BIOCHEMISTRY OF FREE RADICALS
Shock 1998: Oxígeno, Oxido Nítrico y perspectivas terapéuticas
Simposio Internacional, Academia Nacional de Medicina
Buenos Aires, 30 abril 1998
BIOCHEMISTRY OF FREE RADICALS: FROM ELECTRONS TO TISSUES
Laboratorio de Radicales
Libres en Biología y Medicina, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires
Key words: free radicals, reactive oxygen species, nitric
oxide, peroxynitrite, septic shock
radicals are chemical species with an unpaired electron in the outer
valence orbitals. The unpaired electron makes them paramagnetic
(physics) and relatively reactive (chemistry). The free radicals that
are normal metabolites in aerobic biological systems have varied
reactivities, ranging from the high reactivity of hydroxyl radical
(t½ = 10-9 s) to the low reactivity of melanins (t½ = days). The
univalent reduction of oxygen that takes place in mammalian organs
produces superoxide radicals at a rate of about 2% of the total oxygen
uptake. The primary production of superoxide radicals sustains a free
radical chain reaction involving a series of reactive oxygen species
(hydrogen peroxide, hydroxyl and peroxyl radical and singlet oxygen).
Nitric oxide is almost unreactive as free radical except for its
termination reaction with superoxide radical to yield the strong
oxidant peroxynitrite. Nitric oxide also reacts with ubiquinol in a
redox reaction, with cytochrome oxidase competitively with oxygen, and
oxymyoglobin and oxyhemoglobin displacing oxygen. Septic shock and
endotoxemia produce muscle dysfunction and oxidative stress due to
increased steady state concentrations of reactive oxygen and nitrogen
de los radicales libres: del electrón a los tejidos. Los radicales
libres son especies químicas con un electrón solitario en un orbital
externo de valencia. El electrón solitario los hace paramagnéticos
(física) y relativamente muy reactivos (química). Los radicales
libres que son metabolitos normales en los organismos aeróbicos
exhiben reactividades variadas, que van desde la alta reactividad del
radical hidroxilo (t½ = 10-9 s) a la baja reactividad de las
melaninas (t½ = días). La reducción univalente del oxígeno que se
lleva a cabo en los órganos de los mamíferos produce radicales
superóxido a una velocidad aproximada del 2% del consumo de oxígeno.
La producción primaria de radical superóxido mantiene una cadena de
reacciones de radicales libres que involucra a una serie de especies
reactivas del oxígeno (peróxido de hidrógeno, radicales hidroxilo y
peroxilo, y oxígeno singulete). El óxido nítrico es casi
no-reactivo como radical libre, excepto su reacción de terminación
con el radical superóxido que produce al fuertemente oxidante
peroxinitrito. El óxido nítrico también reacciona con el ubiquinol
en una reacción redox, con la citocromo oxidasa competitivamente con
el oxígeno, y con la oximioglobina y la oxihemoglobina desplazando al
oxígeno. El shock séptico y la endotoxemia producen una disfunción
y un estrés oxidativo en el músculo mediados por un aumento en las
concentraciones en estado estacionario de las especies reactivas del
oxígeno y del nitrógeno.
Postal address: Dr. Alberto Boveris, Facultad de Farmacia y
Bioquímica, Junín 956, 1113 Buenos Aires, Argentina. Fax:
54-1-962-7928; E-mail: email@example.com
1. The Chemistry of Free Radicals
A free radical is a chemical species with an unpaired electron in
the outer valence orbitals. Since orbitals are usually filled with a
pair of electrons, an alternative and similar definition is that a
free radical is a chemical species with an odd number of electrons.
The chemical species can be an atom, such as the hydrogen or the
chlorine atom, a transition metal, or a molecule in which case the
unpaired electron is located in a molecular orbital. The unpaired
electron in the outer valence orbital confers a relatively high
reactivity to the molecule due to the strong tendency to acquire a
second electron in the orbital. However, transition metals with an odd
number of electrons and the free radical form of relatively large
organic molecules with delocalized electrons, such as melanins or
nitric oxides in which the nitrogen atoms is in an aromatic ring, are
relatively unreactive and stable.
Free radicals are chemically written with the notation for the
chemical species followed by a dot that indicates the unpaired
electron. For instance, the hydrogen atom is indicated as H• and the
hydroxyl radical as HO•. There are two notation ways to place the
dot, HO• and HO•, the first one is the classic organic and
physical chemistry style and the latter one is the more modern
biochemical style. In our days, notation follows whatever is easier in
the keyboard of the available computer.
