Oxygen reactions in oxidative degradation and oxidative stress

• Air contains 21% dioxygen, O2

• Animals, plants, and aerobic 
  bacteria require dioxygen for 
  efficient production of energy.

•The earliest forms of life were 
  anaerobic.

• Dioxygen  is toxic to all forms of 
  life, whether anaerobic, 
  aerotolerant, or aerobic.

• Life coexists with dioxygen by 
  use  of antioxidant defense 
  systems or by repairing or 
  replacing the components 
  damaged by oxidative stress.
   
   
   
   
  Oxygen reactions in 
respiration

   
 Table 1. Standard Reduction Potential for Dioxygen Species in Water, pH 7,
25o

                         Reaction                                                   EÞ, V, vs. NHE

O2 + e­ → O2­ ­0.33a 

O2­ + e­ + 2 H+ → H2O2                +0.89

H2O2 + e­ + H+ → H2O + OH              +0.38

OH + e­ + H+ → H2O                     +2.31

O2 + 2 e­ + 2 H+ → H2O2           +0.281a 

H2O2 + 2 e­ + 2 H+ → 2 H2O               +1.349

O2 + 4 H+ + 4 e­ → 2 H2O               +0.815a 

aThe standard state used here is unit pressure. If unit activity is used for the

standard state of O2, the redox potential for reactions of that species must be
adjusted by +0.17 V.
   
 1

   
 Kinetics of dioxygen reactions
• Direct reactions of dioxygen tend to be slow because ground state 
   dioxygen is a triplet and most reactants are singlets. 

• Triplet­to­singlet spin conversions are forbidden by quantum 
   mechanics and hence are slow. 

• A collision between two molecules occurs much more rapidly than 
  a spin flip and so cannot be concerted. 

• Instead, the number of unpaired electrons remains the same 
   before and after each elementary step of a chemical reaction, and 
   spin flips must be thought of as kinetically separate steps. 

• For these reasons, we know that it is impossible for a spin 
   forbidden reaction to go in one concerted step.

 3O2 (↑↑) +  1X  (↑↓)                 1XO2 (↑↓)
triplet            singlet 
   
A direct reaction of O2 in which each step is spin allowed:

3
O2 (↑↑) +  1X (↑↓) →  2O2­ (↑)  +  2X+ (↑)

2
O2­ (↑)  +  2X+ (↑) →  2O2­ (↑)  +  2X+ (↓)

2
O2­ (↑)  +  2X+ (↓) →  1XO2 (↑↓)

   
 Reaction of dioxygen with reduced flavins

R H R
N N O N N O

N
N
H
+  O 2 .N
+ N  O
­
2 
.

H
H O O
H
H
R R
(20)
N N O N N O
N N +  H2O2
N H N H
O O
HO O
H

   
 Free radical autoxidation
Initiation: X2  →  2 X.

X. (↓) +  RH  →  XH  +  R. (↓)

Propagation: .
R  (↓) +  O2 (↑↑) →  ROO  (↑) .
ROO. (↑) +  RH  →  ROOH  +  R. (↑)

Termination: R.  +  ROO. →  ROOR

2 ROO. →  ROOOOR  →  O2  +   ROOR
  
(plus other oxidized products such as
 ROOH, ROH, RC(O)R, RC(O)H)

Much more common.
  Very small traces of redox metal ions and peroxide can initiate.
 
 Lipid Peroxidation

   
   
   
   
   
 ROS

Hydroxyl radical and high valent metal oxo species
Highly reactive, indiscriminant oxidants 
Redox metal ions usually involved in generation

Superoxide
Reactive but highly selective 
Most vulnerable are labile Fe­S clusters

Hydrogen peroxide 
Relatively unreactive except as precursor to hydroxyl radicals

Dioxygen itself is not the primary agent of oxidative stress.
It is the precursor of all of the ROS and reacts extremely rapidly with 
organic free radicals, when they are present.
   
 • Hydrogen peroxide itself is a strong oxidant 
   thermodynamically, but its reactions tend to be quite 
   slow in the absence of a catalyst. 

