1. health and fitness
2. disease
3. cholesterol

CHAPTER
4
RATES OF OXIDATION OF PHENOLS BY COMPOUND U OF
HORSERADISH PEROXIDASE AND LACTOPEROXIDASE
AND THEIR DEPENDENCE ON THE REDOX POTENTIALS
4.1
Introduction
Oxidation of para substituted phenols by horseradish peroxidase compound II
(HRP-II) and Lactoperoxidase compound II (LPO-H) were studied using stopped flow
technique. Apparent second order rate constants (kapp) of the reactions of the
oxidisable substrates were determined. In the present study, we have compared the
kinetics of oxidation of phenols by HRP-II and LPO-II, with the redox potentials of
the substrates. Reorganisation energy of electron-transfer in phenols was estimated
from the variation of second order rate constants with the thermodynamic driving
force.
Heme peroxidases are widely distributed in the living matter [1-3]. They
catalyse oxidation of broad variety of organic as well as inorganic substrates (AH2) by
hydrogen peroxide or alkylperoxides, usually but not always, via free-radical
intermediates represented by following equations [1],
E + H20 2
->
E - 1 + AH2
E —I + H 20
->
E - II + AH2
2AH.
E - II + AH.
E + AH. + H20
—»■
Products,
59
(1)
(2)
(3)
(4)
in which E, E-I, E-II represent native peroxidase (HRP/LPO) and its two spectros­
copically and kinetically distinct intermediates, peroxidase-I and peroxidase-II respect­
ively. Among the oxidisable aromatic substrates, phenols are known to react via a free
radical intermediate mechanism depicted [4-14]. Substituted phenols have been shown
to react with HRP -II, in their protonated form and the electron donation to the ferryl
group, (Fe™ = 0), of HRP -II, is accompanied by a concomitant proton donation [5-7],
Reactions of phenols are chemically controlled bimolecular reactions with rapid pre­
equilibrium of the reactants forming a loose precursor complex, which is characterised
by large Michaelis-Menton (Km) constant, particularly at sufficiently high substrate
concentration [5,6,12,15], This is followed by a rate determining redox step [4-14],
Dunford and Adeniran [7] showed that the rates of oxidation of phenols followed
Hammett’s rules. Kinetic and molecular orbital studies showed that the rate of
oxidation also correlated well with the highest occupied molecular orbital energy levels
of phenols [13,14] and showed that the rate of oxidation by HRP-II depends on the
ease of oxidation [7,13,15], Mechanism of electron transfer, however, not fully
understood and also such studies on LPO -II are very limited [16],
HRP-C and LPO were purified by the procedures re[ported in the literature
[16,17], with Rzs 3.2 and 0.94 respectively.
4.2 Results and Discussions :
>
Figure 4.1 shows a plot of kob, vs [S] for HRP-II as well as LPO-H reactions
with phydroxy-benzoic acid as the substrate. It is seen that up to a substrate
concentration of »350 pM, the k ^ values vary linearly with [S], Similar behaviours of
kobs with [S] were observed for the reactions of all the other phenols studied here. This
reaction can be expressed as,
HRPr = 0 +S -» [ HRPFe^ = O - S] -» HRPFem + products (5)
ki, k_i
kET
which gives the relation between kob, and [S] as
60
[S]
OiM)
Figure 4. 1 : Plot of kobsvs substrate (p-hydroxy-benzoic acid) concentration
at pH = 7.0, and at 23° C, for LPO-II and HRP-II. Solid lines are leasts square
fit of the data to equation 7.
kob, =
(6)
kET[S ]/K D+[S]
where KD = [HRP-II][S] / [HRP-II][S] = k-1/ k, = K*
is the dissociation constant (Michaelis constant) of the enzyme substrate
complex. For a loose enzyme-substrate precursor complex, i.e.Ko »
[S], a linear
relationship,
kobs ~ (kET / Kd) [S] « kapp [S]
(7)
is observed. The linearity in kobs vs [S] plots (figure 4.1) is therefore, due to Kd
»
[S]. Apparent second order rate constant, kapp, values were deduced from the kob*
vs [S] plots by least squares fit to equation 7 and also deduced from the slope of the
kot» vs [S] plots, agreed very well.
For a large number of substituted phenols, one electron redox potentials
E(PhO /PhOH) have been measured by pulse radiolysis and are available in the
literature [21-23], Figure 4. 2 shows plot of lnkappvs E(PhO/PhOH) in Volts, the one
electron redox potentials of the phenols used in the measurement. Essentially non­
linear but relatively smooth variation of lnkapp for the reactions of peroxidase-II
(HRP-II/LPO-H) with the substrate redox potentials strongly suggests that the nature
of interaction of phenols with both the enzymes seems to be very similar. Figure 4.2
shows that the lnkapp for phenols with electron donating substituents such as methyl,
methoxy and chloro groups are larger in magnitude relative to electron withdrawing
substituents. Dunford and Adeniran [7] observed that the second order rate constants
o f the reactions of phenols with HRP-II followed Hammett equation (linear
correlation), but the Okomoto-Brown plot of the same rate constants was non-linear
[24],This was explained by a mechanism of phenol oxidation, in which the substrate
simultaneously gives an electron and a proton to HRP- II.
This mechanism was however, questioned on the basis that the Hammett
coefficients used were in error [27], It was suggested that electron transfer is followed
by proton loss by the substrate [27], The redox potentials of the substituted phenols
61
Oxidation Potential
(v)
Figure 4. 2 : Plot of log k.__
vs one electron oxidation potential
® rr
of various p-substituted phenols for LPO II (O) and HRP II (A).
