Oxidation of sulfides by peroxidases. Involvement of radical cations and the rate of the oxygen rebound step

Full text of DOI:10.1021/ja9608003

J. Am. Chem. Soc. 1996, 118, 8973-8974
8973
Table 1. HRP Catalyzed Oxidations of Sulfides 1 and 2 by H2O2
Oxidation of Sulfides by Peroxidases. Involvement
of Radical Cations and the Rate of the Oxygen
Rebound Step
y
Enrico Baciocchi,* Osvaldo Lanzalunga, and
Stefania Malandrucco
Centro CNR di Studio sui Meccanismi di Reazione and
Dipartimento di Chimica
a Absolute yields in µmol. Average of at least two determinations.
b Values in parentheses are referred to the reaction with H2O2 in the
absence of the enzyme. No products are formed when H2O2 is omitted.
UniVersita` “La Sapienza”, I-00185 Roma, Italy
Marcella Ioele and Steen Steenken*
Scheme 1
Max-Planck-Institut fu¨r Strahlenchemie
D-454713 Mu¨lheim, Germany
ReceiVed March 11, 1996
Horseradish peroxidase (HRP) is a hemoprotein peroxidase
capable of catalyzing the oxidation of a large variety of organic
compounds. The reaction mechanism generally involves the
sequential electron abstraction from two substrate molecules,
whereby the ferryl porphyrin radical cation (Por•+-FeIVdO),
compound I, formed by reaction of H2O2 with the ferric enzyme,
is reduced first to Por-FeIVdO, known as compound II, and
then to the resting ferric state.1-3 Typical of these oxidations
is that HRP does not transfer the ferryl oxygen to the substrates.
Thus the finding that HRP can catalyze the oxidation of sulfides
to sulfoxides by a 2-e process involving the ferryl oxygen
transfer to the S atom has recently raised great interest.4-11
Two mechanisms have been proposed to explain this obser-
vation, both of which involve, as the initial step, electron transfer
from the sulfide to compound I to give the radical cation R2S•+
(eq 1).8,10 The mechanisms differ with respect to the conversion
with a pulse radiolysis investigation of the radical cations 1•+
and 2•+. For comparison were investigated the oxidations of 1
and 2 with chloroperoxidase (CPO).
Por•+-FeIVdO (I) + R2S f Por-FeIVdO (II) + R2S•+ (1)
1 and 2 were reacted with HRP using standard procedures.9,11
For example, the sulfide (20 µmol) and HRP (Sigma, type VI)
(0.05 µmol) in 3 mL of 0.1 M phosphate buffer, pH 6, at 25 °C
were magnetically stirred. H2O2 (20 µmol) was added in 10
aliquots at 10 min intervals. The reaction was quenched with
sodium sulfite 2 h after the first addition of H2O2 and the
solution extracted with CH2Cl2. The products in the organic
phase (alcohol and aldehyde) were quantitatively determined
by GC, whereas the sulfoxide in the aqueous phase was analyzed
by HPLC on a reversed-phase column (C8) using 0.1% (v/v)
trifluoroacetic acid in MeOH-H2O (1:1 v/v) as the mobile
phase. As internal standards were used 4-MeO- or 4-HOC6H4-
COCH3 (GC or HPLC).
of R2S•+ to sulfoxide. In one, it is suggested that sulfides bind
to the enzyme at a site different from where the classical HRP
substrates bind, enabling the transfer of oxygen from compound
II to the sulfide radical cation, the oxygen rebound step (eq
2).8 In the other mechanism,10,11 it is proposed that a hydroxyl
Por-FeIVdO (II) + •+SR2 f Por-FeIII + OdSR2 (2)
radical (presumably formed after H+ transfer to oxygen by a
distal group) is released from compound II, which then reacts
with the sulfur radical cation forming the sulfoxide.
The reaction products (Table 1) are the sulfoxides of 1 and
2 (major products) and benzaldehyde and benzyl alcohol from
1 and 4-methoxybenzaldehyde and 4-methoxybenzyl alcohol
from 2. The aldehydes and the alcohols are typical products
of fragmentation of the radical cations, 1•+ and 2•+, from which
they derive by C-H deprotonation to form the benzyl radicals
3 and by C-S bond cleavage to produce the benzyl carbocations
4. Benzaldehyde is obtained by oxidation of 3 followed by
hydrolysis (Scheme 1, X ) H, OCH3, path a), a path resembling
that suggested for the N-dealkylation of amines by HRP.14 The
benzyl alcohols derive from the reaction with water of the benzyl
carbocations (path b).15 The sulfoxides are conceivable as
products of an oxygen rebound step, eq 2.
To get further insight into the mechanism of oxidation of
R2S by HRP, we have now investigated the HRP-induced
oxidation of the water soluble aromatic sulfides 1 and 212 which
can form radical cations capable of undergoing C-H and C-S
bond cleavage,13 reactions that lead to products different from
sulfoxides. The results of this study are presented here together
(1) Dunford, H. B. In Peroxidases in Chemistry and Biology; Everse, J.,
Everse, K. E., Giishman, M. B., Eds.; CRC Press: Boca Raton, FL, 1991;
Vol. II, pp 1-24.