• Very small traces of redox­active metal ions can 
    dramatically catalyze oxidation reactions of H2O2 

Fe2+ + H2O2 + H+ → Fe3+ + H2O + HO.  
Fe3+ + 1/2 H2O2 → Fe2+ + 1/2 O2 + H+
__________________________________________
3/2 H2O2 → H2O + 1/2 O2 + HO.

Hydrogen peroxide should not itself be considered 
a dangerous ROS unless small traces of redox 
metals, especially Fe2+/3+, are present.
   
 Hydroxyl radical, HO . 
• One of the most reactive of the ROS known. 
• It is commonly generated from reaction of H2O2 with 
   reduced Mn+ (Fenton reaction). 
                                         +H+
Fe2+ + H2O2 → [(FeIV=O)2+ + H2O] → Fe3+ + H2O + HO.  

Cu+ + H2O2+ H+ → [(CuIII­OH)2+ + H2O] → Cu2+ + H2O + HO. 

• High valent metal­oxo or hydroxo intermediates, e.g., 
  (FeIV=O)2+ and (CuIII­OH)2+, are also implicated as ROS.

•Hydroxyl radicals and high­valent metal oxo and hydroxo 
  species can act as initiators of free radical autoxidation of 
  lipids and can damage proteins, nucleic acids, 
  carbohydrates, and other organic molecules when they are 
  generated in close proximity to such molecules.
   
HO. +  H­X → H2O + X. 
 SUPEROXIDE, O2­ 

• The pK of HO2 is 4.8 in aqueous solution. 

•Thus the predominant species present in solution at 
  physiological pH is the unprotonated superoxide anion 
  itself.

HO2  O2­ + H+          K  =  1.6 x 10­5 M

• Superoxide itself is a much more sluggish oxidant than 
   hydroxyl radical and hence it is much more selective in 
   the targets that it oxidizes. 

•The best characterized targets are iron–sulfur cluster­
  containing proteins containing single labile iron atoms in 
   
  their clusters. 
• Superoxide disproportionates spontaneously to yield 
   hydrogen peroxide and dioxygen via a pH dependent 
   mechanism involving reactions 1 and 2. 

• Reaction 3 does not occur in the pH range of 0.2­13.

HO2 + HO2 → H2O2 + O2   k = 8.3 x 105 M­1s­1    (1)

                 H+
HO2 + O2­ → H2O2 + O2   k = 9.7 x 107  M­1s­1 (2)

 
O2­ + O2­ → no reaction (3)
   
• Those rare cases in which O2­ is observed to oxidize 
   substrates at high rates occur only when proton 
   transfer is simultaneous with electron transfer, 
   resulting in formation of HO2­ rather than O22­.

X ..... O2­ ..... H­Y →  X+  +  HO2­  +  Y­        

An example of a fast oxidation by superoxide in which 
such proton­coupled electron transfer to superoxide is 
likely to be occurring is the rapid oxidation of 
hydroquinones by superoxide.            

HO         +  O2­                                                           +  HO
OH HO O 2
 ­  

                    
7 ­1 ­1
           k = 1.7 x 10  M s
   
What is “protein oxidation”?

Covalent modification of a protein

induced by ROS or by-products
of oxidative stress.
Reactivity of Proteins with ROS
• Low or no direct reactivity with superoxide or hydrogen
peroxide (in the absence of trace metals or other
catalysts)*

• High reactivity with •OH and free radical oxidants with

similarly high reactivities (can come from H2O2 or from
lipid and other organic peroxides).

• Reactive with products of lipid peroxidation (e.g., HNE,

hydroxynonenal, and MDA, malondialdyhyde)

• Reactive with 1O2

• RNS?

