Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus

Article abstract of DOI:10.1073/pnas.1002785107

The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate (Ar-NH2, where Ar = 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO₂) in the biosynthesis of the antibiotic, aureothin, by Streptomyces thioluteus. It has been reported that this net six-

Full text of DOI:10.1073/pnas.1002785107

Four-electron oxidation of p-hydroxylaminobenzoate
to p-nitrobenzoate by a peroxodiferric complex
in AurF from Streptomyces thioluteus
a,1
a,b,1
a,b,2
a,b,2
Ning Li , Victoria Korneeva Korboukh , Carsten Krebs , and J. Martin Bollinger, Jr.
a
b
Departments of Biochemistry and Molecular Biology and Chemistry, Pennsylvania State University, University Park, PA 16802
Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved July 16, 2010 (received for review March 3, 2010)
The nonheme di-iron oxygenase, AurF, converts p-aminobenzoate
(
Ar-NH , where Ar ¼ 4-carboxyphenyl) to p-nitrobenzoate (Ar-NO )
2
2
in the biosynthesis of the antibiotic, aureothin, by Streptomyces
thioluteus. It has been reported that this net six-electron oxidation
proceeds in three consecutive, two-electron steps, through p-hy-
droxylaminobenzoate (Ar-NHOH) and p-nitrosobenzoate (Ar-NO)
intermediates, with each step requiring one equivalent of O2
and two exogenous reducing equivalents. We recently demon-
strated that a peroxodiiron(III/III) complex (peroxo-Fe2III∕III-AurF)
II∕II
formed by addition of O2 to the diiron(II/II) enzyme (Fe2 -AurF)
effects the initial oxidation of Ar-NH , generating a μ-(oxo)diiron
2
III∕III
(
III/III) form of the enzyme (μ-oxo-Fe2 -AurF) and (presumably)
III∕III
Ar-NHOH. Here we show that peroxo-Fe2 -AurF also oxidizes
Ar-NHOH. Unexpectedly, this reaction proceeds through to the
Ar-NO2 final product, a four-electron oxidation, and produces
Fe2 -AurF, with which O2 can combine to regenerate peroxo-
Fe2 -AurF. Thus, conversion of Ar-NHOH to Ar-NO requires only
II∕II
III∕III
2
II∕II
2
a single equivalent of O and (starting from Fe -AurF or peroxo-
2
III∕III
Fe2 -AurF) is fully catalytic in the absence of exogenous reducing
equivalents, by contrast to the published stoichiometry. This novel
type of four-electron N-oxidation is likely also to occur in the reac-
tion sequences of nitro-installing di-iron amine oxygenases in the
biosyntheses of other natural products.
aureothin ∣ di-iron ∣ N-oxygenase ∣ nonheme ∣ nitroarene
he enzyme AurF from Streptomyces thioluteus converts para-
T
aminobenzoate (Ar-NH , where Ar ¼ 4-carboxyphenyl) to
2
para-nitrobenzoate (Ar-NO ) in the biosynthesis of the antibiotic,
2
Scheme 1. Reactions catalyzed by AurF. (A) Previously proposed stoichiome-
try of the AurF-catalyzed conversion of Ar-NH2 to Ar-NO2 (8–10); (B) stoichio-
aureothin (1, 2). It is structurally similar to the β subunits of class
2
I ribonucleotide reductases and the oxygenase components of
bacterial multicomponent monooxygenases (BMMs), which all
metry of the AurF-catalyzed reaction indicated by this study; and (C) reactions
III∕III
of peroxo-Fe2
-AurF with Ar-NH2 and Ar-NHOH.
use carboxylate-bridged di-iron clusters to activate O (3–5). Fol-
2
lowing some initial controversy over whether the active form of
AurFalso contains a di-iron cluster (6) or, instead, a dimanganese
to Ar-NO (7, 8), Zhao and coworkers reasserted their previous
proposal that Ar-NHOH undergoes direct dehydrogenation to
Ar-NO (10). The basis for this proposal was isotopic labeling stu-
(
7, 8) or manganese/iron cluster (9), two recent studies esta-
blished unequivocally that the di-iron form is active (although
they did not rule out the possibility that the manganese/iron form
could also be active) (10, 11). The first study showed that
Fe -AurF could convert Ar-NH to Ar-NO in the presence of
16
dies, which had shown that reaction of Ar-NH OH in the pre-
18
18
sence of O ðgÞ results in incorporation of only one atom of
O
2
into the Ar-NO (6). This labeling pattern is consistent with the
formation of the presumptive Ar-N O intermediate by a dehy-
drogenation mechanism, which would leave it devoid of any
label, so that the third oxidation step with O would then gen-
2
2
2
2
16
O and a reducing system (although not the native reductase,
2
18
O
which has not yet been identified) (10). It reasserted the pre-
viously proposed reaction sequence comprising three canonical
1
8
2
diiron-oxygenase cycles (6), each involving, first, combination
II∕II
of O with the diiron(II/II) form of the enzyme (Fe
-AurF)
2
2
Author contributions: N.L., V.K.K., C.K., and J.M.B. designed research; N.L. and V.K.K.
performed research; N.L. and V.K.K. contributed new reagents/analytic tools; N.L.,
V.K.K., C.K., and J.M.B. analyzed data; and N.L., C.K., and J.M.B. wrote the paper.
to form an intermediate that oxidizes the substrate (Ar-NH2,
Ar-NHOH, and Ar-NO in cycles one, two, and three, respec-
The authors declare no conflict of interest.
tively) by two electrons and, second, reduction of the resultant
III∕III
diiron(III/III) form of the enzyme (Fe
2
-AurF) back to the
This article is a PNAS Direct Submission.
