Pulse radiolysis investigation on the mechanism of the catalytic action of Mn(II)-pentaazamacrocycle compounds as superoxide dismutase mimetics

Article abstract of DOI:10.1021/jp800690u

The mechanism for the catalytic dismutation of superoxide by the Mn(II) pentaazamacrocyclic compound M40403 ([manganese(II) dichloro-(4R,9R,14R,19R)-3, 10,13,20,26 pentaazatetracyclo [20.3.1.0^4,9.0_14,9] hexacosa1(26),-22(23),24-triene], SODm1) and two 2,21-dimethyl analogues has been investigated using pulse radiolysis. The initial rate of reaction between superoxide and the manganese compounds was found to be dependent on structure and pH, with the resulting transient adducts possessing spectral characteristics of the metal center being oxidized to Mn(III). Values for the pK_a of the transient adducts (pK_a = 5.65 ± 0.05; 5.3 ± 0.1 and <5 for SODml, SODm2 and SODm3, respectively) were obtained from spectrophotometric and conductivity measurements. Reaction of these transient adducts with further superoxide was highly structure dependent with the 25,21S-dimethyl derivative (SODm2) being highly catalytically active at pH 7.4 (k_cat = 2.35 × 10⁸ M^-1 s^-1) compared to SODm1 (k_cat = 3.55 × 10⁶ M^-1 s^-1). In contrast the 2R,21R-dimethyl derivative (SODm3) showed no dismutation catalysis at all. The reaction rates of the initial complexes with HO₂· were significantly lower than with O₂ ^·-, and it is proposed that O₂^·- is the main reactant in the catalytic cycle at pH 7.4. Variable temperature studies revealed major differences in the thermodynamics of the catalytic cycles involving SODm2 or SODm1. In the case of SODm2, the observed high entropic contribution to the activation energy is indicative of ligand conformational changes during the catalytic step. These results have provided the basis for a new mechanism for the catalytic dismutation of superoxide by Mn(II)-pentaazamacrocycle SOD mimetics.

Full text of DOI:10.1021/jp800690u

J. Phys. Chem. A 2008, 112, 4929–4935
4929
Pulse Radiolysis Investigation on the Mechanism of the Catalytic Action of
Mn(II)-Pentaazamacrocycle Compounds as Superoxide Dismutase Mimetics
†
‡
§
†
Andrej Maroz, Geoffrey F. Kelso, Robin A. J. Smith, David C. Ware, and
Robert F. Anderson*
Department of Chemistry, The UniVersity of Auckland, PriVate Bag 92019, Auckland 1142, New Zealand,
Centre for Green Chemistry, Monash UniVersity, P.O. Box 75, Clayton, Victoria 3800, Australia, Department
of Chemistry, UniVersity of Otago, Box 56, Dunedin 9054, New Zealand
ReceiVed: January 23, 2008; ReVised Manuscript ReceiVed: February 28, 2008
The mechanism for the catalytic dismutation of superoxide by the Mn(II) pentaazamacrocyclic compound
4,9 14,9
M40403 ([manganese(II) dichloro-(4R,9R,14R,19R)-3,10,13,20,26 pentaazatetracyclo [20.3.1.0 .0 ] hexacosa-
(26),-22(23),24-triene], SODm1) and two 2,21-dimethyl analogues has been investigated using pulse radiolysis.
1
The initial rate of reaction between superoxide and the manganese compounds was found to be dependent on
structure and pH, with the resulting transient adducts possessing spectral characteristics of the metal center
being oxidized to Mn(III). Values for the pK of the transient adducts (pK ) 5.65 ( 0.05; 5.3 ( 0.1 and <5
a a
for SODm1, SODm2 and SODm3, respectively) were obtained from spectrophotometric and conductivity
measurements. Reaction of these transient adducts with further superoxide was highly structure dependent
8
with the 2S,21S-dimethyl derivative (SODm2) being highly catalytically active at pH 7.4 (kcat ) 2.35 × 10
-1
-1
6
-1 -1
M
s ) compared to SODm1 (kcat ) 3.55 × 10 M s ). In contrast the 2R,21R-dimethyl derivative (SODm3)
•
showed no dismutation catalysis at all. The reaction rates of the initial complexes with HO
2
were significantly
•-
•-
lower than with O
2
, and it is proposed that O
2
is the main reactant in the catalytic cycle at pH 7.4. Variable
temperature studies revealed major differences in the thermodynamics of the catalytic cycles involving SODm2
or SODm1. In the case of SODm2, the observed high entropic contribution to the activation energy is indicative
of ligand conformational changes during the catalytic step. These results have provided the basis for a new
mechanism for the catalytic dismutation of superoxide by Mn(II)-pentaazamacrocycle SOD mimetics.