Chemically, free radicals are characterized for sustaining free
radical chain reactions, a self propagating kind of reactions in which
a free radical reactant yields a product that is also a free radical
and that reacts producing another free radical. These feed-forward
chemical processes are known as propagation reactions and are the core
of the free radical chain reactions. Classically, free radical
reactions are divided in: a) initiation reactions; b) propagation
reactions, and 3) termination reactions. In the initiation reactions a
free radical is formed from stable non-free radical chemical species
(AB + C Þ A• + D + E). In the propagation reactions, a free
radical, also called a reaction center, reacts with a stable molecule
giving another free radical or reaction center as product (A• + CD
Þ AC + D•). In the termination reactions, two free radicals cancel
out their unpaired electrons forming a stable product (A• + B• Þ
The chemical reactivity of free radicals is determined by the whole
molecule bearing the unpaired electron; consequently, reactivity
varies greatly in different free radicals. A way of expressing and
comparing chemical reactivity is by listing the half-life time (t½)
of the chemical species (Table 1). A short t½ indicates a high
reactivity, and then hydroxyl radicals are the most reactive of the
series. It is understood than when HO• is formed it reacts, at
diffusion controlled rates and after a few collisions with water
molecules, with the first or the second organic molecule that it
encounters. Other highly reactive chemical species which are common
biological metabolites, or in other words that are produced in normal
conditions, have similar reactivities although their are not free
radicals. For instance, the electronically excited state of oxygen,
singlet oxygen (1O2) has a t½ of 5 x 10-6 sec and the powerful
oxidant peroxynitrite (ONOO•) has a t½ of 0.05-1 sec.
2. Oxygen free radicals
The oxygen molecule constitutes about 20% of the atmospheric air
and is paramagnetic. Oxygen atoms (1s2, 2s2, 2px2, 2py, 2pz) are
highly reactive and react themselves to form the oxygen molecule.
However instead of forming a pair of s-p ligand orbitals with the two
2py and 2pz forming y-y and z-z bonds, the lowest energy configuration
is one in which there is a right angle rotation and formation of a z-y
s bond; two three electrons bonds are formed between one pair of
electrons of one oxygen atom and a single electron of the other oxygen
atom. This particular chemical bond was described by Linus Pauling1 as
two three-electron bond to explain the electronic configuration of the
oxygen molecule (Fig. 1). Considering the rule of the unpaired
electrons, the oxygen molecule is a biradical, but chemically is
rather stable and has been described as a sluggish radical. Most of
the isolated biomolecules, proteins, DNA, sugars and some lipids are
stable for long time in air (20% O2). However, the oxygen molecule in
quite reactive to combine with the iron atoms of hemoglobin and
cytochrome oxidase (second order reaction constants of 107-108 M-1s-1)
to provide the chemical basis for oxygen transport and respiration.
The difference between non-catalyzed and catalyzed oxidations is
described by Albert Szent-Gyorgi as: “When Tutenkhamon’s grave was
opened, his breakfast, consisting in wheat grains, was found
unoxidized after three thousand years. This represents the
non-catalyzed chemical probability. Had His Majesty risen and consumed
his meal this would have been burned in no time. This is the catalyzed
The oxygen molecule with its two three-electron bonds and its
biradical character can be reduced by four successive transfers of one
electron and the process, advanced by Michaelis3, is called the
univalent reduction of oxygen (Fig. 2). Two of the intermediates,
superoxide and hydroxyl are free radicals. Superoxide radicals are
dissociated at physiological pH (pK = 4.7) and are, consequently,
charged as an anion (O2-). Chemically, superoxide anion radicals are
quite unreactive and biologically behave as a mild reductant reducing
the iron moiety of ferritin, cytochrome c and cytochrome oxidase.
Moreover, being charged its permeability through biomembranes is
highly reduced except for red blood cells that possess a special
system for O2- transport. Hydroxyl radical is one of the most reactive
chemical species and abstracts hydrogen at near diffusion-controlled
rates. Hydrogen peroxide is not a free radical and is chemically
stable; however in biological systems it is easily cleaved
homolytically by transition metals, such as Fe2+ and Cu1+, to yield
hydroxyl radical. Finally, the fourth product of the univalent
reduction of oxygen is water. Biological systems that evolved living
up with O2 in the atmosphere in the past 3 x 109 years have enzymes
that are able to add one, two or four electrons to O2. Water is the
stable product of the tetravalent reduction of oxygen carried out by
mitochondrial cytochrome oxidase, which accounts for about 97% of the
oxygen uptake in higher animals, in a process that is coupled to
energy generation and ATP synthesis.
