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BioMed Research International
Volume , Article ID , pages
Possible Mechanisms of Fullerene C60 Antioxidant Action
V. A. Chistyakov,1Yu. O. Smirnova,2,3 E. V. Prazdnova,1and A. V. Soldatov2
1Research Institute of Biology, Southern Federal University, Rostov-on-Don 344090, Russia
2Research Center for Nanoscale Structure of Matter, Southern Federal University, Rostov-on-Don 344090, Russia
3Department of Physics, Purdue University, West Lafayette, IN 47907, USA
Correspondence should be addressed to Yu. O. Smirnova; firstname.lastname@example.org
Received June ; Revised August ; Accepted September
Academic Editor: Claiton Leonetti Lencina
Copyright © V. A. Chistyakov et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Novel mechanism of antioxidant activity of buckminsterfullerene C60 based on protons absorbing and mild uncoupling of
mitochondrial respiration and phosphorylation was postulated. In the present study we conrm this hypothesis using computer
modeling based on Density Functional eory. Fullerene’s geroprotective activity is suciently higher than those of the most
powerful reactive oxygen species scavengers. We propose here that C60 has an ability to acquire positive charge by absorbing inside
several protons and this complex could penetrate into mitochondria. Such a process allows for mild uncoupling of respiration and
phosphorylation. is, in turn, leads to the decrease in ROS production.
Reactive oxygen species (ROS) are able to cause oxidative
damage to DNA, lipids, and proteins and are known to
be the key regulators of cellular signaling. In spite of the
criticismfromanumberofresearchers free-radical theory
occupies a pivotal position in modern biological concepts
of aging . e ability to retard senescence is typical for
many antioxidants [–]. Well-known ability of fresh vegeta-
bles, fruits, red wines, and spices to stimulate longevity is
largely determined by the existence of compounds such as
deprotonated xanthones , carotenoids , anthocyanins
and pyranoanthocyanins , and avonoids and terpenoids
. ese compounds exhibit a broad spectrum of oxyradical
quenching activity based on reactions of single electron
transfer, hydrogen atom transfer, sequential electron proton
transfer, proton coupled electron transfer, radical adduct
formation, and iron chelation [,–].
IntherecentstudyBaatietal. showed that the oral
administration of the fullerene C60 suspension in olive oil
retards senescence of rats. Herewith, median and maximum
life span increase approximately twice. Moreover, it was
shown that rats treated with fullerene C60 demonstrated high
resistance to carbon tetrachloride. Toxicity of this substance
is mediated by ROS generation . According to this fact and
results of biochemical tests fullerene C60 was proposed to be
of high antioxidant activity in vivo. Due to the free-radical
theory of aging, highly active antioxidant activity can be the
basis for unique antiaging (geroprotective) properties.
Fullerene C60 isknowntobeabletoinactivatehydroxyl
radicals by attaching to double bonds . However, this
mechanism cannot explain sucient (near two times) incre-
ase in lifespan of rats. Such kind of antioxidative activity is
also attributed to natural phenolic antioxidants that do not
possess high senescence retarding activity . We propose
that there is an additional mechanism involved in fullerene
anti-aging activity. Respiratory chain located in the inner
mitochondrial membrane is the main source of superoxide
anion radicals, which lead to a cascade of other toxic ROS.
In this connection mitochondrial-targeted antioxidants like
lipophilic cations (Skulachev ions) with antioxidant load
 are the most eective antiaging agents (geroprotectors)
among synthetic compounds.
Accumulation of Skulachev ions in the mitochondria is
based on the transmembrane potential dierence generated
of inner membrane of mitochondria has positive charge and
the inner side has negative charge. So, lipophilic cations are
concentrating in mitochondria via electric eld forces .
BioMed Research International
F : e results of DFT geometry optimization for one (a) and six (b) protons and fullerene. Initially protons were placed outside the
fullerene and then the conguration that has the minimum value of total energy was found as a result of DFT geometry optimization. As a
result, all protons appeared to be inside the fullerene. For the simulation, GGA-BLYP exchange-correlation potential was used. Carbon atoms
are shown in grey and protons are shown in black.
e lipophilic properties of fullerene C60 are well known .
In addition, Wong-Ekkabut et al. showed using molecular
dynamics simulations thatC
60 fullerene is capable of
penetrating into membrane and accumulates in the middle
of lipid bilayer. However, the simulation does not consider
the possible presence of fullerene and/or membrane charge.
We suppose that fullerene is capable of absorbing protons and
obtaining positive charge, which allows it to be delivered into
the mitochondria. us, superoxide anion-radical generation
is decreased by mild uncoupling of respiration and phos-
phorylation . In the present study we perform theoretical
analysis of the fullerene C60 ability to acquire positive charge
and to absorb protons to prove that the proposed mechanism
indeed may take place.
All the computer simulations were performed within the
framework of Density Functional eory (DFT) for solving
odinger equation , which has been used for the
investigation of antioxidants previously . In the present
work, DFT implemented in ADF code was used .