Introduction
sidered as a disadvantage, as reaction with other radicals can
lead to the loss of SOD activity, as is known for natural
Mammalian cells are constantly exposed to the superoxide
1
2,13
•
-
MnSOD.
In the past decade, another approach to develop
anion (O2 ), which mainly arises as a metabolic byproduct in
1
efficient SOD mimetics, based on Mn(II)-pentaazamacrocyclic
complexes, has been particularly successful and an analogue
has been produced which exhibits specific activity comparable
the respiratory chain. Under normal circumstances, the intra-
•-
cellular level of O2 is controlled by superoxide dismutases
SOD), a group of endogenous enzymes that catalyze the
(
3
,14,15
•-
2
to that of natural SOD.
The SOD activity of these
transformation of O2 into dioxygen and hydrogen peroxide.
complexes is strongly dependent on the ligand structure, and
inversion of only one chiral center can result in a completely
However, during oxidative stress, such as in the case of acute
or chronic inflammation, O2•- production exceeds the level at
1
6
catalytically inactive compound. Early attempts to study the
mechanism by which these complexes dismutate superoxide
which these enzymes can cope. In such cases, treatment with
exogenous SOD to reinforce the natural defensive mechanisms
has been tried. However, the use of exogenous SOD, such as
that sourced as bovine SOD, is limited by both its size (Mr ∼
1
7
have been carried out using the stopped-flow method. How-
ever, this approach does not allow direct observation of
intermediates nor a detailed study of the individual steps of the
catalytic cycle. In this work we have utilized the faster time-
resolution of pulse radiolysis to obtain a more comprehensive
insight into the reactions of superoxide with these Mn(II)-
pentaazamacrocyclic SOD mimetics.
3
0 kDa) and complexity, which affects cell permeability and
3
–6
causes immunological problems.
This has stimulated the
search for synthetic, nonpeptidic compounds which can mimic
the dismutating activity of the natural enzymes and are more
readily bioavailable. Some of the lead compounds in this field
of endeavor are based on Mn(III)-porphyrins with modified
Three different Mn(II)-pentaazamacrocycle complexes
(SODm1-3) were studied (Scheme 1) with the objectives of
determining the nature of the intermediates formed, the rate
constants of the reaction of the compounds with superoxide and
7
–9
3,10
ligands as well as Mn(III) salen complexes.
Several of
the porphyrin SOD mimetics are reported to approach the
9
-1 -1
catalytic activity of natural SOD (kcat ∼ 10 M s ), being
•-
•-
-
the catalytic SOD activities (k ). Our data enable a thermodynamic
active not only toward O2 but also with CO3 , ONOO and
NO . However, nonselective radical scavenging can be con-
cat
•
11
description to be made for the functioning of these complexes.
*
Corresponding author. Fax:(+64)9-3737422. E-mail: r.anderson@
Materials and Methods
auckland.ac.nz.
†
The University of Auckland. E-mail: A.M., a.maroz@auckland.ac.nz;
Materials. All chemicals were of analytical grade and were
used as received. Water for preparation of the solutions was
purified using a Milli-Q system. SODm1 was prepared as
D.C.W., d.ware@auckland.ac.nz.
‡
§
Monash University. E-mail: Geoff.Kelso@sci.monash.edu.au.
University of Otago. E-mail: rajsmith@chemistry.otago.ac.nz.
1
0.1021/jp800690u CCC: $40.75  2008 American Chemical Society
Published on Web 05/13/2008

4
930 J. Phys. Chem. A, Vol. 112, No. 22, 2008
Maroz et al.
