Oxygen may be considered one of the most important element in chemistry. Not counting hydrocarbons, there
is a greater diversity of molecules with oxygen than with carbon. Given its role in the molecular world, very little time
is spend on the chemistry of oxygen in undergraduate chemistry classes. Why is oxygen so special?
Oxygen reacts with atoms of all elements except the Noble gases to form molecules. One of the most important
from a biological sense is water. It :
- provides a perfect solvent for biomolecules
- moderates the earths climate
- is the source of almost all the dioxygen in the air.
- absorbs UV light and allowed life to first develop in the sea
From a chemical point of view, water is a(n):
- nucleophile and electrophile
- acid and base
- oxidizing agent and reducing agent
- a protic solvent that can form H-bonds
The formation of earth and the development of life:
The gaseous and dusty environment from which earth formed contained metals and water, which as you remember
from General Chemistry, can react to form hydrogen gas. H2 reacts with nonmetals (under various conditions of temperature
and pressure) to form H2S, HCl, CH4, and NH3 which contributed to the reducing nature of
the early atmosphere. This kept the transition metals in their lowest oxidation states. Many metals, including the coinage
metals (Cu, Ag, and Au) and the platinum group (Ru, Rh, Pd, Pt) were stable in elemental form.
Then, 2.5 billion years ago, photosynthetic organisms (blue/green algae) developed which could oxidize
water to form dioxygen. Atmospheric dioxygen rose to about 1%. ( Before this time, bacteria oxidized a similar
molecule, H2S to form elemental sulfur.) However, it wasn't until about 1.7 billion years ago that dioxygen was
found in the air in large concentrations (~20%). Why? Because the early atmosphere was reducing, the added oxygen combined
with a large "sink" of reduced metals (like elemental Cu and Fe) or nonmetals (like C and ammonia), preventing a large buildup.
Only after these reduced substances were "titrated" did dioxygen build up to present levels.
Until then, life was restricted to the oceans since there was no ozone to absorb dangerous UV radiation.
The very quick buildup of dioxygen in the air must have led to a great extinction of anaerobic organisms, since as we shall
see, products of oxygen metabolism are very toxic. Some evolved to use dioxygen. Ozone developed, and life could then migrate
from the sea to the land.
This study guide will show that dioxygen is a ground state diradical that has low solubity in aqueuous
solution, reacts in a kinetically sluggish fashion in oxidation reaction, and forms toxic byproducts as it gets reduced. Life
forms hence evolved ways to deal with these problems, including ways to increase its solubility (with dioxygen binding and
transport proteins), and enzymes (that could activate it kinetically and also detoxify oxygen by-products). Dioxygen is toxic
to many cells. Obligate aerobes die in an oxygen environment as many of their cellular components get oxidized by this excellent
oxidizing agent. Several strains of bacteria actually swim away from high levels of dioxygen. A graph showing log of survival
vs log pO2 is linear with a negative slope for a variety of organisms, including mice, fish, rats, rabbits, and
insects. Pure oxygen can induce chest soreness, coughs, and sore throats in people. Premature infants put in pure dioxygen
environments often developed blindness due to retrolental fibroplasia (a build-up fo fibrous tissue behind the lens).
The Properties of Dioxygen
It is important to understand the properties of dioxygen since oxidation reactions using it power not
only our bodies but our entire civilization. We will obviously concentrate on biological reactions, but even these show the
same characterisitics as non-biological ones.
- oxidation of organic molecules by oxygen is thermodynamically favored but kinetically slow.
- pure oxygen environments are toxic to cells and organisms.
First we will try to understand these properties of oxygen, and then we will see how organisms overcome
these problem to use dioxygen.
We can understand both of these properties by looking at the molecular orbitals of oxygen and its reduction
products as shown in the diagrams below. Ground state oxygen is a diradical, which explains the paramagnetic behavior of oxygen.
The two unpaired oxygens each have a spin state of 1/2 for a total resultant spin S of 1, making ground state oxygen a triplet
(2S+1) = 3. Organic molecules typically undergo 2 electron oxidation steps. Consider the stepwise oxidation of methane
below. The oxidation number of C in methane is -4, -2 in methanol, 0 in formaldehyde, +2 in formic acid, and finally
+4 in carbon dioxide, indicating two electron losses in each step.