2
II∕II
O -reactive Fe2
-AurF (Scheme 1A). It purported to detect
Freely available online through the PNAS open access option.
1
the Ar-NO intermediate of the second cycle [Ar-NHOH having
already been detected previously (6, 7)] and discussed the nature
of its formation (10). Whereas the Hertweck group proposed hy-
N.L. and V.K.K. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail: jmb21@psu.edu or ckrebs@psu.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1002785107/-/DCSupplemental.
droxylation of Ar-NHOH to Ar-NðOHÞ followed by dehydration
2
1
5722–15727 ∣ PNAS ∣ September 7, 2010 ∣ vol. 107 ∣ no. 36
www.pnas.org/cgi/doi/10.1073/pnas.1002785107

1
6
18
18
erate Ar-Nð OÞð OÞ with only one atom of O (Scheme S1,
Zhao pathway). The labeling pattern is inconsistent with forma-
1
6
18
tion of Ar-NO by hydroxylation to Ar-Nð OHÞð OHÞ followed
1
6
by dehydration, which should result in a 1∶1 mixture of Ar-N O
1
8
and Ar-N O (assuming no isotope effect nor stereochemical bias
in the dehydration step) and, in the absence of exchange with
solvent of the O-atom from the nitroso group, subsequently result
1
6
18
18
in formation of a 1∶1 mixture of Ar-Nð OÞð OÞ and Ar-Nð OÞ
2
in the final oxidation step (Hertweck pathway).
The second study to confirm the activity of Fe -AurF reported
2
trapping and characterization of a long-lived peroxodiiron(III/
III∕III
III) complex (peroxo-Fe2
-AurF) that is competent to oxidize
Ar-NH (presumably to Ar-NHOH, but this intermediate was not
2
Fig. 1. Sequential-mixing SF-Abs experiments to monitor the reaction of
explicitly verified) (11). The nearly complete conversion of
III∕III
II∕II
peroxo-Fe2
-AurF with Ar-NHOH. (A) A solution of Fe2 -AurF (0.9 mM
Ar-NH to the fully oxidized Ar-NO at low ratios of Ar-NH ∕
2
2
2
III∕III
Fe2) was mixed with an equal volume of reaction buffer (see Experimental
Procedures for composition) containing 1.8 mM O2 (O2∕Fe2 ¼ 2). This solution
was allowed to react at 5 °C for 0.5 s to permit accumulation of peroxo-
peroxo-Fe2
-AurF (<0.3) suggested that the peroxide com-
plex might also be capable of the two subsequent oxidations
Ar-NHOH → Ar-NO → Ar-NO ). In this study, we have con-
(
III∕III
2
Fe2
2
-AurF and was then mixed with an equal volume of an O -free solution
firmed that the two-electron-oxidized substrate species (Ar-NHOH)
does indeed react with peroxo-Fe2III∕III-AurF. The reaction does
not, however, generate Ar-NO and a diiron(III/III) cluster, the
products expected from the published reaction sequence (7, 9,
of Ar-NHOH (in buffer) at concentrations appropriate to give the
Ar-NHOH∕Fe2 ratios indicated in the inset. (B) A solution of Fe2II∕II-AurF
(0.9 mM Fe2) was mixed with an equal volume of reaction buffer containing
either 0.9 mM (black and blue) or 1.8 mM O2 (gray and red). This solution was
allowed to react at 5 °C for 0.5 s to permit accumulation of peroxo-
1
0) and our previous study (11). Rather, the reaction generates
III∕III
II∕II
Fe2
-AurF and was then mixed with an equal volume of an O2-free solution
Fe2
-AurF and the fully oxidized Ar-NO , implying that a four-
2
of buffer (black and gray) or 0.45 mM Ar-NHOH (blue and red).
electron redox process occurs. In the presence of excess O , the
2
II∕II
-AurF so produced regenerates peroxo-Fe2III∕III-AurF,
Fe2
the second mix (Fig. 1B, blue trace). These observations can all
priming for oxidation of another molecule of Ar-NHOH. Thus,
the reaction is catalytic without exogenous reducing equivalents.
The results mandate reformulation of the overall conversion of
Ar-NH to Ar-NO by Fe -AurF (Scheme 1B) and reevaluation
be explained by assuming that the reaction between Ar-NHOH
III∕III
II∕II
and peroxo-Fe2
-AurF generates Fe2
-AurF. With overall
O ∕Ar-NHOH∕AurF ratios of 2∕1∕1, one equiv O remains
2
2
2
2
2
III∕III
after the first has been used to form peroxo-Fe
-AurF. This
of the mechanism of the last two oxidation steps. Specifically, they
2
II∕II
remaining O2 adds to the Fe2
-AurF produced by reaction
suggest that the Ar-NH → Ar-NO conversion proceeds by a se-
2
2
III∕III
of the first equiv of peroxo-Fe2
-AurF with Ar-NHOH,
quence of two consecutive, mechanistically analogous hydroxyla-
tions followed by an inner-sphere, proton-coupled, two-electron
transfer (Scheme 1B), rather than by the alternating, mechanis-
tically distinct hydroxylation, dehydrogenation, and hydroxylation
steps previously proposed (Scheme 1A). The new mechanism,
III∕III
generating a second equiv of peroxo-Fe2
-AurF and yielding
the transient kinetic behavior. With O ∕Ar-NHOH∕AurF ¼
2
2
∕ ≥ 2∕1, sufficient substrate remains to consume the second
III∕III
equiv of peroxo-Fe
and the Fe2
-AurF, O2 is at that point exhausted,
2
II∕II
1
8
-AurF is then stable. With O ∕Ar-NHOH∕
which accounts simply for the aforementioned O isotope label-
2
2
AurF ¼ 1∕1∕1, O is exhausted in the initial formation of
ing experiments (Scheme S1, central pathway), invokes a
2
III∕III
II∕II
peroxo-Fe2
-AurF, and the Fe2
-AurF generated upon
four-electron N-oxidation, which is initiated by a peroxodiiron
reaction with Ar-NHOH is again stable.