SCHEME 1: Structures of Mn(II)-Pentaazamacrocyclic SOD Mimetics Studied in This Work
originally described18 with further modifications. SODm2 and
SODm3 were prepared as reported.
19
radiolytically produced O2•- (1.55 µM). The observed rate
constants were plotted against the concentrations of the com-
plexes to obtain second-order rate constants (Figure 1). The
1
5
Pulse Radiolysis. Pulse radiolysis experiments were carried
out using the University of Auckland’s linear accelerator
producing 4 MeV electron pulses of 200 ns to 1.5 µs duration,
to deliver doses over the range 2-100 Gy. Data acquisition,
dosimetry and radical detection systems have been previously
described. The pH of the solutions was controlled using
phosphate buffers (10 mM) and adjusted to low and high pH
using HClO4 and NaOH, respectively, when required. Non-
buffered solutions were used for conductivity studies and
compared to N2O-saturated solutions containing dimethylsul-
foxide (1 mM) at low and high pH as the reference standard.
Oxygenated solutions were bubbled with pure oxygen for 15
min. tert-Butanol (0.2 M) or sodium formate (25 mM) were
•-
9
results show that O2 reacts with SODm2 (k6 ≈ 1.8 × 10
s ) about 20 times faster than with the other Mn(II)-
pentaazamacrocyclic complexes under investigation (k6 ≈ 9 ×
10 M for SODm1) (Table 1).
-
1
-1
M
7
-1 -1
s
2
0
To determine the nature of the intermediate complex formed
between O2• and the investigated compounds, reaction 6, we
first recorded the absorption spectrum of the transient formed
upon the reaction of O2• with SODm2. This spectrum has a
characteristic band at 310 nm and was found to be different to
-
-
•-
that produced upon one-electron reduction of SODm2 by CO2 ,
which presumably reduces the Mn(II) to Mn(I) in a diffusion-
9
-1 -1
controlled (k7 ) 3.4 × 10 M s ) process, reaction 7, to
give rise to a transient which has a distinctive band centered at
415 nm, Figure 2.
•
used as OH radical scavengers. Experiments were routinely
carried out at room temperature, 22 ( 2 °C.
CO2• + Mn
-
II
L
]2+
f CO + Mn
I
L
]+
(7)
Electrochemistry. Cyclic voltammetry was carried out under
N2 in dry acetonitrile solutions of the complexes (1 mM)
containing [(n-Bu)4N](PF6) (0.1 M) as the supporting electrolyte.
A BAS 100A electrochemical workstation and standard three
electrode cell was used. The BAS-supplied electrodes used were
glassy carbon (working electrode), platinum wire (counter
electrode) and Ag/Ag (3 M NaCl) (reference electrode).
Ferrocene (Fc) was used as an internal standard.
[
[
(
)
(
)
2
The one-electron oxidation of SODm2 was carried out using
the SeO3• radical, which was produced by the scavenging of
-
the e-aq by SeO4 anions, and the OH/H were scavenged by
2-
•
•
2
1,22
added tert-butanol to produce an inert radical.
Oxidation of
Mn(II) to Mn(III) in the complex by the SeO3• radical, has
-
9
-1
occurred rapidly with a rate constant of k ) 3.5 × 10 M
-
1
s , reaction 8.
SeO3 + [Mn (L)] f SeO32- + [Mn (L)]
•-
II
2+
III
3+
(8)
Results
The absorption spectrum of the oxidized complex exhibits a
rising band in the region below 370 nm, similar to that observed
following the reaction of O2•- with the complex. The spectra
Reaction of Superoxide with SOD Mimetics. Superoxide
was produced by the pulse radiolysis (2.5 Gy in 200 ns) of
oxygen-saturated aqueous solutions containing sodium formate
(
25 mM) and phosphate buffers (10 mM). Under these condi-
tions all of the primary radicals formed by the radiolysis of
water (reaction 1, yield values in µM Gy- are given in
parentheses) are converted into superoxide, O2• , reactions 25.
