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
Free 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 species.
Bioquímica 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: firstname.lastname@example.org
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.
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
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
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 
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• 
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 reactive species
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.
NO• + Cyt a3 ® Cyt a3 - NO 
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.
1. Pauling L. The nature of the chemical bonds. New York: Cornell
University Press, 1948.
Free radical t½ (seconds)
Hydroxyl radical HO• 10-9
TABLE 2.– Steady state concentrations of reactive oxygen and nitrogen species
Species Steady state Tissue/cells/ Method*
Superoxide anion O2- 2.5 x 10-11 Rat liver cytosol C
Data taken from ref. 26 for the oxygen reactive species. Data for
NO• from Poderoso’s laboratory.
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
O2-/[1.5 x 10-10] Mitochondria 6.7 x 109 0.03 0.02