2-Phenylpyridine ruthenacycles as effectors of glucose oxidase activity: Inhibition by RuII and activation by RuIII

Article abstract of DOI:10.1007/s00775-013-0999-z

Cyclometalated Ru^II derivatives of 2-phenylpyridine (Hphpy) [Ru(phpy)(bpy)₂]Cl (1a) and [Ru(phpy)(phen)₂]Cl (1b) (bpy is 2,2′-bipyridine, phen is 1,10-phenanthroline) behave as noncompetitive inhibitors of glucose oxidase from Aspergillus niger in the enzyme-catalyzed oxidation of d-glucose by O₂ into the corresponding lactone at pH 5.0 and 25 C. The enzymatic activity has been measured by monitoring the O ₂ consumption. The inhibition constants K _i are 0.036 and 0.017 M for 1a and 1b, respectively, indicating that 1b inhibits the enzymatic activity more efficiently than 1a. The well-known coordination compound [Ru(bpy)₃]Cl₂ (2) behaves, in contrast, as a competitive inhibitor, with K _i = 0.018 M under the same conditions. The monophasic consumption of O₂ in the case of 1a, 1b, and 2 is replaced by a distinct two-phase kinetics in the presence of the cyclometalated Ru ^III compound [Ru(phpy)(bpy)₂]Cl₂ (3), which was obtained from 1a in the presence of a large excess of H₂O ₂ and the iron TAML activator. Interestingly, the rates of the first and the second phases are influenced by 3 in a different way. The rate of the first phase is noticeably higher in the presence of Ru^III, although the dependence is nonmonotonic and maximal acceleration is observed at the lowest loadings of 3. The rate of the second phase decreases monotonically on increasing the concentration of the ruthenium complex in solution. The nonmonotonic action of 3 was confirmed by using the doubly cyclometalated Ru^III derivative [Ru(phpy)₂(bpy)]Cl. The diverse rate variations induced by 3 accounted for acceleration by Ru^III of the O₂ reduction by the reduced form of glucose oxidase during the first phase, which ceases after the enzymatic reduction of Ru^III to the Ru^II species, the latter behaving similarly to 1a as the inhibitor of the enzyme.

Full text of DOI:10.1007/s00775-013-0999-z

J Biol Inorg Chem (2013) 18:547–555
DOI 10.1007/s00775-013-0999-z
ORIGINAL PAPER
2-Phenylpyridine ruthenacycles as effectors of glucose oxidase
activity: inhibition by RuII and activation by RuIII
•
Rafael Omar Saavedra Dıaz Ronan Le Lagadec
•
´
Alexander D. Ryabov
Received: 24 January 2013 / Accepted: 28 March 2013 / Published online: 27 April 2013
Ó SBIC 2013
Abstract Cyclometalated RuII derivatives of 2-phenyl-
pyridine (Hphpy) [Ru(phpy)(bpy)2]Cl (1a) and [Ru(ph-
py)(phen)2]Cl (1b) (bpy is 2,20-bipyridine, phen is 1,10-
phenanthroline) behave as noncompetitive inhibitors of
glucose oxidase from Aspergillus niger in the enzyme-
catalyzed oxidation of D-glucose by O2 into the corre-
sponding lactone at pH 5.0 and 25 °C. The enzymatic
activity has been measured by monitoring the O2 con-
sumption. The inhibition constants Ki are 0.036 and
0.017 M for 1a and 1b, respectively, indicating that 1b
inhibits the enzymatic activity more efficiently than 1a.
The well-known coordination compound [Ru(bpy)3]Cl2 (2)
behaves, in contrast, as a competitive inhibitor, with
Ki = 0.018 M under the same conditions. The monophasic
consumption of O2 in the case of 1a, 1b, and 2 is replaced
by a distinct two-phase kinetics in the presence of the cy-
clometalated RuIII compound [Ru(phpy)(bpy)2]Cl2 (3),
which was obtained from 1a in the presence of a large
excess of H2O2 and the iron TAML activator. Interestingly,
the rates of the first and the second phases are influenced by
3 in a different way. The rate of the first phase is noticeably
higher in the presence of RuIII, although the dependence is
nonmonotonic and maximal acceleration is observed at the
lowest loadings of 3. The rate of the second phase
decreases monotonically on increasing the concentration of
the ruthenium complex in solution. The nonmonotonic
action of 3 was confirmed by using the doubly cyclo-
metalated RuIII derivative [Ru(phpy)2(bpy)]Cl. The diverse
rate variations induced by 3 accounted for acceleration by
RuIII of the O2 reduction by the reduced form of glucose
oxidase during the first phase, which ceases after the
enzymatic reduction of RuIII to the RuII species, the latter
behaving similarly to 1a as the inhibitor of the enzyme.
