Electron%20Transport/%20%20%20%20%20%20%20Oxidation

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Electron Transport/ Oxidation

• Chapter 19

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Glycolysis/Kreb's Cycle

• 1 glu ? 2 pyr ? 2 turns of cycle
• Glycolysis in cytoplasm
• Reduces cofactors
• Cofactors ? mitochondria
• Kreb's cycle in mitochondria
• Reduces cofactors _at_ mitoch
• Flavin cofactors integral to enz's in mitoch

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Mitochondrion

• Outer membrane
• Fluid mosaic lipid bilayer
• Permeable to cmpds MW lt5,000
• Inner membrane
• Fluid mosaic lipid bilayer
• Impermeable
• Specific transporters
• Folds ? cristae
• Components for e- transport/ATP synth embedded

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Mitochondrion contd

• Intermembrane Space
• Between outer, inner membr's
• Impt to ets, ATP synth
• Matrix
• Space enclosed by inner membr
• Enz's of PDC, Kreb's cycle
• Also enz's impt to ox'n FA's, aa's
• All fuel pathways except 1
• Which one?
• Need transporters for metab substrates
• Also transporter for ATP out of matrix

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Fig.19-1
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Cellular Respiration

• Transport e- from cofactors w/in mitoch
• Final e- acceptor O2
• 1/2 O2 2 H ? H2O (w/ ox'n NADH ? NAD
  e-)
• Respiratory chain series of e- carriers
• Embedded in lipid bilayer
• Proteins w/ prosthetic grps, carriers
• Prosthetic grps accept e-, transfer to another
  carrier
• REMEMBER transfer e- by redg equivs
• Direct e- movement H e- as H atom
  H- as hydride ion

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e- Carriers

• Contain 1 or more of 3 prosthetic grps
• Benzoquinone
• Fe-containing porphyrin
• Fe-containing Fe-S protein

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Benzoquinones

• Ubiquinone Coenzyme Q
• Long isoprene chain
• Can accept 1 e- ? semiquinone, or 2 e- ? quinol
• Only carrier not bound/att'd
• Shuttles through mitoch membr bilayer

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Porphyrins Assoc'd w/ Fe

• Cytochromes
• Fe2 (red'd) or Fe3 (ox'd)
• Conj'd ring system
• Fluid p bonds
• Absorb light energy (visible)
• Colored
• 3 Types
• a, b, c
• Differ by side chain
• Att'd to proteins of complexes

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Fig.19-3
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Fe-S Proteins

• Fe coordinated w/ S atoms
• e- transfer causes Fe3 ? Fe2

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Respiratory Chain

• Components organized, arr'd _at_/in inner mitoch
  membr each w/ prosthetic grp(s)
• 4 Complexes CoEQ
• Some bound tightly to membr or each other
• Complex I accepts e- from NADH
• Note H released w/ e- transfer ? intermembr
  space
• e- then transferred ? UQ, Complexes III, IV, O2

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Fig.19-14
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Complex I

• NADHUbiquinone Oxidoreductase Complex
• 42 Polypeptide chains
• Flavoprotein
• FMN (flavin coenzyme)
• Also 6 Fe-S centers
• Overall transfer 2 e- from NADH matrix H2O ?
  UQ
• 2 NADH H UQ ltgt NAD UQH2
• Rotenone inhibits e- flow from Fe-S centers ? UQ

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Fig.19-9
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Complex II

• Succinate Dehydrogenase
• Membr-bound enz of Kreb's cycle
• 4 Subunits
• FAD coenz
• Also 4 Fe-S centers
• Accepts e- w/ rxn succinate ? fumarate
• FAD ? FADH2 redox couple
• Also passes e- to UQ (w/ lipid metab)

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Complex II contd

• Overall Complexes I, II (w/ lipid metab
  cofactor red'n) ? UQ red'd
• REMEMBER UQ is benzoquinone
• Shuttles through lipid bilayer
• UQH2 moves through membr ? Complex III

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Fig.19-8
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Complex III

• Cytochrome bc Complex
• 10 Subunits
• 2 b cytochromes 1 c cytochrome
• Also 1 Fe-S protein
• Integral protein asymmetric

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Fig.19-10
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Complex III contd

• Q Cycle
• QN, QP can bind UQ
• UQH2 loses 1 e- then another
• Overall UQH2 cyt c (ox'd) ? UQ (ox'd)
  cyt c (red'd)
• Also, 2 H w/ ox'n UQH2 rel'd ? intermembr space

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Fig.19-11
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Cytochrome c

• Small, periph membr prot
• Accepts e- from UQH2 ox'n in Complex III
• Diffuses laterally
• Transfers e- ? Complex IV

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Complex IV

• Cytochrome oxidase
• Cyt a, cyt a3
• Also 2 Cu ions
• Impt to e- transfer to O2
• Holds reactive O2
• If partial red'n O2 ? superoxide anion (O2-)
• Reactive, toxic
• When assoc'd w/ H ? H2O not harmful
• Result 4 e- transferred to O2 safely

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Fig.19-13
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Fig.19-14
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O2 Excellent Terminal e- Acceptor

• High affinity for e-
• ? Thermodynamic driving force for ets
• Overall rxn
• NADH H 1/2 O2 ? H2O NAD
• D Go' -220 kJ/mole
• From redox voltage (D G related to E)

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O2

• Approx 90 O2 cell consumption _at_ Complex IV
• Cmpds binding cyt c block cell resp'n
• Poisonous
• CO, cyanide, azide
• Have regen'd ox'd cofactors
• Had been red'd during metabolism
• So what?? What about ATP??

