New PhytoL (1974) 73, 1075-1086.

SUPEROXIDE DISMUTASE, CATALASE AND

GLUTATHIONE PEROXIDASE: SOLUTIONS TO
THE PROBLEMS OF LIVING WITH OXYGEN
BY B. H A L L I W E L L

Department of Biological Sciences., Portsmouth Polytechnic, King Henry I St.,

Portsmouth POi zDY, Hants, U.K.

{Received 13 May 1974)

SUMMARY
Recent research suggests that HjOj is a normal metabolite in both plants and animals and is
not particularly cytotoxic. The impact of HjOj on cell metabolism is discussed with particular
reference to photorespiration. Catalase may function by preventing the formation of excessive
concentrations of H2O2 and by using H2O2 in the peroxidatic oxidation of compounds such as
methanol and formic acid. Radicals (such as OH and O2") and 'excited' oxygen are far more
damaging to living organisms. The formation of these radicals in biological systems is described.
Superoxide dismutase plays a key role in protection against such radicals, but glutathione
peroxidase is also involved.

INTRODUCTION

During photosynthesis, the leaves of many plants show an enhanced rate of respiration
known as 'photorespiration'. This is due to the formation of glycollic acid in the chloro-
plasts and its subsequent oxidative decarboxylation (Zelitch, 1971). Detailed isotopic and
enzymic studies (Tolbert, 1971) have led to the formulation of a metabolic pathway to
account for CO2 release from glycollate (Fig. i). According to this scheme, glycollate is
converted into glycine which is then decarboxylated in the mitochondria. Although this
has been widely accepted (see review by Raven, 1972), there may be other COj-releasing
reactions operative during photorespiration (Zelitch, 1971, 1972a; Halliwell, 1973;
Halliwell and Butt, 1974).
The first enzyme in these pathways, glycollate oxidase, catalyses the 02-dependent
conversion of glycollate to glyoxylate:
CH2OH CHO
I -^O2-1 +H2O2
COOH COOH
In plant tissues, this enzyme is located in a single type of subcellular organelle, the
peroxisome (Tolbert, 1971). Peroxisomes are found in a wide range of plant and animal
tissues (de Duve and Baudhuin, 1966; Halliwell, 1974a) and, in each case, they contain
one or more enzymes which produce H2O2, e.g. D-amino acid oxidase and urate oxidase
are found in many animal peroxisomes. They also contain most, if not all, of the catalase
present in the cell. (Although isoenzymes of catalase have been reported in liver eytosol
(Holmes and Masters, 1972), the evidence is controversial (Mainferme, Wattiaux-de-
1075
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1076 B. HALLIWELL

Coninck and Wattiaux, 1972).) Since peroxisomes contain enzymes which produce
H2O2, it has seemed logical to propose that the function of catalase is to destroy it and
that the high activity of this enzyme leads to immediate destruction of any II2O2
generated. It is often further assumed that this is necessary because H2O2 is highly
toxic. Since recent work has challenged both these assumptions, it is the object of this
review to describe some of this work and to discuss the biological roles of H2O2 and
catalase.

Incoming COg glycollote — glycollote

glyoxylale

glycine
phosphoglycerate-«»-D- glycerafe -
7
D-glycerate J
Ci-tetrahydrofolate
denvotive
hydroxypyruvale
5 / glycine

-senne

CHLOROPLAST PEROXISOME MITOCHONDRION

Fig. I. The 'Tolbert' scheme for CO2 release during photorespiration. The figure also shows
the subcellular location of the enzymes involved (see Tolbert, 1971). Enzymes: (i) glycollate
oxidase; (z) serine-glyoxylate aminotransferase; (3) glutamate-glyoxylate aminotransfc-rase;
(4) glycine decarboxylase complex; (5) serine hydroxymethyltransferase; (6) glyoxylate
(hydroxypyruvate) reductase; (7) glycerate kinase.

H o w EFFICIENT IS CATALASE IN H 2 O 2 DESTRUCTION?

In leaf peroxisomes, the oxidative decarboxylation of glycollate is due to the oxidation

of glycollate to glyoxylate by glycollate oxidase, followed by a non-enzymic reaction of
glyoxylate with H2O2 (Halliwell & Butt, 1974).
CHO
1 +CO2 +H2O
COOH
This occurs despite the presence of high activities of catalase, indicating that this enzyme
does not completely destroy the H2O2 generated inside the peroxisome. A more direct
demonstration of this phenomenon was achieved by Boveris, Oshino and Chance (1972),
who showed that a considerable proportion of the H2O2 produced during oxidation of
urate or D-alanine by rat-liver peroxisomes escaped into the surrounding medium.
Similarly, exposure of erythrocytes to a system generating H2O2 causes oxidation of
haemoglobin, despite the high activities of catalase present in these cells (Keilin and
Hartree, 1945). Indeed, erythrocytes and other animal tissues contain a second enzyme
capable of metabolizing H2O2, glutathione peroxidase.

