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Wink508Biochemo12

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Wink508Biochemo12 Powered By Docstoc
					Redox Biology: Biochemistry
David A. Wink and Murali C. Krishna
National Institutes of Health
National Cancer Institute
Radiation Biology Branch
Bldg. 10, Room B3-B69
Bethesda, Maryland 20892
Biochemical Lecture
        I.   Sources of the instigators
               A. Exogenous sources of radicals
               B. Endogenous
                        NADPH oxidase
                        NADPH P450
             oxidoreductases
                        Xanthine oxidase
                        Mitochondria
                        Nitric oxide Synthase
                        Heme oxygenase
       II. Detoxification:
               A. Enzymatic
                        SOD, CAT, GPx
                        DT-Diaphorase (2-e transfer)
               B. Scavenging
                        GSH
                        Ascorbate
                        Tocopherol
       Formation and Abatement of Oxidants in Redox Biolog

Source of Instigators
                                       NOX
                  XO
         PAO                                                   NOS
                                                 uncoupled

  Xenobiotic            H 2O 2           O 2-
                                  SOD


                                                               NO
Catalase/GPX        oxidation

                                                        HbO2     Cellular
        Prx                                                    Consumption
                                                       GR
               LOOH         Proteinox           GSSG         GSH

  Antioxidant systems            Trx
               Reduction of oxygen to O2- and H2O2
 Outer-sphere electron transfer
        Thus, the determining factor is E and access

 O2 + e-        O 2-   - 0.33 V      O2 + e- + H+      HO2 - 0.46 V

 O2- + e-       H2O2 +0.95 V                           pKa = 4.7

Substance with more negative reduction potential than -0.3
can spontaneously reduce oxygen to superoxide.


Paraquat/methyl viologen + + O2
                       MV                  MV2+ + O2-
                        DE = (+0.45) + (-0.33) = +0.12
           Redox Cycling to form Reactive Oxygen Species
                                                                         Para
Types of Compounds                           O
                       O      O 2-    O2                                 OH
                                                              -
Ubiquinone (CoQ)                                         -e
Menidione
Adriamycin (Doxorubicin)       + e-
Tocopherol              O                    O-       O 2-     H 2O 2
                                                                         OH
                    Quinone             Semi-Quinone                    Diphenol
                                     GSH
                                      GS-Q        Prot-SQ


  Dopamine derivatives, Catecholic Estrogens Ortho
                 O
  Adrenaline                  O          OH
                     O            O -e
                                   -
                                            OH
                                                  -

                                 + e-
                   Quinone            Semi-Quinone           Diphenol
             O
                  CH3
                                Met Hb + SQ-    Thiol
                         HbO2
                                                O 2-
             O
Menadione (Vitamin K3)



     CH2CH2NH3+                  CH2CH2NH3+
                         HO
                                           O2

           OH    hydroxylase          OH

    OH                           OH
dopamine                  6-hydroxydopamine
    Pro-oxidant versus Antioxidant

          +e            +e
Quinone            SQ         QH2


            O 2-        Fe(III) reduction Pro-oxidant



  Pro-oxidant: SQ to Quinone

  Anti-oxidant: reduce the SQ to QH2.

Location is essential and pharmacokinetic stability
             water versus fat soluble
                Vitamins Working in Concert
Vitamin E = TcOH                                Pro-oxidant

  TcOH + OH; LOO or NO2               TcO
                                                    Lipid


       Asc                           Asc          Cytosol

                    DHA and Asc
 Carentiod
                                             CarOH or CarOOL
                                     R
   R                                                   Asc

                     Car + OH; LOO          CarOH or CarOOL

                   NO2     Asc

                     Car
              Xenobiotic Metabolism
 Activation of Quinones, Paraquat etc.