1
-
-
aq
•
•
+
H O f e (0.28), OH (0.28), H (0.06), H O (0.28),
2
3
H O (0.07), H (0.04) (1)
2
2
2
e- + O f O
•-
(2)
(3)
(4)
(5)
aq
2
2
•
•
2
H + O f HO
2
•
OH + HCO f CO2•- + H O
CO • + O f CO + O
-
2
2
•-
-
2
2
2
2
Superoxide was found to react with the complexes to form
transients which absorb in the UV-vis region.
Figure 1. Observed first-order rate constants for the reaction of
superoxide with increasing concentrations of SODm1 (squares), SODm2
(circles), SODm3 (triangles) at pH 7.4 (filled symbols) and pH 8.0
O2•- + [Mn (L)] f [Mn (L)O ]
II
2+
III
+
(6)
2
(
open symbols). Kinetics measured for the absorption increase at 320
The rate constants for reaction 6 were determined under
pseudo-first-order conditions, where the concentration of the
complexes was varied in great excess (20-100 µM) over the
nm following the pulse radiolysis (2.5 Gy in 200 ns) of oxygen-saturated
aqueous solutions of the complexes in the presence of sodium formate
(25 mM) and phosphate buffer (10 mM).

Mechanism of SOD Action of Mn(II)-Pentaazamacrocycles
J. Phys. Chem. A, Vol. 112, No. 22, 2008 4931
TABLE 1: Rate Constants for the Reaction of Superoxide
with the Complexes, k , and Catalytic Activity of SOD
6
Mimetics, kcat, at pH 7.4 and pH 8.0
-
6k
s
10-6k
-6
-6
1
M
0
6
/
6
/
10
M
k
cat
s-1
/
cat
10 k /
M
-
1
-1
-1 -1
-1
-1
s-1
M
s
complex
SODm1
pH 7.4
pH 8.0
pH 7.4
pH 8.0
89 ( 4
90 ( 5
3.55 ( 0.11 2.6 ( 0.1
178 ( 15
no catalysis no catalysis
SODm2 1680 ( 100 1800 ( 100 235 ( 20
SODm3
68 ( 5
66 ( 5
produced upon the reaction of O2• with the complex at pH 7.4
and 3.5 are radically different from the spectrum of the reduced
form (Figure 2) and the slight difference in shape can be attributed
to the protonation state, as these pHs are on the opposite sides of
the estimated pKa of the complex (Figure 3). Overall, the results
presented in Figure 2 indicate that O2• oxidizes the SODm2 to
yield an intermediate complex containing a Mn(III) center, reaction
-
-
6. Similar results were obtained with the other two complexes over
the same pH conditions.
Reaction of Superoxide with SOD Mimetics-pH Studies.
The pH dependence of the reaction of superoxide with the
complexes was studied over the range 3.5-9.0. Transient absorp-
tion changes, observed at 320 nm (Figure 3), indicate that the
intermediate complex, formed as per reaction 6, exists in both
deprotonated and protonated forms (equilibrium 9):
III
+
+
III
2+
[
Mn (L)O ] + H h [Mn (L)OOH]
(9)
2
The pKa values of the intermediate complexes, determined
from the changes in absorption (Figure 3), are 5.65 ( 0.05
(
SODm1), 5.3 ( 0.1 (SODm2) and <5 (SODm3). Due to the
method limitations it is not possible to estimate the pKa for
SODm3 with greater precision, as the absorption changes occur
over wider pH range, resulting in larger error in a sigmoidal
line fit of the experimental data.
The dependence of the second-order rate constants (k6) on pH
for the reaction of superoxide with the complexes is presented in
Figure 4. The reaction rate constants decrease significantly when
Figure 3. Dependence of the change in transient absorbance at 320
nm on pH following the pulse radiolysis (2.5 Gy in 200 ns) of oxygen-
saturated aqueous solutions containing the complexes (0.1 mM), sodium
23
a
formate (25 mM) and phosphate buffer (10 mM). The pK values for
the pH is lower than the pKa (4.8 ) of the superoxide radical. This
SODm1 and SODm2 intermediates of 5.65 ( 0.05 and 5.3 ( 0.1,
is probably due to a reduced reactivity of the complexes with the
respectively, are derived from sigmoidal fits, whereas the pK
a
for
•
protonated form (HO2 ) of superoxide.
