NEW ASPECTS INTO THE CHEMISTRY OF
COENZYME Q10 FAMILY MEMBERS
(calcium binding and transporting)
Rubin Gulaboski, Ivan Bogeski,
Valentin Mirceski, Reinhard Kappl,
Markus Hoth
Rubin Gulaboski, Ivan Bogeski, Reinhard Kappl, Markus Hoth,
„Benzoquinones based Antioxidants”, European Patent Office,
Munich 2010, PATENT No. 09178735.8.
Coenzyme Q10 is a redox mediator in the
mitochondrial electron transfer chain (METC)
contributing to the mitochondrial ATP production
QUINONE (oxidized form)
3HC
3HC
3HC
3HC
2
3
6
5
CH3
CH3
QUINOL (reduced form)
Most of the members of METC
are „one-electron” mediators
Reactive Oxygen Species =
Mitochondrial electron transport chain is a major source
of highly dangerous ROS!!!
Superoxide
dismutase
e
O2
.
O2
-
e
.. ..
. O:O .
.. ..
.. ..
. O:O -:
.. ..
Oxygen
Superoxide
anion radical
Glutathione Peroxidase
Catalase
-
e
H 2O 2
.. ..
H:O:O:H
.. ..
Hydrogen
peroxide
.
e
OH
..
.O:H
..
Hydroxyl
radical
-
H 2O
..
H:O:H
..
Water
Structures of
the most common
Reactive
Oxygen
Species
evaluated from O2
Effective „cure“ against
ROS
are the Antioxidants
Ubiquinol-moderate antioxidant
We started working on this project in 2006
Effect of Ca2+ ions to
the redox process of
2-palmytoilquinonetransfer of Ca2+
across lipid membrane
Structure of
2-palmytoilhydroquinone
This compound can bind and transfer
Ca2+ ions across biomimetic membranes
Our Aim: to see whether CoQ’s have something to do
with Ca2+ transfer across mitochondrial membranes
Why Ca2+?
Ca2+ -is one of the most important
second messengers
Mechanisms of Ca2+ transfer
in mitochondria
???
CH3
H 3C
O
O
O
CH3
H 3C
O
O
Coenzyme Q
CH3
1
n( 2 HC-(
3 HC)C=HC-CH
2)
O
H 3C
O
CH3
O
Coenzyme Q
CH3
10
Experiments with Coenzyme Q1-CoQ1
-3E-05
Cyclic voltammogram of 100 µM CoQ1 in pH of 7.00
I/A
-1.5E-05
-1E-20
1
0.75
0.5
0.25
0
-0.25
E/V
-0.5
-0.75
-1
-1.25
-1.5
-3E-05
pH = 2.0 (black)
pH = 2.7 (blue)
pH = 3.5 (green)
pH = 4.5, 7.4 and 9.2
(orange, pink and brown)
I/A
-1.5E-05
pH increase
0
1
0.75
0.5
0.25
0
-0.25
-0.5
-0.75
-1
-1.25
-1.5
E/V
pH dependence of the redox process of CoQ1-the “yellow form”
in the pH range from 1 to 9
CH3
H 3C
CH3
O
H 3C
OH
O
O
CH3
+
H 3C
O
O
Coenzyme Q
CH3
1
(oxidized)
2e
-
+
2H
CH3
+
H 3C
O
OH
Coenzyme Q
CH3
1
(reduced)
-2.4E-05
I/A
-1.6E-05
+ 2e- + 2Ca2+=?
CV of 100µM CoQ-1-in pH of 7,00 (yellow
native form);
insensitivity to Ca2+;
c(Ca2+)/mM = 0 (black); 1 (green);
10 (orange);50 (black) and 100 (rose)
CoQ1-the yellow form=
insensitive to Ca2+ ions
-8E-06
0
8E-06
0.8
0.6
0.4
0.2
0
-0.2
-0.4
-0.6
-0.8
-1
-1.2
E/V
That is, ...END OF THE STORY, or MAY BE NOT?
-1.4
-0 .0 0 0 0 6
Cyclic voltammograms of CoQ1
recorded in 1 mol/L NaOH.
Scans recorded after 3 min (1) 1 h (2)
3 h (3) and 24 h (4).
Scan rate of 30 mV/s,
c(CoQ1) = 0.075 mmol/L.
Significant changes in the
voltammetric responses have been
observed when
CoQ1 was dissolved in 1M NaOH!
