Single Turnover Reveals Oxygenated Intermediates in Toluene/o-Xylene Monooxygenase in the Presence of the Native Redox Partners

Article abstract of DOI:10.1021/jacs.5b07055

Toluene/o-xylene monooxygenase (ToMO) is a non-heme diiron protein that activates O₂ for subsequent arene oxidation. ToMO utilizes four protein components, a catalytic hydroxylase, a regulatory protein, a Rieske protein, and a reductase. O₂ activation and substrate hydroxylation in the presence of all four protein components is examined. These studies demonstrate the importance of native reductants by revealing reactivity unobserved when dithionite and mediators are used as the reductant. This reactivity is compared with that of other O₂-activating diiron enzymes.

Full text of DOI:10.1021/jacs.5b07055

Communication
pubs.acs.org/JACS
Single Turnover Reveals Oxygenated Intermediates in Toluene/
o‑Xylene Monooxygenase in the Presence of the Native Redox
Partners
Alexandria Deliz Liang and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
*
S Supporting Information
ToMOH. The latter produces a single colorless intermediate,
ToMOHperoxo.
Under single-turnover conditions with limiting [NADH], the
rate of phenol hydroxylation to form catechol by ToMOred and
4
ABSTRACT: Toluene/o-xylene monooxygenase
ToMO) is a non-heme diiron protein that activates O2
for subsequent arene oxidation. ToMO utilizes four
protein components, a catalytic hydroxylase, a regulatory
(
O depends on the ratio of ToMOC to diiron sites of ToMOH,
2
protein, a Rieske protein, and a reductase. O activation
hereafter denoted as ToMOC:diiron (Figure 1). Using 0.01
2
and substrate hydroxylation in the presence of all four
protein components is examined. These studies demon-
strate the importance of native reductants by revealing
reactivity unobserved when dithionite and mediators are
used as the reductant. This reactivity is compared with that
of other O -activating diiron enzymes.
2
ctivation of O by non-heme diiron enzymes generates
2
A
reactive intermediates capable of hydrocarbon hydrox1-
ylation, epoxidation, desaturation, and radical formation.
Because structurally similar active sites catalyze diverse
chemical transformations, the mechanisms by which these
diiron enzymes control reactivity are of particular interest.
Toluene/o-xylene monooxygenase (ToMO) belongs to a
family of diiron proteins termed bacterial multicomponent
Figure 1. Hydroxylation of phenol to catechol at 4 °C during single
turnover by ToMO. The concentrations of the components are as
follows: 50 μM ToMOH (100 μM diiron sites), 100 μM ToMOD, 1
μM (▲) or 100 μM (○) ToMOC, 0.05 μM ToMOF, 90 μM NADH,
2
mM phenol, and approximately 625 μM O (the O -saturated buffer
2 2
monooxygenases (BMMs) that activate O to oxidize hydro-
carbon substrates (Scheme 1). ToMO catalyzes the hydrox-
2
2
contained ∼1.25 mM O at 5 °C; one-to-one mixing yielded 625 μM
O ).
2
2
Scheme 1. General Mechanism for Hydroxylation by BMMs
ToMOC:diiron, 36(3)% of the diiron sites form catechol by 2 s
−1
with a rate constant of 8(1) s . A slow increase in product
formation ensues, and at 10 min, 84(4)% of the diiron sites
produce catechol (Figure S1 in the Supporting Information). In
contrast, using 1 ToMOC:diiron, 70(4)% of the diiron sites
produce catechol by 2 s. These results suggest that inclusion of
stoichiometric ToMOC promotes product formation by
increasing the amount generated rapidly. The rate constant of
each phase is not significantly affected by the ToMOC
concentration, demonstrating that ToMOC does not directly
participate in arene hydroxylation.