H2O2+2GSH->GSSG+2H2O
This enzyme metabolizes a significant proportion of H2O2 in erythrocytes, although
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Solutions to problems of living with oxygen
present at much lower activities than catalase (Cohen and Hochstein, 1963; Nicholls,
1972). A similar result is obtained when H2O2 '^ supplied to a homogenate of rat liver
(Hochstein and Utley, 1968).
An examination of the mechanism of catalase action makes it clear why it does not
destroy H2O2 completely. Fig. 2, based on the studies of Chance (1950, 1952a, b), shows
that the catalatic breakdown of H2O2 requires formation of a catalase-H2O2 complex
(compound I) which then reacts with a second molecule of H2O2, i.e. two molecules of
H2O2 must impinge on the active site of a single catalase molecule. The high con-
centration of catalase present in peroxisomes means that, at constant rates of H2O2

CATALASE + H,0

CATALATIC
ACTION

Fit;. 2. Mode of action of catalase. This scheme is based on the studies of Chance (1950,
1952a, b). In the peroxidatic action of the enzyme, HjO^ oxidizes a secondary electron
donor such as formic acid, methanol, ethanol or HNOj.

generation, compound I accumulates to a steady-state level in dynamic equilibrium

with free catalase and a low concentration of H2O2 (Sies et al., 1973). This situation has
been demonstrated experimentally in rat liver (Oshino et al., 1973a) and in bacterial
cells (Chance, 1952c). In other words, although the maximal velocity (Fn,aJ of catalatic
action is very high, the effective 'Km' is also very high: measured values range from
0.047 *° '-^ ^^ (Jones and Suggett, 1968; Scandalios, Liu and Campeau, 1972). Thus,
other enzymes (such as glutathione peroxidase), other reactions (e.g. with glyoxylate) or
diffusion out of the peroxisome can readily compete for the H2O2 available.

CELLULAR PRODUCTION OF H2O2

Many enzymes and systems which produce H2O2 are located in organelles other than the
peroxisome, e.g. the xanthine oxidase of liver is found in the cytosol. Microsomal and
mitochondrial fractions isolated from liver generate H2O2 (Boveris, Oshino and Chance,
1972; Thurman, Ley and Scholz, 1972). It might be objected that this is merely an
artefact of the in vitro condition, but there are many observations confirming H2O2
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1078 B. HALLIWELL
production by such systems in vivo. For example, H2O2 is generated at a constant rate
in the isolated, perfused rat liver under 'physiological' conditions (Oshino et al., 1973a).
Oxidation of formate to CO2 in animals occurs by a reaction which requires H2O2 (see
below), the H2O2 supply being the rate-limiting step. Rats fed with large amounts of
ethanol contain an increased activity of NADPH oxidase in liver microsomes (Lieher and
de Carli, 1970; Carter and Isselbacher, 1971; Videla, Bernstein and Israel, 1973); this
enzyme system produces H2O2 in vitro (Gillette, Brodie and La Du, 1957; Thurman
et al., 1972). Such rats show an increased ability to oxidize injected formate to CO2,
suggesting increased production of H2O2 in vivo.
Mitochondrial fractions from spinach leaves also generate H2O2 in vitro (Halliwell,
1974b) and the production of H2O2 by isolated chloroplasts is a well-known phenomenon
(Mehler, 1951a, b; Mehler and Brown, 1952; Whitehouse et al., 1971).

H 2 O 2 AND PHOTORESPIRATION

The question of the rate of H2O2 generation by chloroplasts in vivo is of twofold im-
portance with reference to photorespiration. First, H2O2 may be involved in glycollate
production. The transfer of two-carbon (C2) fragments between sugar phosphates by
transketolase, an enzyme of the Calvin cycle, involves the binding of these fragments
to thiamine pyrophosphate at the active site. Shain and Gibbs (1971) who showed that
H2O2 produced by isolated chloroplasts could oxidize this transkctolase-TPP-C2
intermediate, producing glycollate, proposed that this was a source of glycollate in
illuminated chloroplasts. (Pathways of glycollate formation have been discussed in
detail by Tolbert, 1973.) Secondly, Zelitch (1972b), who demonstrated that isolated
chloroplasts could catalyse the rapid decarboxylation of glyoxylate, suggested that
this was a major pathway of CO2 release during photorespiration. These results
were due, at least in part, to production of H2O2 by the chloroplasts, followed by
its non-enzymic reaction with glyoxylate (Elstner and Heupel, 1973; B. Halliwell,
unpublished results).
A knowledge of the rate of H2O2 generation by chloroplasts in vivo is required before
the physiological significance of these two systems can be assessed. That H2C)2 is pro-
duced in vivo is suggested by the observation that some blue-green algae excrete it on
illumination (Patterson and Myers, 1973). H2O2 would also be produced in vivo by the
action of superoxide dismutase in chloroplasts, as discussed below. However, it is very
difficult to assess the rate of H2O2 generation from such data.
Other objections can be raised to the proposal of Zelitch (1972b). Since glyoxylate is
produced in the peroxisomes, it would have to diffuse from peroxisomes into chloro-
plasts before the decarboxylation could occur: this diffusion would compete with con-
version of glyoxylate to glycine (Tolbert, 1971) or direct decarboxylation in the peroxi-
somes (Halliwell and Butt, 1974). That some glyoxylate does enter chloroplasts is sug-
gested by the presence in these organelles of an NADPH-dependent glyoxylate reductase
(Zelitch and Gotto, 1962; Tolbert, Yamazaki and Oeser, 1970). This enzyme has a high
affinity for glyoxylate. Reduction of the latter to glycollate would also be favoured by the
high NADPH/NADP"^ ratio in illuminated chloroplasts in vivo. This reduction would
compete with the decarboxylation for the glyoxylate available to chloroplasts. These
observations do not prove that H2O2-dependent glyoxylate decarboxylation does not
occur in chloroplasts in vivo, but they do suggest that it is not a major contributor to
photorespiratory CO, release.
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Solutions to problems of living with oxygen 1079
H2O2 produced in vivo by mitochondria, chloroplasts or the endoplasmic reticulum
would have to diffuse into peroxisomes before it could be acted upon by catalase, although
in animal tissues it might be metabolized by glutatbione peroxidase in tbe cytosol (Green
and O'Brien, 1970). It is therefore likely that all parts of the cell will be exposed to H^Oj
to some degree. H2O2 migbt also diffuse out of the cell into the surrounding fluid.

