Reaction route control by microperoxidase-9/CTAB micelle ratios

Article abstract of DOI:10.1039/b601671e

Microperoxidases (MP) as water-soluble models attract interest to studying the reaction mechanism of peroxidases because these heme peptides are able to form the same enzyme intermediates during the reaction with peroxides. In this work we have demonstrated that the association of Fe(III)MP-9 and Fe(III)MP-11 with CTAB micelles (MP-9/CTAB and MP11/CTAB) provides a microenvironment with an alkaline interface and a hydrophobic core that exhibits peroxidase behavior. This microenvironment shifts positively the redox potential of microperoxidases by ～100 mV. tert-Butylhydroperoxide (t-BuOOH) when added to the medium, converted Fe(III)MP-9/CTAB to MP-9/CTAB Compound II, a high valence oxidized intermediate of the heme peptide. Subsequent addition of diphenylacetaldehyde (DPAA) to MP-9/CTAB Compound II regenerated the native form of the enzyme, Fe(III)MP-9/CTAB, what characterizes the occurrence of a peroxidase cycle. Fe(III)MP-9/CTAB regenerated during the peroxidase cycle reacted with residual DPAA in the medium to form Fe(II)MP-9/CTAB, which indicates that both Fe(III)MP-9/CTAB and its oxyferryl form can use aldehydes as reducing agents. According to the determined reduction potential, Fe(III)MP-9 and Fe(III)MP-9/CTAB should be able to oxidize DPAA (reduction potential -630 mV). The reaction of MP-9/CTAB with DPAA produced benzophenone as final product, detected by infrared spectroscopy and mass spectrometry. Interestingly, a significant difference was observed in the benzophenone yield according to the micelle/MP-9 molar ratio. the Owner Societies 2006.

Full text of DOI:10.1039/b601671e

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www.rsc.org/pccp | Physical Chemistry Chemical Physics
Reaction route control by microperoxidase-9/CTAB micelle ratiosw
Tatiana Prieto,a Rodrigo O. Marcon,a Fernanda M. Prado,b Antonio C. F. Caires,a
Paolo Di Mascio,b Sergio Brochsztain,a Otaciro R. Nascimentoc and
Iseli L. Nantes*a
Received 3rd February 2006, Accepted 28th February 2006
First published as an Advance Article on the web 14th March 2006
DOI: 10.1039/b601671e
Microperoxidases (MP) as water-soluble models attract interest to studying the reaction
mechanism of peroxidases because these heme peptides are able to form the same enzyme
intermediates during the reaction with peroxides. In this work we have demonstrated that the
association of Fe(III)MP-9 and Fe(III)MP-11 with CTAB micelles (MP-9/CTAB and MP11/
CTAB) provides a microenvironment with an alkaline interface and a hydrophobic core that
exhibits peroxidase behavior. This microenvironment shifts positively the redox potential of
microperoxidases by B100 mV. tert-Butylhydroperoxide (t-BuOOH) when added to the medium,
converted Fe(III)MP-9/CTAB to MP-9/CTAB Compound II, a high valence oxidized intermediate
of the heme peptide. Subsequent addition of diphenylacetaldehyde (DPAA) to MP-9/CTAB
Compound II regenerated the native form of the enzyme, Fe(III)MP-9/CTAB, what characterizes
the occurrence of a peroxidase cycle. Fe(III)MP-9/CTAB regenerated during the peroxidase cycle
reacted with residual DPAA in the medium to form Fe(II)MP-9/CTAB, which indicates that both
Fe(III)MP-9/CTAB and its oxyferryl form can use aldehydes as reducing agents. According to the
determined reduction potential, Fe(III)MP-9 and Fe(III)MP-9/CTAB should be able to oxidize
DPAA (reduction potential ꢀ630 mV). The reaction of MP-9/CTAB with DPAA produced
benzophenone as final product, detected by infrared spectroscopy and mass spectrometry.
Interestingly, a significant difference was observed in the benzophenone yield according to the
micelle/MP-9 molar ratio.
methionine residue. It has been demonstrated that the covalent
Introduction
bonds can be formed by an autocatalytic process involving
reaction of the apoprotein–heme complex with substrates.2,3
Peroxidases are enzymes that contain the heme group as
catalytic center and use peroxides as oxidizing agents to
Peroxidases also differ by the nature of the proximal heme iron
ligand.1–5 These structural characteristics influence the cata-
generate their reactive high-valency intermediates. There are
two superfamilies of peroxidases: the first one encompasses
lytic properties of the peroxidases.1,4,5
plant, fungal and (archae) bacterial peroxidases and the
Interesting models of peroxidases can be obtained by sequen-
second one encompasses the mammalian peroxidases whose
tial pepsin- and trypsin-catalyzed hydrolysis of horse-heart
secondary and tertiary structures as well as the prosthetic
cytochrome c and they are known as microperoxidases. The
group differ significantly from those of plant or fungal perox-
product of the hydrolysis retains the heme group co-
idases.1 A unique characteristic of the mammalian peroxidases
valently attached to cysteine residues 14 and 17.6–8 Four micro-
is the presence of covalent bonds between the heme group and
peroxidase types were identified according to the sequence of
the protein. In myeloperoxidase, lactoperoxidase, eosinophil
native cytochrome c, from which these peptide fragments are
peroxidase, and probably thyroid peroxidase these covalent
derived: microperoxidase-6, (CAQCHT), -8, (CAQCHTVE),
links are two ester bonds between aspartic or glutamic amino
-9 (KCAQCHTVE) and -11 (VQKCAQCHTVE). Microperox-
acid residues and hydroxyl groups on the 1- and 5-methyl
idases exist in the ferric resting states, retain histidine as a fifth
groups of the heme.2,3 Myeloperoxidase has also a third
heme iron ligand but not the methionine residue at the sixth
covalent bond between one of its vinyl groups and the
coordination position which, in this case, at neutral pH, is
occupied by a water molecule. The absence of methionine 80
a
at the sixth coordination position of microperoxidases favors the
peroxidase activity of the hemepeptide.6–8 Scheme 1, below,
shows the structure of microperoxidase-9.