Keywords Ruthenium ꢀ Cyclometalation ꢀ Glucose
oxidase ꢀ Kinetic inhibition ꢀ Activation
Introduction
Studies of various interactions of inorganic and organo-
metallic compounds with glucose oxidase (GO) from
Aspergillus niger, the enzyme that catalyzes the oxidation
of b-D-glucose by O2 to the corresponding lactone via
Eq. 1 [1], have a long history [2].
Dedicated to the memory of a big man, Ivano Bertini, the founder of
Journal of Biological Inorganic Chemistry.
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-0999-z) contains supplementary
material, which is available to authorized users.
OH
OH
O
GO
O
OH
+ O2
+ H2O2
´
R. O. Saavedra Dıaz ꢀ R. Le Lagadec
O
HO
HO
OH
HO
HO
OH
´
Instituto de Quımica, UNAM, Circuito Exterior s/n,
Ciudad Universitaria, 04510 Mexico, D.F., Mexico
´
´
ð1Þ
e-mail: ronan@unam.mx
A. D. Ryabov (&)
Along with other organometallics, ruthenium [3] and
Department of Chemistry, Carnegie Mellon University,
4400 Fifth Avenue, Pittsburgh, PA 15213, USA
e-mail: ryabov@andrew.cmu.edu
osmium [4] complexes appear to be similarly advantageous
compared with earlier introduced ferrocene derivatives [5]
123

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J Biol Inorg Chem (2013) 18:547–555
in their ability to oxidize the reduced active site of GO and
as such appear to be involved in the mediated electron
transfer from the reduced flavin adenine dinucleotide
(FADH2) at an electrode [5]. The mechanism of catalysis
by GO consists of two processes, viz., the oxidation of b-D-
glucose by the enzyme in the resting state (GOox; Eqs. 2, 3)
uptake. Coordinatively saturated, resistant to ligand
exchange cyclometalated RuII complexes have recently
been discovered as potential substitutes for cisplatin in the
cancer therapy [10, 11]. Their inertness in ligand exchange/
substitution allows us to hypothesize on the operative
anticancer mechanism, which may be other than through
the binding of complexes to DNA. It could rather be related
to the activity of caspase 8 and generation of reactive
oxygen species (ROS) [12]. ROS are produced and quen-
ched by redox-active proteins and enzymes. Cyclometa-
lated ruthenium complexes may affect the performance of
these biomolecules in various ways. Knowledge of general
features of these effects might be key for understanding
mechanisms of action of cycloruthenates as anticancer
drugs. Correspondingly, in this work we have considered
GO as a model oxidoreductase enzyme to study the effects
of ruthenium complexes such as 1a, 1b, 2, 3, and 4 (Fig. 1)
on the speed of enzymatic reduction of dioxygen.
followed by the oxidation of the reduced enzyme (GOred
by O2 (Eq. 4), leading to hydrogen peroxide [1].
)
GOox þ b-d-glucose ꢀ fGOox; b-d-glucoseg ðKSÞ
fGOox; b-d-glucoseg ! GOred þ d-gluconolactone ðk1Þ
ð3Þ
ð2Þ
GOred þ O2 ! GOox þ H2O2 ðkO Þ
ð4Þ
ð5Þ
ð6Þ
2
GOred þ 2MLnmþ ! GOox þ 2MLnðmꢁ1Þþ ðkMÞ
GOred þ MLmn þ ꢀ fGOred; MLnmþg ðKMÞ
fGOred; MLmn þg þ O2 ! fGOox;MLnmþg þ H2O2 ðkMO
Þ
2
ð7Þ
Metal complexes—ferrocene derivatives, ruthenium,
and osmium compounds—are capable of reoxidizing
GOred instead of dioxygen via the step shown in Eq. 5,
which is usually a first-order reaction in both GOred and
MLmn ?. Quite remarkably, the second-order rate constants
kM, particularly in the case of cyclometalated ruthenium [6]
Results
General considerations and inhibition of GO
by ruthena(II)cycles 1a and 1b
Provided the mechanism displayed in Eqs. 2–4 is opera-
tive, the rate of dioxygen reduction is given by Eq. 8,
which is a variant of the Michaelis–Menten equation with
Vm ¼ k1kO ½O2ꢂ½Eꢂ=ðk1 þ kO ½O2ꢂÞ and Km ¼ kO KS½O2ꢂ=
and osmium [7, 8] species, may exceed kO , which equals
2
1.6 9 106 M-1 s-1 under the optimal conditions [9]. In
many instances, reduced MLmn ? species are reoxidizable
electrochemically to MLmn ?, and therefore bioamperometric
monitoring of D-glucose, in particular, as well as other
analytes in the presence of corresponding oxidases is feasible.