*Metalloproteins can have other metal-mediated

pathways that give major oxidative damage to that
protein
 General types of protein
oxidative modification
• Sulfur oxidation (Cys disulfides, S-thiolation; Met sulfoxide)
• Protein carbonyls (side chain aldehydes, ketones)
• Tyrosine crosslinks, chlorination, nitrosation, hydroxylation
• Tryptophanyl modifications
• Hydro(pero)xy derivatives of aliphatic amino acids
• Chloramines, deamination
• Amino acid interconversions (e.g., His to Asn; Pro to OH-Pro)
• Lipid peroxidation adducts (MDA, HNE, acrolein)
• Amino acid oxidation adducts (e.g., p-
hydroxyphenylacetaldehyde)
• Glycoxidation adducts (e.g., carboxymethyllysine)
• Cross-links, aggregation, peptide bond cleavage
 Amino acids most susceptible to oxidation
and their main reaction products
Amino Acid Physiological oxidation products
Disulfides, mixed disulfides (e.g., glutathiolation), HNE-
Cysteine
Cys
Methionine Methionine sulfoxide
Tyrosine Dityrosine, nitrotyrosine, chlorotyrosines, dopa
Tryptophan Hydroxy- and nitro-tryptophans, kynurenines
Phenylalanine Hydroxyphenylalanines
Valine, Leucine Hydro(pero)xides
Histidine 2-Oxohistidine, asparagine, aspartate, HNE-His
Glutamyl Oxalic acid, pyruvic acid
Proline Hydroxyproline, pyrrolidone, glutamic semialdehyde
Threonine 2-Amino-3-ketobutyric acid
Arginine Glutamic semialdehyde, chloramines

Lysine a-Aminoadipic semialdehyde, chloramines, MDA-Lys,

HNE-Lys, acrolein-Lys, carboxymethyllysine, pHA-Lys
 Sulfur Oxidations
• In general, Cys and Met are the amino acids that are
most susceptible to oxidation
• Distinguished from other oxidative protein
modifications in that cells have mechanisms to
reverse the oxidation
e.g., methonine sulfoxide reductase
e.g., glutathione or thioredoxin redox systems
• Hence may serve a regulatory function
• Reversible oxidation/reduction of methionine may
protect proteins from more damaging forms of
oxidative modification (e.g., carbonyl formation)*

* Stadtman, E. R., Moskovitz, J., Berlett, B. S., and Levine, R. L. (2002) Mol. Cell. Biochem. 234-235,
3-9
Peptide bond cleavage due to reaction with hydroxyl radical

Peptide Bond Cleavage.

OH, generated by either radiolysis of water or the metal-

catalyzed cleavage of H2O2 can abstract hydrogen atoms from
the -CH(R)- group of the polypeptide backbone (reactions a, b).

The alkyl radical thus formed may react with oxygen to form the
alkylperoxy
  radical (reaction c) or  with another alkyl radical to
form inter- or intraprotein cross-linkages (reaction p).
The protein peroxy radical can be converted to the alkyl peroxide by
either

•reaction with free peroxy radical (reaction d),

•reaction with Fe2+ (reaction e), or
•abstraction of a hydrogen from another source (not shown).

Irrespective of how it is formed, the protein alkyl peroxide can be

converted to the alkoxy protein derivative by either

•dismutation (reaction o),

  •reaction with free peroxy radical  (reaction f), or
•reaction with Fe2+ (reaction g).
Finally, the alkoxy radical may undergo conversion to the hydroxy derivative
(reactions i, j),

which will undergo peptide bond scission by the so-called α-amidation

pathway (reactions k, l).

Alternatively,
  the alkoxy radical may undergo
  peptide bond cleavage by the
so-called diamide pathway (reaction m).
 A little more about protein carbonyls
• Carbonyl groups are stable (aids detection and
storage)
• Present at low levels in most protein preparations
(~1 nmol/mg protein ~ 0.05 mol/mol ~ 1/3000
amino acids)

• See 2- to 8- fold elevations of protein carbonyls

under conditions of oxidative stress in vivo

• Induced in vitro by almost all types of oxidants

(site-specific metal catalyzed oxidation, γ-
irradiation, HOCl, ozone, 1O2, lipid peroxide
adducts)

• Sensitive assays are available (≤ 1 pmol)