       NADPH Cytochrome P450 Oxidoreductases
 X                                                       X-
                  + 1 electron


     Detoxification of Quinones, Paraquat etc.
             DT-Diaphorase or Quinone Reductase
             ( NQ1, P450 reductase super family)
   X                                               XH2
                  + 2 electrons

Broccoli extracts induce DT-Diaphorase (NQ1) expression
                        Mitochondrial Sources of ROS                        O2

Site I           Site II             Site III             Site IV
                                                                         H 2O
                                O2              O 2-   Cytochrome c oxidase
NADH


                                                       O 2-        H 2O 2
                     -0.04 V
Ubiquinone (Q)                  Ubisemiquinone (SQ)                 Ubiphenol (H2Q)
                                                              -0.25 V
  HO2            O 2-      O2
         pKa = 4.7




  Polarization/depolarization of the mitochondria can effect reduction
  where the proton gradient can be important to reduction of O 2.
             Energetics of ROS formation from Site III

Eo = -0.33
Eo = 0.04
DE = -0.29
                           SQ +O2
                                      e-       Water reduction
DG ~6 kcal         K = 0.01                          at Site IV
K =0.01
         Aconitase        Q +O2- +NO
                                       ONOO-      NO3-
                  MnSOD         e-


                             H 2O 2        Spin trap

 Over expression of MnSOD                  DHR
 leads to increase DCHF oxidation          DCHF
         Examples of Inner-sphere reduction of Oxygen

Bleomycin                                   BLMFe(II)
                                 e-
       BLMFe(III)                                 O 2-
                                                                   Fe(II)O2 or Fe(III)O2-
 Substrate                             H 2O 2                 e-
 oxidation


      OH and Fe(IV)                             Fe(V) or Fe(III)O2H+
                                                2 electron, oxygen transfer
    1 electron oxidants
                                                hydroxylation


Prolyl Hydroxylase
                                      +e-                          O2
                   Fe(III)                          Fe(II)                  Fe(II)O2
   Hydroxylation
   of proline                         +e-
                             -
                   Fe(O2 )                       Fe(III)O2-
        Different reduction mechanism of O2 by P450 system

Cytochrome P450                 Fe2+ + O2       Fe2+O2

                        +e-
                                                       +e-
    Resting state              Peroxide shunt
                 Fe3+                            Fe3+ -O22-
                              +H2O2


  Substrate
                               Fe5+=O           H 2O
  oxidation
                                1.4 -1.6 V

P450 reductase

NADPH           FAD              FMN                   P450

           Q                     O2
                          SQ
Enzymatic generation of Reactive Oxygen Species



    Purpose:

       •   As an antimicrobial agent

       •   as a by product of metabolism

       •   Part of signal transduction mechanisms
        Examples of Hydrogen peroxide formation

Glucose Oxidase (Glc oxidase: Bacterial)
         O2 + Glucose --> gluconolactone + H2O2
Polyamine oxidase (PAO)
         O2 + spermine ---> acrolein + putrescine + H2O2

Xanthine Oxidase (XO)
         O2 + Xanthine --> Urate and O2-/H2O2

         O2 + Hypoxanthine --> Xanthine and O2-/H2O2

NADPH oxidase (NOX)

         NADPH + 2 O2 --> 2 O2- + NADP+
                 Xanthine Oxidase

                  Purpose:    Purine metabolism
                              Secretion of milk drops
                              Detoxification of Aldehydes
                              Generation of ROS


           XD
                    Uric acid + NADH       Normal (XD)
Xanthine

           XO       Uric acid + O2-/H2O2 Disease and injury
                                               (XO)
    XO
HX --> X + ROS
                             Xanthine Oxidase
Homodimer
                                         Inhibited by allopurinol (abundant in goats milk
        HX or X

                        Mo                                       O2

                                     FAD          FADH2
            Urate
                                 Fe2S2                                O2-/H2O2


                S
       O
            Mo                                    Mouse utilizes XO more than humans
                    S        O
H 2O                    H
            S                        H
                        N        N
3-
     O3PO
                    O   N
     Moco               H
                 NADPH oxidase (NOX)
                         Purpose:
Phagocytic (Phox)              Antimicrobial
                               Regulation of cell-surface signalin
                               regulation of physiological functio
Membrane bound
         gp91phox
         p21phox                NOX-1 colon VSMC, prostate
                                NOX-2 innate immune system
                                NOX-3`inner ear
 Cytosolic regulators           NOX-4 kidney
           p47phox              NOX-5 spleen (human only)

           p67phox
           p40phox


Small GTPase/rac1/Rac2           Vascular NOX
NADPH oxidase                                  O 2-
                                    O2


cytochrome b558                     -Fe-

                                        -Fe-

                             gp91phox      FAD

        gp91 p22                    NADPH
                         P
  rac          p47
        gp67             P
    P          p40

                     P
                   Nitric Oxide Synthase
    Homodimer

NOS-1 nNOS or neuronal NOS    Constitutive and calcium sensitive
NOS-3 eNOS or endothelial NOS

NOS-2 iNOS or inducible NOS    Induced and calcium insensitive



P450 reductase domain          NADPH        Calmodulin binding
                               FMN
                               FAD


Heme oxidase domain            O2
                               Arginine/N-hydroxyarginine (NOHA)
                    Nitric Oxide Synthase

130kd


  Arginine
                  Heme Cm Reductase domain
               oxygenase              BH4 BH
                                             4
              BH4     BH4
Citrulline                             Heme
+                 Reductase domain Cmoxygenase
NO
               FMN               FAD


    NOSox
                                           NADPH
    NOSred NADPH-dependent P450 reductase


 In cells can exist as monomers which then are converted to dimers
                     NOS Biochemistry
                                         NO
      Arginine
      Oxygen                     NOS
                                         NOHA
      NADPH
                                         O 2-
                 Calcium         PZT
                 BH4             HSP90   H 2O 2

                                         HNO


Fe(NO)             Fe(II) + NO
 NO       iNOS>> nNOS or eNOS
Ki

     O2
Km          eNOS > iNOS > nNOS
       Heme Oxygenase

       HO-1
                                          Heme
       HO-2


        M       V   M       V M       V   M       V
                                                            + CO
    O                                                 O
            N           N         N           N
            H                     H           H                    Guanylyl cyclase
                                                          NADPH
Biliverdin
        M           M       V M       V   M       V       NADP+
                V

                                                      O
                                                                      Antioxidant
   O        N           N         N           N
            H           H         H           H

 Bilirubin
Biochemistry of Prevention of Oxidative or Nitrosative str


                 Different classes


  Preventive
      Inhibition of production of instigators


  Interception and Quenching

           Scavenging of reactive intermediates formed
1) Want the product to be innocuous
2) The product could be biologically recycled
3) Prevent chain reactions from occurring (chain breakin
          Biochemistry Reactive Oxygen Species
                  XO             NOX


                         O 2-              Peroxidases
         NOSunc
                                           Cl-      NO2-
                                                Br-
                                         HOCl HOBr NO2

Fenton                  H 2O 2            Antimicrobial


LOOH
                                  MAPK
                                  NFkB
           Aconitase              p53
           inhibition   COX
                        LipOX
                        Chemical Biology of Nitric Oxide

Direct
                                    NOS        Radical scavenging
Inhibition of Respiration
                                               Antioxidant properties

         Guanylyl                                Inhibition of P450
                                    NO
         cyclase

                          O 2-
Indirect

                            +NO                    N 2O 3
            ONOO    -                NO2


Antitumor
Antipathogen
                        Oxidation                Nitrosation
                        Nitration
               Preventive Mechanisms
                       O 2-

                          SOD
  Catalase
                      H 2O 2              Peroxiredoxin (Prx)
Heme protein
                                          Isoforms: Prx 1-6
 peroxisome
                                          Contains reactive thiol
                                          1-4 two cys
                                          5 one cys
                                          6 one cys
           Glutathione peroxidase (GPX)
           Selenium containing protein    Location
           Cytosol and mitochondria          Cytosol
                                             Mitochondria
                                             Peroxisome
                                             plasma

                         LOOH
    Scavenging of NO (Prevention of RNOS formation)


                     NOS
                                    General Cellular
          HbO2                      Consumption
          MbO2
NO3-                 NO             Mechanism to be
                                    determined
                  Fe=O
                                     Mitochondria?
k > 107 M-1 s-1
                     NO2-


   Critical to compartmentalizing NO effects
          Biochemistry of Glutathione Peroxidase
NADPH

  Glutathione
  reductase (GR)               GPX
                    GSSG                  H 2O 2
                               Prot-Se-   LOOH
            GSH                           ONOO-

                                                      H 2O
     Prot-SeSG + H2O                      Prot-Se-OH +LOH
                                GSH                   NO2-



  Selenium compound Eblselen
                 Peroxiredoxin (Prx)
                            SH
                      Prx                         Prx (1-4)
                            SH
                                         H 2O 2

                 Trx(S-S)
                                                       S
                                             Prx
NADPH        Trxred                                    S
                              Trx(SH)2

Trx = Thioredoxin (ER)
Trxred = thioredoxin reductase (FAD)

       NADPH          FADH2         Reduced S-S

      Both GR and Trxred use hydride transfer to reduce
            the disulfides to sulfides
             Reduction of a seleno-cysteine




A selenylsulfide in a protein is reduced by a cysteine-
exchange reaction and the resulting disulfide is then reduced
by electron transfer. This example shows the reduction of
thioredoxin (Trx) by thioredoxin reductase (TrxR).
Jacob C, Giles GI, Giles NM, Sies H. (2003) Sulfur and Selenium: The Role of Oxidation State in Protein
Structure and Function. Angewandte Chemie International Edition. 42: 4742-4758.
Glutathione Metabolism

                                      SOD              GPX
                                             H 2O 2           H 2O
                               O 2-

                GSSG
  NADPH                                O2
      GR
                    GSH
                            GSSG             GSH
  GSH ---> GS
                                      ONOO-
    Oxidant O                                         GSH
      OH/Fe=O   2
                     GSOO              GSOH                  GSSG
      NO2
                                             SOD             GPX
                          O2                       H 2O 2            H 2O
                                      O 2-
           Abatement of Nitrosative Stress
                   NO                          Both NO and HNO
                                            toxicity is abated by GSH
                        O2
                   N 2O 3

              GSH           Cu(I)ZnSOD      Cu(II)ZnSOD


                  GSNO                           GSH + NO
           GSH
                                     ProteinS-SG
      GSSG +HNO
                                Protein SNO +GSH
        GSH
                     GSH                   GR
GS(O)NH2   GSNOH              GSSG                  GSH
                                         NH2OH
       Glutathione and Ascorbate Pools Communicate


                      GSSG     2 GSH


                Protein Disulfide Isomerase (PDI)
                     Glutaredoxin (cytosol)         Trxred

                                                    NADPH
            -
                  Y      Asc
     AscH                                DHA
                 -0.28          +0.17

 Reduce metals


Increase Metal-mediated Oxidation
 Urate and Uric Acid             Human Plasma 0.2-0.4 mM
     Xanthine
XD or XO




      OH             NO2
                           107
                 9
                                     Urea and HOC-CHO
           >10
                                         Glyoxylic acid

                                          Oxalic acid
             Interaction of NO and ROS      ?
                               CO and Bilirubin
                 HO-1

                 Heme       NOX
                                           Lipid Peroxidation
      NOS        NO          O2- SOD


                    RNOS          H 2O 2         LOOH
Ferrochelatase
                   Ox
                                                     GPX
                                                     Prx
                   GSSG           GPX
                   GR             Prx              ROH
                    GSH
   Heme and iron regulation is critical to the redox balance
          All enzymes require Oxygen and NADPH

                         O2



                                           HO
         XO
                                NOS
                  NOX

                   NADPH versus NADH
     Metabolism of iron, oxygen and glucose will be
        critical factors in the cellular redox state


Now the adventure begins in biology

				
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posted:5/12/2014
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