free radical is?
a species which contains an unpaired e- in its outer orbital
b/c e- want to pair up, free radicals are very reactive
charge of free radicals?
1. negative charged, i.e. superoxide anion radical
2. positively charged, i.e. methonium radical
3. neutral, i.e. glutathione radical
why is molecular oxygen important?
it is used in aerobic metabolism, which produces more ATP than anaerobic metabolism does, 32 vs. 2 for glucose oxidation
net of the e- respiratory chain?
how does this occur?
what is the result?
O2 is reduced by 4 e- to 2 molecules of water
small amounts of O2 can be reduced by 1 or 2 e- transfers to raective oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals
dangerous b/c these react w/ celllarmacromolecules and disrupt their function or structure
what diseases are ROS implicated in?
toxicity of oxygen, inflammation, sepsis, carcinogenesis, cardiovascular diseases/atherosclerosis (LDLs and foam cells), aging (free radical theory of aging: we rust w/ age), ischemia/stroke/infarction, reperfusion/transplantation injury, neurodegenerative diseases like Parkinson and Alzheimer, radiation injury, smoking injury xenobiotic and drug toxicity, alcohol toxicity, iron and asbestos toxicity, vitamin deficiency g vitamin E and C, mineral deficiency eg selenium, zing, manganese, cataracts & retinopathies, others
all b/c oxygen radicals react w/ macromolecules and disrupt their structure and/or function
what is oxidative stress?
imbalance btwn oxidants and antioxidants either b/c of increased production of ROS or decreased levels of antioxidants or both
ROS which can cause tissue damage, & whose levels may be increased by certain drugs, infection, external exposures, tissue injury
inhibit either formation of ROS or remove/scavenge the generated ROS
what are RNS?
reactive nitrogen species
derived from nitric oxide, which can be produced from arginine by nitric oxide synthases
can also cause tissue injury, like ROS
what is the state of molecular oxygen in the ground state?
O2 is a diradical
6 e- surrounding each of the O atoms that make up O2, so 1 unpaired e- on each oxygen atom and 2 unparied e- in outer orbital
although a radical, is unreactive b/c the unpaired e- are spinning in the same direction, and to make a covalent bond w/ another atom, they have to spin in opposite directions
what is required of unpaired e- for covalent bond formation?
unpaired e- must be spinning in opposite directions
what produces the singlet ground state of oxygen?
what can it do?
absorption of energy, eg UV light or radiation, by triplet oxygen
this inverts the spin of 1 of the e- to produce the very reactive singlet ground state of oxygen
singlet oxygen can insert into double bonds or aromatic amino acids of proteins (Phe, Tyr, Try), of unsautrated fatty acids, or purine and pyrimidine bases of DNA and RNA
what causes skin lesions?
sunlight-induced singlet oxygen formation
what blocks singlet oxygen formation?
B-carotene, a vitamin A derivative
it is a potent scavenger of singlet oxygen, and common ingredient in sunscreens & skin blister treatments
how is the superoxide anion radical formed?
what is its pKa?
how is it at physiological pH?
triplet oxygen is reduced by a single e-
has a negative charge, = anion
its pK = 4.8
it is unprotonanted at physiological pH
what forms the perhydroxyl radical?
if superoxide anion radical is protonated
what forms peroxide?
how is peroxide at physiological pH?
if 2 e- are added to triplet oxygen
done by enzymes, mostly peroxisomal enzymes like urate oxidase, glycolate oxidase, D-amino acid oxidase, fatty acyl oxidase
is protonated and in the form of H2O2, hydrogen peroxide, at physiological pH
is H2O2 a radical?
no - has no unpaired e-
however, it's a potent oxidizing agent, and therefore reactive and toxic
what is a dismutation rxn?
ex of it w/ superoxide radical?
dismutation: when 2 identical compounds interact, 1 becoming reduced & the other oxidized - 1 loses an e- and the other gains the e- lost by the first superoxide
occurs non-enzymatically or w/ enzymes w/ superoxide
what are SODs, superoxide dismutases?
why do they matter?
enzymes that can catalyze superoxide dismutation reaction
important antioxidant enzymes b/c they remove superoxide
what is the most powerful ROS? why?
can react w/ any biochemical or macromolecule
reacts and inactivates or disrupts proteins, lipids, DNA and RNA
what is the fenton reaction?
what must occur for it to continue?
explains dominant way that hydroxyl radical is produced in cells
H2O2 reacts w/ Ferric iron (Fe2+), which is reduced by superoide to Ferrous iron (Fe3+)
for it to continue, Fe3+ must be reduced back to Fe2+ which occurs by superoxide which is good at reducing metals:
Fe3+ + O2- radical -> Fe2+ + O2
what is the Haber-Weiss reaction?
what can catalyze it?
way that it was believed OH radical was produced, from direct reaction between hydroxyl radical and hydrogen peroxide
iron or copper can catalyze it, but iron's more plentiful and more reactive
how can toxicity associated w/ ROS be treated? think haber-weiss and fenton rxns
toxicity of ROS and its role in many diseases can be mitigated in part by removal or chelation of iron since they are involved in generated hydroxyl radicals
what biological systems produce ROS?