II
I
3 m in u te s
I/A
-0 .0 0 0 0 2
II '
0 .0 0 0 0 2
I'
0 .0 0 0 0 6
- 0 .0 0 0 0 8
6 0 m i n u te s
II
- 0 .0 0 0 0 4
I/A
I
0
I I'
I'
0 .0 0 0 0 4
- 0 .0 0 0 0 8
II
1 8 0 m in u t e s
- 0 .0 0 0 0 4
I /A
I
0
I'
0 .0 0 0 0 4
I I'
-0.00008
24 hou rs
II
I /A
-0.00004
0
II'
0.00004
0.2
0
-0.2
-0.4
E / V
-0.6
-0.8
-1
UV-Vis spectrum of 10 µM CoQ1 recorded in kinetic mode in 1 M NaOH
-3E-05
I/A
-2E-05
-1E-05
100 µmol/L CoQ-1 in pH of
7.
Black curve-yellow form of
CoQ1
Red curve-CoQ1
firstly dissolved in NaOH for
0
1E-05
0.6
0.4
0.2 1E-15 -0.2 -0.4 -0.6 -0.8
-1
-1.2 -1.4
E/V
Comparison of the cyclic voltammograms of CoQ1 directly dissolved
in pH of 7.00, and of CoQ1 initially dissolved in 1 M NaOH for 45 min
and retitrated afterwards to pH of 7.00
pH increase
Effect of pH to the redox processes of CoQ1. In this situation
CoQ1 was in contact with 0.1 M NaOH for 60 minutes
Next logical task: DETERMINE the MECHANISM
and the STRUCTURE of the New Benzoquinone Product
Electron Paramagnetic resonance-EPR-suitable technique for structure
evaluation by the radical species
(many quinones form radicals when dissolved
in alkaline media)
Simulated EPR spectrum of the CH3
radical
EPR spectrum of CoQ1
after reduction with
half-equimolar amount
of NaBH4 in pH of 7.00
EPR spectrum of CoQ1
obtained in 0.1 M NaOH but
without using any reductant!!
CH2
Nine-line EPR spectrum od parent CoQ1
corresponds to presence of one CH3 and one CH2 group in the structure
I
NEXT STEP to determine the structure-NMR EXPERIMENTS
NMR spectrum of CoQ1 in D2O
Protons of O-CH3
CoQ1-5 minutes in NaOD
retitrated afterwards to
Protons of O-CH3 pD of 7.00
Signal from the protons
of METHANOL
Protons of O-CH3
CoQ1-10 minutes in NaOD
Retitrated afterwards to
pD of 7.00
Signal from the protons
of METHANOL
Protons of O-CH3
CoQ1-25 minutes in NaOD
Retitrated afterwards to
pD of 7.00
Signal from the protons
of METHANOL
Protons of O-CH3
CoQ1-60 minutes in NaOD
Retitrated afterwards to
pD of 7.00
Signal from the protons
of METHANOL
Where METHANOL does
come from, when CoQ is
dissolved in alkaline media?
-from the methyl group?
or
-from the METHOXY
O-CH3 group?
By using methyl benzoquinone
derivatives we found that the
methyl group
CAN NOT BE
CLEAVED
from the aromatic
ring!!!
Tetramethyl benzoquinone
2-methyl-benzoquinone
(duroquinone)
CH3
CH3
O
H 3C
O
H 3C
O
O
-
????
CH3
+
H 3C
O
O
CH3
C o enzym e Q 1
2O H
-
H 3C
O
-
+
2C H 3O H
O
O -d e m e th y la te d C o e n z y m e Q
(2,3-dihydroxo-5-methyl-6-isoprenyl-benzoquinone)
1
Ratio of the signal of Methanol vs Methoxy groups from
NMR experiments of CoQ1 in NaOD
Color of solutions of
Benzoquinones with
2 OH groups in its structure
Color of solutions of
Benzoquinones with
1 OH group in its structure
Benzoquinones with 2-OH groups
are able to bind earth-alkaline cations
in ratio 1:2
Benzoquinones with 1-OH group
are NOT able to bind (at least not strongly)
earth-alkaline cations
New product is able to bind Ca2+ ions
in stochiometric ratio 1:2 (L:M2+)
concentration of Ca2+ increase
New form of CoQ-1, sensitivity to Ca2+ ions
The slope of 59mV indicates a complex
of a type 1:2 (L to M)
Dependence of the mid-peak potential of the cyclic voltammograms
of new form of CoQ1
on the logarithm of Ca2+ concentration
CH3
CH3
O
H 3C
O
H 3C
O
O
-
CH3
+
H 3C
O
O
CH3
C o enzym e Q 1
2O H
-
H 3C
O
-
+
2C H 3O H
O
O -d e m e th y la te d C o e n z y m e Q
1
EXPERIMENTS with Coenzyme Q10-CoQ10
Native CoQ10 is absolutely
insoluble in water, but...