The reduced enzymatic active sites are reoxidized by
MLnm? although ML(nm-1)? species are inactive. Corre-
spondingly, MLmn ? complexes are, to a certain extent,
substrates of enzymes, which possess a definite affinity for
the enzymatic active sites. Inactive reduced complexes of
the form ML(nm-1)? are structurally similar to MLmn ?; they
differ just in the overall charge. This structural similarity
suggests that reduced ML(nm-1)? species may bind revers-
ibly to enzymes and affect their catalytic activity, partic-
ularly the ability to reduce dioxygen. It is thus desirable to
know to what extent ML(nm-1)? complexes can retard the
activity of oxidoreductases. Therefore, in this work we
report results of studies of inhibition of dioxygen uptake by
GO from Aspergillus niger by cyclometalated RuII com-
plexes 1a and 1b (Fig. 1), which in contrast to their RuIII
counterparts cannot reoxidize GOred. The structurally
similar RuII complex 2, which does not have a cyclo-
metalated ligand, has also been investigated for compari-
son. We have found that RuII compounds 1a, 1b, and 2
retard the reduction of O2, although the RuIII derivative of
1a, complex 3 (as well as 4), increases the speed of O2
2
2
2
ðk1 þ kO ½O2ꢂÞ:
2
k1kO2 ½O2ꢂ
½Eꢂ½Sꢂ
þ ½Sꢂ
d½O2ꢂ
dt
k1þkO2 ½O2ꢂ
ꢁ
¼
ð8Þ
kO2 KS½O2ꢂ
k1þkO2 ½O2ꢂ
This allows us to use a standard approach based on the
Michaelis–Menten equation for analysis of the inhibition of
GO activity by the RuII compounds presented in Fig. 1.
Figure 2a shows dependencies of steady-state rates of O2
consumption as a function of D-glucose concentration in
the absence and in the presence of different concentrations
of ruthena(II)cycle 1a. There is a distinct retardation of
the activity of GO by 1a, the effect increasing with
increasing inhibitor concentration. The corresponding
Lineweaver–Burk plots shown in Fig. 2b confirm that (1)
the reaction follows Michaelis–Menten kinetics and (2) the
noncompetitive mechanism of inhibition is operative
because the straight lines intersect at a common point on
the x axis [13]. The values of the calculated effective
maximal rates (Vm,eff) and effective Michaelis constants
(Km,eff) in Table 1 confirm the mechanistic conclusion
because the Km,eff values are essentially independent of the
concentration of 1a, but the Vm,eff values decrease
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549
N
N
N
RuII
N
N
RuII
N
II
N
N
N
Cl
Cl2
Cl
Ru
N
N
N
N
N
N
N
1a
1b
2
N
N
N
N
RuIII
RuIII
N
N
Cl2
Cl
N
N
N
3
4
Fig. 1 Complexes used in this work: ruthenium(II) cyclometalated compounds 1a and 1b with a r Ru–C bond, coordination compound 2, and
ruthenium(III) cyclometalated compounds 3 and 4
Fig. 2 a Steady-state rates of dioxygen uptake by glucose oxidase
(GO) (1 9 10-8 M) as a function of D-glucose concentration at
various concentrations of 1a (0, 0.001, 0.004, 0.009, and 0.016 M
from a to e, respectively) at pH 5.0 (0.05 M phosphate) and 25 °C.
b The corresponding double reciprocal plot showing that the lines
intersect the x axis at a single point, indicative of a noncompetitive
inhibition
systematically with increasing 1a concentration. The
inverse of Vm,eff is a linear function of the concentration
of 1a (Fig. S1) and thus an inhibition constant Ki of
slightly better inhibitor of GO than 1a, as revealed by a
smaller value of Ki (0.017 M) calculated from the corre-
sponding linear plot included in Fig. S1.
-1
0.036 M was calculated because (Vm,eff
)
= (Vm)-1
(1 ? [RuII]/Ki) [13]. The value of Ki indicates
comparable affinity of 1a and D-glucose toward GO
because the Km,eff values for D-glucose are in the range
of 0.01–0.03 M [1], which agrees with value of 0.016 M
found in this work at zero concentration of 1a (Table 1).
