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 O2­ + e­ + 2 H+ → H2O2 H2O2 + e­ + H+ → H2O + OH OH + e­ + H+ → H2O O2 + 2 e­ + 2 H+ → H2O2 H2O2 + 2 e­ + 2 H+ → 2 H2O O2 + 4 H+ + 4 e­ → 2 H2O                     ­0.33a 
               +0.89

             +0.38 +2.31           +0.281a                              +1.349 +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

3

 

 

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 N N H R N N H N N O N N O H R N
+

+  O 2

.N
R N N

N O H N O N O

O

 O2­  N
.

H O H

H

(20) +  H2O2

O O HO H

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                     H+ HO2 + O2­ → H2O2 + O2     O2­ + O2­ → no reaction
   

(1) (2) (3)

k = 9.7 x 107  M­1s­1

• 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                                 k = 1.7 x 10  M s
   
7 ­1 ­1

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., phydroxyphenylacetaldehyde) • Glycoxidation adducts (e.g., carboxymethyllysine) • Cross-links, aggregation, peptide bond cleavage

Amino acids most susceptible to oxidation and their main reaction products
Amino Acid
Cysteine

Physiological oxidation products

Disulfides, mixed disulfides (e.g., glutathiolation), HNECys 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 Arginine Lysine 2-Amino-3-ketobutyric acid Glutamic semialdehyde, chloramines 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 metalcatalyzed 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).

• 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, γirradiation, HOCl, ozone, 1O2, lipid peroxide adducts) • Sensitive assays are available (≤ 1 pmol)

A little more about protein carbonyls

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 protein
H2O2 Fe activated  neutrophil DNP

oxidized  protein

DNPH

DNP­  protein

Absorbance  at 370 nm

2. Immunoassays for protein carbonyls  e.g., Western blot, ELISA, immunohistochemistry 
* O oxidized  protein DNPH * * * * * * * *

DNP DNP­  protein Anti­DNP antibody

DNP DNP­  protein

Proteins that contain iron-sulfur clusters play an important role in biological systems

Rieske iron-sulfur proteins [2Fe-2S]

Aconitase family [4Fe- 4S] cluster

 

[3Fe-4S] cluster

 

Aconitase
Catalyzes isomerization of citrate to isocitrate

From Garrett & Grisham

 

 

Iron­sulfur center in aconitase

4 3 1 2

Basic residue
   

(keeps the citrate in active site)
From Lehninger Principles of Biochemistry

 

 

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

Chemical Mutagens

         ­ 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.

 

 

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 

Categories of Mutations

 

 

          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    

 

 

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