1. Mito respiratory chain: Complex I, III
2. Heme enzymes which react w/ molecular oxygen, like hemoglobin & myoglobin
3. Cytochrome P450 mixed function oxidase electron transport chain - CYP2E1, which oxidizes drugs & xenobiotics
4. Autooxidation, direct oxidation by O2, of cellular biochemicals like catecholamines (epinephrine, norepinephrine, dopamine), serotonin, flavins, tetrahydrobiopetrin
what enzymes produce ROS?
superoxide and H2O2 produced by:
cyclooxygenases - produce prostaglandins, leukotrines
what's a major source of ROS in the brain?
monamine oxidase uses O2 to oxidize important neurotransmitters like epinephrine, norepinephrine, dopamine, serotonin
this all produces H2O2
major source of ROS in most tissues?
mito electron transfer chain
produces superoxide from the oxidation of reduced flavin in Complex I and autooxidation of reduced ubisemiquinone
how does the autodoxidation of quinones form oxygen radicals?
Oxidized Q, Ubiquinone + 1 e- from either complex 1 or 2 forms Ubisemiqunone or Semiquinone radical
Semiquinone radical + 1 e- forms reduced Q or ubiquinol. This radical passes into Complex 3, reduces. OR can directly react w/ O2 to give Oxidized Q plus Superoxide
This regenerates Oxidized Q, which can again be reduced by 1 e- to Ubisemiquinone, and reaction can repeat, producing lots of superoxide
what inner source can form oxygen radical? (think enzymes)
in heme enzymes such as cytochrome P450s, hemoglobin, myoglobin, etc., Ferric iron (Fe3+) of heme is reduced to Ferrous iron (Fe2+)
Fe2+ can bind O2 to form heme ferrous-O2 complex
resonance structur for this is obtained by transferring just 1 e- from Fe2+ to O2, and producing Fe3+-O2-*
some of this decays to yield ferric heme enzyme and superoxide radical
external sources of ROS?
drugs and xenobiotics
in what ways are ROS toxic to proteins?
1) esp. hydroxyl radical oxidize SH group of cysteine residues of proteins to the disulfides or to sulfoxide (SO) or sulfonic acid (SOOH) - lose enzymatic activity dependent on cysteine, protein tertiary structure's disrupted
2) CH3S of methionine is readily oxidized to methionine sulfoxide, CH3SO, which interferes w/ methionine fxn like methylation reactions
3) Oxidize aromatic rings of Phe and Tyr or indole ring of Try, open up the rings, form protein carbonyls & cause loss of enzymatic activity and structure
4) disrupt peptide bonds, covalent and noncovalent interactions
how are ROS toxic to nucleic acids?
1) cleave phosphodiester bonds, break chain structure of RNA and DNA
2) cause base excision
3) oxidize bases eg dG -> 8OHdG - 8C position is v. sensitive
4) cause base mispairing mutations via deaminations from adenine or guanine to form hypoxanthine or xanthine, or remove amino group from cytosine to form uracil
what occurs to nucleic acid base pairing if ROS deaminate them?
H, hypoxanthine, pairs w/ G, so A:T base pair changes to H:G
this is major cause of mutations
what is lipid peroxidation, when and why does it occur
ROS oxidation of polyunsaturated fatty acids = lipid peroxidation
it disrupts normal membrane structure
rancidity of foods is due to lipi peroxidation as they age
steps of lipid peroxidation?
1) initiation: unsaturated fatty acids in lipid become lipid radical
2) propagation: lipid can be peroxidized by oxygen to make a lipid peroxyl radical; can extract a H from another lipid
3) degredation: if lipid radical takes on another H, it becomes unstable, v. reactive
4) termination: propagation steps eventually are halted by Vitamin E
what do lipid radicals formed during propagation step bind to?
amino groups of amino acids, purines and pyrimidines, and SH groups of cysteines
this inactivates enzymes and disrupts protein, RNA, and DNA structure
what does fragmentation of LOOH do?
destroys the phopholipid bilayer holding membranes together
is major reason for toxicity of ROS - i.e. mitochondria cannot generate a protein motor force, permeability of plasma membrane's altered, ligand interactiosn w/ receptors are lost, etc
what is vitamin E's role in lipid peroxidation?
reacts w/ lipid and lipid alkoxyl radicals to restore lipid and form a Vitamin E radical
Vitamin E then is regerated from E radical by ascorbic acid (Vitamin C)
when are ROS beneficial?