A series of subcellular organelles are able to partially reduce the O2
molecule to O2- and H2O2 (Fig. 3)4. The primary production of both
products of the partial reduction of oxygen, O2- and H2O2, and the
secondary production of HO• constitute the molecular mechanism of
oxygen toxicity5. Mitochondria produce primarily O2- which dismutates
by the enzymatic action of Mn-superoxide dismutase (Mn-SOD)
specifically located in the mitochondrial matrix. Endoplasmic
reticulum, by autoxidation of the flavoprotein NADPH-cytochrome P-450
reductase and cytochrome P-450, produce both O2- and H2O2. Similarly,
other cytosolic enzymes, such as xanthine oxidase, produce both O2-
and H2O2. Peroxisomes generate hydrogen peroxide into the peroxisomal
core by two-electron transfer from the flavin oxidases to the oxygen
molecule. Mitochondria, present in all aerobic cells, are the most
important physiological source of superoxide radicals. In hepatocytes,
the well developed endoplasmic reticulum affords an equally important
source of O2- and other subcellular sources are relevant in some
cellular types. The semiquinone form of two components of the
mitochondrial respiratory chain, ubisemiquinone and the flavin
semiquinone of the NADH-dehydrogenase, produce O2- by autoxidation in
a vectorial reaction directed to the mitochondrial matrix.
Ubisemi-quinone autoxidation is known as the Boveris-Cadenas
reaction6-7 and the autoxidation of the flavin seiquinone of
NADH-dehygrogenase as the Boveris-Turrens reaction8. Superoxide anions
are not permeable through the inner mitochondrial membrane and are
consequently confined into the matrix where Mn-SOD and NO are the O2-
co-reactants to yield H2O2 and ONOO- as final products, respectively,
in a two very fast, diffusion-controlled, reactions. The mitochondrial
production of O2- accounts for about 2% of the total O2 uptake of
perfused rat liver. Similarly, the mitochondrial production of H2O2
accounts for about 2% of the total O2 uptake of perfused rat liver and
3. The nitrogen free radical
Nitrogen molecules account for 79% of the atmospheric air and are
formed by two nitrogen atoms, that as free atoms have three unpaired
electrons (1s2, 2s2, 2px, 2py, 2pz) and that form three full covalent
bonds (s and 2 p) in making the stable and inert nitrogen molecule.
When a nitrogen atom, with its three unpaired electrons, combines with
an oxygen atom, with its two unpaired electrons, the nitric molecule
(NO) is formed with an odd total number of electrons. A full N=O
double bond (s-p) is formed and an unpaired delocalized electron is
left in the molecule that defines the free radical (NO•) character
of the NO molecule (Fig. 1). Nitric oxide is a physical free radical
in terms of the unpaired electron. The chemical free radical character
of NO is restricted; no propagation reactions of NO• are known to
occur in condensed systems but NO• readily reacts with O2- to yield
ONOO- in a classical termination reaction.
4. The physiological free radical chain reaction
4a. The Fenton-Haber-Weiss reactions of oxygen free radicals. The
primary production of O2- and H2O2 is able to initiate and sustain a
free radical chain reaction under physiological conditions that
encompasses the reactions of lipoperoxidation. Both O2- and H2O2 are
the reactants of the initiation process (reactions 1 and 2) in which
the reactive HO• is generated. Reactions 1 and 2 are known as the
Fenton-Haber-Weiss chemistry (originally to describe H2O2
decomposition by iron salts)4. Moreover, the biological protective
action of superoxide dismutase and catalase is understood as to keep
at the slowest rate possible the generation of HO•. The concept has
been frequently recognized as the Fridovich dogma9 of the antioxidant
effect of both superoxide dismutase and catalase. Hydroxyl radicals
are able to start
O2- + Fe3+ ® O2 + Fe2+ 
H2O2 + Fe2+ ® HO• + Fe3+ + HO- 
propagation reactions with unsaturated fatty acids (RH) to yield
the stable hydroperoxides (ROOH) (reactions 3, 4 and 5). The peroxyl
radicals (ROO•) are able to yield termination reactions with
formation of electronically excited products such as singlet oxygen
(1O2) and aldehydes (RHO) and ketones (RO) with excited carbonyl
groups(=CO*) (reactions 6 and 7). Reactions 6 and 8 provide, through
chemiluminescence, the chemical and molecular basis of an assay to
determine the rate of the free radical chain reaction of
lipoperoxidation under physiological conditions10.