´
Centro Interdisciplinar de Investigac¸a˜o Bioquımica-CIIB
Universidade de Mogi das Cruzes-UMC, Mogi das Cruzes – SP,
Brazil. E-mail: ilnantes@umc.br; Fax: +55-11-4798-7068;
Tel: +55-11-4798-7102
b Depto de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o
Because these structural characteristics, microperoxidases
have been useful to study structural and catalytic properties of
peroxidases and cytochromes9,10 as well as to develop elec-
trode sensors.11
Paulo, USP-Sa˜o Paulo – SP, Brazil
c
´
Grupo de Biofısica, Instituto de Fısica de Sa˜o Carlos, Universidade
de Sa˜o Paulo, USP-Sa˜o Carlos, Sa˜o Carlos – SP, Brazil
´
w Electronic supplementary information (ESI) available: Electroche-
mical data of microperoxidases in phosphate buffer and CTAB
micelles (Tables S1–S4; Fig. S1). See DOI: 10.1039/b601671e
Similarly to biological peroxidases, microperoxidase-8 is
able to convert a wide variety of organic compounds at the
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Scheme 1 The structure of MP-9.
expense of hydrogen peroxide (H2O2) in a type of peroxidase
chemistry.12 Similarly to horseradish peroxidase (HRP), mi-
croperoxidases can react with peroxides and produce the
intermediate compounds 0, I and II of HRP.13,14 Compound
0 has been detected for HRP at low temperature and corre-
sponds to the porphyrin iron-hydroperoxide (Por-
Fe(III)–OOR) species.13 This species precedes the formation
of Compounds I (Por+Fe(IV)QO) and II (PorFe(IV)QO)
which are well-known high-valent intermediates of peroxi-
dases.14,15 Compound II can react with a reducing agent
leading to the regeneration of the native peroxidase form.
This reaction pathway is known as peroxidase cycle. The first
step of the peroxide cleavage promoted by peroxidases is the
formation of Compound 0 in which the deprotonated water at
the sixth coordination position was replaced by the hydroper-
oxide [Scheme 2, eqn (1) and (2)]. After the formation of
Compound 0, there are two possible mechanisms for the
cleavage of peroxides (ROOH): a heterolytic cleavage gener-
ating the alcohol derivative (ROH) and Compound I (Scheme
2, eqn (3)), which reacts with another peroxide molecule
generating Compound II and the peroxyl radical (ROOꢂ)
(Scheme 2, eqn (5)) or a homolytic cleavage of ROOH to
form Compound II directly (Scheme 2, eqn (4)). Whatever the
mechanism for peroxide cleavage, native Fe(III)peroxidase can
be regenerated after the reaction of Compound II with another
ROOH molecule or another reducing agent (Scheme 2,
eqn (6)).13–15
It is known that protons in the distal heme active site
influence the formation of the catalytic reactive species for
both peroxidases and cytochrome P450.16 In HRP, the distal
His42 ligand acts as an acid/base catalyst that favors the
deprotonation of the hydroperoxide substrate at the enzyme
active site (Compound 0) and the subsequent heterolytic
cleavage.17–19 Therefore, the deprotonation of the hydroper-
oxide in Compound 0 is a crucial step in the catalytic cycle of
peroxidases and cytochrome P450.
A competitive effect of protons has been demonstrated for
the reaction of Fe(III) or Mn(III)MP-8 with hydrogen peroxide,
suggesting that Compound 0 precedes the formation of a high-
valent intermediate of Fe(III) or Mn(III)MP-8.20 In comparison
with HRP, MP-8 in aqueous medium does not exhibit a site
pocket with a distal basic residue participating in the peroxide
deprotonation and favoring the O–O peroxide bond cleavage.
These characteristics could somehow be responsible for the
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Scheme 2 Proposed mechanism for the benzophenone production from DPAA oxidation by MP-9/CTAB.
low reactivity of MP-8 at low pH. In aqueous medium, alka-
line pH leads to deprotonation of the MP-8 bound water,
which then either assists concerted hydrogen peroxide depro-
tonation and coordination of the hydroperoxo group to the
metal center or is directly oxidized by hydrogen peroxide to a
metal-hydroperoxo MP-8.20
solutions were prepared with deionized water (mixed bed of
ion exchanger, Millipore) and the pH was measured using a
combined glass electrode (Orion Glass pH SURE-FLOWt).
The reference electrode (ROSSt, model 8102) was filled with
Orion Filling Solution (ROSSt). The pH meter was calibrated
using METREPAK pHydrion standard buffer solutions
(Brooklyn, NY).