2) cell signaling
3) enzymatic reactions
how are ROS important re: signaling agents in biological systems?
H2O2 can catalyze"mild" oxidations which can activate or inactivate enzymes or transcription factors, including Hrf2, NF-kB, Ap-1
these are catalyzed by H2O2-dependent oxidation of Cysteine SH to Cystine Disulfied
These txn factors subsequently activate many genes, some of which code for cellular antioxidants
SO low levels of ROS can protect vs. High levels of ROS
what are phagocytes? how do they contribute to ROS?
a type of WBC
contain the enzyme NADPH oxidase which is activated when phagocytes come into contact w/ bacteria and other cells, ie tumor cells
NADPH oxidase catalyzes a respiratory burst b/c oxygen uptake is increased
what occurs in a respiratory burst?
rapid, large production of superoxide and other ROS
activates the PPP pathway to regenerate NADPH
what is myeloperoxidase? its action?
enzyme in phagocytes
catalzyues interaction of H2O2 w/ chloride anion, produces the powerful oxidant hypochlorite, HCLO
HCLO is active oxidant in bleach, destroys bacteria & other organisms
what causes chronic granulomatous disease?
if WBC phagocytes are deficient in some of the subunits which make up the NADPH oxidase which is crucial to a respiratory burst
if cannot mount a burst, cannot deal w/ invading organisms
this makes individuals very sensitive to infection
often is fatal b/c of sepsis
what does superoxide dismutase do?
catalyzes the removal of 2 moles of superoxide via a dismutation reaction - oxidizes 1 mole to O2 and 1 mole to H2O2
what superoxide dismutases do humans have?
1) copper/zinc SOD in the cytosol and space between the outer and inner mito membranes
2) a manganese SOD in the matrix of the mito
what kind of SOD do bacteria have?
what metals are considered antioxidants, why?
they are needed for SOD activity
effect of low amounts of ROS on SODs?
increases levels of SODs to protec against large levels of ROS
what does chemical inhibition of SOD or siRNA against SOD cause?
increases the sensitivity of cells to ROS
proves that SOD protect cell from ROS
function of catalase?
heme-containing enzyme that catalyzes removal of 2 moles of H2O2
has a high Km for H2O2 and very high Vmax
function of glutahion peroxidase?
in cytosol and mito
functions in conjunction w/ GSH, NADPH and glutathione reductase to get rid of ROS
what is GSH
most important antioxidant in cells
tripeptide made of glutamate + cysteine + glycine
glutamate and cystine linked by gamma carboxyl
contains selenium at its active site, so low amounts of selenium are important for antioxidant protection
family of enzymes found to have potent H2O2 removal activity
heme oxygenase, Vitamins E and C, Carotenoids, melatonin, uric acid, ...
how is iron usually in cells? result re: antioxidant action?
there's little free iron available in cells - most is stored in ferritin
iron stored in ferritin cannot catalyze Haber-Weiss or Fenton reactions
cellular repair enzymes' fxn re: oxidative stress?
some can repair or remove oxidized amcromolecules from the cell, such as DNA repair enzymes and proteasome complex
what foods have antioxidants?
plant extracts - flavonoids, polyphenols, catechinds, curcumin
how do ROS play a role in neurodegenerative diseases?
brain is a major target for ROS
Parkinson, Alzheimer, other nd diseases thought to have ROS involvement
1. brain is v enriched in polyunsaturated fatty acids and iron - perfect for LP
2. monoamine neurotransmitters are substrates for oxidation by mono amine oxidase, with formation of ROS, mainly H2O2
how does smoke cause oxidant stress
smoke has many oxidants
these inactive anti-proteases like tripsin inhibitor found in high amounts in lung tissue
this inactivation allows proteases to be highly active, cause damage to lung tissue
results in emphysema
how can drugs cause ROS-induced issues
drugs w/ a quinone-like structure can cause ROS-induced tissue injury b/c high levels of such drugs can easily undergo a 1 e- reduction by cellular reductases in the mito and microsomal e- transfer chains
this forms the semiquinone drug, which can react w/ oxygen to regenerate the drug and superoxide
this is redox-cycling results in ROS-induced tissue injury
what is adriamycin? action re: ROS?