 Detection of protein carbonyls
• Measure total protein carbonyls levels after reaction with
DNPH followed by spectroscopy (A370), ELISA, or
immunohistochemistry
• Measure carbonyl levels in individual proteins within a
mixture of proteins (tissue samples, cell extracts) by
reaction with DNPH followed by Western blot
immunoassay
        Measurement of total carbonyls

1. Spectrophotometric DNPH assay

O DNP

H2O2 Fe
oxidized  DNPH DNP­  Absorbance 
protein
activated  protein protein at 370 nm
neutrophil

2. Immunoassays for protein carbonyls 
e.g., Western blot, ELISA, immunohistochemistry 
* *
* *
* *
* *
O *
DNP DNP
oxidized  DNPH DNP­  Anti­DNP DNP­ 
protein protein antibody protein
 Proteins that contain iron-sulfur clusters play an
important role in biological systems

Rieske iron-sulfur proteins Aconitase family

[2Fe-2S] [4Fe- 4S] cluster

   
[3Fe-4S] cluster
 Aconitase
Catalyzes isomerization of citrate to isocitrate

From Garrett & Grisham

   
 Iron­sulfur center in aconitase

3
1

Basic residue
(keeps the citrate
in active site)
   
From Lehninger
Principles of Biochemistry
   
 Chemical Mutagens
A chemical mutagen is a substance that can alter a base that is 
already incorporated in DNA and thereby change its 
hydrogen­bonding specificity.

  Three Powerful Chemical Mutagens
    1. HNO2 (Nitrous acid)
         – converts amino groups to keto groups by “oxidative deamination” :
                C                U (Uracil)

                

                A                 H (Hypoxanthine) 

                
                G                  X (Xanthine)

         ­ these bases can form the base­pairs: U.A, H.C, and X.C
         ­ the changes are G.CA.T and A.T­G.C as cytosine and adenine 
            are deaminated.
   
2. Hydroxylamine
       ­ reacts with C and converts it to a modified base that 
         pairs only with A, so that G.C pair ultimately becomes  
         A.T pair.

                     +  NH2OH → (modified base) ­ 

       
        cytosine (C)                                                adenine (A)

3. Ethylmethyl sulfonate 
      ­ an alkylating agent

   
 Mutagenesis and Carcinogenesis

• Mutagenesis
b) A change in the genetic code which may or may not 
have an effect on the organism.
c) Changes in chromosomal structure such as breaking 
off of part of chromosome or translocation of an arm 
known as clastogenesis
d) Uneven separation of chromosome during cell 
division known as aneuploridization.

   
 Categories of Mutations
1.) Spontaneous mutation­ occurs without the introduction of an exogenous 
                                            mutagenic agent.
        Examples:
          Sugar­Base cleavage by ROS.
             a. Deamination­ C­ U
             b. Methylation ­ mostly affected, G and A
             c. Mistake during replication ­ endogenous enzymes involved in DNA 
                                                              repair
             d. Isomerization of bases ­ Enol form of T binds with G instead of A
             e. Addition or Deletion of base sequences during DNA replication.

2.) Induced Mutation ­ introduction of exogenous agents or physical agents such as 
                                      radiation into cell.
             a. Base­analog substitution­ e.g. 5­bromouracil is similar in structure to T.         
          
                          During DNA replication T is replaced by 5­bromouracil, which does 
                          not bind with T but binds with G.
            b. Base modification ­ substances such as epoxides, nitrogen mustards and 
                                                aldehydes can modify the base
            c. Base intercalation ­ e.g. Actinomycin D can intercalate between G and C and 
   
                                               forms hydrogen bond with G, resulting to either deletion 
   
          d. UV radiation
               <i.> Induces dimerization
               <ii.> Causes single and double strand breaks
                         ** Single strand break can be repaired.
                          ▫ Three possible outcome for double strand breaks:
                            <i.> The molecule will be connected with no error
                            <ii.> The molecule is repaired incorrectly and produces 
                                      a mutated DNA molecule
                           <iii.> The DNA molecule is not repaired.