Recently we have demonstrated that the association of
microperoxidases-8 and -9 to CTAB micelles provides a
protein-like hydrophobic environment to the heme group,
which allows this complex to exhibit the catalytic properties
of the peroxidases.21,22 The association of Fe(III)MP-8 with
CTAB micelles provides a microenvironment with an alkaline
interface and a hydrophobic core that gives special character-
istics to the Fe(III)MP-8/peroxide (tert-butylhydroperoxide
(t-BuOOH) or H2O2) reaction as compared to homogeneous
medium. The reaction of Fe(III)MP-8 with t-BuOOH and
H2O2 produced alkoxyl and hydroxyl radicals as the initial
radicals, respectively. These free radicals are produced pre-
sumably by homolytic scission of the O–O bond by Fe(III)MP-
8/CTAB. The UV-visible spectral changes detected for
Fe(III)MP-8 during the reaction with peroxides point to the
formation of Compound II as the species that exhibits sub-
sequent bleaching. The Km and the maximal conversion rate
(k2) of CTAB-bound Fe(III)MP-8 into the corresponding
Compound II were determined for the reaction with peroxides
in 20 mM CTAB, at pH 7.4 and 9.1. For both substrates, the
Preparation of micellar CTAB solutions
CTAB micellar solutions were prepared by dissolving CTAB
in an appropriate buffer with stirring at 37 1C. The CMC
(critical micelle concentration) was determined in the usual
fashion from plots of the surface tension vs. log [CTAB].
Surface tensions were measured with DeNouy tensiometer
¨
equipped with a Pt ring.
Cyclic voltammetry measurements
Cyclic voltammetry was performed by using AUTOLAB
PGSTAT30 potentiostat. All measurements were carried out
at room temperature (25 ꢃ 2 1C) in a three-compartment
electrochemical cell consisting of a glass carbon disk (0.07 cm2
area) as a working electrode, a platinum auxiliary electrode
and a Ag/AgCl reference electrode in 3 M KCl solutions.
Argon bubbling was used to remove oxygen from the solutions
in the electrochemical cell. The diffusion coefficient (D0) was
calculated from the slope of the curve obtained from the plot
of ipc (A) vs. the square root of the sweep rate (mV) according
the Randles–Sevick equation (eqn (1))
K
m values increased at pH 9.1 without significant changes in k2
values, indicating alteration on the affinity of the substrates for
CTAB-bound Fe(III)MP-8.21 In another work we reproduced
the HRP catalytic cycle for MP-9 associated to CTAB mi-
celles. The addition of t-BuOOH to the medium containing
MP-9 associated to CTAB micelles led to the occurrence of a
peroxidase cycle, indicating that this aggregate behaves as a
true enzyme.22 In this work, for the first time, we present the
reduction potential of MP-9 in CTAB micelles followed by a
study of the one electron oxidation of diphenylacetaldehyde
by MP-9 associated to CTAB micelles.
ipc = (2.687 ꢄ 105)n3/2 v1/2 D1/2 AC
where 2.687 ꢄ 105 is a constant when the temperature is 298.15
K (C molꢀ1 Vꢀ1/2), n = number of electrons appearing in half-
reaction for the redox couple, v is the rate at which the
potential is swept (V sꢀ1), A = the electrode area (cm2) and
C (mol cmꢀ3) is the concentration of the redox species.
UV-visible measurements
Solutions of Fe(III)MP-9 and the corresponding H2O2 or
t-BuOOH solutions were both buffered with 5 mM sodium
phosphate. The concentration of Fe(III)MP-9 was verified by
using e397 = 1.57 ꢄ 105 Mꢀ1 cmꢀ1 for Fe(III)MP-8 at pH 7.0.23
The concentration of Fe(III)MP-9 in CTAB micelles was
calculated by using e400 = 1.13 ꢄ 105 Mꢀ1 cmꢀ1, determined
Materials and methods
Chemicals
Microperoxidase-9, t-BuOOH and CTAB were obtained from
Sigma Chemical Co. (St Louis, MO, USA). All aqueous
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from a standard curve obtained by registering the spectra of
Fe(III)MP-8/CTAB at different hemepeptide concentrations.22
Time-resolved spectra were recorded on a Shimadzu Model
1501 MultiSpec (Tokio, Jp), employing the photodiode array
scan mode. The spectral resolution was 0.5 nm and the spectra
were obtained with a time interval of 1 s. According to
microperoxidase concentration, the optical path length was
0.1 or 1 cm. The data sets represent the average of three
independent measurements. The kinetic data were recorded at
417 nm for Fe(III)MP-9/CTAB, the region of strong absor-
bance of the Compound II species.
Heme iron EPR measurements
Direct EPR measurements of Fe(III)MP-9 (100 mM) were
obtained in a Bruker ELEXSYS EPR system E-580 under
the following conditions: gain 5 ꢄ 103, modulation amplitude
1.0 mT, microwave power 4 mW, temperature 11 K, time
constant 20.48 ms and conversion time 81.92 ms. After mixing,
solutions were quickly introduced into an EPR quartz tube
that was previously cooled in liquid nitrogen. After freezing
the sample was introduced into the microwave cavity at low
temperature and the EPR measurements were performed.
Fig. 1 Cyclic voltammetry of MP-9 and MP-11 in the absence and in
the presence of CTAB (dashed line: MP-9 in phosphate buffer, dotted
line: MP-9 in CTAB micelles, thin solid line: MP-11 in phosphate
buffer and thick solid line: MP-11 in CTAB micelles). The peptides
were present at a concentration of 0.1635 mg mLꢀ1. Phosphate buffer
(0.02 M) at pH 7.4 was present in all samples. In the absence of CTAB
25 mM of MgCl2 was added to the samples. The data were acquired
over the potential range of 100 to ꢀ600 mV vs. Ag/AgCl, with sweep
rate of 50 mV sꢀ1 and temperature of 25 1C. The electrode area was
0.07 cm2. The inset shows a plot of ip vs. (scan rate).2
Fourier transform infrared (FTIR) spectrometry
The spectra of the samples were recorded on a Perkin-Elmer
spectrometer mod. Spectrum One. The benzophenone spectra
were obtained by using KBr pellets technique in the region of
4000–400 cmꢀ1 with a resolution of 4 cmꢀ1. The spectra of MP
Schiff bases were measured by using ATR technique, in the
region of 4000–650 cmꢀ1 with a resolution of 2 cmꢀ1. A
monocrystal of ZnSe, 451, was utilized as optical element of
internal reflexion.
in the presence and in the absence of CTAB, both MP-11 and
MP-9 exhibit ipc proportional to the square root of the scan
rate and thus the redox process of microperoxidases at the
electrode surface could be considered diffusion-controlled.