The two electrons lost by the organic substrate are added to oxygen, but since the two lost electrons
are spin paired, a spin flip must occur to allow the electrons to enter the unfilled oxygen orbitals. Alternatively, energy
can be put into ground state dioxygen to produce excited state singlet oxygen (S=0, 2S+1 = 1). The source of the large activation
energy required (about 25 kcal/mol) to flip the electron spin accounts for the kinetic sluggishness of reactions of dioxygen
with organic reactants.
A traditional Lewis structure for ground state dioxygen can not be easily written since the electrons
are added in pairs, and dioxygen is a diradical. There are 6 electrons in the sigma molecular orbitals from second shell electrons
(two each in s2s, s2s*,
and s2p,) and 6 electrons in the pi molecular orbitals from second
shell electrons (two each in two different p2p orbitals, and one
electron each in two different p2p*), so the net number
of electrons in bonding orbitals is 4, giving a bond order (or number of 2). In contrast it is
easy to write the Lewis structure of singlet, excited state oxygen, since all electrons can be viewed as paired, with two
net bonds (1 sigma, 1 pi) connecting the atoms of oxygen. This Lewis structure will be used to represent single, excited
oxygen, which should react more quickly with organic molecules.
The Reductions of Dioxygen
When oxygen oxidizes organic molecules, it itself is reduced. By adding electrons one at a time to the
molecular orbitals of ground state dioxygen we produce the step-wise reduction products of oxygen. On the addition of one
electron, superoxide is formed. A second electron produces peroxide. Two more produces 2 separated oxides since no bonds connect
the atoms (the number of electrons in antibonding and bonding orbitals are identical). Each of these species can react with
protons to produce species such as HO2, H2O2 (hydrogen peroxide) and H2O. It is
the first two reactive reduction products of dioxygen that make it potentially toxic.
How are the potential problems in oxygen chemistry dealt with biologically?
Kinetic sluggishness: Enzymes that utilize dioxgen must activate it in some way, which decreases
the activation energy. Enzymes that use dioxygen typically are metalloenzymes, and often heme-containing proteins. Since metals
such as Fe2+ and Cu2+ are themselves free radicals (i.e. they have unpaired electons), they react readily
with ground state oxygen which itself is a radical. The molecular orbitals of the metal and oxygen combine to produce new
orbitals which for oxygen are more singlet-like in nature. Likewise, dioxygen reacts more readily with organic molecules which
can themselves form reasonably stable free radicals, such as flavin adenine dinucleotide (FAD), as we shall see later.
Dioxygen toxicity: Since toxicity arises from the reduction products of oxygen, enzymes that use
oxygen have evolved to bind oxygen and its reduction products tightly (through metal-oxygen bonds) so they are not released
into the cells where they can cause damage. In addition, enzymes which detoxify free dioxygen reduction products are widely
found in nature. For example:
- superoxide dismutase catalyzes the dismutation (self-redox) of 2 superoxides into dioxygen and
- catalase converts hydrogen peroxide into water and oxygen;
- peroxidase catalyzes the reaction of hydrogen peroxide with an alcohol to form water and
- peroxiredoxins react with peroxides and thioredoxin (a small electron donor) to form water and
oxidized thioredoxin .
Finally free radical scavengers such as vitamins A, C, E, and selenium can react with reactive free radicals
to produce more stable free radical derivatives of the vitamins and Se. More on this later.
The Reactions of Dioxygen and its Reduction Products
1. Triplet O2 - Ground State:
Figure: Triplet O2 - Ground State
- Metals ions - Metal ions are radicals themselves, so can react with dioxygen. Ex:
+ O2 <--> [ Fe2+-- O2 <--> Fe3+-- O2-.] <-->
Fe3+ + O2-. (superoxide)
- Autoxidation of organic molecules to produce peroxides. In this free radical reaction, several reactions
RH ---> R. (Initiation)
R. + O2 --->
ROO. + RH ---> R. + ROOH
R. + R. ---> R--R (Termination)
ROO. + ROO.