(
III/III) intermediate and is, to the best of our knowledge, unpre-
cedented.
Evaluation of Di-iron Products in Reaction of Peroxo-Fe2III∕III-AurF with
Ar-NHOH by Mössbauer Spectroscopy. Mössbauer spectroscopic
experiments were conducted to test the conclusion that reaction
Results
Testing for a Reaction Between Peroxo-Fe2III∕III-AurF and Ar-NHOH
III∕III
II∕II
of peroxo-Fe2
-AurF with Ar-NHOH produces Fe2
-
by Stopped-Flow Absorption (SF-Abs) Experiments. Our recent
II∕II
study showed that mixing Fe
-AurF with O results in rapid
AurF, which is stable when O2 is limiting and transient when
2
2
III∕III
formation of peroxo-Fe2
-AurF, which can be monitored by its
O2 is in excess. The 4.2-K∕53-mT spectrum of a sample prepared
−
1
−1
II∕II
absorption at 500 nm with molar absorptivity of ∼500 M cm
by reacting Fe2
that peroxo-Fe2
-AurF with limiting (0.75 equiv) O2 reveals
III∕III
(
11). Subsequent mixing of the intermediate with Ar-NH results
-AurF is the predominant species in the sam-
2
in a rapid decrease in absorbance at this wavelength (A500),
ple (vertical bars in Fig. 2A). The red line plotted over the data is
a “reference spectrum” of the intermediate complex, which was
generated by analysis of the experimental spectrum of a sample
that was prepared so as to yield a maximum fraction of the inter-
mediate (see Fig. S1 for the reaction conditions, explanation of
the analysis, and the Mössbauer parameters). The reference spec-
trum accounts for 62% of the total intensity of the experimental
spectrum in Fig. 2A. (We estimate an uncertainty of ꢀ3 on this
and all other percentages of total absorption area given in the
III∕III
reflecting oxidation of Ar-NH by peroxo-Fe
-AurF. A500
-
2
2
versus-time traces from similar, sequential-mixing, SF-Abs experi-
III∕III
ments, in which peroxo-Fe
-AurF was formed by an initial
2
II∕II
mix of Fe2
-AurF with O2 and then exposed to Ar-NHOH
in the second mix, show that the two-electron-oxidized substrate
species also reacts efficiently (Fig. 1). Revealingly, the kinetic
behavior depends markedly on the relative concentrations of
AurF, O , and Ar-NHOH. When the initial mix to form
2
III∕III
peroxo-Fe2
-AurF delivers ∼2 equiv O and the second mix
text.) As expected, use of limiting O2 results in a significant frac-
2
II∕II
delivers 1 equiv Ar-NHOH, A500 decreases rapidly, reaches a
minimum after ∼50 ms, and then increases to approximately
the original value (red traces in Fig. 1 A and B). By contrast to
this transient behavior, A500 decreases and remains stable when
tion (36%) of unreacted Fe2
-AurF starting material (δ ¼
1.23 mm∕s, ΔEQ ¼ 3.08 mm∕s; blue line). A minor fraction
III∕III
(6%) of μ-oxo-Fe2
-AurF is also present (δ ¼ 0.54 mm∕s,
ΔE ¼ 1.86 mm∕s, green line). Treatment of an identical sample
Q
∼
2 equiv O is delivered in the first mix and ≥2 equiv Ar-NHOH
with one equiv Ar-NHOH for 45 ms or 400 ms prior to rapid
freezing (freeze-quenching) results in marked spectral changes
2
is delivered in the second mix (Fig. 1A, blue and green traces).
Similarly, A500 decreases and remains stable if ≤1 equiv O is de-
(vertical bars in Fig. 2 B and C, respectively, and Fig. S2). The
2
III∕III
livered in the first mix and ≥1 equiv Ar-NHOH is delivered in
features of peroxo-Fe2
-AurF decay (to 33% and 8%, respec-
Li et al.