From the slope of the curves, the values of Do for both MP-
11 and MP-9 diffusion coefficient were calculated. In the
absence of CTAB, Do was the same for both microperoxidases
(1.5 ꢄ 10ꢀ5 cm2 sꢀ1), and in the presence of CTAB, Do values
obtained were 3.7 ꢄ 10ꢀ6 and 5.6 ꢄ 10ꢀ6 cm2 sꢀ1 for MP-9
and MP-11, respectively.
Mass spectrometry analysis of benzophenone
Benzophenone was analyzed by a Quatro II Micromass mass
spectrometer with Z-sprayt ion source (Manchester, UK).
The analysis of benzophenone was done by using acetonitri-
le–formic acid 0.1% (1 : 1, v/v) by electrospray ionization
(ESI) in the positive ion mode. The source and desolvation
temperatures were 120 and 150 1C, respectively. The para-
meters of the equipment were: sample cone voltage at 30 V,
extraction cone voltage at 5 V and capillary potential at 3.5
kV. Full scan data were acquired over a mass range of 100–300
m/z. The data were processed by the Mass Lynx NT data
system, Version 3.2 (Micromass, Manchester, UK).
The redox potential exhibited by MP-11 and MP-9 in
homogeneous and heterogeneous media suggests that these
hemepeptides in the native form (Fe(III)) and in the high
valence forms, i.e., Compound I and Compound II, should
be able to oxidize aldehydes such as diphenylacetaldehyde
(DPAA) with E1/2 = ꢀ630 mV (cyclic voltammogram not
shown).
The addition of t-BuOOH to
a medium containing
Fe(III)MP-9 associated to CTAB micelles (Fig. 2A, thin solid
line) led to the conversion of the native form of the hemepep-
tide to its high valence intermediate Compound II (oxoferryl
form, thick solid line in Fig. 2A). According to previous
work,22 in this experimental condition, MP-9 Compound II
returned to the native form but the regeneration of the native
form was accompanied by discrete bleaching of the Soret band
(Fig. 2A, dashed line), probably due to the attack of free
radicals produced in the course of the reaction. However,
when 10 or 600 mM diphenylacetaldehyde (DPAA) was added
to the medium after the accumulation of MP-9/CTAB Com-
pound II (Fig. 2B and C, thick solid lines), the regeneration of
Fe(III)MP-9/CTAB (Fig. 2B and C, dotted lines) was faster
Results
Fig. 1 shows the cyclic voltammograms of 100 mM MP-9 and
MP-11 in the absence and in the presence of CTAB (dashed,
dotted, thin solid and thick solid lines, respectively). The
presence of CTAB micelles shifted the MP-11 E1/2 from
ꢀ346 to ꢀ276 mV. Similar results were obtained with MP-9.
MP-9 exhibited E1/2 = ꢀ346 mV in the absence of CTAB and
this value changed to ꢀ231 mV in the presence of CTAB
(Fig. 1).
In the presence of CTAB micelles, the minimal DEp was
B57 mV obtained at 10 mV sꢀ1 suggesting a fully reversible
one-electron-transfer reaction. The inset of Fig. 1 shows that
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Fig. 2 (A) MP-9 spectral changes during the peroxidase cycle. Thin solid line: Fe(III)MP-9 associated to CTAB micelles. Thick solid line: MP-9
Compound II associated to CTAB micelles, 20 s after addition of 10 mM t-BuOOH. Dashed line: Fe(III)MP-9 associated to CTAB micelles
recovered 3.5 min after addition of 10 mM t-BuOOH. (B) MP-9 spectral changes during the peroxidase cycle. Thin solid line: Fe(III)MP-9 associated
to CTAB micelles. Thick solid line: MP-9 Compound II associated to CTAB micelles, 20 s after addition of 10 mM t-BuOOH. Dashed line:
Fe(III)MP-9 associated to CTAB micelles recovered 50 s after addition of 10 mM DPAA. Dotted line: Fe(III) and Fe(II)MP-9 associated to CTAB
micelles obtained 20 min after the addition of 10 mM DPAA. (C) MP-9 spectral changes during the peroxidase cycle. Thin solid line: Fe(III)MP-9
associated to CTAB micelles. Thick solid line: MP-9 Compound II associated to CTAB micelles, 20 s after addition of 10 mM t-BuOOH. Dashed
line: Fe(III)MP-9 associated to CTAB micelles recovered 50 s after addition of 600 mM DPAA. Dotted line: Fe(II)MP-9 associated to CTAB
micelles obtained 20 min after the addition of 600 mM DPAA. The experiments were carried out with 20 mM CTAB, 2.5 mM MP-9, in 5 mM
phosphate buffer, pH 7.4 at 25 1C by using a 1 cm optical path length cuvette. (D) Kinetic profiles of MP-9/CTAB catalytic cycle obtained in the
conditions described in (A) (thin solid line), (B) (dashed line) and (C) (thick solid line).