Initially from one up to seven protons were placed outside the
fullerene and then the most probable atomic conguration
was found by minimizing the total energy of the system
during the process of geometry optimization, that is, nding
a stable conguration of the system that corresponds to
the minimum of total energy. For the exchange-correlation
part of molecule potential General Gradient Approximation
(GGA) was used in both GGA-BLYP  and GGA-BLYP-
D [,] forms, but all nal results were obtained using
GGA-BLYP potential. Basis sets are DZ (double-𝜁)withinthe
calculations including water molecules around C60 and TZP
(triple-𝜁) within the calculations without taking into account
the water molecules around “C60 plus-protons” system.
At rst step an interaction between single proton and
fullerene was simulated. e proton was placed outside the
C60 above one of the pentagons at the distance about ˚
from the pentagon plane. As a result, the proton transfers
fullerene at a distance about . ˚
A from the nearest carbon
atom (Figure (a)). Next, more protons were added to this
system; some of them were initially placed above pentagons,
but most were placed above hexagons. e rst two protons
were placed at maximum possible distance from each other.
All others were equally distributed around the fullerene. In all
cases protons were “absorbed” by the fullerene, and it was so
until the seventh proton was added to the system—it repulsed
from the fullerene. So, the maximum amount of protons
inside the fullerene consists of six protons (Figure (b)).
It is crucial to know the distribution of charge over C60 for
each conguration of protons. Figure shows the distribu-
tion for two, four, and six protons inside the fullerene. It can
be seen that when there are two protons inside the surface of
are added the fullerene surface obtains positive charge.
Table provides information about binding energies and
VDD charges  for each proton added to the system. Both
charges on protons and relatively big C-H distances allow us
to suppose that protons interact with fullerene according to
donor-acceptor mechanism and do not form strong chemical
It is important to know whether the presence of other
molecules near fullerene will impact the ability of protons
to penetrate into fullerene or not. For this purpose we
performed a simulation involving water molecules which are
in the presence of both protons and water hydronium ions
will appear, water molecules can be chosen. An exchange
of protons between hydronium ions takes place in such
environment, so for some small period of time protons are
e simulation was carried out for a fullerene with single
proton placed above a pentagon and water molecules
randomly distributed around the fullerene. It was shown
that solvent molecules do not inuence the capability of a
fullerene to absorb the proton.
BioMed Research International
F : e distribution of charge for two, four, and six protons inside the fullerene. e charge of fullerene with two protons inside is
about zero (red color) while fullerenes that have four or six protons inside obtain positive charge (green and blue color). Protons lose their
positive charge starting from positive charge (blue color) to almost zero (orange color).
T : Binding energies and VDD charges for dierent amounts
of protons added to fullerene.
e Voronoi Deformation
60 accumulating in
mitochondria provides high radical scavenging activity in
this subcellular compartment, called by Skulachev the “dirt-
iest place in the cell” . Another eective antioxidant
mechanism is based on mild uncoupling of respiration and
phosphorylation. Respiratory chain obtains electrons from
electron reduction of oxygen. But the transfer of one or two
electrons could produce the radicals that are dangerous to
cells (such as superoxide or peroxide anions).
e specic feature attributable to the generation of
ROS by mitochondria is related to the fact that the higher
is the membrane potential (the larger is the dierence in
the concentration of protons inside and outside the mito-
chondria), the higher is the level of the superoxide anion
production. As it was shown , there is steep depen-
dence of mitochondrial superoxide-anion-radical generation
on transmembrane potential (Δ𝜓). Even a small (–%)
decline of Δ𝜓 resulted in tenfold lowering of ROS production
erefore, the so-called mild uncouplers of oxidative
phosphorylation are the substances which can move some
of the protons inside the mitochondria and can possess
an excellent oxygen-protective eect, although they are not
antioxidants in terms of chemistry .
DFT simulations allowed us to propose the following
mechanism. C60 fullerene molecules enter the space between
inner and outer membranes of mitochondria, where the
excess of protons has been formed by diusion. In this com-
partment fullerenes are loaded with protons and acquire
positive charge distributed over their surface. Such “charge-
loaded” particles can be transferred through the inner mem-
brane of the mitochondria due to the potential dierence
generated by the inner membrane, using electrochemical
mechanism described in detail by Skulachev et al. [,]. In
this case the transmembrane potential is reduced, which in
turn signicantly reduces the intensity of superoxide anion-
e proposed ability of C60 fullerenes to acquire positive
charge allows ascribing them to the mitochondrial-targeted
compounds. e key role of mitochondria in the cellular
regulation makes such “charge-loaded” fullerenes be of great
interest along the route for novel classes of drugs develop-
V. A. Chistyakov and Yu. O. Smirnova contributed equally to
e authors are grateful to Dr. V. S. Lysenko and Dr. Igor
Alperovich for valuable comments that improved the paper.
BioMed Research International
e support by Southern Federal University of Russia grants
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