PNAS
∣
September 7, 2010
∣
vol. 107
∣
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∣
15723

Fig. 2. 4.2-K∕53-mT Mössbauer spectra of samples in which peroxo-Fe III∕III-AurF was reacted with Ar-NHOH or Ar-NH2. In all cases, the red, blue, and green
2
III∕III
II∕II
III∕III
lines illustrate the fractional contributions of the reference spectra of peroxo-Fe2
-AurF, Fe2 -AurF, and μ-oxo-Fe2
-AurF, respectively, to the experi-
II∕II
mental spectrum, as described in the text. (Left) A solution of Fe2 -AurF (1.2 mM Fe2) was mixed with 0.5 equivalent volume of buffer solution containing
1
.8 mM O2 (O2∕Fe2 ¼ 0.75). This solution was allowed to react at 5 °C for 0.11 s to permit accumulation of peroxo-Fe2III∕III-AurF. (A) The solution was then
directly freeze-quenched. (B and C) The solution was then mixed with one-sixth equivalent volume of an O2-free solution of 3.6 mM Ar-NHOH
(
Ar-NHOH∕O2 ¼ 1), and this solution was allowed to react for 45 ms (B) or 400 ms (C) prior to being freeze-quenched. D and E are the difference spectra
III∕III II∕II
B-A and C-B, respectively. The black line in D is the sum of the contributions of peroxo-Fe
the difference spectrum B-A for comparison. (Middle) A solution of Fe2 -AurF (1.8 mM) was mixed with two equivalent volumes of a buffer solution contain-
-AurF (−28%) and Fe2 -AurF (28%). The black line in E is
2
II∕II
III∕III
ing 1.8 O2 mM O2 (O2∕Fe2 ¼ 2). This solution was allowed to react at 5 °C for 110 ms to permit accumulation of peroxo-Fe2
-AurF. (F) The reaction was then
directly freeze-quenched. (G and H) The solution was then mixed with one-sixth equivalent volume of an O2-free buffer solution containing 3.6 mM Ar-NHOH
(
Ar-NHOH∕O2 ¼ 0.5), and this reaction was allowed to proceed at 5 °C for 45 ms (G) or 2 s (H) prior to being freeze-quenched. I and J are the difference spectra
II∕II
G-F and H-G, respectively. The black line in J is difference spectrum C–B, scaled by a factor of −0.8 for comparison. (Right) A solution of Fe2 -AurF (1.2 mM) was
mixed with 0.5 equivalent volume of a buffer solution containing 1.8 mM O2 (O2∕Fe2 ¼ 0.75), and the reaction was allowed to proceed at 5 °C for 110 ms to
III∕III
permit accumulation of peroxo-Fe2
-AurF. (K) The reaction was directly freeze-quenched. (L) The resulting solution was then mixed with one-sixth equiva-
lent volume of an O2-free buffer solution containing 0.91 mM Ar-NH2 (Ar-NH2∕O2 ¼ 0.25), and the reaction was allowed to proceed for 4 s before being freeze-
III∕III
II∕II
quenched. M is the difference spectrum L–K. The black line in M is the sum of the contributions of peroxo-Fe
-AurF (−29%), Fe2 -AurF (12%), and
2
III∕III
μ-oxo-Fe2
-AurF (17%).
tively; red lines), and features attributable to Fe2II∕II-AurF grow
tra strongly suggests that the process being monitored has only
one kinetically significant step (two states, reactants and pro-
ducts), because a sequence of two or more steps (three or more
states) ought not to give a constant difference spectrum from 0 to
45 ms and 45 ms to 400 ms. The kinetics of the conversion of
III∕III
in. The contribution from μ-oxo-Fe2
-AurF remains constant.
The changes are best illustrated by the difference spectrum gen-
erated by subtracting 2A from 2B (Fig. 2D, vertical bars). In this
presentation, the features pointing upward are associated with
III∕III
III∕III
II∕II
the decaying species (peroxo-Fe2
-AurF; red line), and the
peroxo-Fe2
-AurF to Fe2
-AurF reflected in the Möss-
II∕II
features pointing downward with the developing species (Fe2
-
bauer data are reasonably consistent with those determined by
AurF; δ ¼ 1.23 mm∕s, ΔE ¼ 3.00 mm∕s; blue line). We note
SF-Abs. One minor inconsistency is that 8% of peroxo-
Q
III∕III
that the measured quadrupole splitting parameter, ΔE , of the
Fe2
-AurF remains at 400 ms, whereas the SF-Abs data indi-
Q
II
developing Fe species (3.00 mm∕s) is slightly different from that
cate that decay should be essentially complete by this reaction
II∕II
of the reactant Fe2
-AurF complex (ΔE ¼ 3.08 mm∕s), sug-
time (Fig. 1B, blue trace). We attribute this remaining peroxo-
Q
III∕III
III∕III
II∕II
gesting that cycling through the peroxo-Fe
might cause a conformational change at the Fe2
-AurF state
Fe2
-AurF to the reaction of a small fraction of the Fe2
-
2
II∕II
cluster, as
AurF product with O from the air during passage through the
2
was observed for the diiron-carboxylate oxidase, stearoyl-acyl
freeze-quench reaction hose and into the cryosolvent.