(rate = 2.7 ꢄ 10ꢀ3 DA415nm
(rate = 0.4 ꢄ 10ꢀ3 DA415nm
s
ꢀ1) than in the absence of DPAA
ꢀ1). Thus, the comparison of the
CTAB (Fig. 2C, dashed line). The kinetic profiles of the MP-9
catalytic cycle in the absence and in the presence of 10 and 600
mM diphenylacetaldehyde are shown in Fig. 2D, thin solid,
dashed and thick solid lines, respectively. Similar results were
obtained in different MP-9 and CTAB micelle concentrations
(not shown). The reaction of MP-9/CTAB with t-BuOOH and
DPAA, in a micelle/peptide ratio of 6.0, was also accompanied
by continuous-wave EPR of heme iron. Similarly to previous
reports,21,22 the heme iron EPR spectra obtained after
t-BuOOH addition exhibited decrease of the signal compatible
with the formation of compound II, an EPR silent species. In
the absence of DPAA, the signal of the Fe(III) heme iron was
recovered, coherent with the occurrence of the peroxidase
catalytic cycle.22 However, in the presence of DPAA the
EPR signal was lost again concomitantly with the formation
of Fe(II)MP-9/CTAB, also an EPR silent species (not shown).
Considering that oxygen consumption was detected during
the reaction of DPAA with MP-9/CTAB (Fig. 3), we propose
a reaction mechanism in which benzophenone is the final
reaction product (Scheme 3).
s
regeneration rate in the presence and in the absence of the
aldehyde revealed that DPAA accelerated the enzyme catalytic
cycle. Fe(III)MP-9 recovered at the cycle end, when the
t-BuOOH should be depleted, reacted with residual DPAA
in a concentration dependent manner to form Fe(II)MP-9/
However, the reaction of DPAA with the amino groups
present in the MP-9 structure to form Schiff base adducts
could not be overlooked. In this regard, interesting differences
were observed by comparing the reaction carried out in the
Fig. 3 Oxygen consumption during the reaction of MP-9/CTAB with
DPAA. The experiment was carried out at 25 1C, in 5 mM phosphate
buffer, pH 7.4, with 2.5 mM MP-9, 20 mM CTAB and 600 mM DPAA
added to the medium at the time indicated by the arrow.
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Scheme 3 Oxidation of diphenylacetaldehyde by Compound II MP-9/CTAB and native MP-9/CTAB.
presence of micelle/MP-9 molar ratio B66.0 with the reaction
in the presence of micelle/MP-9 molar ratio B6.0. In the
presence of the micelle/MP-9 molar ratio B66.0, the addition
of DPAA led to a fast increase in the absorbance at 254 nm
(thin solid line in Fig. 4A), accompanied by a concomitant
increase in the fluorescence of the sample (inset). In the
presence of the micelle/MP-9 molar ratio B6.0, the addition
of DPAA led to the enhancement of the absorbance at 260 nm
(thin solid line) with a kinetic profile similar to that observed
for the heme iron reduction (thick solid line in Fig. 4B). Fig.
4C shows the spectral differences observed for the MP-9/
CTAB complexes in different micelle/hemepeptide ratios.
Fig. 4C shows that the decrease of the micelle/hemepeptide
ratio led to the red shift of Soret and Q bands, suggesting the
formation of aggregates with at least one pair of MP-9 per
micelle. Fig. 4D shows the corresponding EPR spectra of
MP-9/CTAB in the micelle/MP-9 ratios 0.5 and 5.0. The
parameters obtained from the EPR spectra (see Discussion)
corroborate that different MP-9/CTAB high spin states are
favored in different micelle/MP-9 ratios. The results described
above suggest that benzophenone production was predomi-
nant in the presence of micelle/MP-9 molar ratio B6.0,
whereas Schiff base adduct was predominant in the presence
of micelle/MP-9 molar ratio B66.0. In fact, during the reac-
tion of MP-9 CTAB with DPAA, when the micelle/MP-9
molar ratio was B6.0, it was evident the precipitation of
crystals at the bottom of the tube (Photo as Fig. 1 of Supple-
mental Materials). The production of benzophenone as the
final product was corroborated by infrared spectroscopy (Fig.
5) and mass spectrometry analysis (Fig. 6) of the material that
precipitated during the reaction. Fig. 5 shows the IR spectra of
pure benzophenone (solid line) and the spectrum of the
material that precipitated and in which benzophenone and
residual DPAA were probable present. The vibrational spec-
trum of pure benzophenone shows a strong absorption band
(4.3% T) at 1652 cmꢀ1, due to the normal vibrational mode of
the functional group –CQO stretching. The aromatic ring
conjugation changes the absorption of this group to a lower
frequency comparatively to aliphatic ketones. Bending ap-
pears as a sharp band at 1323 cmꢀ1 (12.7% T). Absorption
due to vibrational normal modes ofQC–H stretching for sp2
C–H out-of-plane can be observed in the 3087–3055 cmꢀ1
region. A pair of ring stretch absorption is noted at 1593 and
1573 cmꢀ1. The IR spectrum of the product precipitated
during the reaction and containing a mixture of benzophenone
and DPAA exhibits a strong absorption band (51.5% T)
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Fig. 4 (A) Kinetic profile of the MP-9/CTAB heme iron reduction concomitant with the formation of Schiff base adduct obtained with
MP-9/CTAB molar ratio B66.0 by using 20 mM t-BuOOH and 0.6 mM DPAA. Thick solid line corresponds to alterations in the Soret band
intensity at 415 nm due to changes in the heme iron redox state. The thin solid line corresponds to alterations in the absorbance intensity at 254 nm
due to the formation of Schiff base adducts. (B) Kinetic profile of the MP-9/CTAB heme iron reduction concomitant with the formation of Schiff
base adduct obtained with MP-9/CTAB molar ratio B6.0 by using 300 mM t-BuOOH and 20 mM DPAA. The thick solid line corresponds to
alterations in the Soret band intensity at 415 nm due to changes in the heme iron redox state. Thin solid line corresponds to alterations in the
absorbance intensity at 260 nm due to the formation of benzophenone. The addition of the peroxide and the aldehyde is indicated by the arrows.