FREE RADICALS
For those unfamiliar with the subject of radicals, a more detailed consideration of the
species discussed in this section is presented in an Appendix (p. 1082).
Many enzymes and enzyme systems catalyse oxidations in which electrons are trans-
ferred from the substrate to O2. Transfer of two electrons to O2 gives H2O2, i.e.

In several enzymic reactions, however, electrons can be transferred singly,

followed by H O 2 + e - + H + -.H2O2
The species HO2 is acidic and dissociates to give O2", the 'superoxide' ion. Since the
pKa of HO2 is 4.8, O2" is the main form present at 'physiological' pH values. There is
good evidence for the production of 02~ during the reduction of O2 by several enzymes
(Knowles et al., 1969; Pederson and Aust, 1972; Fridovich, 1973), including some present
in mitochondria and in the endoplasmic reticulum. It is also formed during oxidation
of the reduced forms of several compounds found in biological systems, including
ferredoxin, flavins and haemoglobin (Fridovich, 1973). Of particular interest to plant
biochemists are the observations that isolated chloroplasts generate O2" on illumination
(Asada and Kiso, 1973a, b; Allen and Hall, 1973; Elstner and Kramer, 1973; Epel and
Neumann, 1973). This seems to be produced by the light-driven reduction of added or
endogenous electron acceptors, followed by their auto-oxidation to give O^".
O2~ can act either as a reducing agent, giving up its extra electron, or as an oxidizing
agent, becoming reduced to H2O2. For example, it reduces cytochrome c,
cytochrome c (F"e-^*) +O2" -•cytochrome c (Fe^*) +O2
but it oxidizes ascorbic acid
20i ~ +ascorbate + aH ••" -•dehydroascorbate + 2H2O2
The latter ability may well be responsible for the effects of ascorbate on O2 uptake
by isolated chloroplasts (Allen and Hall, 1973; Elstner and Kramer, 1973; Epel and
Neumann, 1973).
However, in comparison with other oxygen radicals, O' " is rather unreactive, with a
lifetime in the msec range. In the absence of anything for it to react with, it reacts with
itself.
o r + O r + 2 H + -^H2O2+O2
The O2 produced by this reaction is in the 'excited' (singlet) state. Singlet oxygen is far
more reactive than normal, 'ground state' (triplet) oxygen and rapidly oxidizes com-
pounds such as unsatunited fatty acids. Khan (1970) showed that O^" can react with
H2O2, which is probably present in most subcellular organelles, as discussed above.
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
io8o B. H A L L I W E L L
+-0H+02
•OH is a very powerful oxidizing agent, able to attack many organic molecules.
O j " formed in vivo could easily damage the cell, either hy direct reaction with its
components or by the generation of -OH and singlet oxygen. Its relatively long lifetime
means that it can diffuse away from the site of formation, leading to the destruction of
other structures. There is considerable evidence that pcroxidation of membrane lipids,
causing damage to and loss of integrity of the membrane and inactivation of membrane-
bound enzymes, involves O^", although this need not be the radical which actually
attacks the lipid (Fee and Teitelhaum, 1972; Pederson and Aust, 1972, 1973; Fong et al.,
1973; Zimmermann et al., 1973; Goda et al., 1973). E.xposure of membranes to a
source of O2~ causes peroxidation. Bacteria and viruses exposed to 02~ are rapidly
killed (Lavalle, Michelson and Dimitrijevic, 1973; Gregory, Yost and Fridovich, 1973;
Gregory and Fridovich, 1974). Production of O j " may be responsible for the effects of
bipyridyl herbicides (Farrington et aL, 1973) and for the nerve degeneration caused by
injecting animals with 6-hydroxy-dopamine (Heikkila and Cohen, 1973).
It is therefore clear that organisms need to protect themselves against free-radicals.