Ruthena(II)cycle 1b behaves similarly to 1a, as expec-
ted (Fig. S2a). The noncompetitive inhibition scheme was
similarly confirmed (Fig. S2b, Table 1). It is though a
Inhibition of GO by tris(2,20-bipyridine)
ruthenium(II) (2)
The rate versus D-glucose concentration profiles in Fig. 3a
look slightly different from those in Figs. 2a and S2a for
ruthena(II)cycles 1a and 1b . The Lineweaver–Burk plots
in Fig. 3b highlight the origin of dissimilarity, which is a
switch from the noncompetitive to competitive inhibition
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J Biol Inorg Chem (2013) 18:547–555
increase with increasing concentration of 2 (Fig. S3,
Table 1). In the case of the competitive inhibition,
Km,eff = Km (1 ? [RuII]/Ki) [13], and therefore Ki = 0.018
M was calculated from the plot of Km,eff versus RuII con-
centration (Fig. S3). Despite different inhibition schemes
being realized, the values of the inhibition constants Ki
summarized in Table 1 are similar for cycloruthenated spe-
cies 1 and noncyclometalated compound 2. It is also worth
mentioning that the affinity of ruthenium compounds 1 and 2
in terms of Ki is similar to that of D-glucose in terms of Km.
Table 1 Effective maximal rates and Michaelis constants for the
oxidation of D-glucose by O2 catalyzed by glucose oxidase from
Aspergillus niger calculated using the Lineweaver–Burk routine from
the measured dependence of O2 consumption in the D-glucose con-
centration range from 0.0025 to 0.1 M
[RuII]
(M)
107 9 Vm,eff
(M s-1
102 9 Km,eff
(M)
Ki (M)
)
1b
0
10.2 0.1
9.2 0.2
7.6 0.1
6.5 0.3
1.58 0.04
1.76 0.06
1.71 0.04
1.82 0.09
0.017 0.002a
0.0025
0.0075
0.009
Activation of GO by ruthena(III)cycle 3
1a
0
10.2 0.1
9.2 0.1
8.1 0.1
7.9 0.1
6.6 0.1
1.58 0.04
1.59 0.02
1.54 0.01
1.64 0.04
1.58 0.02
0.036 0.005a
The kinetic curves for the GO-catalyzed reduction of O2
both in the absence and in the presence of RuII complexes 1
and 2 are typical of enzymatic processes and contain sig-
nificant steady-state portions as exemplified in Fig. 4.
However, the kinetics change noticeably in the presence of
ruthena(III)cycle 3. The entire kinetic curve appears to be
divided into two phases by a break point (Fig. 4). The rate
of O2 consumption during the first phase, which becomes
longer on increasing the concentration of 3, is higher than
the rate in the absence of the RuIII complex. The rates as a
function of D-glucose concentration were measured at
variable loadings of RuIII. The data in Fig. 5 indicate that
RuIII enhances the GO activity, although the activating
effect is nonmonotonic, which is very unusual. In fact, the
maximal acceleration is observed at the lowest loading of
RuIII (Fig. 5). As before, the data were analyzed using the
Lineweaver–Burk routine, and the corresponding values of
Vm,eff and Km,eff are summarized in Table 2. Although
these values are determined with a lower accuracy than
those in the case of the inhibiting RuII compounds (for
reasons, see below), one may conclude that RuIII increases
0.001
0.0038
0.009
0.016
2
0
21.8 0.3
21.3 0.1
2.31 0.03
2.58 0.01
2.67 0.03
2.85 0.04
3.24 0.03
0.018 0.006b
0.0025
0.00375 20.2 0.2
0.0050
0.0075
20.0 0.3
20.4 0.2
The conditions were as follows: 1 9 10-7 M glucose oxidase for 1a
and 1b and 2 9 10-7 M glucose oxidase for 2, pH 5.0 (0.05 M
phosphate), 25 °C
a
Noncompetitive scheme
b
Competitive scheme
mechanism. In fact, the straight lines intersect virtually at a
single point on the y axis (Fig. 3b, inset), the Vm,eff values
are unaffected by the presence of 2 (Fig. S1), but the
effective Michaelis constants Km,eff have a tendency to
V
m,eff although Km,eff remains virtually unchanged as in the
Fig. 3 a Steady-state rates of dioxygen uptake by GO (2 9 10-8 M)
as a function of D-glucose concentration at various concentrations of 2
(0, 0.0025, 0.0037, 0.0050, and 0.0075 M) at pH 5.0 (0.05 M
phosphate) and 25 °C. b The corresponding double reciprocal plot
showing that the lines intersect the y axis at a single point, which is
consistent with a competitive inhibition
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J Biol Inorg Chem (2013) 18:547–555
551
The rates of the second phase, which were measured as
shown in Fig. 4, reveal the opposite trend. As in the case of
complexes 1a and 1b introduced to the reaction mixture
directly, there is systematic rate retardation on increasing the
ruthenium loading (Fig. 6). Similarly determined values of
Vm,eff and Km,eff are collected in Table 2 as well. If these data
are compared with the corresponding data for 1a in Table 1,
one may see a clear match, i.e., the values of Km,eff do not
change although the Vm,eff values decrease on increasing the
effector concentration as anticipated for the noncompetitive
mechanism. In other words, the second phase in the case of
RuIII resembles the inhibiting effect of RuII complex 1a and
therefore the break in Fig. 4 occurs presumably because of the
conversion of RuIII, which is an activator of GO activity, into
RuII, which is an inhibitor of GO activity.