9
carrier protein Δ desaturase (12). The summation of the appro-
The 4.2-K∕53-mT Mössbauer spectrum of a sample prepared
II∕II
priately weighted reference spectra for the decaying peroxo-
by reacting Fe2
-AurF with ∼2 equiv O per diiron cluster
2
III∕III
II∕II
Fe2
-AurF (−28%) and the developing Fe
-AurF (28%)
(Fig. 2F, vertical bars) indicates the presence of 82% peroxo-
2
III∕III
III∕III
agrees well with the experimental difference spectrum (compare
solid black line and vertical bars in Fig. 2D). The difference be-
tween the spectra of the 400-ms and 45-ms samples (C-B, Fig. 2E
vertical bars) is, within the experimental uncertainty, identical to
Fe2
and Fe2
-AurF (red line) and 9% each of μ-oxo-Fe2
-AurF
II∕II
-AurF. The spectra of identical samples that were
subsequently mixed with one equiv Ar-NHOH and then allowed
to react for 45 ms (Fig. 2G) or 2 s (Fig. 2H) before being freeze-
quenched (see Fig. S3 for additional spectra corresponding to
the B-A difference spectrum (Fig. 2E, solid line) and again
III∕III
demonstrates conversion of 28% of peroxo-Fe
-AurF to
different reaction times) confirm the conclusion from the SF-Abs
2
II∕II
III∕III
2
8% of Fe2
-AurF. The similarity of these two difference spec-
experiments that peroxo-Fe2
-AurF initially decays (to 52%
1
5724
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www.pnas.org/cgi/doi/10.1073/pnas.1002785107
Li et al.

in Fig. 2G, red line) and is subsequently regenerated (74% in
substrate.) The chromatogram from a control sample in which
Fig. 2H, red line). The G-F difference spectrum (Fig. 2I, vertical
450 μM Ar-NHOH was exposed to O under the conditions of
2
III∕III
II∕II
bars) reveals the conversion of 28% of peroxo-Fe2
-
the enzyme reaction but in the absence of Fe2
-AurF (green
II∕II
AurF (red line) to 20% Fe
-AurF (blue line) and 6%
trace) shows the prominent peak of the synthetic compound
and two small peaks from unknown contaminants or decay pro-
ducts. This control confirms that Ar-NHOH is relatively stable on
this time scale in the absence of the enzyme. The chromatogram
of the experimental sample containing the enzyme (blue trace)
shows that >95% (>430 μM) of the Ar-NHOH has been con-
2
III∕III
III∕III
μ-oxo-Fe2
-AurF (green line). The μ-oxo-Fe2
could result from the reaction of some of the peroxo-Fe2
-AurF
III∕III
-
AurF with small amounts of either Ar-NH or Ar-NO present
2
in the Ar-NHOH substrate solution (see Fig. S4). The H-G dif-
ference spectrum (Fig. 2J, vertical bars) reveals clean conversion
II∕II
III∕III
of 20% Fe2
-AurF (blue line) to 20% peroxo-Fe2
-AurF
sumed and ∼450 μM Ar-NO has been produced (estimated by
2
(
red line; note that the reformation of peroxo-Fe2III∕III-AurF is
comparison of the peak area to that in the chromatogram of the
standard mixture). Thus, the enzyme has accomplished 28–30
turnovers in the absence of any obvious source of reducing
equivalents. Catalytic oxidation of Ar-NHOH under these condi-
tions is inconsistent with the previous formulation of the Ar-
reflected by inversion of the peaks). This difference spectrum
is very similar to the difference spectrum 2E from the limiting-
O reaction (solid line in Fig. 2J, scaled by −0.8). Recovery of
2
III∕III
the peroxo-Fe2
-AurF is nearly complete (73% of total Fe,
8
9% of the peroxo-Fe2III∕III-AurF initially present). This obser-
NHOH → Ar-NO → Ar-NO oxidation steps (Scheme 1A) but
2
vation, together with the Mössbauer and SF-Abs data on the
entirely consistent with our reformulation (Scheme 1 B and C).
reaction with limiting O , confirm that Ar-NHOH effects a
2
III∕III
II∕II
Testing for Reduction of μ-oxo-Fe2III∕III-AurF by Ar-NHOH. The surpris-
four-electron reduction of peroxo-Fe2
-AurF to Fe2
-
III∕III
AurF, presumably with concomitant four-electron oxidation of
ing ability of Ar-NHOH to reduce peroxo-Fe2
-AurF by four
II∕II
Ar-NHOH to Ar-NO (confirmed below).
electrons to Fe2 -AurF and the known ability of hydroxylamine
compounds to reduce Fe complexes suggested the possibility
2
III
Verification of Catalytic Oxidation of Ar-NHOH by Fe II∕II-AurF. Our
III∕III
that Ar-NHOH might also reduce μ-oxo-Fe2
-AurF to
2
II∕II
reformulation of the AurF six-electron-oxidation sequence elim-
inates the previously proposed requirement for a total of four
Fe2
-AurF, thereby possibly permitting the six-electron-oxida-
tion sequence to proceed without net input of any exogenous
exogenous electrons in the last two steps (compare Scheme 1
electrons and with only a single equivalent of O . However, pro-
2
II∕II
III∕III
A and B). Fe2
-AurF should, therefore, be capable of multiple
longed incubation (10 min at 22 °C) of as-isolated μ-oxo-Fe2
-
turnovers when oxidizing Ar-NHOH (Scheme 1C, right side). To
AurF with one equiv (1.1 mM) Ar-NHOH yielded less than 10%
II∕II
II∕II
verify this prediction, 15 μM Fe
-AurF was incubated with
reduction to Fe2
-AurF (Fig. S5), implying that two exogenous
2
4
50 μM Ar-NHOH (Ar-NHOH∕Fe ¼ 30) and ∼0.9 mM O
electrons and two equiv O are indeed required for the complete
2
2
2
(
O ∕Ar-NHOH ∼ 2). After 20 min at 0 °C, the small-molecule
six-electron oxidation sequence.