SUPEROXIDE DISMUTASE

Enzymes ('superoxide dismutases') catalysing the breakdown of O2~ to O2 and H2O2

are found in a wide range of aerobic organisms (Fridovich, 1973). The O2 produced, in
contrast to that formed by the non-enzymic dismutation of O2~, is in the ground
(triplet) state. The presence of superoxide dismutase activity in aerobes but not in
anaerobes suggests a role in protection against oxygen free-radicals. In experiments in
vitro, superoxide dismutase has been shown to protect subcellular organelles and bac-
teria against oxidative damage (Pederson and Aust, 1972, 1973; Fee and Teitelbaum,
1972; Zimmermann et al., 1973; Lavalle et al., 1973; Gregory et al., 1973).
Recent experiments suggest that superoxide dismutase may be able to catalyse the
removal of singlet oxygen as well as of O^" (Paschen and Weser, 1973). Removal of
O " should prevent formation of -OH and singlet oxygen by the reactions described
above: ability to scavenge the latter might be important as a 'second line of defence'
against O2" and also in protection against singlet oxygen formed by reactions other than
the dismutation of O j " .
There is considerable evidence that superoxide dismutase helps to protect against
free radicals in vivo. Certain bacteria grown under air contain no catalase, but dismutase
is present and increases in activity when the organisms are grown at increased partial
pressures of O2 (Gregory and Fridovich, 1973a). The dismutase activity of Escherichia
coli cells is increased by growth under 100% O2, whereas that of Bacillus subtilis is not.
Cells of Escherichia coli grown under 100% O2 were much more resistant to hyperbaric
O2 than cells grown under air, but this was not true for Bacillus subtilis (Gregory and
Fridovich, 1973b). Cells of Escherichia coli B depleted of periplasmic superoxide dis-
mutase by growth on an iron-deficient medium were much more rapidly killed by 0 2 "
than normal cells (Gregory et al., 1973). Mutants of E. coli temperature-sensitive in
superoxide dismutase activity show a comparable defect in their ability to grow in O2
(Gregory and Fridovich, 1974).
In animal tissues, superoxide dismutases have been reported to he present in mito-
chondria (Weisiger and Fridovich, 1973) and in the cyto.sol (Rotilio et al., 1973). At
least part of the dismutase activity of spinach leaves is found in the chloroplasts (Asada,
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Solutions to problems of living with oxygen 1081
Urano and Takahashi 1973; Lumsden and Hall, 1974). Since both mitochondria and
chloroplasts contain systems which can generate 02~, it seems likely that the dis-
mutases function to remove it. This dismutation would be a source of H2O2.
No dismutase activity has been found in tbe endoplasmic reticulum, altbougb micro-
somal lipids peroxidize rapidly and O^" is involved (Pederson and Aust, 197^, 1973;
Fong et al., 1973). However, the enzyme glutathione peroxidase, which acts on H2C)2i
can also act on lipid peroxides (designated R.OOH)
R.OOII+2GSH-^GSSG+R.OH+Il2O
The hydroxy-acids (ROH) produced are stable and, unlike the peroxides, do not give
rise to free radicals in the membrane (I,ittle and O'Brien, 1968; Christophcrsen, 1968,
1969). It may well be that glutathione peroxidase in the cytosol of animal cells helps to
control lipid peroxidation in the endoplasmic reticulum and possibly other organelles
(Christophersen, 1966; Sies et al, 1972; Flohe et al, 1973). In agreement with this,
animals deficient in selenium, an essential component of glutathione peroxidase, show a
variety of symptoms consistent with extensive lipid peroxidation (Flohe, Gunzler and
Schock, 1973: Rotruck et al., 1973; Smith, Tappell and Chow, 1974).

Is H 2 O 2 A USEFUL METABOLITE?
I have argued so far that H2O2 is not itself especially cytotoxic, but it can give rise to
free radicals, against which cells must be protected. Tbe observation that superoxide
dismutase protects organelles and cells against O2" again shows that H2O2, a product of
this enzyme, is far less toxic.
Under certain circumstances, the controlled generation of free radicals from H2O2
might be advantageous. For example, polymorphonuclcar leucocytes produce II2O2 and
the rate of production increases markedly during phagocytosis (Iyer, Islam and Quastel,
1961). This II2O2 can be used in an antibacterial system involving myeloperoxidasc, an
enzyme present in these cells, and either halide ions or thiocyanate. It is likely that this
system operates by forming free-radicals in the vicinity of the ingested bacterium (Kleb-
anoff, 1968, 1970). Indeed, O j " appears to be formed by leucocytes during phagocytosis
(Babior, Kipncs and Curnuttc, 1973). (An analogy may perhaps be drawn here to the
generation of free radicals during the enzymic oxidation of phenols in damaged plant
tissues, leading to modification of the proteins of any invading pathogen (Pierpoint,
1969).) Of course, the occurrence of such systems may well be rather restricted, but they
do show that H2O2 is not necessarily just a waste product. A further role for H2O2 is
considered below.

WHAT ARE PEKOXISOIXIES FOU?

As we have seen, peroxisomal catalase does not completely destroy H2O2, but it prob-
ably does prevent the concentration from rising too high. High levels of H2O2 can
damage organelles and can also inactivate superoxide dismutase (Fielden et al., 1973;
Beauchamp and Fridovich, 1973).
Catalase compound I can react with electron donors other than H2O2, including
methanol, ethanol, and formic acid (Fig. 2). This 'peroxidatic' action of catalase accounts
for a large part of methanol metabolism in animals (Mannering et al., 1969), formate
oxidation in both animals (Oro and Rappoport, 1959) and plants (Leek et al,
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
io82 B. H A L L I W E L L
Halliwell, 1974b) and it may also make a small contribution to ethanol oxidation in
animals (Oshino et al., 1973b), although this has heen disputed (Carter and Isselhacher,
1972). It may be that the function of catalase is to use H2O2 in such peroxidatic oxida-
tions: this would be facilitated by the concentration of catalase in peroxisomes, which
leads to accumulation of compound I. Further, compound I is readily converted to an
inactive form (compound II) at steady rates of H2O2 generation (Fig. 2) (Chance, 1950).
Thus, a constant flow of H2O2 or oxidizable metabolites to the peroxisomes in vivo might
be expected to lead to a loss of most of the catalase activity. The presence of peroxidatic
electron donors prevents this (Oshino et al., 1973a) and so their oxidation may be essen-
tial for the maintainence of catalase activity, and hence the control of cellular concentra-
tions of H2O2, which in turn will affect superoxide dismutase activity and GSH/GSSG
ratios.