HO• + RH ® R• + H2O 
R• + O2 ® ROO• 
ROO• + RH ® R• + ROOH 
ROO• + ROO• ® RHO + RO + 1O2 
ROO• + ROO• ® = CO* + RO + O2 
1O2 + 1O2 ® 2 O2 + hv 
4b. The Beckman-Moncada reactions of nitrogen free radical. The
recognition of the production of NO• by the nitric oxide synthase
(cNOS) of the endothelium as the ERF11-12 and of the reaction of O2-
with NO•13-14 opened a new line of thought in free radical
biochemistry. Moreover, the recent discovery of NO• production by a
mitocondrial NOS (mtNOS) located in the inner membrane of rat liver
mitochondria15-17 has started a revolution in terms of both regulation
of tissue oxygen uptake18-23 and of free radical toxicity. Nitric
oxide is produced by a series of NOS (cNOS, iNOS, mtNOS) that share
the common property of utilizing arginine and NADPH2 as substrates;
the reaction is in terms of free radical chemistry and initiation
reaction in which the free radical NO• is produced (reaction 9). The
very fast, diffusion-controlled (k = 6.7 x 109 M-1.s-1), termination
reaction of the radicals O2- and NO• (reaction 10) is easily
understood after considering a collision between the two molecules
with unpaired and delocalized electrons that results in bond formation
(ON•/•O2- Þ ONOO-). Delocalized molecular electrons move
thousands times faster than a molecular collision.
arginine + NADPH2 + O2 ® citrulline + NADP + NO• 
NO• + O2- ® ONOO- 
ONOO- + RH + H+ ® NO2 + H2O + R• 
In addition, ONOO- has been reported as able to abstract hydrogen
atoms from unsaturated fatty acids, acting as a “crypto-HO•” or
apparent hydroxyl generator24 (reaction 11), and to initiate the
propagation reactions of lipid peroxidation25.
5. The steady state concentrations of oxygen and nitrogen
The steady state approach in which the rate of production of a
chemical species is equaled to its rate of utilization or
disappearance (i.e., + d.[O2-]/dt = -d.[O2-]/dt) and the utilization
of the corresponding differential equations allow the estimation of
the steady state concentrations of the chemical species. By a
combination of measurements and calculations the steady state
concentrations of the chemical species participating in the free
radical chain reaction of reactive oxygen and nitrogen species are
estimated (Table 2).
6. The utilization pathways of nitric oxide
Nitric oxide has been recognized to react with a series of relevant
biomolecules which are ubiquitous in mammalian tissues and organs. The
physiological actions of NO• would depend ultimately on the relative
ratios of the reaction rates of NO• with the target molecules. In
some cases a strong biological effect is to be expected. The reaction
of NO• with O2- (reaction 10) is the link between the reactions of
oxygen and nitrogen free radicals. However, the rate of O2-
utilization by this reaction, calculated for rat heart mitochondria
under physiological conditions (taking from Table 2 [3 x 10-8 M NO•]
and [1.5 x 10-10 M O2-] and k = 6.7 x 109 M-1.s-1) results equal to 3
x 10•8 M O2-/sec. This rate is about 30 times slower than the rate
of O2- utilization by the dismutation reaction. This latter rate can
be calculated as d.[O2-]/dt = [O2-].[SOD].k (with [SOD] as 3 x 10-6 M
and k = 2.4 x 109 M-1.s-1) and it equals 1.1 x 10-6 M O2-/sec.
There are five important metabolic reactions that utilize NO• in
heart and muscles; three of the reactions occur in the mitochondria:
a) with cytochrome oxidase (reaction 12), b) with ubiquinol (reaction
13), and c) with O2- (reaction 10). The other two are the reactions
with d) cytosolic myoglobin (reaction 14) and e) with extracellular
hemoglobin (reaction 15). Some of these reactions have important
biological significance. The reaction of NO• with cytochrome oxidase
inhibits the main pathway of O2 uptake and energy production18-23 and
the reaction with ubiquinol produces ubisemiquinone that by
autoxidation produces O2- and operates as free radical initiation
NO• + Cyt a3 ® Cyt a3 - NO 
NO• + UQH2 ® UQH• + H+ + NO- 
NO• + MbO2 ® metMb + NO3- 
NO• + HbO2 ® metHb + NO3- 
The reactions of NO• with MbO2 and HbO2 yield the met-derivatives
which are subsequently reduced by the NADPH2-dependent reductases and
no biological effect is to be expected from a mild oxidation of the
two hemoproteins. The steady state concentrations of the five
biomolecules that react with NO• in rat heart under physiological
conditions, the reaction constants and the expected reaction rates are
given in Table 3. As it can be seen the reactions with the
hemoproteins are highly favored.