ylation of toluene to form o-, m-, and p-cresol and can also
perform the catalytic, regiospecific hydroxylation of phenol to
form catechol. Four protein components are required to carry
3
out efficient catalysis, a dimeric hydroxylase (ToMOH), a
Although some diiron proteins require substrate to bind prior
regulatory protein (ToMOD), a reductase (ToMOF), and a
6
2a
to reduction, preincubation of toluene monooxygenases with
Rieske protein (ToMOC). Previous O -activation studies
2
4,7
reductants provided the reduced hydroxylase. In the single-
with ToMO utilized a simplified reaction comprising ToMOH,
turnover experiments reported here, product quantification
requires rapid protein precipitation, which releases product
bound at the active site. Thus, ToMOC must influence steps(s)
between reduction of the diiron site and product release. These
ToMOD, and sodium dithionite as a reductant with methyl
4
viologen (2e-MV) as an electron-transfer mediator. Reduction
of ToMOH by ToMOC is important for efficient arene
2a,5
hydroxylation, however. Here we sought to determine how
single turnover and O2 activation by the native system,
ToMOred (ToMOH, ToMOD, ToMOC, ToMOF, and
NADH), differs from that observed using 2e-MV to reduce
Received: July 7, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b07055
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
4
,7
steps could include O activation, aromatic substrate binding,
substrate hydroxylation, and any conformational changes
necessary for the execution of each step.
unreacted reduced enzyme. Species 420A is stable for >6 s.
Because of its long lifetime, we initially questioned whether this
species is a diiron−O intermediate. Diiron−O adducts with
2
2
2
11
In the presence of 0.01 ToMOC:diiron, O activation by
lifetimes from 3 s to 3 h have been reported, however.
2
ToMOred generates absorption changes at 420 and 675 nm
Given the significant difference in the absorption profiles of
420A and the colorless intermediate ToMOHperoxo, these
intermediates must differ. Altering the reaction pH did not
quench the absorbance corresponding to 420A (Figure S7),
showing that excess protons alone cannot induce formation of
ToMOHperoxo rather than 420A. Thus, other changes must be
required, such as a conformational change.
(Figure 2) that are not observed when 2e-MV is used as the
Generation of 420B and formation of Hox occurs with rate
constants significantly smaller than steady-state turnover
−1
12
(
toluene hydroxylation, ∼0.5 s ), suggesting that these
steps are not relevant to catalysis. Analysis of intermediate 420B
is provided in the Supporting Information. The final species has
negligible absorbance at 420 and 675 nm, consistent with the
diiron(III) resting state, Hox.
The absorption changes were also monitored in the presence
of excess toluene (Figure 3). The decay of the first species,
Figure 2. Analysis of O activation upon mixing of dioxygen with
2
ToMOred at 5 °C. (left) Time-resolved stopped-flow UV−vis
spectroscopy of the reaction between ToMO containing 0.01
red
ToMOC:diiron. The absorption changes at 420 and 675 nm are
shown in orange and gray, respectively. Fits to the absorption traces
are shown in black. (right) Mossbauer spectroscopy of the ToMOH
̈
diiron site at 0.2 s isolated by rapid freeze-quenching of a reaction
containing 0.1 ToMOC:diiron. The data were recorded at 80 °C. The
̈
Mossbauer signal is shown in gray, and the simulated fits are shown in
3
+
light blue for the Fe intermediate (δ = 0.54 mm/s and ΔE = 0.67
Q
2+
mm/s, 80%) and in red for unreacted Fe starting material (δ = 1.31
mm/s and ΔE = 2.88 mm/s, 20%).
Q
4
reductant. These absorption changes are absent when either
hydroxylase or O is omitted (Figure S2), indicating that the
2
Figure 3. Analysis of O activation upon mixing of O and toluene
with ToMOred at 5 °C. (left) Time-resolved stopped-flow UV−vis
spectroscopy under the following conditions: 1 diiron, 1 ToMOD,
2
2
features result from reaction of O with the hydroxylase.
2
Because ToMOC contains a redox-dependent chromophore,
higher ToMOC:diiron ratios were not suitable for monitoring
0
.01 ToMOC, 0.0005 ToMOF, 0.9 NADH, 20 toluene, 6.25 O . The
2
8
O activation by the diiron center.