APPENDIX

Eree radicals involving oxygen

Introduction
A covalent bond consists of a pair of electrons shared between the bound atoms. If a
covalent bond is split to leave one electron associated with each atom, the products are
called radicals or free radicals, e.g. when water is exposed to ionizing radiation an O—H
bond is split:

H—O—H """'->H- + OH
The symbol, •, is used to denote the spare electron. H- is a hydrogen radical; -OH is a
hydroxyl radical.
Radicals are highly reactive. They can combine with one another to form covalent
bonds, e.g.

or they can attack bonds in other molecules. This often leads to production of new
radicals, setting up a chain reaction, e.g.
>C = C< + OH-^>C—C<

OH
Oxygen
The oxygen atom has eight electrons. In the oxygen molecule, O2, it has been shown
that only one electron pair is shared and there are two unpaired electrons, i.e. O2 is not
0 0 but O—O- and is itself a radical.
The two unpaired electrons occupy molecular orbitals which are referred to as n*
orbitals {antibonding orbitals). Usually, these two electrons occupy different 7t* orbitals
but they have the same spin quantum number (a quantity which has only two values).
This form of O2 is referred to as being in the triplet state and it is the state of least
energy for O2, i.e. the ground state.
Two other states, of higher energy, are known in which the 2 electrons have opposite
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Solutions to problems of living with oxygen 1083
spin. These are illustrated in Fig. Ai. Because an input of energy is needed to generate
them, these are excited states and O2 in these states is far more reactive than ground state
oxygen. Superoxide, Oi~, (see below) produces Oj in the 'Sg^ state (Khan, 1970), a
singlet state.

Superoxide
Superoxide is the product of i-electron reduction of O^: the extra electron enters
one of the n* orbitals. It is usually written as O2 " or O2 ~. Like other anions, it can form
ionic bonds, e.g. potassium superoxide (K^^O^") is a white, crystalline solid.
At low pH values, it becomes protonated to give

'*2py.2pz © © ; e © 0 ®
" 2py.2pz e © 1 O O (Q) (Q)
ENERGY
•'2PX
o ; (Q)
Type of orbiial GROUND STATE ' EXCITED STATE EXCITED STATE

(Eriergy 92 kJ (Energy 155 I<J

above ground slate) obove ground sfaCe)
Fig. A I . Molecular orbital scheme for oxygen.

If a further electron is supplied to 0 ^ " , then O^^' results, in which both n* orbitals
are completely filled. O^^" readily combines with H^ to yield hydrogen peroxide.

o
H
H2O2 is a much weaker acid than is HO2. At physiological pH values, the species
present will be mainly H2O2 and Oi~ respectively.

ACKNOWLEDGMENTS

I am grateful to Dr P. C. Jocclyn for information about glutathione peroxidase and to

Mr J. A. Smith for bibliographic help.