(C) Effect of the micelles/hemepeptide ratio on the MP-9 Soret and Q bands. Dotted line: Fe(III)MP-9 absence of CTAB at pH 7.4 The spectrum
was obtained with 2.5 mM MP-9 and presented in e units. Thick solid line: Fe(III)MP-9/CTAB when the CTAB/hemepeptide ratio was 50.0. The
spectrum was obtained with 2.5 mM MP-9 and presented in e units. Thin solid line: Fe(III)MP-9/CTAB when the CTAB/hemepeptide ratio was 5.0.
The spectrum was obtained with 100 mM MP-9 and presented in e units. Dashed line: Fe(III)MP-9/CTAB when the CTAB/hemepeptide ratio was
0.5. The spectrum was obtained with 100 mM MP-9 and presented in e units. Dashed-dotted line: Fe(III)MP-9/CTAB when the CTAB/hemepeptide
ratio was 0.25. The spectrum was obtained with 100 mM MP-9 and presented in e units. (D) Direct EPR measurements of Fe(III)MP-9 (100 mM) in
the presence of CTAB and in the micelle/MP-9 ratio = 0.5 and 5.0. The spectra were obtained in a Bruker ELEXSYS EPR system E-580 under the
following conditions: gain 5 ꢄ 103, modulation amplitude 1.0 mT, microwave power 4 mW, temperature 11 K, time constant 20.48 ms and
conversion time 81.92 ms. For the ratioof 0.5, the frequency was 9.474 45 GHz, g-values were 6.019, 5.949 and 2.009 with 320.0, 750.0 and 80.0/
g(D)H (MHz) and the line was adjusted with a Lorentzian shape. For the ratio of 5.0, the frequency was 9.472 98 GHz, the g-values 6.079, 5.820
and 2.006 with 450.0, 440.0 and 80.0/g(D)H (MHz) and the lines were adjusted with Lorentzian shape.
relative to functional carbonyl group of the aldehyde at 1724
cmꢀ1 and a stretch of aldehyde hydrogen (–CHO) at 2849
cmꢀ1 (41.0% T). However the presence of the high intensity
band (56.0% T) at 1658 cmꢀ1 (n –CQO of aromatic ketones),
as well as the absorption band at 2849 cmꢀ1 (41.0% T) can be
assigned to the presence of high concentration of benzophe-
none mixed with residual DPAA.
spectrometry recorded in the positive ion mode with a cone
voltage of 10 V exhibiting signals corresponding to MP-9
molecular ion, [M + H]+ = 1635 and the corresponding
adduct with DPAA molecular ion, [M + H]+ = 1834 (spectra
not shown). The IR analysis of MP-9 after the reaction with
DPAA, in conditions in which the formation of Schiff base
adducts was favored (Fig. 7), revealed a strong band at
1634 cmꢀ1 attributed to the vibrational normal mode of the
Fig. 6 shows the mass spectrum of the material that
precipitated during the reaction of MP-9/CTAB (micelles/
MP-9 ratio = 6.0) with DPAA. The material was recorded
in the positive ion mode with a cone voltage of 30 V exhibiting
a peak with m/z 183 which corresponds to the pseudomole-
cular ion of benzophenone ([M + H]+ = 183) and the peak
with m/z 167 that represents the loss of the oxygen atom
([(M–O) + H]+ = 167). The formation of MP-9 Schiff base
adducts during the reaction with DPAA when the micelle/
hemepeptide ratio was 66.0 was also corroborated by mass
–CQN– group of the Schiff bases.24 Aromatic Schiff bases
25
absorb in IR region near 1630 cmꢀ1
and generally, the
CQN bands exhibit signal with intensity similar to that
observed for CQO and CQC bands. The vibrational spec-
trum of DPAA-modified MP-9 revealed also an intense ab-
sorption band at 857 cmꢀ1
, characteristic of angular
deformation of the C–H groups in the phenyl rings. Another
intense band, attributed to the CQC stretching of the phenyl
rings, was detected at 1538 cmꢀ1. The results described above
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Fig. 5 Comparative FTIR spectra of benzophenone and the product
of the reaction between MP-9/CTAB (hemepeptide/micelle ratio
= 6.0) and DPAA. The dashed line represents the spectrum of
the product that precipitated at the end of the reaction between
MP-9/CTAB and DPAA. The solid line represents the spectrum of
pure benzophenone.
Fig. 7 Comparative FTIR spectra of MP-9 (solid line) and MP-9
with Schiff base obtained after the reaction with DPAA when heme-
peptide/micelle ratio = 66.0 (dashed line).