SODm3 is estimated at <5.
Conductivity Measurements. Changes in conductivity fol-
lowing the pulse radiolysis of solutions containing the complexes
were compared to that of solutions containing dimethyl sulfoxide
sulfinic acid (pKa ) 2.28), which is formed upon irradiation of
(
DMSO) as a standard under N2O saturated conditions. The
an aqueous solution of DMSO, results in known changes in
24
conductivity under acidic and basic conditions. When aqueous
oxygenated solutions of SODm1 and SODm2 were irradiated
at pH 4, a rapid increase in conductance followed by a decrease
to near prepulse levels was observed (Figure 5). This observation
is indicative of an inner sphere mechanism, where the superoxide
enters the coordination sphere to bind to the metal center to
give a transient intermediate which is protonated at pH 4 and
hence has the same charge as the starting complex. At pH 10,
immediately after the pulse, the conductivity decreased followed
by a further smaller decrease (Figure 5). This is explained as
an initial rapid neutralization of the protons (formed by the
radiation pulse) by the hydroxide ions in solution and the
decrease in conductivity of the transient intermediate formed
in reaction 6 due to the reduced charge relative to the starting
complex. The further decrease in conductivity at pH 10,
observed at the millisecond time scale is attributed to the slow
protonation of the complex.
Figure 2. Absorption spectra observed at ca. 20 µs following the pulse
radiolysis (2.5 Gy in 200 ns) of aqueous solutions of SODm2 (0.1 mM)
containing phosphate buffer (10 mM) and (a) sodium formate (25 mM),
pH 7.4, oxygen saturated (O); (b) sodium formate (25 mM), pH 3.5,
oxygen saturated (2); (c) sodium formate (25 mM), pH 7.4, deaerated
Catalytic Activity of SOD Mimetics. The rate constants for
•-
the catalytic removal of O2 , kcat, were determined from the
(
0); and (d) sodium selenate (25 mM), tert-butanol (0.2 M), pH 7.4,
-
deaerated (9).
measured half-lives of O2• in the absence and increasing concen-

4
932 J. Phys. Chem. A, Vol. 112, No. 22, 2008
Maroz et al.
Figure 5. Kinetic traces, showing changes in conductivity following
the pulse radiolysis (3 Gy in 200 ns) of oxygen-saturated aqueous
solutions of the complexes (0.1 mM) containing tert-butanol (0.2 M)
at (a) pH 4 and (b) pH 10. Conductivity changes are compared to that
observed following the pulse radiolysis of N O-saturated aqueous
2
solutions containing dimethylsulfoxide (1 mM) under the same
conditions.
Figure 4. Dependence of the second-order rate constants for the
reaction of superoxide with the complexes on pH. Experimental
conditions are as in Figure 1. The reaction rate constants decrease
23
significantly when the pH is lower than the pK
a
(4.8 ) of the superoxide
radical, because of a reduced reactivity of the complexes with the
•
2
protonated form (HO ) of superoxide.
trations of SODm1 (Figure 6) and SODm2 (Figure 7). To separate
pseudo-first-order decay of O2• by reaction with SOD complexes
-
•-
from second-order decay of O2 by reaction with itself, the inverse
•
-
•-
in half-life of O2 was plotted against the concentration of O2
Figure 6a). The intercepts give inverse half-lives for the pseudo-
(
•-
25
first-order decay of O2 in reaction with SOD complexes. The
rate constants derived from these half-lives were plotted against
concentration of the SOD complex (Figure 6b). The slope of the
fitted line provides the value for kcat. Assuming a two-step scheme,
II
2+
•-
viz. oxidation of the [Mn (L)] by O2 to form the transient
III
+
[
Mn (L)O2] intermediate (k6, reaction 6) followed by reaction
•-
II
2+
with a second O2 to reform the original [Mn (L)] together with
O2 and H2O2, k10:
Figure 6. (a) Kinetic plots of the dependence of the inverse in half-
life of superoxide, measured at 270 nm in the absence and with
increasing concentrations of SODm1 at pH 7.4. (b) Dependence of rate
constants for the reaction of SODm1 with superoxide (derived from
intercepts of (a)) on the concentration of SODm1. The slope of the
fitted line (b) provides the value for kcat (Table 1). Data obtained in the
pulse radiolysis (0-100 Gy in 1.5 µs) of oxygen-saturated aqueous
solutions containing SODm1, sodium formate (25 mM) and phosphate
buffer (10 mM) pH 7.4.
III
+
•-
+
II
2+
[
Mn (L)O ] + O + 2H f [Mn (L)] + O + H O
2 2 2 2
2
(10)
the dismutation rate can be described with the following
equation:
d[O • ]/dt ) (2k k /(k + k ))[[Mn (L)] ][O ] )
-
II
2+
•-
-
2 6 10 6 10 2
k [[Mn (L)] ][O •-]
II
2+
cat
2
Both SODm1 and SODm2 showed significant catalytic
activity, even at micromolar concentrations, whereas SODm3
did not influence the decay of superoxide at both pH 7.4 and
8.0 when added in concentrations up to 50 µM (Table 1).

Mechanism of SOD Action of Mn(II)-Pentaazamacrocycles
J. Phys. Chem. A, Vol. 112, No. 22, 2008 4933
for the overall catalytic process, a different temperature
dependence was observed for SODm1 and SODm2. The kcat
values for SODm2 exhibit an apparent decrease with temper-
ature and this behavior is reflected in the thermodynamic
parameters, presented in Table 2.
II
Redox Properties of Initial Mn Complexes. To determine
the redox properties of the metal center in the complexes, quasi-
reversible cyclic voltammetry measurements were carried out
under N2 using acetonitrile as the solvent (Figure 9). Ferrocene
(
Fc) was used as an internal standard so that the half-wave
potential of the complexes could be directly compared to the
+
half-wave potential of the Fc /Fc couple. The final redox
potential values vs NHE were obtained using the known value
+
for the ferrocene couple in acetonitrile (E°(Fc /Fc) vs SCE )
2
6
4
25 mV ), and the relation between the two reference elec-
2
7
trodes: SCE vs NHE ) 242 mV. The measured redox
3
+
2+
potentials E1/2(SODm Mn /Mn ) were found to be 525, 464
and 452 mV for SODm1, SODm2 and SODm3, respectively.
Given the similarity between the values obtained for the active
(
SODm2) and inactive (SODm3) catalysts, it is concluded that
metal center oxidation is not a crucial (rate-determining) step
in the catalytic process.
Discussion
The present study has relevance to the mechanism of catalytic
action of Mn(II)-pentaazamacrocyclic compounds as super-
oxide dismutase mimetics (Scheme 1) at physiological pH. The
pH values of 7.4 and 8.0 are typical for intracellular and
intramitochondrial environments, respectively, where natural
SOD activity is operative. Our data support the proposal, that
at pH > 7.4 the catalytic cycle proceeds through the inner sphere
mechanism. Introduction of the O2• into the coordination sphere
of the metal center is accompanied by the formation of Mn(III);
i.e., metal center oxidation occurs. The directly observed
Figure 7. (a) Kinetic plots of the dependence of the inverse in half-
life of superoxide in the presence of increasing concentrations of
SODm2 at pH 7.4. (b) Dependence of rate constants for the reaction
of superoxide with SODm2 (derived from intercepts of (a)) on the
concentration of SODm2. The slope of the fitted line provides the value
for kcat. Experimental conditions are as in Figure 6.
-
Thermodynamics of the Catalytic Process. The reaction
•
-
of O2 with SODm1 and SODm2, as well as the catalytic
activity of these complexes, were studied over the temperature
range 15-45 °C (Figure 8). Eyring plots of k6 for both SODm1
and SODm2 are very similar with the rate constants increasing
with temperature. However for kcat, which is the rate constant
III
+
intermediate complex, described as [Mn (L)O2] , has a pKa <
6, which is supported by both optical and conductivity measure-
ments. Thus, at natural pH levels the intermediate exists
predominantly in a deprotonated state.