---> ROOR + O2 (Termination)
ROO. + R. ---> ROOR (Termination)
initiation step above occurs mostly at C atoms which can produce the most stable free radicals (allylic, benzylic position,
and 3o > 2o >> 10 carbons). Hence unsaturated fatty acids are extra reactive at
the methylene C that separate the double bonds.
Figure: unsaturated fatty acids are extra reactive at the methylene C that separate the double bonds
2. Single O2 - Excited State.
Figure: Single O2 - Excited State
It can be made from triplet oxygyen by photoexcitation. Alternatively, it can be made from triplet
oxygen through collision with an excited molecule which relaxs to the ground state after a radiationless transfer of energy
to triplet oxygen to form reactive singlet oxygen. (This later process accounts for photobleaching of colored clothes when
the conjugated dye molecules absorb UV and Vis light, relax by transferring energy to triplet oxygen to form singlet oxygen,
which then chemically reacts with the conjugated double bonds in the dye. )
- Alkenes react with oxygen to form hydroperoxides, potentially through a epoxide intermediate
- Dienes reacts with oxygen in a Diels-Alder like reaction to form endoperoxides
- Dismuation: O2-. + O2-. ---> H2O2
- Acid/Base: HO2. ----> O2-. + H+
(pKa = 4.8)
- With metal ions: Fe3+ (as in heme) + O2-. ---> O 2
In contrast to dioxygen which contains multiple bonds between the O atoms, peroxide has only one bond.
In fact, it is quite weak and requires only 38 kcal/mol to break it. Remember, bonds can be broken in a heterolytic
way (both electrons in a bond go to one of the atoms, or in a homolytic fashion, in which the one electron goes to each atom.
- Acid/Base: H2O2 ---> HO2- ---> O22-
(pKa1 = 11.8; pKa2 > 14)
- Reaction with Fe2+ - The Fenton Reaction: (similar to reaction of triplet O2 with
Fe2+ + OOH- <--> Fe2+-- OOH <--> Fe2+--
O + OH- <--> Fe3+-- O . <--> Fe3+ + OH. (last
step a proton is added). In this reaction, a homolytic cleavage of the O--O bond occurs generating
OH- and the hydroxy free radical, OH., which will react with any molecule it encounters.
- thermal or photochemical homolytic cleavage of peroxide. This forms alkoxide free radicals which
reacts like the hydroxy free radical.
- Reactions with alkyl groups in the presence of metal ions such as Cu, Co, or Mn:
RH + R'OOH ---->
5. Hydroxy free radical:
Figure: Hydroxy free radical
As mentioned above this species is extremely reactive. It will react with any molecule it encounters and
does so immediately. It can abstract a H atom leaving another free radical. For example, the hydroxy free radical could
extract a hydrogen atom from a polyunsaturated fatty acid to from a carbon-centered radical. A particularly nasty
reaction is the insertion of the hydroxy radical into bases in DNA, as shown in the diagram.
Dioxygen Reduction Products and Disease
Significant evidence suggests oxygen free radicals are linked to aging and diseases. Mutations
caused by hydroxylation reactions (presumably from the generation of hydroxyl free radicals as shown above) can potentially
lead to cancer. Recently it has been shown that mitochondrial DNA is more susceptible to oxidation that is nuclear DNA.
Human mitochondria has its own small genome (16.5 kb compared to the nuclear genome of 3 Gb)
which code 13 protein subunits involved in respiration, 22 tRNAs and two ribosomal RNAs. (The mitochondria presumably
are vestiges of a bacteria which invaded an early cell and established a symbiotic relationship with the cell). A recent
study has shown that there is an inverse correlation of oxidized mitochondrial DNA [8-oxoG] with maximal life span of an organism,
but this correlation is not seen with nuclear DNA. Presumably the nuclear DNA is somewhat protected from oxidative damage
since it is bound to histone proteins (which form nucleosome core particles with DNA) and by DNA repair enzymes, both of which
are missing from the mitochondria. Also, examination of human bladder, head and neck and lung primary tumors reveals
a high frequency of mitochondrial DNA mutations. In addition most dioxygen use by the cell occurs in the mitochondria.