REFERENCES
Ai.LKN, J. F. & HALL, D . O . (1973). Superoxide reduction as a mechanism of ascorbate-stimulated oxygen
uptake by isolated chloroplasts. Biochem. Biophys. Res. Commun., 5a, 856.
AsADA, K. & Kiso, K. (1973a). The photo-oxidation of epinephrine by spinach chloroplasts and its inhibi-
tion by superoxide dismutase: evidence for the formation of superoxide radicals in chloroplasts. Agr.
biol. Chem., 37, 453.
AsADA, K. & KISO, K . (1973b). Initiation of aerobic oxidation of sulphite by illuminated spinach chloro-
plasts. Eur.jf. Biochem., 33, 253.
AsADA, K, URANO, M . & TAKAHASHI, M . (1973). Subcellular location of superoxide dismutase in spinach
leaves and preparation and properties of crystalline spinach superoxide dismutase. Eur. Jf. Biochem.,
35i 257-
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
1084 B. HALLIWELL
BABIOR, B . M . , KIPNES, R. S. & CURNUTTE, J. T. (1973). Biological defence mechanisms. The production
by leucocytes of superoxide, a potential bactericidal agent. J. din. Invest., 52, 741.
BEAUCHAMP, C . O . & FRIDOVICH, I. (1973). Isoenzymes of superoxide dismutase from wheatgerm. Biochim.
biophys. Acta, 317, 50.
BovERis, A., OSHINO, N . & CHANCE, B . (1972). The cellular production of H2O2. Biochem. J., ia8, 617.
CARTER, E. A. & ISSEI.BACHER, K . J. (1971). The role of microsomes in the hepatic metabolism of ethanol.
Ann. N.Y. Acad. Sci., 179, 282.
CARTER, E. A. & ISSFXBACHHH, K . J. (1972). Hepatic microsomal ethanol oxidation. Mechanism and physio-
logic significance. Lab. Invest., 27, 283.
CHANCE, B . (1950). The reactions of catalase in the presence of the notatin system. Biochem. J., 46, 387.
CHANCE, B . (1952a). Effect of pi I upon the reaction kinetics of the enzyme-.substrate compounds of
catalase. X biol. Chem., 194, 471.
CHANCE, B. (ig52b). The effect of pH upon the equilibria of catalase compounds. J. biol. Chem., 194, 483.
CHANCE, B. (1952c). The state of catalase in the respiring bacterial cell. Science, 116, 202.
CHRLSTOPHI'RSEN, B. O . (1966). Oxidation of reduced glutathione by subcellular fractions of rat liver.
Biochem. J., 100, 95.
CHRISTOPHERSKN, B . O . (1968). Formation of monohydroxy-polyenoic fatty acids from lipid peroxides by
a glutathione peroxidase. Biochim. biophys. Acta, 164, 35.
CHRISTOPHERSEN, B . O . (1969). Reduction of Iinolenic acid hydroperoxide by a glutathione peroxidase.
Biochim. biophys. Acta, 176, 463.
COHEN, G . & HOCHSTEIN, P. (1963). Glutathione peroxidase as the primary agent for the elimination of
H2O2 in erythrocytes. Biochemistry, 2, 1420.
DuVE, C. DE & BAUDHUIN, P . (1966). Peroxisomes—microbodies and related particles. Physiol. Rev., 46,
323.
ELSTNKR, E . F . & HEUPEL, A. (1973). On the decarboxylation of a-keto acids by isolated chloroplasts.
Biochim. biophys. Acta, 325, 182.
ELSTNER, E . F . & KRAMER, R. (1973). Role of the superoxide free radical ion in photosynthetic ascorbate
oxidation and ascorbate-mediated photophosphorylation. Biochim. biophys. Acta, 314, 340.
EPEL, B . L . & NEUMANN, J. (1973). The mechanism of the oxidation of ascorbate and Mn^* by chloro-
plasts. The role of the radical superoxide. Biochim. biophys. Acta, 325, 520.
FARRINGTON, J. A., F.RERT, M . , LAND, E. J. & FLETCHER, K . (1973). Pulse radiolysis studies of the reaction
of paraquat radical with oxygen. Implication for the mode of action of bipyridyl herbicides. Biochim.
biophys. Acta, 314, 372.
FEE, J. A . & TEITKLBAUM, H . D . (1972). Evidence that superoxide dismutase plays a role in protecting red
blood cells against pcroxidative haemolysis. Biochem. biophys. lies. Commun., 49, 150.
FiHLDEN, E. M., ROBERTS, P. B., BRAY, R. C . & ROTILIO, G . (1973). Mechanism and inactivation of super-
oxide dismutase activity. Biochem. Soc. Trans., i, 52.
FLOHE, L . , GUNZLER, W . A. & SCHOCK, H . H . (1973). Glutathione peroxidase—a seleno-enzyme. FEBS
left., 3a, 132.
FONG, K . , MCCAY, P. B. POYER, J. L. KEELE, B . B . & MISRA, II. (1973). Evidence that peroxidationof ly-
sosomal membranes is initiated by hydroxyl free radicals produced during flavin enzyme activity. J.
Biol. Chem. 248, 7792.
FRIDOVICH, I. (1973). Superoxide radical and superoxide dismutase. Biochem. Soc. Trans., i, 48.
GILLETTE, J. R., BHODIE, B. B. & LA DU, B. N . (1957). The oxidation of drugs by liver microsomes: on
the role of TPNH and Oj. J. Pharmacol. Exp. Ther., 119, 532.
GODA, K . , CHU, J., KIMURA, T . & .SCHAAP, A. P. (1973)- Cytochrome c enhancement of singlet molecular
oxygen production by the NADPH-dependent adrenodoxin reductase-adrenodoxin system. The role
of singlet oxygen in damaging adrenal mitochondrial membranes. Biochem. biophys. Res. Commun.,
5a, 1300.
GREEN, R. C . & O'BRIEN, P. J. (1970). The cellular localization of glutathione peroxidase and its release