(2) Dunford, H. B.; Stilman, J. S. Coord. Chem. ReV. 1976, 19, 187.
(3) Ortiz de Montellano, P. R. Annu. ReV. Pharmacol. Toxicol. 1992,
32, 89.
(4) Kobayashi, S.; Nakano, M.; Goto, T.; Kimura, T.; Schaap, A. P.
Biochem. Biophys. Res. Commun. 1986, 135, 166.
(5) Kobayashi, S.; Nakano, M.; Kimura, T.; Schaap, A. P. Biochemistry
1987, 26, 5019.
(6) Doerge, D. R. Arch. Biochem. Biophys. 1986, 244, 678.
(7) Doerge, D. R.; Cooray, N. M.; Brewster, M. E. Biochemistry 1991,
30, 8960.
(8) Harris, R. Z.; Newmyer, S. L.; Ortiz de Montellano, P. R. J. Biol.
Chem. 1993, 268, 1637.
(9) Ozaki, S.-I.; Ortiz de Montellano, P. R. J. Am. Chem. Soc. 1994,
116, 4487. Ozaki, S.-I.; Ortiz de Montellano, P. R. J. Am. Chem. Soc.
1995, 117, 7056.
(12) Compounds 1 and 2 were obtained by the reaction of benzyl and
4-methoxybenzyl chlorides with 4-mercaptobenzyl sulfonic acid12a,b in the
presence of anhydrous potassium carbonate, in refluxing acetone.12c (a)
Beringer, F. M.; Falk, R. A. J. Am. Chem. Soc. 1959, 81, 2977. (b) Kawai,
H.; Sakamoto, F.; Taguchi, M.; Kitamura, M.; Sotomura, M.; Tsukamoto,
G. Chem. Pharm. Bull. 1991, 39, 1422. (c) Baciocchi, E.; Intini, D.;
Piermattei, A.; Rol, C.; Ruzziconi, R. Gazz. Chim. Ital. 1989, 119, 649.
(13) Baciocchi, E.; Rol, C.; Scamosci, E.; Sebastiani, V. J. Org. Chem.
1991, 56, 5498.
(10) Perez, U.; Dunford, H. B. Biochim. Biophys. Acta 1990, 1038, 98.
(11) Colonna, S.; Gaggero, N.; Carrea, G.; Pasta, P. J. Chem. Soc., Chem.
Commun. 1992, 357.
(14) Okazaki, O.; Guengerich, F. P. J. Biol. Chem. 1993, 268, 1546;
Guengerich, F. P.; Okazaki, O.; Seto, Y.; Macdonald, T. L. Xenobiotica
1995, 25, 689.
S0002-7863(96)00800-1 CCC: $12.00 © 1996 American Chemical Society

8974 J. Am. Chem. Soc., Vol. 118, No. 37, 1996
Communications to the Editor
In order to check these ideas, 1•+ and 2•+ were produced by
chemical oxidation using SO4•- (eq 4). Thus, a deoxygenated
solution of 1 or 2 (0.1 mM), K2S2O8 (2 mM), and t-BuOH (0.1
M) at pH 4 was irradiated with a 60Co γ source (product
analysis) or with 0.4 µs pulses of 3 MeV electrons delivered
•-
by a van de Graaff accelerator (pulse radiolysis). SO4 was
obtained via the radiation-chemically generated hydrated elec-
tron, e-aq (eq 3). The products from 1 were benzyl alcohol and
benzaldehyde in a 0.9:1 ratio; those from 2 were the corre-
sponding 4-methoxy compounds in a 2.5:1 ratio (the sulfoxides
of 1 and 2 were not produced).16 These ratios are similar to
those from the enzymatic oxidation (see Table 1).
2-
e-aq + S2O82- f SO4•- + SO4
(3)
(4)
SO4•- + 1 (2) f SO42- + 1•+ (2•+)
Figure 1. Absorption spectra recorded on pulse radiolysis (dose ) 4
Gy) of an Ar-purged aqueous solution (pH 4.0) of 2 (0.1 mM), K2S2O8
(2 mM), and t-BuOH (0.1 M) at the times after the pulse as indicated
by the symbols. Inset a shows the decay of 2•+ at 550 nm; inset b
shows the rise of conductance resulting from the decay of 2•+ at pH
4.5 (dose ) 1.6 Gy); inset c shows the formation and decay (by radical-
radical reaction) of the carbon-centered radical 3 (X ) OMe) at 360
nm, in the absence of O2. From inset d it is evident that 3 is scavenged
by O2. The decay at 360 nm has the same rate as that of 2•+ at 550
nm which shows that 2•+ has a weak absorption at 360 nm.