2
absorption changes at 420 and 675 nm are shown in orange and gray,
respectively. Fits to the absorption traces are shown in black. (right)
Analysis of intermediates at 0.2 s by rapid freeze−quench Mossbauer
̈
spectroscopy under the following conditions: 1 diiron, 1 ToMOD, 0.1
When the concentration of O was varied between 375 and
25 μM, the observed kinetics was unchanged (Figure S3 and
2
6
Table S1), indicating that the interaction of ToMOH with O2
ToMOC, 0.0005 ToMOF, 0.9 NADH, 8 toluene, 1.25 O . The
must be faster than the first observable step, as found for related
2
9
Mo
̈
ssbauer signal is shown gray, and the simulated species are shown
diiron enzymes. The simplest interpretation of these data
in light blue (δ = 0.52 mm/s and ΔE = 0.67 mm/s, 52%), green (δ =
Q
invokes the formation of four species: 675A, t ≤ 2 ms, k
max
decay
0
.845 mm/s and ΔE = 2.01 mm/s, 27%), and red (δ = 1.26 mm/s
−1
−1
Q
=
40(5) s ; 420A, t = 200 ms, k
= 23(1) s ; 420B,
max
formation
and ΔE = 2.79 mm/s, 20%).
−1
Q
tmax ∼ 70 s, k
= 0.023(2) s ; and H , t > 1000 s,
formation
ox max
−
1
kformation = 0.0018(2) s .
The maximum accumulation of 675A occurs before the
mixing time of the instrument (2 ms), which limits the ability to
fully characterize this species by rapid mixing techniques.
Intermediates 420A and 420B were characterized by rapid
675A, is unaffected by toluene, indicating that this species is not
responsible for oxidizing aromatic substrates. In contrast,
addition of toluene alters the absorption trace at 420 nm.
The absorption trace at 420 nm is well-described by an A → B
10
−1
−1
freeze-quench (RFQ) Mo
bauer reactions were conducted at 0.1 ToMOC:diiron and 1
ToMOC:diiron. The two sample conditions yielded similar
̈
ssbauer spectroscopy. RFQ Mo
̈
ss-
→ C → D model with rate constants of 40(5) s , 15(3) s ,
−1
and 0.61(4) s . On the basis of the wavelength of maximum
absorbance, rate of formation, and Mossbauer parameters, we
assign the first species as intermediate 420A. The decay of 420A
̈
̈
Mossbauer parameters (Figure S4).
−1
Species 420A has a broad absorption band at 420 nm (Figure
S5) that accumulates with a rate constant similar to that
occurs rapidly in the presence of substrate (15 s ), consistent
with 420A being responsible for substrate oxidation. Instead of
forming Hox directly, 420A decays to generate a species with
tmax ∼ 0.5 s (420-Ar), corresponding to toluene oxidation. The
diiron(III) species 420-Ar is not observed in the absence of
substrate and absorbs at both 420 and 675 nm. The rate
constant for formation of 420-Ar is similar to that observed for
hydroxylation of phenol in our single-turnover experiments
above. Binding of phenolic derivatives to the diiron active site
increases the absorbance at 420 and 675 nm (Figure S8). On
−1
observed for ToMOHperoxo (∼26 s ), the colorless inter-
mediate previously reported when 2e-MV was used to reduce
4
ToMOH. For a sample frozen after 0.2 s, approximately 80%
̈
of the iron sites are iron(III) with Mossbauer parameters δ =
0
.54 mm/s and ΔE = 0.67 mm/s (Figure 2). The isomer shift
Q
and quadruple splitting are identical to those reported for
ToMOHperoxo (Figure S6). The remaining 20% is iron(II) (δ =
4
1
.31 mm/s and ΔEQ = 2.88 mm/s), corresponding to
B
DOI: 10.1021/jacs.5b07055
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
the basis of these observations, we assign 420-Ar as a cresol−
diiron adduct resulting from hydroxylation of toluene.