Figure 8. (a, c) Eyring plots for the reaction of superoxide with SODm1 (b) and SODm2 (O) over the temperature range 15 -45 °C. The rate
constants, k , were determined as described in Figure 1. (b, d) Eyring plots for overall catalytic process. The rate constants, kcat, were determined
as described in Figures 6 and 7.
1

4
934 J. Phys. Chem. A, Vol. 112, No. 22, 2008
Maroz et al.
redox potential of the Mn(III)/Mn(II) couple is similar for two
complexes with vastly differing SOD activities. It is clear from
our work that the reaction of the initial Mn(II) compound with
•
-
III
+
O2 is an important step, as it produces the [Mn (L)O2] , but it
does not determine the overall catalytic rate. Furthermore, we sug-
III
+
gest that the [Mn (L)O2] intermediate oxidizes the next molec-
ule of O2 , and this process is a crucial point in the catalytic cycle.
•-
Investigation of the conformational and coordinational changes
of Mn-pentaazamacrocyclic complexes, using computational
methods, has revealed differences in the geometry of Mn(II)-
(
pentagonal bipyramidal) and Mn(III)-(pseudooctahedral) com-
33
pounds. These data, obtained using a molecular mechanics
method, which necessarily incorporates substantial approximations,
were compared to solid state crystallography data that may not
truly reflect the conformation of the ligand in the aqueous
environment. Nonetheless, this approach was very useful for
identifying structural features that enhance SOD activity and, in
particular, indicated that flexibility in the ligand geometry could
be a significant factor in the reaction. We propose, that the ability
of a ligand to accommodate Mn(III) pseudo-octahedral coordination
affects the second step of catalytic process and not the initial metal
15
center oxidation as previously suggested. This aspect is fully
supported, and indeed enhanced, by our results (Table 2), which
show the major influence of an entropy factor on the second stage
of the catalytic process for the most effective (SODm2) catalyst.
On the basis of the data obtained, we propose the following reaction
mechanism (Scheme 2):
Figure 9. Examples of cyclic voltammetry measurements in acetonitrile
for SODm1 and SODm2. Solutions contained the complexes (1 mM),
[
(
4 6
(n-Bu) N](PF ) (0.1 M) as the supporting electrolyte and ferrocene
ca. 1 mM) as an internal standard, to determine the potential vs NHE.
Measurements were taken with a scan rate of 50 mV/s. Redox potentials
SCHEME 2: Proposed Catalytic Cycle
3
+
2+
(
vs NHE) are calculated to be E1/2(SODm1 [Mn /Mn ]) ) 525 mV;
3
+
3+
2+
E
1/2(SODm2 [Mn /Mn2+]) ) 464 mV; E1/2(SODm3 [Mn /Mn ]) )
4
52 mV.
The mechanism by which Mn(II)-pentaazamacrocyclic com-
pounds act as SOD mimetics, is very different from that of the
native enzyme. Early pulse radiolysis studies show that the
active center of native MnSOD contains Mn(III). The reduction
of the metal center by O2• is believed to occur as the first step
of catalysis, immediately converting one equivalent of super-
oxide to oxygen without the formation of a complex. This was
determined by indirect observation of the superoxide decay and
-
2
8
loss of Mn(III) band at 480 nm.
The overall mechanism consists of three steps:
It has been stated in earlier studies that the SOD catalytic rate
of Mn(II)-pentaazamacrocyclic compounds is governed by the
rate of oxidation of the metal center, as being the rate-determining
. Activation of the complex by reaction with O2• (fast):
-
1
29,30
O2•- + [Mn (L)] f [Mn (L)O ]
II
2+
III
+
step in the mechanism.