2
components were separated from the protein (by passage through
a molecular weight filter, requiring an additional 10 min at 4 °C)
and were analyzed by reverse-phase high-performance liquid
chromatography (RP-HPLC; see Experimental Procedures and
SI Text for details). The chromatogram of a standard solution
containing 400 μM each of Ar-NH , Ar-NHOH, and Ar-NO
Reevalution of the Diiron Products from the Reaction of peroxo-
III∕III
Fe2
-AurF with Limiting Ar-NH . Scheme 1C predicts that reaction
2
III∕III
of peroxo-Fe2
-AurF with limiting Ar-NH in the absence of
2
excess O should generate equal quantities of two distinct diiron
2
2
2
products (highlighted in boxes). The first oxidation should pro-
III∕III
(
Fig. 3, red trace) shows that the compounds are efficiently sepa-
duce Ar-NHOH and μ-oxo-Fe2
-AurF. The Ar-NHOH so
III∕III
rated by this procedure. Injections of the individual compounds
allowed the elution peaks to be assigned as indicated by the
chemical structures. (Note that, because of differences in their
molar absorptivities, the peak height and area for the Ar-NO2
product is less than that for an equivalent quantity of Ar-NHOH
produced should then dissociate from μ-oxo-Fe2
-AurF and
III∕III
react with a second equivalent of peroxo-Fe2
-AurF to
II∕II
generate Ar-NO and Fe2
-AurF. In our previous study (11),
2
O was present in excess, which (we now understand) must have
2
II∕II
converted the Fe2
-AurF (the reduced product of the Ar-
III∕III
NHOH → Ar-NO2 oxidation) back to peroxo-Fe2
-AurF
(
red arrows), thereby preventing detection of the reduced en-
zyme form. We therefore tested the prediction of Scheme 1C
by Mössbauer spectroscopy on samples prepared with limiting
O . A sample was enriched in peroxo-Fe
of Fe2
III∕III
-AurF by reaction
2
2
II∕II
-AurF with 0.75 equiv O . The Mössbauer spectrum of
2
III∕III
this sample (Fig. 2K, vertical bars) confirms that peroxo-Fe2
-
AurF is the major species (72%, red line). The spectrum of an
identical sample that was subsequently treated with 0.19 equiv
Ar-NH and allowed to react to completion (4 s) prior to being
2
freeze-quenched shows marked differences (Fig. 2L). Analysis of
the L-K difference spectrum (Fig. 2M, vertical bars) indicates that
III∕III
2
9% of peroxo-Fe2
-AurF (red line) is converted to 17%
III∕III
II∕II
μ-oxo-Fe2
-AurF (green line) and 12% Fe2
-AurF (blue
line). The sum of these spectral contributions (Fig. 2M, black
solid line) reproduces the experimental difference spectrum well.
III∕III
The total loss of intensity attributable to peroxo-Fe2
-AurF is
only 76% of the theoretical value: 0.19 equiv Ar-NH should con-
2
Fig. 3. Reversed-phase high-performance liquid chromatography (RP-HPLC)
of the small-molecule reactants and products following incubation of
III∕III
sume 0.38 equiv peroxo-Fe2
-AurF complex, resulting in a
loss of 38% (compared to the observed 29%) of total intensity.
II∕II
II∕II
Fe2 -AurF with excess Ar-NHOH and O2. Fe2 -AurF (15 μM) was incubated
with 450 μM Ar-NHOH and ∼0.9 mM O2 for 20 min at 0 °C. Small molecules
were separated from the enzyme and analyzed as described in SI Text (blue).
A control experiment was carried out under identical conditions, except for
III∕III
The yield of μ-oxo-Fe2
− AurF is within experimental error
of the theoretical value (17% compared to 19%), but the yield of
II∕II
Fe2
-AurF is only ∼60% of the theoretical value (12% com-
II∕II
II∕II
omission of Fe2 -AurF (green). A solution containing 400 μM each of
pared to 19%). As argued above, the observed yield of Fe
-
2
III∕III
Ar-NH2, Ar-NHOH, and Ar-NO2 was also analyzed (red).
AurF and observed consumption of peroxo-Fe2
-AurF are
Li et al.
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most likely diminished from their theoretical values by exposure
with initial UV absorption detection followed by mass spectro-
metric (MS) detection of a fragment ion of appropriate mass-
to-charge ratio (m∕z) to be the decarboxylation product of the
Ar-NO molecular ion (10). We suggest that the Ar-NO detected
to atmospheric O in the freeze-quench procedure, which results
2
II∕II
in conversion of a fraction of the Fe2
-AurF product (∼7% of
the total absorption intensity, essentially the same as in Fig. 2C)
III∕III
back to peroxo-Fe2
-AurF. The important point is that both by Zhao and coworkers could have arisen from nonenzymatic oxi-
products predicted by Scheme 1C are readily detected.
dation or disproportionation of Ar-NHOH (a true accumulating
intermediate in the AurF sequence) rather than as a product of
the AurF reaction. Supporting this view, we have repeatedly de-
tected by HPLC-MS measurements on solutions of Ar-NHOH
Discussion
2
AurF catalyzes the six-electron oxidation of Ar-NH to Ar-NO2
(2). All previous studies assumed that this conversion entails
dissolved in O -containing buffer (with or without AurF) a spe-
2
three sequential two-electron oxidations (Scheme 1A) (8–10). Of
the two proposed intermediates in this sequence, Ar-NHOH and
Ar-NO, the former compound was unequivocally identified (6)
cies of the correct m∕z to be the Ar-NO parent ion (Fig. S4). Our
inference is also corroborated by the Hertweck group’s detection
of the dimerized substrate species, azoxybenzol-4,4′-dicarboxylic
acid, which, they proposed, could have formed nonenzymatically
via an Ar-NO intermediate (7).