There is nothing unexpected in the reduction of RuIII to RuII
by the GO/D-glucose system via Eq. 5. More interesting is the
activation of GO by RuIII. Unusual is the effect itself and its
dependence on the concentration of RuIII. Wishing to find out
how general the effect is, we tested doubly cyclometalated RuIII
complex 4, which differs from 3 by a significantly reduced, by
about 0.6 V, reduction potential for the RuII/III couple [14]. The
data in Fig. 7show that complex 4 behaves similarly to complex
3. It splits the entire process into two phases. The first phase is
noticeably faster in the presence of RuIII and its smaller loadings
cause larger accelerations, i.e., the effect is nonmonotonic.
The activation of GO by RuIII for both 3 and 4 is observed
untilallRuIII isenzymaticallyreducedtoRuII. Thismeansthat
the activation by RuIII is monitored in the presence of a certain
amount of RuII in the system. Therefore, the activation is
always realized along with inhibition. Two opposite phe-
nomena may thus cause lowered accuracy in determining
Vm,eff and Km,eff during the first phase of dioxygen con-
sumption in the presence of RuIII.
Fig. 4 Dioxygen uptake by GO (2 9 10-8 M) in the presence of RuII
and RuIII complexes (0.0025 M 2 and 0.0005 M 3) at 0.1 M
D-glucose, pH 5.0 (0.05 M phosphate), and 25 °C. Solid lines indicate
the how the corresponding rates of O2 uptake were measured
Fig. 5 Rates of the first phase of dioxygen uptake by GO
(2 9 10-8 M) as a function of D-glucose concentration at various
concentrations of RuIII complex 3 (0, 0.0005, 0.001, and 0.0015 M) at
pH 5.0 (0.05 M phosphate) and 25 °C
Discussion
case of 1a (Table 1). This observation suggests that RuIII
does not compete with D-glucose for the binding site but
enhances the effective catalytic rate constant.
Cycloruthenated compounds 1a and 1b and tris(bpy)
ruthenium(II) complex 2 (bpy is 2,20-bipyridine) are
Table 2 Effective maximal rates and Michaelis constants for the first
and second phases of the oxidation of D-glucose by O2 catalyzed by
glucose oxidase from Aspergillus niger in the presence of RuIII
complex 3 calculated using the Lineweaver–Burk routine from the
measured dependence of O2 consumption in the D-glucose concen-
tration range from 0.0025–0.1 M
[RuIII] (M)
1st phase
2nd phase
107 9 Vm,eff (M s-1
)
102 9 Km,eff (M)
107 9 Vm,eff (M s-1
)
102 9 Km,eff (M)
0
17.3 0.3
1.51 0.09
2.6 0.4
17.3 0.3
11.8 0.5
11.5 0.7
11.4 0.8
1.51 0.09
1.0 0.1
1.3 0.2
1.4 0.3
0.0005
0.0010
0.0015
64
64.8 0.4
22
2
4
1
5
1.8 0.6
The conditions were as follows: glucose oxidase concentration equal to the concentration of 3, pH 5.0 (0.05 M phosphate), 25 °C
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J Biol Inorg Chem (2013) 18:547–555
There is an extra feature that differentiates complexes
1a and 1b from 2. The IC50 values with respect to certain
cell lines are four orders of magnitude lower for ruthena-
cycles 1a and 1b [11] compared with 2 [17, 18].1 In fact,
this makes the former promising anticancer drugs. The data
reported here suggest that the ability of molecules to retard
enzymatic activity may not correlate at all with IC50
because the Ki values summarized in Table 1 are close to
each other. There might be a relation between IC50 and the
mechanism of inhibition, although much deeper studies are
required to claim this with certainty.