With pulse radiolysis, on reaction of 1 and 2, strong
absorptions at 320 and 520-600 nm were observed 8 µs after
the initiating pulse (as seen in Figure 1 for the case of 2). The
transients are assigned to the corresponding radical cations, in
the monomeric form.17 Both 1•+ and 2•+ decay by a first-order
process with a rate constant of 2.6 × 103 s-1 (1•+) and 3.6 ×
103 s-1 (2•+), respectively.18 In the inset b of Figure 1, it is
shown that the decay of the radical cation is accompanied by
an increase of conductance, which is due to the production of
H+. The radical cation undergoes two decomposition reac-
tions: deprotonation with formation of the benzyl-type radical
3 (X ) H, OCH3), which has an absorption band at 360 nm19
(see Figure 1, 220 and 750 µs after the pulse) and which can
be scavenged by O2 (see inset d), and cleavage of the C-S
bond with formation of the arylthiyl radical 5, which is
recognizable by the absorptions at 310 and 420-540 nm
observed after 220 and 750 µs.20 The lifetimes of the radical
cations or of 5 were not influenced by O2.
If in the enzymatic reaction the sulfide radical cations undergo
partitioning between oxygen rebound and fragmentation, as
illustrated in Scheme 2, it is possible to estimate the rate of the
oxygen rebound step, kr, from the overall fragmentation rate
(kf) of the radical cation and the product distribution. The
fragmentation rate in the absence of enzyme is that measured
by the pulse radiolysis technique, and it is assumed that it is
the same in the presence of the enzyme.
Scheme 2
in view of the differences in microenvironment between the
chemical and the enzymatic systems.
In order to compare HRP with another enzyme, we studied
the oxidation of 1 and 2 as catalyzed by CPO.21 The CPO-
induced oxidations lead exclusively to the formation of sulfox-
ides; i.e., there was no evidence for the formation of the
fragmentation products of the radical cation. Clearly, if sulfide
radical cations are at all involved in this reaction, the oxygen
rebound step must be much faster with CPO than with HRP, a
conclusion in line with the greater accessibility of the ferryl
oxygen in the former enzyme which makes possible a direct
oxygen transfer to the radical cation.22 However, the possibility
cannot be excluded that with CPO the O is transferred to the
sulfide without the intervention of free radical intermediates, a
path that is likely in view of the recent report suggesting an
oxygen insertion mechanism in CPO-induced alkane hydroxy-
lation.23
Then, from the kf-value and the ratio of the yield of sulfoxide
and that of the fragmentation products in the enzymatic reaction
(see Table 1), the rate constant of the oxygen rebound process
leading to sulfoxide from the radical cation (see Scheme 2)
follows as kr ) kf[ArCH2(Ar′)SdO]/[ArCH2OH + ArCHO] )
6.3 × 103 and 7.2 × 103 s-1 for 1•+ and 2•+, respectively. Of
course, these numbers have to be considered with some caution,
(15) The reaction of the (incipient) benzyl cation with water may be
concerted with the fragmentation of the C-S bond.
Acknowledgment. Financial contributions of the European Union
(CEE ERBSC1-CT91-0750) and the Italian Ministry for the University
and Scientific Research (MURST) are gratefully acknowledged.
(16) The benzyl alcohols and benzaldehydes were also formed on
chemical oxidation with CoIIIW12O40
.
5-
(17) The same signal was observed when the aromatic sulfide radical
cation was generated by reaction of e-aq with the sulfoxide (cf. Engman, L.;
Lind, J.; Mere´nyi, G. J. Phys. Chem. 1994, 98, 3174), i.e., in the absence
of any sulfide which could possibly lead to the formation of a dimeric radical
cation.
JA9608003
(21) The sulfide (20 µmol) and CPO (Sigma) (0.3 nmol) were magneti-
cally stirred in 5 mL of 0.1 M citrate buffer, pH 5, at 25 °C. H2O2 (20
µmol) was added in 5 aliquots at 5 min interval. The reaction was quenched
with sodium sulfite 45 min after the first addition of H2O2 and the solution
extracted with CH2Cl2. No products were detected by GC analyses of the
organic phase, whereas the sulfoxides (2 and 5.4 µmol from 1 and 2,
respectively) were observed and quantitatively determined by HPLC
analyses of the aqueous phase.
(22) Griffin, B. W. In Peroxidases in Chemistry and Biology; Everse,
J., Everse, K. E., Giishman, M. B., Eds.; CRC Press: Boca Raton, FL,
1991; Vol. II, pp 123-126.
(23) Ortiz de Montellano, P. R.; Choe, Y. S.; De Pillis, G.; Catalano, C.
E. J. Biol. Chem. 1987, 262, 11641.
(18) Due to second-order contributions, the measured rate constants are
somewhat dose dependent. To correct for this effect, dose variations were
performed, on the basis of which the rate constants for the decomposition
of 1•+ and 2•+ were extrapolated to dose ) 0.
(19) The benzyl radicals 3 can be selectively produced by reaction of 1
or 2 with O•- at pH 13.5 or by reaction of 1•+ and 2•+ with a high
concentration of base such as OH- or HPO4 (Ioele, M.; Steenken, S.;
2-
Baciocchi, E. Unpublished results).
(20) The spectrum of the authentic arylthiyl radical 5 was obtained by
oxidizing 4-HSC6H4CH2SO3 with Tl2+ at pH 3.6, using pulse radiolysis
-
(N2O-saturated aqueous solution, 1 mM Tl2SO4, 0.1 mM HSC6H4CH2-
SO3K).