it can be dissolved in
NaOH...and
it can be transformed
into a new form in
presence of
OH- anions present
in the organic phase
Coenyzme Q10 in presence of 1 M NaOH
5 µM Coenyzme Q10 in presence
of 1 M NaOH and retitrated to pH of 7.0
after 3 weeks
The membrane-bound enzyme
Cytochrome P450 does the same
Effect of Ca2+ to the voltammetric
response of CoQ10 in presence of CYP450
in pH of 7.40
O
O
H 3C
H 3C
-
O
+
O
CH3
O
CH3
2OH
3 OH
-
CYP450
R
2CH
+
-
O
R
O
O
2. Stabilization of the CH3-cleaved CoQ10 products in water
O
O
H
O
-
CH3
O
H
-
+
O
CH3
O
R
H 2O
-
O
-
R
H
O
O
H
O
3. Reduction of the CH3-cleaved CoQ10 product and binding of
-O
O
-
-
+
-
O
2e
R
CH3
O
2Ca
2+
-
O
R
O
-
+
-
2+
-O
CH3
O
CH3
O
Ca
Ca2+
O
-O
R
-O
Ca
2+
Experiments with CoQ10 embedded in organic membrane in presence
of organic Hydroxide to show whether the new form of CoQ10
can transfer Ca2+ ions across biological membranes
Ca2+ out
Lipid membrane
CoQ10 embedded
+ electrode
OH-
WT+Ca+Cyt
WT+Ca
1.8
Ratio (340/380)
1.2
0.6
0
0
30
60
90
Time (min)
120
150
180
105%
Mitochondrial Ca
2+
influx (slope in %)
***
100%
95%
90%
85%
80%
75%
Mito WT
Mito CoQ10 KO
Experiments with wild types of mitochondria and knockout mitochondria
depleted of CoQ10 in absence and in presence of CYTP450. Effect of Ca2+ ions.
SUMMARY
Plenty of CoQ family members are present in the living systems
The chemistry and most of the functions of the native forms of Coenzyme Q family members are mainly portrayed in the
features of the 2e-/2H+ redox reaction (electron and proton transfer) that leads to reversible transformation of the quinone
to quinol forms.
If the Coezyme Q structures are in contact with high concentration of OH- ions or CYP450 enzymes, quite different
quinonic forms can be obtained.
CYP450 and NaOH can both induce scission of the both O-CH3 (methoxy) groups in the structure of the Coenzyme Q
family members, thus creating so called „O-demethylated“ quinones that bear charge of „2-“.
These new Coenzyme Q structures formed in alkaline media (or in presence of CYP450) are more polar than their parent
compounds, while also having much stronger antioxidative features.
The inherent properties of the new Coenzyme Q structures to bind the earth-alkaline cations upon their reduction classify
these compounds as potential facilitators for transferring of metal ions across biological membranes.
-3E-05
I/A
-2E-05
-1E-05
0
1E-05
0.6
0.4
0.2 1E-15 -0.2 -0.4 -0.6 -0.8
E/V
-1
-1.2 -1.4
Main people involved in this project
A. in Homburg
I. Bogeski
M. Hoth
Bernd Morgenstern
Barbara Kutzky
R. Kappl
Richi Kohler
D: MKD
B. Saarbrücken
C. UNi K‘Lautern
Prof. Hermann
ACKNOWLEDGMENT also to
Alexander von Humboldt
Foundation
Prof. Mirceski
Experiments with Coenzyme Q10-CoQ10
Solubility of CoQ10 in water is bellow 10 pM!!!
Impossible to perform experiments in water media!
CoQ10 studied voltammetrically in Thin-film voltammetry set-up
Electron conductor, graphite electrode
droplet of org.
Organic
water
solution
of an
immiscible
electroactive
solvent
compound
CoQ10 + 2eCoQ10
CoQ102H+ H+
OH- H+
OH- H+
Cat2+
Liquid-Liquid interface
Cat2+ 2An-
-E/V
NADH
-0.2 _
CoQ
Complex III
0.0 _
Complex IV
0.2 _
The processes in the mitochondrial electron transfer chain are driven
by the differences in the standard redox potentials of the contributors