M. Kinne et al. / Biochemical and Biophysical Research Communications 397 (2010) 18–21
19
2.2. Reaction conditions
acid was formed (Supplementary Fig. 1B). In reactions with 4-
nitrotoluene as the starting substrate, the reaction sequence was
not as apparent (Fig. 1), but other experiments with 4-nitrobenzyl
alcohol or 4-nitrobenzaldehyde as starting substrates showed the
same precursor-product relationships as in the experiments with
toluene (Supplementary Fig. 1C and D).
Typical reaction mixtures (0.5 ml) contained purified peroxy-
genase (4 U mlÀ1), potassium phosphate buffer (50 mM, pH 7.0),
acetonitrile (5% vol/vol) and the substrate (0.5 mM). The reactions
were started by the addition of H2O2 (1 mM).
An 18O-labeling study established that H2O2 supplied the oxy-
gen incorporated during AaeAPO-catalyzed oxidation of the two
toluenes. (Fig. 2). When we conducted the reaction with toluene
and H218O2, mass spectral analysis of the resulting benzyl alcohol
showed that its principal ion had shifted from the natural abun-
dance m/z of 108 to m/z 110. Similarly, the reaction with 4-nitrotol-
uene and H218O2 yielded 4-nitrobenzyl alcohol in which the
principal ion had shifted from m/z 153 to m/z 155.
2.3. Product identification
The reaction products benzoic acid and 4-nitrobenzoic acid
were analyzed by high performance liquid chromatography (HPLC)
using an Agilent Series 1100 instrument equipped with a diode ar-
ray detector and an electrospray ionization mass spectrometer on a
Luna 5-lm-pore-size C18 column (Phenomenex). The column was
We also observed incorporation of 18O from H218O2 in the benz-
aldehyde and benzoic acid formed from toluene in these experi-
ments. To clarify this finding, we performed labeling experiments
using each of the intermediate products as AaeAPO substrates,
and thus showed that 18O was incorporated from H218O2 at each
oxidation step. With benzyl alcohol as the substrate, some of the
resulting benzaldehyde shifted from its natural abundance m/z of
106 (100%) to m/z 108 (22%), which indicates 18% incorporation
of 18O from H218O2. The m/z values for the benzoic acid formed
in this experiment also shifted, in this case from m/z 121 to m/z
123 (100%) and m/z 125 (9.5%). When benzaldehyde was used as
the substrate instead, the resulting benzoic acid shifted quantita-
tively from its natural abundance m/z of 121 to m/z 123 (Fig. 3).
The same trend was apparent with 4-nitro-substituted sub-
strates. In reactions started from 4-nitrobenzyl alcohol, some of
the resulting 4-nitrobenzaldehyde shifted from the natural abun-
dance m/z of 151 (100%) to m/z 153 (15.5%), thus indicating 13%
incorporation of 18O from H218O2. The m/z values for the 4-nitro-
benzoic acid formed in this experiment also shifted, in this case
from m/z 166 to m/z 168 (100%) and m/z 170 (9.5%). The shift from
the natural abundance m/z of 166 to m/z 168 was quantitative for
4-nitrobenzoic acid when 4-nitrobenzaldehyde was used instead
as the starting substrate (Fig. 3).
eluted at 40 °C and 0.35 ml minÀ1 with an aqueous ammonium for-
mate solution (0.1% vol/vol, pH 3.5)/acetonitrile, 95:5 (70:30 for ni-
tro-substituted compounds), for 5 min, followed by a 25-min linear
gradient to 100% acetonitrile. Products were identified relative to
authentic standards, based on their retention times, UV absorption
spectra, and [M–H]- ions.
The reaction products benzyl alcohol, 4-nitrobenzyl alcohol,
benzaldehyde and 4-nitrobenzaldehyde were analyzed by gas
chromatography (GC) of benzene extracts, using a Hewlett Packard
6890 chromatograph equipped with a Hewlett Packard 5973 mass
spectrometer. GC was performed with a temperature program
starting at 40 °C for 2 min and then increasing at 15 °C minÀ1 to
220 °C, using helium as the carrier gas at a column flow rate of
1 ml minÀ1 on a 5% polysiloxane column (Zebron ZB-5, 250
diameter by 30 m length, 0.25 m film thickness, Phenomenex).
lm
l
The products were identified relative to authentic standards by
their retention times and by electron impact MS at 70 eV. For each
m/z value, the average total ion count within the product peak was
used after background correction to generate the ion count used
for mass abundance calculations. Calculation of 18O-incorporation
was performed by dividing the sum of the natural species abun-
dance and the isotope abundance with the isotope abundance.
The 10% of natural abundance H2O2 in the H218O2 preparation
was taken into account in these calculations.
4. Discussion
2.4. Kinetics experiments
Our results show that AaeAPO can convert toluenes to benzoic
acids via sequential two-electron oxidations, and that the interme-
diate benzyl alcohols and benzaldehydes are released from the en-
zyme active site. In addition to side chain oxidation, AaeAPO also
catalyzes the oxygenation of the aromatic ring of toluene (but
not of 4-nitrotoluene) leading to mixtures of p- and o-cresol and
their oxidation products [9]. As reported earlier, these reactions
may compete with side chain oxidation. In the present study,
where the focus has been on side chain oxidations, ring oxygena-
tion of toluene was ignored.
The time course of product release during AaeAPO-catalyzed
hydroxylation of toluene and 4-nitrotoluene was analyzed in stir-
red reactions (0.20 ml, 23 °C) that contained 1 U mlÀ1 of the perox-
ygenase, potassium phosphate buffer (50 mM, pH 7.0), and
0.50 mM of the substrate. The reactions were initiated with
1 mM H2O2 and stopped with 0.02 ml of 50% (w/vol) trichloroace-
tic acid after 5, 10, 30, 60 and 120 s. The reaction products were
quantified by HPLC as described above.
3. Results
The 18O-labeling experiments establish that the oxygens intro-
duced during oxidations originate from H2O2. The results support
a mechanism similar to that envisaged for the peroxygenase activ-
ity of P450s [14,15] and for ether cleavage catalyzed by AaeAPO [8],
in which the enzyme heme is oxidized by H2O2 to give a ferryl oxy-
gen intermediate [16] that carries one of the peroxide oxygens and
can be depicted formally as (FeO)3+. The latter (very probably a
Compound I-type intermediate) abstracts a hydrogen from the
benzylic carbon to give an enzyme-bound benzylic radical, after
which rebound of an ꢀOH equivalent occurs to introduce a new hy-
droxyl group on the same carbon (Fig. 4).
AaeAPO hydroxylated toluene and 4-nitrotoluene to give the
corresponding benzyl alcohols, benzaldehydes and benzoic acids.
The reactions proceeded rapidly with total conversions of 93% for
toluene and 12% for 4-nitrotoluene (Fig. 1). The low extent of 4-
nitrotoluene oxidation is attributable to inhibition of the enzyme
by the substrate, which has also been observed during P450-cata-
lyzed oxidations of nitroaromatics [12,13]. The initial product of
toluene oxidation was benzyl alcohol, which then declined with
concomitant production of benzaldehyde, which in turn declined
with concomitant production of benzoic acid. When benzyl alcohol
was used instead of toluene as the starting substrate, the products
were benzaldehyde and benzoic acid (Supplementary Fig. 1A),
whereas with benzaldehyde as the starting material, only benzoic
According to this model, oxygen incorporation from H2O2
should be quantitative when a toluene is oxidized to a benzyl alco-
hol, and our data agree with this picture. When the substrate is a
benzyl alcohol instead, the enzyme-bound intermediate will be
Kinne, Matthias