In the final phase, 420-Ar decays with a rate constant of 0.61
correspond to any one of many (hydro)peroxo−diiron
̈
conformations. Because 420A is long-lived and has Mossbauer
properties similar to those observed and calculated for μ-1,2-
−1
11d,16,17
s , and the absorbance at 420 and 675 nm decreases. These
changes are consistent with product release from the active site.
The rate constant associated with this step is much smaller than
that observed in our single-turnover experiments but is similar
peroxodiiron intermediates,
as such a species.
we tentatively assign 420A
Previous O -activation experiments reported half-sites
2
4
reactivity with respect to the hydroxylase dimer. The current
12
to the rate constant for steady-state turnover. Thus, release of
product may be rate-limiting for steady-state turnover, as
previously proposed for the analogous BMM toluene 4-
monooxygenase (T4MO) on the basis of X-ray crystallographic
work shows greater than 50% reactivity. This difference in
activity may be a result of including ToMOC or of the ratio of
O to ToMOH used in our experiments. Here, 1.25 and 6.25
2
equiv of O per diiron site are present in the Mo
̈
ssbauer and
̈
2
1
3
data. O activation in the presence of phenol proved to be
single-turnover studies, respectively. In previous Mossbauer and
2
4
much more complex than that observed with toluene (Figure
S9), probably because of the formation of both diiron−phenol
and diiron−catechol adducts.
single-turnover studies, we estimate that the ratios were 0.6
and 1.5, respectively, on the basis of the O saturation methods
2
(see the Supporting Information).
Three important conclusions are evident from this work: (i)
ToMOC is critical for the formation of an active hydroxylating
species; (ii) the previously undetected species, 420A, decays
rapidly upon addition of toluene, consistent with arene
oxidation; (iii) ToMOH is capable of greater than half-sites
reactivity.
In summary, we have demonstrated the importance of the
natural reduction system for efficient hydroxylation and O2
activation by the multicomponent diiron protein ToMO. Using
this knowledge, we were able to identify a previously
unobserved species, 420A, during O activation by the diiron
2
protein ToMO. A proposed scheme that accounts for the
The difference in reactivity between the natural system and
the 2e-MV simulation is surprising given that the BMM soluble
methane monooxygenase (sMMO) undergoes efficient single
observed O -activation kinetics is given in Figure 4B. Further
2
insight into the effects of ToMOC at a molecular level would
benefit from high-resolution structural data of the oxidized and
reduced complexes between ToMOC and ToMOH.
14
turnover using 2e-MV as the reduction system (Figure S10).
The non-heme diiron protein stearoyl-acyl carrier protein
desaturase (Δ9D) also forms an inactive peroxo species when
the diiron reducing protein is replaced with sodium
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b07055.
■
*
S
1
5
dithionite. For both ToMO and Δ9D, the reductases clearly
promote activity, but it is also possible that dithionite inhibits
activity, resulting in low turnover when dithionite present in
2
a,15
Experimental details, evaluation of 420B, Figures S1−
S10, and Tables S1−S3 (PDF)
excess.
A recent X-ray structure of the hydroxylase and Rieske
protein complex for T4MO hints at a possible mechanism for
Rieske protein function. In the 2.05 Å structure of the complex,
residue E104 adopts a different conformation than those
AUTHOR INFORMATION
Corresponding Author
*lippard@mit.edu
■
16
previously reported for BMMs (Figure 4A). The new position
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH Grant GM032134 from the
National Institute of General Medical Sciences (NIGMS).
A.D.L. was supported in part by the NIH NIGMS
Biotechnology Training Program Grant T32 GM008334. The
authors acknowledge Tsai-Te Lu and Woon Ju Song for helpful
discussions.
Figure 4. Influences of ToMOC on the activity of ToMO . On the
red
basis of the Rieske−hydroxylase complex of T4MO, we propose that
the unusual E104 conformation (A) may be responsible for the activity
seen in the presence of ToMOC. A mechanistic scheme consistent
with the activity of ToMOred is provided in (B).
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DOI: 10.1021/jacs.5b07055
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX