REACTIVE OXYGEN

SPECIES

REDOKS & ROS

 • The Earth was originally anoxic
• Metabolism was anaerobic
• O2 started appearing ~2.5 x 109 years ago

Anaerobic metabolism-glycolysis
Glucose + 2ADP + 2Pi Lactate + 2ATP + 2H2O

O2 an electron acceptor in aerobic metabolism

Glucose + 6O2 + 36ADP + 36Pi 6CO2 + 36ATP + 6H2O
 Reactive Oxygen Species (ROS)
◼ Oksigen ada di mana saja dan terdistribusi dalam
berbagai organ
◼ Oxigen punya 2 elektron tidak berpasangan

: :
: :
. O:O .

◼ Di atom oksigen bersifat diradikal

◼ Oksigen yang menerima elektron tunggal → molekul
tak stabil → ROS
◼ ROS, free radikal: superoksid, hidroksil (sebuah atau
sekelompok atom yang mempunyai elektron tak
berpasangan)
◼ ROS, non radikal: H2O2, singlet oksigen
 Basics of Redox Chemistry
Term Definition

Oxidation Gain in oxygen

Loss of hydrogen
Loss of electrons

Reduction Loss of oxygen

Gain of hydrogen
Gain of electrons

Oxidant Oxidizes another chemical by taking

electrons, hydrogen, or by adding oxygen

Reductant Reduces another chemical by supplying

electrons, hydrogen, or by removing oxygen
 ROS
Free Radicals: R3C. Carbon-centered
▪ Any species capable of independent
R3N. Nitrogen-centered
existence that contains one or more
unpaired electrons R-O. Oxygen-centered
▪ A molecule with an unpaired electron
in an outer valence shell R-S. Sulfur-centered

H2O2 Hydrogen peroxide

Non-Radicals:
▪ Species that have strong oxidizing
HOCl- Hypochlorous acid
potential O3 Ozone
▪ Species that favor the formation of
1O Singlet oxygen
strong oxidants (e.g., transition 2
metals) ONOO- Peroxynitrite
Men+ Transition metals
 Reactive Oxygen Species (ROS)
Radicals: Non-Radicals:
O2.- Superoxide H2O2 Hydrogen peroxide
OH. Hydroxyl HOCl- Hypochlorous acid
O3 Ozone
RO2. Peroxyl
1O Singlet oxygen
2
RO. Alkoxyl
ONOO- Peroxynitrite
HO2. Hydroperoxyl

Reactive Nitrogen Species (RNS)

Non-Radicals:
ONOO- Peroxynitrite
ROONO Alkyl peroxynitrites
Radicals: N2O3 Dinitrogen trioxide
N2O4 Dinitrogen tetroxide
NO. Nitric Oxide HNO2 Nitrous acid
NO2. Nitrogen dioxide NO2+ Nitronium anion
NO- Nitroxyl anion
NO+ Nitrosyl cation
NO2Cl Nitryl chloride
 “Longevity” of reactive species
Reactive Species Half-life

Hydrogen peroxide
Organic hydroperoxides ~ minutes
Hypohalous acids

Peroxyl radicals ~ seconds

Nitric oxide

Peroxynitrite ~ milliseconds

Superoxide anion
Singlet oxygen ~ microsecond
Alcoxyl radicals

Hydroxyl radical ~ nanosecond

 Radical-mediated reactions
Addition
R. + H2C=CH2 R-CH2-CH2.

Hydrogen abstraction
R. + LH RH + L.

Electron abstraction
R. + ArNH2 R- + ArNH2.+

Termination
R. + Y. R-Y

Disproportionation
CH3CH2. + CH3CH2. CH3CH3 + CH2=CH2
 ROS
◼ Reaksi H2O2 + Fe2+ (logam transisi lain) →
membentuk radikal hidroksil
◼ Radikal hidroksil menginisiasi reaksi rantai radikal yg
bersifat autokatalitik
◼ Singlet oksigen: terbentuk bila dioksigen
mengabsorbsi energi yg cukup utk memindahkan
satu elektronnya ke orbital yang lebih tinggi
◼ ROS pada sel fagosit: menghancurkan dinding sel
bakteri agar dapat di degradasi oleh enzym lysosom
 Hydroxyl radical (.OH)

O2.- + Fe3+ O2 + Fe2+ (ferrous)

Fenton H2O2 + Fe2+ OH- + .OH + Fe3+ (ferric)
Haber-Weiss O2.- + H2O2 OH- + O2 + .OH