(formation of Compound I) and the reducing agent (formation
of Compound II and regeneration of the native form) of the
prosthetic group, a phenomenon previously observed for
peroxidases. Therefore, associated to CTAB micelles, micro-
peroxidases behave as true peroxidases. In this regard, the
hydrophobic core provided by CTAB micelles changed the
redox potential of the microperoxidases to more positive
values and made the lipoenzyme a better oxidizing agent as
compared with microperoxidases in homogeneous medium
(Fig. 1). The decrease of the catalytic efficiency (kcat/KM)
previously observed for the cleavage of peroxides was due to
the partition of these compounds inside and outside the
micelles. In the case of DPAA, the highly hydrophobic
compound, the partition occurs preferentially inside the mi-
celles and the redox potential of the enzyme was the limiting
factor of the catalytic efficiency.
corroborated the formation of DPAA-promoted Schiff bases
in the MP-9 structure when the micelle/hemepeptide ratio
was 66.0.
Discussion
The association of microperoxidases with CTAB micelles
improves the catalytic activity of the enzyme.
The occurrence of peroxidase catalytic cycle during the reac-
tion of MP-9/CTAB with t-BuOOH shows that this aggregate
hemepeptide/micelle22 behaves as an artificial enzyme that we
have named lipoenzyme. The catalytic cycle observed when
peroxide (in this case, t-BuOOH) was the only substrate
available implicates that the peroxide acts as the oxidizing
Literature data have shown that, differently from metallo-
proteins that need the presence of appropriate promoters such
as 4,-40 bipyridyl to achieve best electron transfer at the
electrode,26 microperoxidases exhibit rapid, reversible electron
transfer at the electrode in the absence of mediators or
promoters. This behavior has been attributed to the simpler
and disordered conformation of microperoxidases, in which
the heme group is exposed to the solvent. In the case of MP-11
it was previously reported that the hemepeptide takes part in a
rapid one-electron reaction at the glassy carbon electrode in
the absence of mediators. In this condition, the smallest DEp
value obtained was about 90 mV indicating that a fully
reversible one-electron transfer reaction was not achieved.
However, a reversible electrode process was achieved for
MP-11 in the presence of 25 mM Mg2+ (ref. 26 and Table
S1–S4 in the electronic supplemental materialw). This fact was
expected as, at neutral pH, both microperoxidases (pI = 4.7)
and the glassy carbon surface are negatively charged. Thus, it
was postulated that Mg2+ forms a transient bridge between
the negatively charged peptide and the electrode surface. The
fully reversible electrode process obtained for MP-9 and
MP-11 associated to CTAB micelles, in the absence of
Mg2+, suggests that the cationic surfactant could also
Fig. 6 ESI mass spectrum of the product obtained after the reaction
between MP-9/CTAB (hemepeptide/micelle ratio = 6.0) and DPAA.
The analysis was recorded in the positive ion mode with sample cone
voltage and extraction cone voltage at 30 and 5 V, respectively.
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establish a transient bridge with the electrode surface. The
diffusion coefficients of MP-9 and MP-11 associated to CTAB
micelles were almost identical to that reported for CTAB
micelles (5.5 ꢄ 10ꢀ6 cm2 sꢀ1, ref. 27). The diffusion coefficient
values obtained in the presence of CTAB were lower than that
reduce Fe(III)MP-9 it was expected that the high valence forms
of the lypoenzyme could also oxidize this aldehyde increasing
the rate of the Compound II to Fe(III)MP-9 conversion.
During the reaction of MP-9/CTAB with DPAA, the yield of
the product, benzophenone, was dependent of the micelle/MP-9
molar ratio.
obtained in water (1.5 ꢄ 10ꢀ5 cm2
s
ꢀ1) indicating that the
peptides were associated to the micelles and diffused to the
electrode more slowly than in water.
Interestingly, as previously reported and according to UV-
visible spectral changes observed when microperoxidases were
associated to CTAB micelles,21,22 the increase of B100 mV in
The proposed reaction mechanism (Scheme 3) was corrobo-
rated by the identification of benzophenone as the final
reaction product (Figs. 4B, 5 and 6). Interestingly, a significant
difference in the benzophenone yield could be observed ac-
cording to the micelle/MP-9 molar ratio (Fig. 4A and B). This
difference could be assigned to the accessibility of the amino
group to the aldehyde. The analysis of Fig. 4C suggests that
the decrease of the micelle/MP ratio favors the association of
hemepeptide pairs into the micelle. Only the assumption that
at least a couple of MP molecules was occupying one CTAB
micelle could explain the MP-9 UV-visible spectra obtained in
the CTAB micelle/MP-9 ratio = 0.50 and 0.25. In this regard,
the UV-visible spectrum of MP-9 obtained with CTAB mi-
celle/MP-9 ratio = 5.0 seems to be a composite of the
spectrum of monomeric MP-9 inside the micelles and dimeric
or polymeric MP-9 also inside the micelles. The decrease of the
CTAB micelle/MP-9 ratio led to a change of the equilibrium
favoring the latter aggregates. In a dimeric association with
CTAB micelles, Lys13 amino groups could be forced to locate
out of the micelle where the concentration of DPAA was
significantly lower due to the partition of the aldehyde, pre-
ferentially inside the micelle core. In the monomeric form the
Lys13 amino groups could be found inside the micelle core and
thus be prone to react with DPAA, leading to the formation of
Schiff base adducts. In the monomeric form, the peptide chain
of MP-9, being preferentially inside the micelle, could be also
prone to being attacked by the intermediate free radicals
formed in the course of the reaction. The trapping of the
intermediate radicals would decrease the yield of benzophe-
none without the impairment of the heme iron reduction, a
process that precedes the formation of these radicals (Fig. 4A
and B). The proposed model was corroborated by the com-
parison of the MP-9 heme iron EPR spectra obtained with the
micelle/MP-9 molar ratio 5.0 and 0.5 (Fig. 4D). In both
conditions, MP-9 was predominantly in the high spin form
that is in accordance with the proposal that Lys13 a- and e-
amino groups could be out of the micelle and not in the micelle
core where the reaction intermediate free radicals are gener-
ated. The EPR spectra (Fig. 4D), similarly to what was
suggested by the UV-visible spectra (Fig. 4C), indicated that
different high spin species are favored in different micelle/MP-
9 molar ratios. The MP-9/CTAB EPR spectrum obtained at
micelle/MP-9 molar ratio = 5.0 reveals higher distortion
degree of the axial symmetry of the heme iron as compared
with the EPR spectrum obtained at micelle/MP-9 molar
ratio = 0.5 since the increase of the micelle/MP-9 molar ratio
changed the g shift from 0.070 to 0.259. Scheme 4 illustrates
the putative structures of MP-9/CTAB aggregates obtained in
low and high micelle/hemepeptide ratios.