and reasonably presumed to form in the rapid reaction of peroxo-
III∕III
Fe2
-AurF with Ar-NH (11). In this study, we investigated
2
the reaction of this first intermediate, Ar-NHOH, with peroxo-
Whereas it is possible that the substrate, Ar-NH (Ar-NHOH),
2
III∕III
IV∕IV
Fe2
-AurF. Surprisingly, this reaction couples the oxidation
could either rapidly trap a more reactive (e.g., Fe2
) complex
III∕III
of Ar-NHOH by four electrons (to Ar-NO ) to the complete re-
2
with which peroxo-Fe2
-AurF rapidly interconverts or, upon
III∕III
II∕II
duction of peroxo-Fe2
-AurF to Fe2
-AurF (Scheme 1B). binding, could trigger the rapid conversion of the peroxide inter-
The proposed intermediates in this reaction, Ar-NO or
mediate to the N-oxygenating complex, we presume for the
III∕III
Ar-NðOHÞ and a Fe
cluster, apparently do not accumulate
purposes of this discussion that the peroxide complex is itself
2
2
substantially during the reaction (even in the active site during a
single turnover), as implied by the near identity of the Mössbauer
difference spectra at different reaction times (Fig. 2, spectra D, E,
I, and J) and the ability to account for these spectral changes by
summation of the spectra of only the reactant and product states
the N-oxygenating species. The hydroxylation of Ar-NH is likely
2
to involve nucleophilic attack of the amine on the peroxide moi-
III∕III
ety of peroxo-Fe2
-AurF (11). Hydroxylation of Ar-NHOH
could also proceed by nucleophilic attack of the N-atom on
the peroxide O-atom. Indeed, Ar-NHOH is expected to be even
more nucleophilic than Ar-NH2 as a result of the so-called
III∕III
II∕II
(
peroxo-Fe2
-AurF and Fe2
-AurF).
This finding can explain several published observations and
alpha effect (13). Examination of the published structure of
III∕III
has implications for the mechanism of the Ar-NHOH →
the μ-oxo-Fe2
-AurF • Ar-NO complex (10) suggests a rela-
2
Ar-NO conversion by AurF. The reaction entails, formally, trans-
tively simple trajectory for this hydroxylation mechanism
2
III∕III
fer of an O-atom from the peroxide moiety of peroxo-Fe2
-
(Scheme 2). The peroxide ligand is depicted in a μ-1,1 or dis-
2
2
AurF to Ar-NHOH and transfer of two H-atoms from Ar-NHOH
to the diiron cluster. The Hertweck group proposed that this
conversion might proceed by a sequence of hydroxylation of
torted μ-η ∶η coordination mode, because the spectroscopic
III∕III
properties of peroxo-Fe2
well-characterized μ-1;2-peroxo-Fe2
However, the peroxide moiety could also be protonated (i.e.,
-AurF are different from those of
III∕III
complexes (14, 15).
Ar-NHOH to Ar-NðOHÞ , elimination of water to form Ar-NO,
2
III∕III
and transfer of a second O-atom to yield Ar-NO (Scheme S1,
in a μ-1, 1-hydroperoxo-Fe2
complex), as we previously
2
right branch) (7, 8). They attempted to identify the proposed
Ar-NO intermediate but failed to do so, despite employing a sen-
sitive assay. They concluded that “rapid and possibly spontaneous
proposed (11). Nucleophilic attack of the bound Ar-NHOH on
the uncoordinated or more distally coordinated O-atom of the
(hydro)peroxide moiety would cleave the peroxide O-O bond
and transfer an O-atom to the substrate. The product would
be the Ar-NðOHÞ2 intermediate proposed by the Hertweck
turnover” of this intermediate to Ar-NO could rationalize their
2
failure to detect it (7). Their result is consistent with our observa-
tion that the presumptive intermediates in the four-electron
group, or perhaps its deprotonated form, bound to the resulting
III∕III
conversion of Ar-NHOH to Ar-NO do not accumulate.
Fe2
-AurF form. Transfer of two electrons and two protons
2
III∕III
The Zhao group proposed a different mechanism, founded on
the presumption that Ar-NO is an obligatory intermediate on the
from Ar-NðOHÞ to the Fe
cluster (formally, a dehydro-
2
2
genation), rather than the sequence of dehydration followed
pathway to Ar-NO (Scheme 1A) and the observation that oxida-
by O-atom transfer proposed by the Hertweck group, could then
2
1
8
18
tion of Ar-NHOH in the presence of O gas results in produc-
give Ar-NO directly, with the O-isotope labeling pattern ob-
2
2
1
8
tion of Ar-NO2 with at most one atom of O (6, 10). As
explained by Scheme S1, the authors interpreted this result to
imply that the oxidation of Ar-NHOH to Ar-NO must be a direct
dehydrogenation, rather than the hydroxylation-dehydration
sequence proposed by Hertweck. The presumed intermediacy
of Ar-NO also understandably led the Zhao group to search
for this intermediate, which they purported to identify by HPLC
served by Zhao and coworkers (Scheme 2 and Scheme S1, mid-
dle). The new proposal appears to accommodate all available
experimental data. The latter steps might conceivably occur by
deprotonation of the Ar-NðOHÞ intermediate, perhaps by the
2
μ-oxo bridge (as depicted in Scheme 2) or one of the protein
carboxylate residues (16), followed by inner-sphere electron
transfer steps.