Hence this organelle probably faces the highest concentration of toxic oxygen reduction products. Recently, the crystal
structure of an enzyme, adenine DNA glycosylase (MutY), that repairs 8-oxyG modified DNA has been determined in complex
with the oxidatively damaged DNA. If not repaired, the 8-oxyG base pairs with adenine instead of cytosine, causing a
GC to AT mutation on DNA replication.
Oxidative Modification of Proteins:
Figure: Oxidative Modification of Proteins
Oxidized levels of proteins (as evidenced by increased levels of aldehydes) increase dramatically with
age (especially after age 40 ). The reactions seem to be catalyzed by metals and may proceed by generation of hydroxy free
radicals. Diseases associated with premature aging (Werner's Syndrome, Progeria) show very high levels of oxidized proteins at an early age. Fibroblasts from 10 yr. old children with progeria have
levels of oxidized proteins usually not seen until the age of 70. Beta amyloid protein deposits (found in Alzheimer's and
Down's Syndrome) cause neurotoxicity and death, partly by causing increased superoxide production by endothelial cells,
causing vasoconstriction/dilation, and ultimately disease progression. Beta amyloid aggreagates appear to increase H2O2
levels, in a process facilitated by Fe2+ and Cu+. Free radical scavengers (antioxidants) help
to prevent this damage. Recent studies suggest that vitamin E may delay the symptoms of Alzheimers.
Lou Gehrigs Disease (Amyotrophic Lateral Schlerosis) is a disease of progressive motor neuron degeneration,
which affects 1/100,000 people, and is 10-15% familial. Of the familial cases, about 25% have a mutation in superoxide dismutase
I, a copper-zinc enzyme. About 2-3% of ALS patients carry 1 of 60 different dominant mutations in this enzyme.
Mutations often decrease the stability of the protein which decreases Zn2+ affinity 5-50 fold. The A4V mutation
(valine at amino acid 4 substituted for Ala) has the weakest Zn affinity and causes rapid disease progression. In the
absence of Zn2+, the apoprotein somehow seems to induce cell death in neurons. This superoxide dismutase
also expresses a second activity. It also acts as a peroxidase which takes ROH + H2O2 to form an RHO
(an aldehyde) plus water. In some cases, the enzyme retains normal activity against superoxide but altered peroxidase
Oxidative Modification of Lipids:
Figure: Oxidative Modification of Lipids
The initial stages of cardiovascular disease appear to involve the development of fatty acid streaks under
the artery walls. Macrophages, an immune cell, have receptors which appear to recognize oxidized lipoproteins in the blood,
which they take-up. The cells then become fat-containing foam cells which form the streaks. Fatty acids in lipoproteins
appear to be oxidized (possibly by ozone) to produce lipid peroxides which can lead to protein oxidation in lipoproteins.
Cortical neurons from fetal Down's Syndrome patients show 3-4 times levels of intracellular reactive O2
species and increased levels of lipid peroxidation compared to control neurons. This damage is prevented by treatment of the
neurons in culture with free radical scavengers or catalase.
Figure: oxidized LDL uptake
A recent study of peroxiredoxins by Neumann et al showed the importance of these gene products in mice. Peroxidredoxins
are small proteins with an active site cysteine and are found in most organisms. Transcription of the mammalian peroxiredoxin
1 gene is activated by oxidative stress. They inactivated the gene which produced a mouse that could reproduce and appeared
vital, but which had a shortened lifespan. These mice developed severe hemolytic anemia and several types of cancers.
High levels of reactive oxygen species and resulting increased levels of oxidized proteins were found in red blood cells of
the knockout mice with anemia. High levels of 8-oxoguanine, resulting from oxidative damage to DNA, were found in tumor
Oxidative damage to biomolecules might not be the events that initiates aging and disease processes ,
but rather is associated and correlated with these processes. Reversing or preventing oxidative damage might then slow
the progression of aging and disease. Aging is a complex feature of organisms and would be expected to have complex
causes and biological effects. At the organismal level, aging has been studied in the round worm C. elegans which lives
for only a few weeks. Genetic analyses can be easily used to find gene alterations associated with premature aging.