7. Septic shock and the free radical chain reaction
It is apparent that septic shock is associated with high NO•
levels in blood and tissues. It was accepted that the
cytokine-dependent expression of macrophage iNOS is part of the
response to septic shock and endotoxin administration. Recently, it
has been found that NO• synthetized by iNOS of rat diaphragm after
administration of Escherichia coli endotoxin participates in the
development of diaphragm contractile failure28. The increased iNOS
activity of the endotoxin treated animals increased the NO• steady
state concentration in diaphragm to 0.47 µM from a level of 0.02 µM
in the control animals29. Diaphragm mitochondria isolated from rats
treated with E. coli endotoxin, at times similar to the ones that
produce the contractile failure of diaphragm fibers, show: a) partial
uncoupling and decrease in respiratory control29, b) increase in H2O2
production29-30, and c) nitration of mitochondrial proteins29.
Muscle is a target organ in septic shock and for E. coli endotoxin.
Increased spontaneous muscle chemilu-miniscence is an early indicator,
simultaneous with hypothermia, of the multiple dysfunction of septic
shock in rats30. Muscle oxidative stress, as detected by in situ organ
chemiluminescence, clearly precedes liver oxidative stress. Muscle
dysfunction plays a key role in the circulatory and respiratory
failure of septic shock.
Acknowledgements: This study was supported by grants from Universidad
de Buenos Aires and Agencia Nacional de Promoción Científica y
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TABLE 1.– Estimated half-lives of free radicals in biological
Free radical t½ (seconds)
Hydroxyl radical HO• 10-9
Alcoxyl radical RO• 10-6
Nitric oxide NO• 1-10
Peroxyl radical ROO• 10-1
Ubisemiquinone UQH• 10-2-l
Melanins Complex days
Semiquinones (tar) Complex days
TABLE 2.– Steady state concentrations of reactive oxygen and
Species Steady state Tissue/cells/ Method*
concentration (M) organelles
Superoxide anion O2- 2.5 x 10-11 Rat liver cytosol C
0.8 x 10-10 Rat liver mitochondria M/C
1.5 x 10-10 Rat heart mitochondria M/C
Hydrogen peroxide H2O2 0.5 x 10-8 Rat liver mitochondria M/C
0.6 x 10-8 Rat heart mitochondria M/C
1 x 10-8 Rat liver cytosol C
4 x 10-9 Rat liver peroxisomes M
1 x 10-7 Perfused rat liver M/C
1 x 10-7 Liver cells and slices M
Hydroxyl radical HO• 6 x 10-18 Liver C
Alkyl radical R• 6 x 10-16 Liver C
Peroxyl radical ROO• 2 x 10-9 Liver C
Singlet oxygen 1O2 1 x 10-15 Isolated hepatocytes M/C
1 x 10-16 Liver M/C
Nitric oxide NO 5 x 10-8 Liver C
2 x 10-8 Muscle M
1 x 10-7 Rat heart (+ bradikynin) M
Peroxynitrite ONOO- 1 x 10-8 Heart and liver mitochondria C
Data taken from ref. 26 for the oxygen reactive species. Data for
NO• from Poderoso’s laboratory.
* C: calculated; M/C: production rates measured and steady state
calculated; M: measured by diffusion equilibrium
TABLE 3.– Relative rates of reaction of nitric oxide in rat. A
physiological steady state concentration of 3 x 10-8 M NO• is
assumed. The steady state concentrations of the coreactants are given
in each case
Species/[M] Location Reaction constant Reaction rate Relative rate
(M-1.s-1) (µM/s) (%)
O2-/[1.5 x 10-10] Mitochondria 6.7 x 109 0.03 0.02
Cyt a3/[2 x 10-5] Mitochondria 108 30 22
UQH2/[1 x 10-4] Mitochondria 1.2 x 104 0.036 0.03
MbO2/[1.5 x 10-4] Cytosol 107 45 33
HbO2/[2 x 10-4] Extracellular 107 60 44
Fig. 1.– Electronic configuration of oxygen and nitric oxide
molecules. The lines indicate full covalent bonds with a pair of
electrons and the dots indicate single electrons. The oxygen molecule
has two three-electrons bonds and is a biradical. The nitric oxide
molecule has one single unpaired electron and is a free radical.
Fig. 2.– The univalent reduction of the oxygen molecule according to
Fig. 3.– Intracellular production of the products of the partial
reduction of oxygen. Taken from Chance, Sies and Boveris4.