Scheme 2. Proposed mechanism of the four-electron oxidation of Ar-NHOH to Ar-NO2 by peroxo-Fe III∕III-AurF. The scheme is derived from the X-ray
2
III∕III
crystal structure of the μ-oxo-Fe2
-AurF • Ar-NO2 complex, Protein Data Bank identification code 3CHT (10).
1
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Li et al.

By contrast, the first step of the Zhao pathway (6, 10), dehy-
drogenation of Ar-NHOH, is distinct from the aforementioned
tional experiments will be required to distinguish among these
and other mechanistic possibilities.
nucleophilic attack of the substrate on the electrophilic
III∕III
Experimental Procedures
peroxo-Fe2
complex. Distinct reactivities of intermediates
with similar structures are well documented for the mononuclear
non-heme-iron enzymes, in which the Fe -oxo (ferryl) unit can
Materials. AurF was prepared as previously described (11).
Ar-NHOH was synthesized according to a published procedure
(22). Its purification and characterization are detailed in SI Text
and Figs. S6 and S7.
IV
act either as electrophile (17), transferring its O-atom to an
electron-rich substrate, or as hydrogen-atom-abstractor, initiating
hydroxylation, halogenation, desaturation, and cyclization out-
comes (18). One-electron oxidation of Ar-NHOH by peroxo-
AurF Turnover Assay. Catalytic conversion of Ar-NHOH to
Ar-NO2 was demonstrated by using RP-HPLC to resolve the
two compounds and UV absorption to detect them. Comparison
of the chromatograms of assay and control samples to that of a
standard mixture of Ar-NH , Ar-NHOH, and Ar-NO was used
III∕III
Fe2
-AurF with deprotonation and coordination of the result-
•
ing aminoxyl radical (Ar-NHO ) to the Fe cluster, which is
2
formally an H-atom abstraction akin to those effected by the
ferryl intermediates, seems a conceivable reaction pathway on
the basis of precedent from inorganic chemistry (19, 20). We
2
2
to quantify the Ar-NHOH substrate remaining and Ar-NO pro-
2
anticipate that this step would cleave the O-O bond and generate
duct generated (Fig. 3). The standard mixture was prepared by
dissolving the solids in 50 mM HEPES buffer (5% glycerol,
pH 7.5) to a final concentration of 400 μM of each. The reaction
was carried out in the same buffer, which was saturated with O2 at
III∕IV
a high-valent (hydr)oxo-bridged Fe
-cluster, given that the
2
III∕III
related peroxo-Fe2
intermediate in the I100W variant of
toluene∕o-xylene monooxygenase converts to a Fe2
III∕IV
complex
upon transfer of an electron from the introduced tryptophan
residue (21). Several decay pathways for the hypothetical
0
°C by vigorous stirring on ice under 1.1 atm of the gas. Imme-
diately before initiation of the reaction by addition of enzyme,
III∕IV
•
ðhydrÞoxo-Fe2
-AurF∶Ar-NHO complex can be envisaged.
Ar-NHOH was dissolved in the O -saturated buffer to a final
2
Transfer of another proton [presumably to one of the bridging
oxygenic ligands or a protein carboxylate residue (16)] and
another electron via an inner-sphere mechanism would yield for-
II∕II
concentration of 450 μM. Fe2
-AurF (1.0 mM) was prepared
by treating as-isolated AurF with one equiv sodium dithionite for
3
0 min at room temperature in the absence of O . After a brief
2
III∕III
III∕III
mally Ar-NO and a Fe2
cluster. In the final step, this Fe2
II∕II
(∼30 s) exposure to air the Fe2
-AurF was added to the reac-
cluster would have to serve as an O-atom donor to generate
tion system to a final concentration of 15 μM. The solution was
stirred on ice for 20 min and then filtered through an Amicon
Ultra-0.5 centrifugal filter (10,000 molecular weight cut-off;
Millipore) at 4 °C (13,000 rpm, 10 min). The filtered solution
II∕II
Fe2
-AurF and Ar-NO . Alternatively, the order of these
2
steps (electron transfer, proton transfer, and O-atom transfer)
III∕IV
•
within the ðhydrÞoxo-Fe
-AurF∶Ar-NHO complex could be
2
different.
was analyzed by RP-HPLC as described in SI Text. An otherwise
Of the two general pathways described above, nucleophilic
attack of Ar-NHOH on the peroxide moiety versus formal
H-atom transfer from Ar-NHOH to the peroxide, we prefer
the former pathway (Scheme 2), because it most simply accounts
for the outcome of the reaction, implies more similar reactivities
II∕II
identical sample lacking only Fe2
for this reaction.
-AurF served as the control
ACKNOWLEDGMENTS. We thank Tyler L. Grove and Denise A. Conner for
assistance in preparation and characterization of the synthetic Ar-NHOH,
Gang Xing for assistance in the RP-HPLC analysis, and Megan L. Matthews
for preparation of Fig. 3 and Scheme S1 and for advice on Schemes 1 and 2.
of the Ar-NH2 substrate and Ar-NHOH intermediate toward
III∕III
peroxo-Fe2
-AurF, and accounts for all available data. Addi-
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