3. Large Mutation­ Deletion, inversion, and translocation are processes 
that involve hundreds to thousands of base pairs and several 
different genes, producing changes in large segments of the DNA.
            a. deletion ­ occurs at any point along the chromosome and results 
                                in fewer bases in the chromosome.
            b. Inversion ­ chromosomal aberration in which segment of the 
                                  DNA is inverted 180 degrees.
            c. Translocation ­ occurs when segments are transferred to a 
                                         different part of the same chromosome.
   
4. Point Mutation ­ caused by the reaction of a genotoxic substance 
                                with DNA that may involve either base 
substitution 
                                or frameshift mutation.
       Type of Base Substitution:
         a. Transition ­ Purine replaced by purine
                                Pyrimidine replaced by Pyrimidine
         b. Transversion­ purine replaced by pyrimidine
or vice versa

2 Transitions: AT­GC; GC­AT
4 Transversion: AT­TA; AT­GC; GC­CG; GC­AT

5. Frameshift Mutation­ base pairs are added or deleted and their 
number is other than three or a multiple of three.  The triplet code 
is misread entirely and the result is a radical change of the protein 
structure.
   
 Interaction of Chemical with DNA
1. Akylation
     a. Methylation at O­6 of G causes a change in its tautomeric 
form so that it will resemble A
     b. Aflatoxin B, upon metabolic activation to 2,3­epoxide reacts 
with N­7 of G or N­6 of A­ leads to frameshift mutation
     c. Alkylation by benzo(a)pyrene with G causes frameshift 
          mutation
     d. Alkylation of OH group in phosphate­ leads to the formation 
           of 3­OH and 5­P. 

2. Intercalating Agents ­ insertion of aromatic compounds between 
                                        stacked bases of DNA
                                      ­ interferes with the action of  
Topoisomerase II­ catalyzes transient double strand breaks of 
DNA for purposes such as replication and transcription, leads 
 
to frameshift mutation.  
 Intercalation of Acridine

   
 3. Non­alkylating Agents
      a. Nitrous acid­ deaminates bases
            A Hypoxanthine
            G Xanthine
            C Uracil

4. UV radiation­ causes strand breaks via radical formation

   
 Reactivity of Nucleic Acids

1. Pi Bond Order ­ highest pi bond order means most reactive for addition 
                               reactions.
                 e.g. T­ most reactive at 5­6 positions
            Possible reactions:
              a. Reaction with with Br2, O3, R.
              b. Effect on Ionizing Radiation
              c. Effect on UV radiation
2. Free Valence ­ highest polarizability of pi electrons
               e.g. C­8 of G and A; G> A
                      C­6 of T, C­5 of C
               ** Can form adduct with H2N­­­­
3. Dipositivity of N­C bonds
       e.g. N9­C(ribose) of G – most reactive
                  ­ ease of cleavage during mutagenesis
                  ­ alkylation at N­7 of G, enhances cleavage of N­C glycosidic 
   
                     bond
4. Availability of Lone Pairs on N: N­7 of G and A
             ­Alkylation may lead to apurinic site
        A has more available lone pairs than G because it only 
        forms 2 H bonds; will react easily with HNO2 and 
        formaldehyde

5. Availability of Lone pairs on O: O­6 of G prone to 
alkylation (favors formation of tautomer which leads to 
base mispair.

   
 Molecular Aspects of Carcinogenesis
Cancer ­ a disease in which altered cells  divide 
               uncontrollably (neoplastic growth) resulting in 
               tumors (neoplasm)

BASIC TERMINOLOGY
A. Tumor ­ a swelling; could be due to any number of causes
B. Dysplasia ­ alterations in size, shape and staining 
                        characteristics of cells in nonneoplastic
                         tissue.
C. Neoplasia ­ a relatively autonomous growth of tissue; the growth 
                        of which exceeds and is uncoordinated with that of 
                        normal tissue and persists in some manner after 
                        cessation of the inducing stimulus.

   
   
1. Initiation Stage­ reactivity of carcinogen with DNA; may lead to base 
changes, deletions, small insertions and chromosomal changes such as 
  invertions and translocations