one of most commonly prescribe anti-cancer agents
can only be used for short periods of time b/c it causes signfiicant heart injury due to its ability to redux cycle
what is free radical theory of aging
posits that our cells fill up w/ damaged oxidized molecules, which accumulate over a lifetime of metabolism and oxidation
some antioxidants may decrease w/ age
posits taking extra Vitamins E and C would help slow aging ramifications
what does increased metabolic rate cause in lower organisms
increased metabolic rate = increased oxygen uptake = lessens life span whereas decreased metabolic rate = decreased oxygen uptake = enhanced life span
what is ischemia
lack of oxygen
occurs in heart tissue during an MI or in brain tissues during storke b/c of prevention of blood flow
have immediate need to restore oxygen by moreval of the blockage
what is a reperfusion injury
when resumption of blood flow and delivery of oxygen to ischemic tissue causes tissue damage
due to an oxygen paradox by which ROS are produced during reperfusion
reasons for reperfusion injury?
1) during ischemia, mito etc is in a highly reduced state b/c of lack of oxygen; when reperfusion's initiated & oxygen's re-introduced, there's a burst of ROS from eg reduced ubisemiquinone, which may overwhelm tissue antioxidant defense
2) XDH normally oxidizes purine bases to uric acid for excretion, uses NAD+ for this oxidation and produces NADH; if XDH is converted to oxygenase form, XO, it uses oxygen instead of NAD to oxidize, making a superoxide product instead of NADH
why does a XDH conversion occur during ischemia
XDH -> XO
b/c certain Ca-activated proteases are increased, since calcium floos ischemic tissue due to lack of ATP production that prevents calcium from being pumped out of the cell
ATP also isn't made due to lack of oxygen, so it breaks down to hypoxanthine, and GTP breaks to xanthine
when reperfusion provides Oxygen, the tissue is flooded w/ a brust of ROS via the XO reaction
how can reperfusion or organ transplant occur w/o issue?
to avoid a reperfusion injury, load the blood or soak the organ in fluid w/ :
antioxidants, esp. GSH, and Vitamins E and C,
and chelators of Iron such as desferrioxamine,
and an inhibitor of Xo and XDH called allopurinol
what is a free radical?
why is it reactive?
an atom with an unpaired electron in its outer orbital. It’s reactive because of that unpaired e-, which wants to pair up.
it tends to make very destructive molecules in the process as well as messes up proteins
what is the state of molecular oxygen at ground state?
what would happen if added 1 e-?
Molecular oxygen is a diradical triplet in the ground state – it has 2 unpaired e-, but their spin is the same, so it’s unreactive.
In the singlet ground state, the spin of 1 of these e- on molecular oxygen is inverted, so it becomes very reactive. This is formed if 1 e- is added to the triplet state, forming the superoxide anion radical.
If another e- is added to this, the perhydroxyl radical forms.
If another e- is added, then it’s peroxide.
is ROS production always destructive?
no - protective sometimes
ie low levels of ROS initiate production of some antioxidants, so these can mount a larger response in the presence of even more ROS
Also a really important part of the immune system. Useful for destroying bacteria when engaged in a controlled fashion.
what do these metals do re: ROS? pro-oxidant or antioxidant?
Iron, Copper, Selenium, Zinc, Manganese
Iron and copper catalyze the formation of the OH radical.
Selenium is in the active site of GSH, so it’s an important antioxidant.
Zinc SOD is in the cytosol and space between the outer and inner mitochondrial membranes in humans – it’s an important antioxidant.
Manganese SOD is in the matrix of the mitochondria in humans – also thus an important antioxidant.
what are redox cycling agents re: drugs? what does this cause?
drugs w/ a quinone-like structure that can cause ROS-induced tissue injury because high levels of the drug can easily undergo a 1 e- reduction that makes them into the semiquinone-like structure, which can react with oxygen to regenerate the drug and superoxide.
This causes tissue injury.
eg: adriamycin and heart tissue injury
what is ROS impact on: nucleic acids, enzymes, lipids, proteins
ROS cause mutations by breaking purine rings and causing base mismatch (A:H instead of A:T).
ROS inhibit enzyme activity by inhibiting SOD, which are important in protecting cells from ROS.
The chain reaction of Lipid Peroxidation is how ROS oxidation of polyunsaturated fatty acids occurs – this causes membrane structure disruption, for example, which can be very dangerous for the cell.
Proteins: ROS oxidize cysteine’s SH groups, disrupting any enzyme dependent on cysteine.
what is ischemic reperfusion injury?
how can it be avoided?
when ischemic tissue is reperfused with oxygen and this causes ROS production
injury is minimizable by putting antioxidants in the blood used for transfusion, or the solution in which an organ for transplantation is soaked, to minimize the ROS production
This can occur via GSH, Vitamin E and C addition, chelators of Iron like desferrioxamine (since Iron is important in ROS formation), or an inhibitor of XO and XDH, allopurinol