jr.; Krebs, C.; Vicol, A.; Chen, S.; Ley, B. A.; Edmondson,
16
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9
17
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acyl carrier protein ∆ desaturase, and soluble methane monooxy-
18
genase hydroxylase (sMMOH). The intermediates in these proteins
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19
2
2
have µ-1,2-peroxo (or perhaps µ-(η ,η )-peroxo, for the case of
(
19) Skulan, A. J.; Brunold, T. C.; Baldwin, J.; Saleh, L.; Bollinger, J. M., Jr.;
20
sMMOH ) diiron cores. The properties of the AurF complex are more
similar to those of a peroxo-Fe
toluene/o-xylene monooxygenase (ToMO). In this enzyme, the
peroxide complex is thought to be attacked as an electrophile by the
π-system of the substrate. A similar mechanism for the first step in
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III/III
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2
intermediate recently detected in
21
JA9064969
J. AM. CHEM. SOC. 9 VOL. 131, NO. 38, 2009 13609