One hormonal system that has recently been associated with aging in eukaryotes (and in C. elegans) involves the signaling
pathways for insulin and insulin growth factor I (IGF-1), which regulate carbohydrate, lipid, and reproductive pathways in
C. elegans. Mutations that decrease signaling from this pathway increase C. elegans life span. These mutations
lead to increased activity of the DAF16 transciption factor, which upregulates the expression of many genes. In contrast, wild type organisms, when exposed
to insulin or IGF-1, decrease the activity of DAF16. Using DNA microarrays, investigators determined which DAF16-controlled
genes were upregulated in mutant worms in the mid-life point of the organism. These genes included, among others,
peroxisomal and cytosolic catalase, Mn-superoxide dismutase, cytochrome P450s, metallothionein-related Cd-binding protein,
and heat shock proteins. We will investigate the function of several of these gene products in the next section, but
needless to say, they are all involved in cellular responses to stress, often involving dioxygen metabolites.
Biological Uses for Reactive Oxygen Species - ROS
ROS can be used in the body as defense mechanisms. The can be generated by immune cells that phagocytize
(engulf) bacteria and other foreign substances. After uptake, these immune cells undergo an oxidative burst, increasing
the amount of dioxygen that they consume, leading to production of superoxide and hydrogen peroxide which can be used to kill
the engulfed bacteria. Recent articles by Wentworth et al. go even farther. They demonstrated that antibody
molecules (protein which bind to foreign molecules and target them for clearance or further immune response) can also generate
ROS when they bind to their target. Such an outcome was totally unexpected, but not inconsistent with the observation
that antibodies can have catalytic activity if made against transition state analogs of hydrolyzable substrates. Over
100 different antibodies were found to generate hydrogen peroxide through the reaction of singlet oxygen (possibly generated
by phagocytotic neutrophils during oxidative bursts) with water to form H2O2. (We have previously
discussed the idea that singlet O2 can be generated from ground state O2 by excitation through UV light
or through collisional activation with an excited state chromophore - a conjugated alkene or aromatic molecule). They
postulate the following reaction to form hydrogen peroxide. The oxygen in the peroxide comes from water, as shown by
labeling water with 18O:
2H2O + 1O2 (singlet oxygen)
--> H2O3 + H2O --> H2O2
Antibodies show this effect to a much greater extent that other proteins studied. Using UV irradiation
as a source of energy, the rate of production of H2O2
was linear with time, but decreased from the initial rate as H2O2
concentrations increased. If it was removed with catalase, the initial rate returned to normal, suggesting that
H2O2 was a product inhibitor of the reaction.
The reactions also saturated with dioxygen as well. The maximal wavelength of UV irradiation to produce H2O2 was identical to the maximal wavelength of absorbance for tryptophan
in the protein. The excited state tryptophan presumably deexcites by energy transfer to triplet dioxygen (3O2
) in much the manner described earlier for photobleaching. A significant source of electrons needed to be found to account
for the production of so much H2O2 which if
it was made from singlet oxygen would require 2 electrons for each H2O2.
These numbers of electrons could not come from photooxidation of aromatic amino acids in the protein. Hence they theorized
that water would add as a nucleophile to the singlet oxygen, providing the source of electrons required to make H2O2. This reactions would go through the H2O3 intermediate.
In another recent study (Nov. 02), the same team discovered that antibodies can not only produce H2O2 but also ozone. O3 is used in killing pathogens
but also might lead to inflammation. In their previous work described above, they generated singlet oxygen in a "nonphysiological"
way - through UV irradiation of the antibodies. They also didn't show that the ROS produced can kill bacteria without
any other immune mediators. They used a new singlet oxygen generating system which led to insufficient peroxide formation
to account for the extensive killing of E. Coli that they observed (95%). This led them to look for the generation of
a more potent oxidizing agent - which they believe is consistent with the formation of ozone. They also showed that
neutrophils were the source of the required singlet oxygen as described above.
In an additional study (Dec 02), the showed that antibodies in the presence of single oxygen and in the absence
of immune cells or other immune proteins (like complement proteins) can "destroy antigenic target". They also conform
the ozone is produced by antibodies during bacterial killing by neutrophils (which produce sinlget oxygen) and in inflammatory
states. Signaling through ozone might amplify other immune molecules that signal the activation (or hyperactivation) of the
Their latest study (Nov. 03) questioned the involvement of ozone (generated from antibodies in the presence
of singlet O2 from neutrophils) in the oxidation of lipoproteins, a process which has been associated with atherosclerosis
in coronary arteries. First they tested if ozone could be generated by atherosclerotic lesions treated with phorbol
myristate acetate. 14/15 plaques produced ozone, as evidenced by the photobleaching of the dye indigo carmen to isatin
sulfonic acid. This conversion, known to be specific for ozone, is shown below.