1
the microperoxidases E was compatible with the lower degree
2
of heme exposure28 and indicates that inside the CTAB
micelles, microperoxidases are better oxidant species than
exposed to the aqueous medium. Accordingly, the decrease
in the diffusion coefficient in the presence of CTAB could only
be explained by the presence of the redox entity inside a
micellar aggregate.
The MP-9/CTAB and its oxoferryl form can use an aldehyde as
reducing agent
The significant increase in the rate of the conversion of MP-9
Compound II to the native form observed when DPAA was
added to the MP-9 oxoferryl species shows that, similarly to
peroxidases as HRP,29 MP-9 Compound II can use dipheny-
lacetaldehyde as reducing agent. At the end of the catalytic
cycle, when the peroxide was depleted, Fe(III)MP-9/CTAB was
able to oxidize DPAA, being converted to the ferrous form,
i.e., Fe(II)MP-9 (Fig. 2C and D). HRP was unable to react
with DPAA in the absence of peroxides29 but ferric cyto-
chrome c could be reduced by DPAA in a pH-dependent
manner.30 In the case of cytochrome c it was verified that two
ionizable groups of the protein with pKa = 8.9 and pKa =
11.4, probably Tyr67 and Tyr72, are related to the electron
transfer from DPAA to heme iron. Probably, for cytochrome c
reduction, the stacking of DPAA phenyl rings with tyrosine
phenol groups (one ionized and the other protonated) is
crucial to electron transfer from DPAA to heme iron. While
protonated, cytochrome c tyrosine residues stabilize the re-
duced form of DPAA. On ionization of one cytochrome c
tyrosine residue, the DPAA enol form increases its electron-
donating capability, favoring electron transfer to cytochrome
c heme iron. Therefore, for cytochrome c, the DPAA stacking
and the ionization of tyrosine residues are the limiting step in
the electron transfer process and not the potential of the
aldehyde and the heme iron. In the case of HRP, the ineffi-
ciency of DPAA to reduce the enzyme should be expected as
the one electron reduction of the heme iron, in non deaerated
medium, should lead to the formation of HRP Compound III
(ferrous/dioxy/ferric-superoxide complex), an unstable HRP
intermediate that rapidly decays to the ferric form.31 The
lypoenzyme MP-9/CTAB is stable in the reduced form and
the reaction is favored probably because the hydrophobic core
of the micelle accumulates DPAA, a molecule with low polarity
degree. In this regard, the UV-visible spectrum of Fe(II)MP-9/
CTAB suggests that the reduced heme iron is coordinated with
molecular oxygen as the Q bands are well-defined (Fig. 2C,
dashed line) similarly to oxyhemoglobin.32,33 Since DPAA can
The existence of dimeric MP-9 associated to CTAB micelles
in low CTAB micelles/MP ratios was reinforced by the heme
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Scheme 4 Proposed model for the reaction path of DPAA with MP-9 in CTAB micelles according to the CTAB/MP-9 ratio.
iron EPR spectrum obtained in this condition.22 In the pre-
sence of CTAB (micelle/MP ratio = 6.0), only the high spin
form of MP-9 was found but with different EPR parameters
compared to the high spin state observed in aqueous medium.
In CTAB micelles, the prosthetic group exhibits a subtle
rhombic distortion, probably due to interactions between the
porphyrin rings paired inside the micelle. The characterization
of CTAB micelles associated to a pair of microperoxidases
concerning the structure and physico-chemical behavior of the
aggregate was not the scope of the present work and is under
investigation in our laboratories.
ꢂ
The exclusive ability of both Compound II and
Fe(III)MP-9/CTAB to oxidize DPAA contrary to HRP that
can only do it in the high valence states and cytochrome c, only
the alkaline pH values.
ꢂ The high yield of benzophenone production obtained with
the catalyst MP-9/CTAB, which was not observed for the pre-
viously studied enzymes: HRP and cytochrome c. In this regard it
is important to extend the future investigation to other substrates
concerning application of this catalyst in nanotechnology.
ꢂ The possibility to modulate the reaction mechanism of
aldehydes’ oxidation according to the MP/CTAB ratio, also
important for future application of this catalyst.
Conclusion
Acknowledgements
Important findings can be outlined from this work:
ꢂ The determination of the redox potential of the previously
described lipoenzyme MP/CTAB.
We are grateful for the Brazilian research funding agencies
FAPESP (Fundac¸ ao de Amparo a Pesquisa do Estado de Sao
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Paulo), FAEP-UMC (Fundac
Pesquisa da Universidade de Mogi das Cruzes), CAPES
(Coordenadoria de Aperfeicoamento de Pessoal de Ensino
¸
ao de Amparo ao Ensino e
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