from mitochondria during swelling. Biochim. biophys Acta, 197, 31.
GREGORY, E. M . & FRIDOVICH, I. (1973a). Inductionof superoxide dismutase by molecular O j . ^ . Bacteriol,
" 4 . 543.
GREGORY, E. M . & FRIDOVICH, I. (1973b). Oxygen toxicity and the superoxide dismutase. J'. Bacteriol.,
114, 1193.
GREGORY, E . M . & FRIDOVICH, I. (1974). Oj metabolism in Lactobacillus plantartim. J. Bacteriol., 117,
166.
GREGORY, E. M . , YOST, F . J. & FRIDOVICH, I. (1973). Superoxide dismutases of E. coli.: intracellular
localization and functions. X BiicterioL, 115, 987.
HALLIWELL, B. (1973). The role of formate in photorespiration. Biochem. Soc. Trans., 1, 1147.
HALLIWELL, B . (1974a). Marker enzymes of plant cell organelles. In: Methodological Developments in
Biochemistry, vol. 4 (Ed. by E. Reid), in the pre.ss.
HALLIWELL, B. (iq74b). Oxidation of formate by peroxisomes and mitochondria from spinach leaves.
Biochem. y., 138, 77.
HALLIWELL, B . & BUTT, V. S. (1974). Oxidative decarboxylation of glycollate and glyoxylate by leaf
peroxisomes. Biochem. J., 138, 217.
HEIKKILA, R. E . & COHEN, G . (1973). 6-hydroxydopamine. Evidence for superoxide radical as an oxidative
intermediate. Science, i8r, 456.
HOCHSTEIN, P. & UTLEY, H . (1968). Roles of glutathione peroxidase and catalase in liver. Mol. Pharmacol.,
4. .S74-
HOLMES, R . S . & MASTERS, C . J. (1972). Species-specific features of the distribution and multiplicity of
mammalian liver catalase. Arch. Biochem. Biophys., 148, 217.
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Solutions to problems of living with oxygen 1085
IYER, G . Y. N . , ISLAM, D . M . F . & QUASTEL, J. H. (1961). Biochemical aspects of phagocytosis. Nature,
Lond., 193, 535.
JoNKS, P. & SUGOETT, A. (1968). The catalase-HjOj system. Kinetics of catalatic action at high substrate
concentrations. Biochem. y., n o , 617.
KEILIN, D . & HAHTRER, E . F . (1945). Properties of catalase. Catalysis of coupled oxidation of alcohols.
Biochem. y., 39, 293.
KHAN, A. U. (1970). Singlet molecular oxygen from superoxide anion and sensitized fluorescence of
organic molecules. Science, 168, 476.
KLEBANOFF, S . J . (1968). lVIyeloperoxidase-halide-H2O2 antibacterial system. X BacterioL, 95, 2131.
KLEBANOFF, S . J. (1970). Myeloperoxidase: contribution to the microbicidal activity of intact leucocytes.
Science, 169, 1095.
KNOWLES, P . F . , GIBSON, J. F., PICK, F . M . & BRAY, R . C . (1969). Klectron-spin-resonance evidence for
enzymic reduction of Oi to a free radical, the superoxide ion. Biochem. y., i i i , 53.
LAVALLE, F., MICHELSON, A. M. & DIMITRIJEVIC, L . (1973). Biological protection by superoxide dismutase.
Biochem. Biophys. Res. Commun., 55, 350.
LEEK, A. E., HALLIWELL, B . & BUTT, V. S. (1972). Oxidation of formate and oxalate in peroxisomal
preparations from leaves of spinach-beet. Bioehim. hiophys. Acta, 286, 299.
LiEBER, C. S. & DE CARLI, L . M . (1970). NADPH oxidase: activity enhanced by ethanol consumption.
Science, 170, 78.
LITTLE, C . & O'BRIEN, P. J. (1968). An intracellular glutathione peroxidase with a lipid peroxide substrate.
Biochem. Biophys. Res. Commun., 31, 145.
LuMSDEN, J. & HALL, D . O . (1974). Soluble and membrane-bound superoxide dismutases in a blue-green
alga {Sprulina) and spinach Biochem. Biophys. Res. Commtm., 58, 35.
MAINFERME, F . , WATTIAUX-DE-CONINCK, S. & WATTIAUX, R. (1972). Comportcmcnt de la catalase du foie
de rat lors d'une <-lectrophor&se en gradient de pH (^lectrofocalisation) realis^e sur des cxtraits de
difftSrentes fractions subcellulaires. Arch. Int. PhysioL Bioehim., 80, 609.
MANNERINO, G . J . . VAN HARKEN, D . R . , MAKAR, A. B., TEPHLY, T . R . , WATKINS, W . D . & GOODMAN, J. I.
(1969). Role of the intracellular distribution of hepatic catalase in the peroxidative oxidation of
methanol. Ann. N. Y. Acad. Sci., 168, 265.
MEHLER, A . H . (1951a). Studies on reactions of illuminated chloroplasts. Mechanism of the reduction of
O2 and other Hill reagents. Arch. Biochem. Biophys., 33, 65.
MEHI.FR, A. H . (1951b). Studies on reactions of illuminated chloroplasts. Stimulation and inhibition of the
reaction with molecular Oj. Arch. Biochem. Biophys., 34, 339.
MEHLER, A. H. & BROWN, A. H. (1952). Studies on reactions of illuminated chloroplasts. Simultaneous
photoproduction and consumption of O2 studied with oxygen isotopes. Arch. Biochem. Biophys., 38,
36.S.
NlcnoLl.s, p. (1972). Contributions of catalase and glutathione peroxidase to red cell peroxide removal.
Bioehim. biophys. Acta, 279, 306.
ORO, J. & RArpoPORT, D. A: (1959). Formate metabolism by animal tissues. The mechanism of formate
oxidation. J . Biol. Chem., 234, 1661.
OsHiNO, N., CHANCE, B., SIES, H . & BUCHER, T . (1973a). The role of H2O2 generation in perfused rat