•Transition metal catalyzed

•Other reductants can make Fe2+ (e.g., GSH, ascorbate, hydroquinones)
•Fe2+ is an extremely reactive oxidant
 Biological Pathways for Oxygen Reduction

From: McMurry and Castellion “Fundamentals of general, organic and biological chemistry”
 Oxidative Stress

Antioxidants

Prooxidants

“An imbalance favoring prooxidants and/or disfavoring

antioxidants, potentially leading to damage”
ROS
◼ ROS → oksidatif stress → inaktifasi enzym,
depolimerisasi polisaccarida, merubah DNA, auto-
oksidasi fosfolipid membran dsb
◼ stres oksidatif → setidaknya 100 penyakit: kanker,
kelainan cv, neurologi
◼ transfer elektron di RC, elektron diterima satu
persatu → carrier elektron atau oksigen tak stabil
(radikal) → normal: tidak keluar dari mitokondria
◼ Antioksidant: superoksid dismutase, katalase,
glutation peroksidase, bilirubin, asam urat, β caroten
/ vit A, vit C, vit E
Important Enzyme-Catalyzed Reactions
 Oxidative Damage in Biological Systems

Oxidative stress and cell damage

• High doses:
directly damage/kill cells

• Low doses/chronic overproduction of oxidants:

activation of cellular pathways
stimulation of cell proliferation
damage to cellular proteins, DNA and lipids
Consequences of lipid peroxidation
• Structural changes in membranes
alter fluidity and channels
alter membrane-bound signaling proteins
increases ion permeability
• Lipid peroxidation products form adducts/crosslinks
with non lipids
e.g., proteins and DNA
• Cause direct toxicity of lipid peroxidation products
e.g., 4-hydroxynonenal toxicity
• Disruptions in membrane-dependent signaling
• DNA damage and mutagenesis
 Protein targets for ROS
NH2 H NH2
HS CH2CHCOOH H3C S CH2CH2 C COOH HO CH2CHCOOH

NH2
Cysteine Methionine Tyrosine

Oxidized proteins and amino acids found in biological systems

N NH2

CH2CHCOOH
HN

Histidine

NH2

HN CH2CHCOOH

Tryptophan
Consequences of protein thiol oxidation

Oxidation of catalytic sites on proteins

loss of function/abnormal function
BUT(!): sometimes it is gain in function!
Formation of mixed sulfide bonds
Protein-protein linkages (RS-SR)
Protein-GSH linkages (RS-SG)
Alteration in 2o and 3o structure
Increased susceptibility to proteolysis
 DNA oxidation products
O NH2 NH2

N N N
HN N N
OH OH OH

N N N HO N N
H2N N
H
R R
R
8-hydroxyguanine 8-hydroxyadenine 2-hydroxyadenine

O O
NH2
CH3 CH2OH
H
HN OH N
N OH

OH OH
O N
O N H O H
H H
5,8-dihydroxycytosine thymidine glycol 5-hydroxymethyluracil
 Oxidation of deoxyribose (DNA backbone)
O B O B B
O O O
O
. .
H
. O O O
R Strand
O P OR O P OR O P OR
Breaks
O- O- O-

O2

O
O O B B
O O
. OO O
O +B +
O
O P OR O P OR
.O O
O
O- O-

Apurinic/apyriminic sites Aldehyde products

Consequences of DNA oxidation

• DNA adducts/AP sites/Strand breaks

mutations
initiation of cancer
• Stimulation of DNA repair
can deplete energy reserves (PARP)
imbalanced induction of DNA repair enzymes
induction of error prone polymerases
activation of other signaling pathways
 Sources of ROS
Endogenous sources of ROS and RNS
Microsomal Oxidation,
Flavoproteins, Myeloperoxidase
CYP enzymes (phagocytes)

Xanthine Oxidase,
NOS isoforms Endoplasmic Reticulum Transition
metals
Cytoplasm Lysosomes
Fe
Cu

Oxidases, Peroxisomes
Flavoproteins Mitochondria

Plasma Membrane
Lipoxygenases,
Electron transport
Prostaglandin synthase
NADPH oxidase
 Exogenous sources of free radicals

• Radiation
UV light, x-rays, gamma rays
• Chemicals that react to form peroxides
Ozone and singlet oxygen
• Chemicals that promote superoxide formation
Quinones, nitroaromatics, bipyrimidiulium herbicides
• Chemicals that are metabolized to radicals
e.g., polyhalogenated alkanes, phenols, aminophenols
• Chemicals that release iron
ferritin