The activation of GO by RuIII complex 3 is an unex-
pected challenge. All previous knowledge of interactions of
GOred with RuIII suggests rather the opposite effect, i.e., a
decrease in the rate of O2 uptake. If both O2 and RuIII
compete for the reduced active site of the enzyme (Eqs. 4,
5, respectively), the steady-state rate of the enzymatic O2
reduction should be given by Eq. 9:
Fig. 6 Rates of the second phase of dioxygen uptake by GO
(2 9 10-8 M) as a function of D-glucose concentration at various
concentrations of RuIII complex 3 (0, 0.0005, 0.0010, and 0.0015 M)
at pH 5.0 (0.05 M phosphate) and 25 °C
k1kO2 ½O2ꢂ
½Eꢂ½Sꢂ
d½O2ꢂ
dt
k1þkO2 ½O2ꢂþkM½Mꢂ
kO2 KS½O2ꢂ
ꢁ
¼
ð9Þ
½Mꢂ þ ½Sꢂ
k1þkO2 ½O2ꢂþkM
Under the saturating concentrations of D-glucose,
ð½Sꢂ ꢃ kO KS½O2ꢂ=ðk1 þ kO ½O2ꢂ þ kM½MꢂÞÞ, Eq. 9 reduces
2
2
to Eq. 10, which predicts a hyperbolic decrease of the
reaction rate with increasing concentration of RuIII (M in
Eqs. 9, 10):
d½O2ꢂ
dt
k1kO ½O2ꢂ
2
ꢁ
¼
½Eꢂ
ð10Þ
k1 þ kO ½O2ꢂ þ kM½Mꢂ
2
The effect of RuIII could formally be interpreted in terms
of the mechanism summarized by Eqs. 2–4, 6, and 7. For
simplicity, Eq. 5 is excluded from the consideration for a
moment. The additional steps shown in Eqs. 6 and 7
assume that RuIII is also capable of binding to GOred
somewhere away from the active site, i.e., it does not
reoxidize GOred, and the {GOred, MLmn ?} associate reduces
Fig. 7 Dynamics of dioxygen uptake by GO (1 9 10-8 M) in the
absence and in the presence of RuIII complex
4 at various
concentrations at 0.1 M D-glucose, pH 5.0 (0.05 M phosphate), and
25 °C. Solid lines indicate how the corresponding rates of O2 uptake
were measured
structurally similar but differ in the overall charge, which is
?1 for 1a and 1b and ?2 for 2. Compounds 1a, 1b, and 2
contain RuII. An extra feature that makes compounds 1a,
1b, and 2 different is the nature of their highest occupied
molecular orbital. It is essentially based on the ruthenium
atom in the case of 2 [15] but in 1a and 1b it is significantly
extended at the phenyl ring, which is bound to ruthenium
via a r M–C bond, i.e., at the cyclometalated ligand [16]. It
is, however, difficult to tell precisely which of the two features
causes which mechanism of inhibition is operative, i.e., non-
competitive for 1a and 1b and competitive for 2. Increasing
the overall charge of 1a by going to the RuIII derivative 3 does
not make ruthenacycle 3 a competitive inhibitor but rather
makesitapromoteroftheenzymaticactivity.Thus, thecharge
of ?2 is probably not a decisive factor for the competitive
mechanism of inhibition.
O2 with rate constant kMO . The steady-state rate of O2
reduction is then described by Eq. 11:
2
k1ðkO2 KM þ kMO ½MꢂÞ½O2ꢂ
2
½Eꢂ½Sꢂ
þ ½Sꢂ
d½O2ꢂ
dt
k1KM þ k1½Mꢂ þ kO2 ½O2ꢂKM
ꢁ
¼
ð11Þ
kO2 ½O2ꢂKSKM
k1KM þ k1½Mꢂ þ kO2 ½O2ꢂKM
As above, at the saturating concentrations of D-glucose
([S] ? ?) and provided k1KM ꢄ k1½Mꢂ þ kO ½O2ꢂKM,
2
Eq. 11 reduces to Eq. 12, which predicts the activating
effect of RuIII because of the kMO ½Mꢂ term in the
2
numerator:
1
We are grateful to the reviewer for directing our attention to the
IC50 issue.
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J Biol Inorg Chem (2013) 18:547–555
553
[19–22]. This well-recognized phenomenon is insuffi-
ciently understood at a molecular level. The effects of RuIII
on GO activity described here exemplify also a nonmon-
otonic dose response. Perhaps understanding this minor
case will help to resolve a significantly more fundamental
problem aimed at avoiding hazards from chemicals tar-
geted at the human endocrine system.