However, our results indicate that the
2
oxidation of the metal center is not the rate-determining step, as
•-
The resulting intermediate Mn(III) complex is present in its
deprotonated form at pH 7 and above (pKa ≈ 5.5, Figures 3
and 4).
the moderately active catalyst, SODm1, reacts with O2 at a rate
similar to that of the ineffective catalyst, SODm3. An alternative
31
mechanism, which has been proposed recently, suggests a rate-
determining role for the Cl- T H2O ligand exchange in the
complexes. However, recent studies on the ligand exchange
mechanism with seven-coordinate Mn(II) compounds question the
2. Reduction of the activated intermediate complex by a
further O2• , which is influenced greatly by its 3-dimensional
structure. The metal center is reduced back to the initial Mn(II),
although the intermediate pseudoctahedral geometry is main-
tained:
-
32
potential rate-determining role of this process. Moreover, our
cyclic voltammetry measurements clearly demonstrate that the
TABLE 2: Thermodynamic Properties for the Reactions of Superoxide with the Complexes at pH 7.4, Derived from Eyring
Plots Presented in Figure 8
k
cat, catalytic O •
2
-
kcat, catalytic O
2
•-
•
-
•-
k
6
, O
2
+ SODm1
dismutation by SODm1
k
6
, O
2
+ SODm2
dismutation by SODm2
θ
-1
∆
∆
∆
H /kJ mol
27.4
-1
27.6
38.8
11
35.5
13.7
-34
23.8
-14.9
-135
25.3
θ
-1
-1
S /J mol
K
θ
-1
G298 /kJ mol

Mechanism of SOD Action of Mn(II)-Pentaazamacrocycles
J. Phys. Chem. A, Vol. 112, No. 22, 2008 4935
III
+
•-
II
(10) Gonzalez, P. K.; Zhuang, J.; Doctrow, S. R.; Malfroy, B.; Benson,
P. F.; Menconi, M. J.; Fink, M. P. J. Pharmacol. Exp. Ther. 1995, 275,
[
Mn (L)O ] + O f [Mn (L)O ] + O
2 2 2 2
7
98.
3
. Proton-assisted release of the H2O2 with concomitant
(
11) Spasojevi c´ , I.; Batini c´ -Haberle, I.; Stevens, R. D.; Hambright, P.;
conformational relaxation of the Mn(II) complex to yield the
starting compound with pentagonal-bipyramidal coordination
sphere:
Thorpe, A. N.; Grodkowski, J.; Neta, P.; Fridovich, I. Inorg. Chem. 2001,
40, 726.
(
12) Muscoli, C.; Cuzzocrea, S.; Riley, D. P.; Zweier, J. L.; Thiemer-
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(
II
+
II
2+
[
Mn (L)O ] + 2H f [Mn (L)] + H O
2 2 2
(
This mechanism supports the view that the catalytic activity
W. L.; Rivers, W. J.; Aston, K. W.; Sample, K. R.; Rahman, H.; Ling,
C. S.; Shieh, J. J.; Busch, D. H.; Szulbinski, W. Inorg. Chem. 1996, 35,
of the Mn(II)-pentaazamacrocyclic complexes is governed
5
213.
(
III
+
by the intermediate geometry of the [Mn (L)O2] and its
reactivity with the second approaching O2•-. In regard to the
folding ability (pentagonal bipyramidal f pseudooctahedral)
15) Aston, K.; Rath, N.; Naik, A.; Slomczynska, U.; Schall, O. F.; Riley,
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(18) Salvemini, D.; Wang, Z. Q.; Zweier, J. L.; Samouilov, A.;
(
1
5
as previously proposed, this ligand modification strategy is
still viable, but structural modifications to the initial Mn(II)
complexes should be dictated by the desirable structure of
(
III
+
Macarthur, H.; Misko, T. P.; Currie, M. G.; Cuzzocrea, S.; Sikorski, J. A.;
[
Mn (L)O2] intermediate. As the catalytic activity of the
Riley, D. P. Science 1999, 286, 304.
9
-1 -1
natural enzyme (kcat ∼ 10 M s ) is still unmatched by the
existing SOD mimetics at physiological pH, there is scope for
improvement to be made, possibly by changing the charge
distribution in the ligand and substituent groups to increase its
(
19) Riley, D. P.; Neumann, W. L.; Henke, S. L.; Lennon, P.; Aston,
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•
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.
(
21) Neta, P.; Huie, R. E.; Ross, A. B. J. Phys. Chem. Ref. Data 1988,
Acknowledgment. This work is supported by grant UOA
407 of the Marsden Fund from the Royal Society of New
Zealand.
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