Figure: Conversion of indigo carmen to isatin sulfonic acid
They then looked for the telltale signature of the reaction of ozone with plaque cholesterol (cleavage of the
D5,6 double bond to form 5,6-secosterol as shown below and found it. This reaction is known
not to occur with other oxygen oxidants, including both triplet and singlet oxygen, superoxide, and the hydroxyl free
radical. A secondary reaction product aldol condensation product was also produced. They named these new products
Figure: Cholesterol/ozone reaction
Figure: Ozonolysis mechanism
Significant increases in secosterol was found in plaques treated with the phorbal ester. 6 of 8 patients
with atherosclerosis had elevated plasma levels of the secosterol, while only 1 of 15 of control patients did. They
also showed that these ozone products caused normal macrophages to fill with cholesterol (presumably from oxidized LDL), and
that the structure of the LDL apoprotein B100 changed. These findings might provide a link between the immune system
and cholesterol in the generation of cardivascular disease.
Just Say NO - The Chemistry of Nitric Oxide
In the last decade, the role of another gaseous free radical, nitric oxide (.NO) has become
apparent. This molecule is synthesized biologically through the action of an inducible heme enzyme, nitric oxide synthase,
which forms NO by the oxidation by dioxygen of the guanidino group of Arg, which gets converted to citrulline. .NO
is soluble and can diffuse through cell membranes into the cytoplasm, where it has a myriad of effects in signal transduction
pathways. However, it can also be metabolized to form reactive nitrogen intermediates (much as with dioxygen) which
can be deleterious to the body, if they damage native biomolecules, or advantageous, when they are used by immune cells like
macrophages in the destruction of engulfed bacteria.
A molecular orbital diagram of .NO shows it to have a bond order of 2.5 and one unpaired electron
in a p2p* antibonding orbital (hence the notation .NO). Here are some of
the relevant reactions of NO and its reactive nitrogen intermediates (RNI):
The high energy electron p2p*can leave on oxidation of NO to form the nitrosyl ion,
NO+. The bond order in NO+ is three suggesting strong interactions between the N and O atoms.
.NO (oxidation number of N of +2) is thermodynamically unstable and can disproportionate
or dismutate (like superoxide) in a self redox reaction to form nitrous oxide (N2O) and nitrogen dioxide
(.NO2), with oxidation number for N of +1 and +4, respectively.
In acidic condition .NO can be oxidized to nitrite (NO2-)
which can be protonated to form nitrous acid (HNO2).
Nitrous acid can disproportionate or dismutate to form .NO and the radical .NO2
which can be protonated to ONOOH with a pKa of 6.5.
.NO can react with superoxide (.O2-) faster than any
other molecule to form peroxynitrite, ONOO- .
.NO and superoxide (.O2-) are both produced by cells during
infllammation, leading to increased levels of peroxynitrite.
The O-OH bond in protonated peroxynitrite (ONO-OH) can be cleaved either heterolytically to produce NO3-
and H+ (70%) or homolytically to produce the radical .NO2 and the highly reactive
.OH free radical (30%).
- The deprotonated form of peroxynitrite (ONOO-) can oxidize organic molecules, especially thiols and CO2.
When it reacts with CO2, it can form either NO3- and CO2 (65%) or CO3.-
and .NO2 (35%). These latter products can oxidize organic molecules in one electron steps.
Peroxynitrite can also oxidize protein, DNA, and lipids.
- .NO2 and peroxynitrite can interact with Tyr side chains in proteins to form nitrated proteins
(Tyr-NO2), whose presence reflects inflammation.