d½O2ꢂ k1ðkO KM þ kMO ½MꢂÞ½O2ꢂ½Eꢂ
2
2
ꢁ
¼
ð12Þ
dt
KMðk1 þ kO ½O2ꢂÞ
Strictly speaking, the step shown in Eq. 5 should also
2
not be ignored completely because, as mentioned earlier,
RuIII reduces eventually to RuII as suggested by the two-
phase kinetics such as shown in Figs. 4 and 7. If the step
shown in Eq. 5 is taken into account, the activation by RuIII
In conclusion, the results obtained using a ‘‘model’’ GO
enzyme suggest that the cyclometalated RuII and RuIII
complexes have a variety of options in tuning the enzy-
matic activities of oxidoreductases. GO is an example of a
‘‘fine’’ tuning in terms of the values of Ki, which are not
large. However, the result is not striking because the Ki
values are of the same order of magnitude as the Michaelis
constant Km for b-D-glucose, a natural substrate of GO. We
speculate here that the effects could be significantly larger
for enzymes with much higher affinity toward their natural
substrates. An example is prolyl hydroxylase domain
(PHD) dioxygenase. It is an iron-containing oxidoreduc-
tase, the biological role of which is to hydroxylate proline
residues of a hypoxia-inducible factor protein in the pres-
ence of a-oxoglutarate and dioxygen [23]. High activity of
PHD is crucial for suppressing the growth of tumor cells,
but its activity is suppressed by ROS. ROS oxidize FeII of
PHD to FeIII and the enzyme loses the ability to activate
could still be qualitatively observed provided kMO [ kM.
2
The main conclusions drawn from this investigation are
presented schematically in Fig. 8. Complexes structurally
similar to tris(bpy)ruthenium(II) interact/bind with/to dif-
ferent sites of GO and affect differently its ability to cat-
alytically reduce dioxygen. The kinetic data suggest that
tris(bpy)ruthenium(II) itself (complex 2) as a competitive
inhibitor should bind somewhere close to the binding site
of b-D-glucose. Ruthena(II)cycles 1a and 1b, the charge of
which is ?1 as opposed to ?2 for 2, should bind at some
distance from the binding site of b-D-glucose because they
behave as noncompetitive inhibitors, i.e., 1a and 1b do not
affect the affinity of the substrate for the enzyme.
Ruthena(III)cycles 3 and 4, particularly in low doses,
enhance the enzymatic activity. Therefore their localization
in the proximity of the b-D-glucose binding site is unlikely.
In this case the basic role of RuIII species is to facilitate an
electron transfer from FADH2 of GOred at O2 and therefore
we hypothesize that the RuIII complexes may accomplish
this via binding closer to the izoalloxazine ring of FADH2
and serving as a functional bridge between FADH2 and O2.
Needless to say, the exact mechanism of activation of GO
by cyclometalated RuIII complexes is not clear at the
moment and more effort should be made to clarify it. The
effect found here is perhaps related to the recently estab-
lished phenomenon of ‘‘nonmonotonic dose–response
curves.’’ This is when low concentrations of endocrine-
disrupting chemicals produce a stronger effect on a human
organism than when they are used at higher concentrations
O2. Interestingly, Km for a-oxoglutarate is 2.2 9 10-7
M
[24], which indicates that the affinity of a-oxoglutarate
toward PHD is five orders of magnitude higher than that of
D-glucose toward GO. Thus, ruthenacycles similar to 1a
and 1b, which revealed exceptional anticancer activity
[11], could be potential efficient effectors of the enzymatic
activity of PHD. This statement is now under extensive
investigation.
Materials and methods
Chemicals
GO from Aspergillus niger was obtained from Sigma
(211 U mg-1) and D-glucose was obtained from Acros.
KH2PO4, RuCl3, 2-phenylpyridine, 1,10-phenanthroline,
bpy, KPF6, and a Dowex 1X8 200 ion exchange resin were
obtained from Sigma-Aldrich. All solvents used were of
high-performance liquid chromatography grade from
Fischer. Stock solutions of the ruthenium complexes
(0.015 M) were prepared by dissolving complexes 1a, 1b,
2, 3, and 4 in 3.5 mL MeOH and this solution was added to
31.5 mL phosphate buffer of pH 5 (0.05 M). Two stock
solutions of D-glucose (0.03 and 0.3 M) in the pH 5 buffer
were allowed to stand overnight at 25 °C to ensure equil-
ibration between a and b anomers before measurements. A
stock solution of GO (1.5 9 10-7 M) in phosphate buffer
Fig. 8 Various locations of ruthenium complexes within a GO
molecule resulting in competitive (2) and noncompetitive (1a, 1b)
inhibition along with increasing (3 and 4) the ability of the enzyme to
reduce O2
123

554
J Biol Inorg Chem (2013) 18:547–555
of pH 5 (0.05 M) was prepared after determination of the
active flavin adenine dinucleotide (FAD). All solutions
were kept in a refrigerator. The water-soluble chloro
complexes 1a and 1b were made from the corresponding
[Ru(phpy)(NN)2]PF6 (Hphpy is 2-phenylpyridine, NN is
bpy or 1,10-phenanthroline) precursors using a previously
described method [25]. The corresponding [RuII(ph-
py)(NN)2]PF6 complex (400 mg) was dissolved in 3 mL of
a 1:1 (v/v) MeOH–CH3CN mixture and the solution was
brought on a column charged with 2 g Dowex 1X8 200 ion
exchange resin in a Cl- form. The product was eluted with
MeOH. The purple fraction was collected, the solvent was
evaporated, and the solid was dried in a vacuum. Anion
exchange was quantitative (more than 98 %) and 1H NMR
coefficient used was 1.41 9 104 M-1 cm-1; the amount of
active FAD found was 91 %.