The solid line drawn to highlight the non-linearity of the variation.
however, satisfy linear Hammett correlation using Okamoto-Brown (a+) substituent
constants [22,24-26]. Figure 4.2, therefore, represents Okamoto-Brown plot of both
LPO-II and HRP-II reactions which are non-linear. The mechanism of oxidation of
phenols occurs by Dunford mechanism.
The reactivity of the enzyme towards substrate is related to the
thermodynamic driving force.
Since one electron redox potentials of the unstable
organic radicals of the substrates are known [21-23], it is possible to estimate the
reorganisation energy parameter for the electron transfer using Marcus treatment [2829],
Thermodynamic driving force for reaction of peroxidase -II with the reducing
substrate (equation 3) is the difference between the mid-point potentials of the
peroxidase-II / peroxidase (HRP-II / HRP) or (LPO-II / LPO) and substrate radical /
substrate (S**tT / SH) redox couple.
AE
= E (peroxidase-II / peroxidase) - E (S**H" / SH)
(8)
Mid-point potentials are defined as the potential at which the concentration of
the reduced and the oxidised forms of the couple are equal [30], E(HRP-II/HRP =
0.903 V at pH = 7.6 has been reported by He at al. [31], but no such data is available
for LPO-H/LPO couple. Figure 4.3 shows the variation of the rate of reduction of
HRP-n on the thermodynamic driving force of the reactions of phenols.
>
The thermodynamic free energy of the electron transfer reaction (AG°) is equal
to the -nFAE (n number of electrons transferred, F is the Faraday constant). The
increase in lnkapp on increasing the thermodynamic force (AE) as in figure 4.3,
therefore, indicates that the electron transfer rate (or activation energy) is
predominantly regulated by the redox potential difference between the substrate and
HRP-II and the reaction can be described by an “outer-sphere” mechanism [27,28,31].
This is also consistent with the observation by Dunford and others [5,6,12,15] that the
phenol oxidation reactions are predominantly chemically controlled.
According to Marcus treatment [28,29] of electron transfer reactions, the
parabolic curve (figure 4.3) representing the dependence of the logarithm of the rate
62
18
16 -
14 -
dde,
- *
c
12
-
10
-
8
-
6 i ----------------------1----------------------1----------------------1----------------------1---------------------- \
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
E ( HRP II / HRP ) - E ( S ~ H+ / S H )
Figure 4.3 : Variation of rate of reduction of HRP II to HRP on the
thermodynamic driving force of the reaction . Natural logarithm
of electron transfer rate constant (In kapp) are plotted against
AE. The solid curve calculated using parameters obtained by
leasts square fit of the data to equation 11. (see Text)
constant (IiiIcet) on the thermodynamic driving force (AE) is associated with the
reorganisation energy X as given by
kET = kET0 exp ( -AG* / RT )
where
k ET°
(9)
is the rate of activationless
electron transfer in the enzyme-
substrate complex and AG* is the activation free energy, given by;
AG* =
X( 1-(FAEA.) f 4
(10)
Combining equations 7, 9 and 10 to yield,
lnkapp = lnkapp° - ( X-AE)274 kB X T
(11)
where kB is Boltzmann constant. The reorganisation energy X (in units of eV) is
related to the internal nuclear, as well as solvent molecule, rearrangements that must
occur prior to electron transfer between the donor and the acceptor atoms [28,29],
The solid line in figure 4.3 shows the least squares fit to equation (11)
(correlation coefficient, r2 = 0.85) using two variable parameters lnkapp0 and X. The
values are X = 0.2± 0.1eV and lnkapp0 = 16.0 ±0.8. X values reported for typical
electron transfer proteins are in the range of 0.1-1.75 eV [29], Relatively low value of
0.2 eV obtained here, may be due to electron transfer which occurs in a loose enzymesubstrate complex.
The HRP structure shows that the heme edge is particularly
oriented for formation of such a complex, which may be helpful to minimise the donoracceptor distance. It also reflects rigidity of the electron accepting moiety, the heme
group. Figure 4 shows a plot for LPO-II reactions with the phenols. The similarity in
the nature of variation of lnk*pp vs thermodynamic force indicates that the mechanism
of electron in both these peroxidases is same. Here we have used the same E(HRPII/HRP) = 0.903 V at pH 7.6 to calculate the thermodynamic driving force as this
quantity is not known. The values of the parameters estimated by the least squares fit
to the equation 9 are, X = 0.09 ± 0.05 and lnv = 15.4 ± 0.8
and correlation
coefficient r2 = 0.31. The reason for low correlation coefficient may be the E(LPOn/LPO) value used may be in error. Also the measurements need to be extended on
63
app
E (LPO 117 LPO ) - E ( S ~H+ / SH )
Figure 4 .4 : Variation of rate of reduction of LPO II to LPO on the
thermodynamic driving force of the reaction . Natural logarithm
of electron transfer rate constant (In kaDD) are plotted against
" H r
0.903 - v values of HRP I I I HRP. The solid curve calculated
using parameters obtained by leasts square fit of the data to
equation 11. (see Text)
more reducing phenols.
However, the increase in the electron transfer rate with
increasing thermodynamic driving force is similar as in HRP-II. Considerably low
reorganisation energy compared to HRP-II reactions may be that LPO with large
molecular size [32] has heme moiety in very rigid [33] and deeply buried in the protein
fold [34],
64
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