Macrophages make use of RNIs in the killing of engulfed bacteria. How does one of humankinds greatest
foes, Mycobacteria tuberculosis, the causative agent of tuberculosis, avoid this killing mechanism? It is estimated
that the bacteria resides persistently in latent form in 2 billion people. If it becomes activated, it becomes one of
the greatest killers. Consider the following table:
Killer Diseases through time (Scientist, June 2, 2003)
|Justinian Plaque (6th centr.)
||142 million (on 70% mort)
||about 100 mill|
|China Plaque (Bubonic) 3rd Pandemic (1896-1930)
|Spanish flu 1918-19
People infected with the bacteria but without clinical symptoms must mount a sufficient enough immune response
to restrain proliferation of the bacteria, but not enough to clear it from the body. Immune-compromised people (transplant
recipients taking anti-rejection drugs or AIDS patients) have more difficulty in holding the bacteria at bay. One immune
response mediator which facilitates restrains bacterial growth is the soluble protein interferon g
(a protein cytokine released by immune cells). This proteins induces synthesis of nitric oxide synthase, producing .NO,
which through the reactions listed above, can damage bacterial macromolecules.
Bacteria are "digested" in acidic phagosomes of the macrophage. Nitrite formed in the acid conditions
from .NO (generated by inducible NO synthase) forms .NO2 and peroxynitrite which have antibacterial
properties (better than anti-Tb drugs). Mutant mice that can not synthesize inducible NO synthase have little defense
against the bacteria. Darwin e al. exposed the bacteria to sources of nitrite at pH conditions typical of macrophage
phagosomes and found mutants sensitive to the RNIs. The mutations involved protein of the bacterial proteasome, a complex
multi-protein complex which proteolyzes unwanted presumably damaged) cellular proteins.
Bacterial genes encoding proteins associated with the bacterial proteasome seem to confer resistant to the
effects of macrophage-inducted RNI production. The macrophage proteasome, like other eukaryotic proteasomes, is a cytoplasmic
protein complex which degrades damaged cytoplasmic proteins. Although the mechanism is uncertain, the bacterial proteasome
may rid the bacteria of nitrated or otherwise oxidized (damaged) proteins or remove the nitrate and facilitate refolding of
the damaged protein.
On Line Literature: Nitric Oxide Synthases: Structure, Function, and Inhibition
Antioxidant Protection From Oxidative Modification
If oxidative damage by dioxygen reduction products can cause disease, maybe antioxidant vitamins (E, C,
A), which can form reasonably stable free radical, can protect the body from their effects.
Figure: antioxidant vitamins (E, C, A)
It has been shown that these vitamins can help protect white blood cells from DNA damage arising from
hydroxy free radicals. Vitamin E, a fat-soluble vitamin carried in circulating lipoproteins, can reduce the risk of cardiovascular
disease, presumably by preventing oxidation of lipids and proteins in lipoproteins. Vitamin E and C, along with a common food
additive, butylated hydroxytoluene (BHT) can form stable free radicals (formed possibly by abstraction of a hydrogen atom
by hydroxyl free radicals) since the lone electron is stabilized by resonance and the the O-centered resonant form is sterically
restricted from intermolecular interactions which could propagate the free radical chain reactions.
How do cells respond to low O2 ?
We have spend much time studying how the body deals with and utilizes the toxic byproducts of dioxygen
reduction. What happens when the body doesn't get enough dioxygen - a condition call anoxia (no dioxygen) or hypoxia
(too little dioxygen)? This might occur under muscles undergoing vigorous exercise, and in the brain and heart when
clots occlude blood flow to these organs (as occurs in most strokes and heart attacks). Under low dioxygen concentrations,
a family of protein transcription factors called hypoxia-inducible factor (HIF) become activated. The functional
proteins appears to be a dimer of HIF-a and HIF-b. In contrast to the concentration of the beta form, the activity of HIF-a is increased under low dioxygen concentrations. HIF-a concentration
is regulated not at the transcriptional level, but through proteolysis of the protein. In the presence of abundant dioxygen,
HIF-a is hydroxylated at two Pro residues by the enzyme prolyl hydroxlase.
This post-translational modification targets the protein for proteolysis (through ubiquitination by a the VHL protein
and subsequent cleavage by the proteasome). An second independent pathway in rapidly growing tissue leads to increased
expression of HIF-a even in the presence of dioxygen. This could be
beneficial to cells since rapidly growing tissue, especially tumor cells, might be expected to experience low oxygen
Figure: Cell response to hypoxia