Kinetics of enzymatic O2 uptake
The measurements were made using a homemade Clark
electrode with a Mylar surface (YSI, Yellow Springs, OH,
USA) equipped with a YSI 5331 probe in a 1.5-mL cell, the
temperature of which was controlled at 25 °C. The cali-
bration was performed as follows. The cell was filled with
distilled water at 25 °C with stirring and the voltage was
recorded. Then the cell was cleaned and filled with the
previously deoxygenated water (in a vacuum and bubbled
with argon) and a lower voltage was recorded. The drop of
0.06 V was referred to 1.21 9 10-3 M O2 (O2 concentra-
tion in water at 25 °C). The voltage was related to the
concentration of O2 in solution as suggested elsewhere [27]
using the following equation:
-
spectra were similar to those of the PF6 compounds. IR
-
analysis showed the disappearance of the PF6 band at
831 cm-1. [RuIII(phpy)2(bpy)]Cl (4) was prepared as
described elsewhere [14].
0:00121 ðMÞ
½O2ꢂ ðMÞ ¼ ðVr ꢁ V0Þ
0:16 ðVÞ
Synthesis of [RuIII(phpy)(bpy)2]Cl2 (3)
Here Vr and V0 are the observed potential and the potential
at zero O2 concentration, respectively. The buffer was first
added to the cell, followed by a ruthenium complex solu-
tion and GO. The solution was stirred, and the recording
device was turned on. The reaction was initiated by the
addition of a solution of D-glucose after 20 s, and the cell
was immediately sealed. Time intervals between readings
were in the range from 3 to 10 min depending on the con-
centration of D-glucose, which was varied in the range from
0.0025 to 0.1 M. Concentrations of O2 were plotted as a
function of time. The corresponding kinetic curves contained
typically significant steady-state portions, i.e., O2 concentra-
tions varied linearly with time (the duration of this phase
depends on the concentration of D-glucose). The slopes of the
linear portions were associated with steady-state rates d[O2]/
dt (see Fig. 4) and were used for making the Michaelis–
Menten and Lineweaver–Burk plots. The concentrations of
ruthenium complexes used are indicated in the figure legends.
Each measurement was made at least in duplicate.
Complex 1a (150 mg) and 7.5 mg of iron TAML as a
catalyst (obtained from GreenOx Catalysts, Pittsburgh, PA,
USA) were dissolved in 20 mL water. A saturated solution
of NaCl (2 mL) was added, followed by a slow dropwise
addition of 30 % H2O2 (1.5 mL). The mixture was stirred
overnight, and the product was extracted three times with
10 mL CH2Cl2. The organic layers were dried over sodium
sulfate, and the solvent was evaporated to dryness under a
vacuum. The resulting paramagnetic purple-red solid was
used without further purification. Yield 63 mg (40 %).
Positive electrospray ionization M? = 568 m/z. The
extinction coefficient in the UV–vis spectrum was
7,400 M-1 cm-1 at 540 nm in MeOH.
Methods
The UV–vis spectra were obtained using an HP 845x
diode-array spectrophotometer. 1H NMR measurements
were made using a Bruker 300 MHz instrument. Fourier
transform IR data were collected on a Bruker Alpha-P
using ATR. Mass spectra were recorded using a Thermo
Fisher LCQ ESI? ion trap mass spectrometer.
Acknowledgments We thank DGAPA (PAPIIT project IN204812)
and CONACyT (project 153151 and scholarship to R.-O. S. D.) for
financial support. Jose David Vazquez Cuevas is thanked for solving
computer-related problems. We are grateful to Michael P. Hendrich
(Carnegie Mellon University) for allowing us to use his research
facilities and for helpful suggestions.
Titration of GO
Enzyme concentrations are expressed as molarity of cata-
lytically active bound FAD. Active FAD was determined
by UV–vis spectroscopy using the method of Swoboda and
Massey [26]. The absorbance of a 10 lM solution of the
GOox in phosphate buffer of pH 5 (0.05 M) was measured
at 450 nm. D-Glucose (20 lL, 0.3 M) was added and the
absorbance of GOred was determined. The extinction
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