liver and the reaction of catalase compound I and hydrogen donors. Arch. Biochem. Biophys., 154,
OsHiNo, N., OsHiNO, R. & CHANCE, B. (1973b). The characteristics of the peroxidatic reaction of catalase
in ethanol oxidation. Biochem. J., 131, 555.
PASCHEN, W . & WESER, U . (1973). Singlet oxygen decontaminating activity of erythrocuprem (superoxide
dismutase). Bioehim. biophys. Acta, 327, 217.
PATTER.SON, C. O. P. & MYERS, J. (1973). Photosynthetic production of H^Oj by Anacystts nidulans.
Plant PhysioL, 51, 104. • 1 j ,
PEDER.SON, T. C. & AtJST, S. D. (1972). NADPH-dependent lipid peroxidation catalysed by purified
NADPH-cytochrome c reductase from rat liver microsomes. Biochem. Biophys. Res. Commun., ^9, 789.
PEDERSON, T . C . & AUST, S. D . (1973). The role of superoxide and singlet oxygen in lipid peroxidation
promoted by xanthine oxidase. Biochem. Biophys. lies. Commun., S^i io7i-
PlERfOiNT, W. S. (1969). o-Quinones formed in plant extracts. Their reactions with bovine serum albumin.
Biochem. y., 112,619. , • --,
RAVEN, J. A. (1972). I'^ndogenous inorganic carbon sources in plant photosynthesis. Companson of total
CO2 production in the light with measured COj evolution in the light. Netv PhytoL, 71, 995.
RoTiLto, G., CALABRESE, L . , FJNAZZI-AORO, A . . ARGENTO-CERU, M . P., AUTORI, F. & MONDOVI, B . (1973).
Intracellular location of superoxide dismutase and its relation to the distribution and mechanism of
HjOj.producing enzymes. Bioehim. biophvs. Acta, 321, 98.
RoTRucK, J. T., PoPF., A. L., GANTHER, H . IC., SWANSON, A. B., H.\FEMAN, D . G . & HOF.KSTRA. W . G .
(1973). Selenium: biological role as a component of glutathione peroxidase. Science, 179, 588.
ScANiiAi.ios, J. G., LIU, F . H . & CAMPEAU, M . A. (1972). The effects of intragenic and intergenic comple-
mentation on cataliise structure and function in maize: a molecular approach to heterosis. Arch.
Biochem. Biophys., 153, 695. • • • . , ,
SHAIN, Y. & Gimis, M. (1971). Formation of glycollate by a reconstituted spinach chloroplast preparation.
Plant PhysioL, 48, 325.
SIES, H . , BtirHEn, T., Osumo, N. & CHANCE, B . (1973). Haem occupancy of catalase in haemoglobin-free
perfused rat liver and of isolated rat liver catalase. Arch. Biochem. Biophys., 154, 106.
SIES, H . , GKRSTENECKER, C , MF,NZEL, H . & FLOHE, L . (1972). Oxidation in the NADP system and release
of GSSG from haemoglobin-free perfused rat liver during peroxidatie oxidation of glutathione by
hydroperoxides. FEBS lett., 27, 171-
 14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
io86 B. HALLIWELL
SMITH, P. J., TAPPEL, A. L. & CHOW, C. K . (1974). Glutathione peroxidase activity as a function of dietary
selenomethionine. Nature, Lond., 247, 392.
THURMAN, R. G . , LEY, H . G . & SCHOLZ, R. (1972). Hepatic microsomal ethanol oxidation. H2O2 formation
and the role of catalase. Eur. J. Biochem., 25, 420.
ToLBERT, N. E. (1971). Microbodies-peroxisomes and glyoxysomes. Annu. Rev. Plant PhysioL, 22, 45.
ToLBERT, N. E. (1973). Glycollate biosynthesis. In: Current Topics in Cellular Regulation (Ed. by B. L.
Horecker & E. R. Stadtman), 7, 21-50.
ToLBERT, N. E., YAMAZAKI, R. K . & OESER, A. (1970). Localization and properties of hydroxypyruvate
and glyoxylate reductases in spinach leaf particles. J. hiol. Chem., 245, 5129.
ViDELA, L., BERNSTEIN, J. & ISRAEL, Y. (1973). Metabolic alterations produced in the liver by chronic
ethanol administration. Increased oxidative capacity. Biochem. Jf., 134, 507.
WEISIGER, R. A. & FRIDOVICH, I. (1973). Mitochondrial superoxide dismutase. Site of synthesis and intra-
mitochondrial localization. _7. hiol. Chem., 248, 4793.
WHITEHOUSE, D . G . , LUDWIG, L . J. & WALKER, D . A. (1971). Participation of the Mehler reaction and
catalase in the O2 exchange of chloroplast preparations. Jf. exp. Bot., 22, 772.
ZKLITCH, I. (1971). Photosynthesis, Photorespiration and Plant Productivity. Academic Press, New York.
ZELITCH, I. (1972a). Comparison of the effectiveness of glycollic acid and glycine as substrates for photo-
respiration. plant PhysioL, 50, 109.
ZELITCH, I. (1972b). The photo-oxidation of glyoxylate by envelope-free spinach chloroplasts and its
relation to photorespiration. Arch. Biochem. Biophys., 150, 698.
ZELITCH, I. & GOTTO, A. M. (1962). Properties of a new glyoxylate reductase from spinach leaves. Biochem.
.7-, 84, 541-
ZiMMERMANN, R., FLOHE, L . , WESER, U . & HARTMANN, H . J. (1973). Inhibition of lipid peroxidation in
isolated inner membrane of rat liver mitochondria by superoxide dismutase. FEBS lett., 29, 117.
14698137, 1974, 6, Downloaded from https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.1974.tb02137.x by Indian Institute Of Technology Guwahati, Wiley Online Library on [17/08/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License