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The Importance of a Space

• W/ each e- transfer (except Cmplx II), gen'd H
• H released to intermembr space
• ? electrical, chemical difference between mitoch
  matrix intermembr space
• Intermembr space now has ?? H
• pH?
• Matrix now has relatively ?? H
• pH?

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Space -- contd

• ? electrical, chemical difference between mitoch
  matrix intermembr space (contd)
• Electrochemical gradient results
• Diff's in concent chem (H) across membr
• Diff's in charge () across membr
• ? energy potential w/ these diff's due to sep'n
  across membr

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Proton Motive Force

• Due to
• Chem potential energy
• Diff in a molecule (H) on two sides of membr
• Sep'd can't equilibrate
• Electrical potential energy
• Diff in charges () on two sides of membr
• Sep'd can't equilibrate

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Thermodynamics (yet again!!)

• Can calc D G for energy inherent in sep'ns
• Includes factors for concent diff's charge
  potentials
• Find 200 kJ energy conserved in proton membrane
  differential
• Ox'n NADH ? NAD rel'd 220 kJ
• 20 kJ used to move H ? intermembr space

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Fig.19-15
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Yet again

• If H could flow from area of high concent ? low
  concent
• Free energy (200 kJ) gen'd
• Could do work
• BUT inner mitoch membr impermeable, even to H
  ions

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And again

• Mitoch has structure that channels H across
  membr
• Proton motive force
• H (proton)
• Channeled to move (motive) from intermembr space
  to matrix
• Releasing potential energy (force)

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ATP Synthase

• Integral protein across inner mitoch membr
• Subunits form channel
• Called Fo
• For H movement
• Other catalytic subunits assoc'd
• Called F1
• Catalyze rxn ADP Pi nHP ? ATP
  H2O nHN
• Form high energy phosphate bonds
• Use energy of proton motive force gen'd by
  channelling H through assoc'd subunits

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Fig.19-16
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F1 of ATP Synthase

• Catalyzes ATP synth from ADP Pi
• 9 Subunits
• 3 Each (a and b), alternating
• 3 Others (g, d, e)

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F1 contd

• a and b structure forms 3 active sites
• Binding and catalytic
• b -- binding
• Positive cooperativity
• REMEMBER allosteric proteins (like hemoglobin?)
• Binding ADP Pi _at_ one subunit ? conforml change
• ? Release ATP _at_ nearby subunit promoted

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Fig.19-22
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Fo of ATP Synthase

• 3 subunits (a, b, c) "link" F1 w/ Fo
• c of small, hydrophobic aa's
• In what part of an integral protein would you
  expect to find c?
• Proton (H) channel

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Fig.19-22
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Fo contd

• Proton flux through Fo fuels interconversion of
  binding substr's _at_ F1, so fuels catalysis at F1
• Protons move through channel
• ? Rotation of c subunits
• ? Conform'l change in d subunit
• ? d subunit contacts 1 b subunit
• ? Conform'l change in b subunit
• ? Release ATP

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Fo contd

• Exper'l data
• F1 can catalyze ADP Pi ? ATP w/out proton flux
• F1 cannot release ATP w/out proton flux
• Stays tightly bound
• Proton flux impt to conform'l changes that allow
  release ATP

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Fig.19-22
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Fig.19-23
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ATP Now in Matrix

• Good for rxns that need ATP in matrix
• BUT need ATP elsewhere in cell
• Ex need ATP for glycolysis
• Where does glycolysis take place?
• Also, need ADP, Pi avail in matrix to make more
  ATP by synthase

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ATP Now in Matrix -- contd

• Also need to get NADH from glycolysis, Kreb's
  cycle ? matrix
• Needed for ets Complex I
• BUT inner mitoch membr impermeable (even to H)
• Can only move mol's across inner mitoch membr by
  transporters

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Adenine Nucleotide Translocase

• Integral protein antiporter
• Exchanges
• ATP matrix ? intermembr space
• ADP intermembr space ? matrix
• Works ONLY when can exch
• So ADP ? matrix ONLY when ATP avail to move out
  of matrix
• Activity favored by electrochem gradient

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Fig.19-25
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Phosphate Translocase

• Integral protein symporter
• Moves 1 H2PO42- 1 H into matrix

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Malate-Aspartate Shuttle

• Moves reducing equivalents across membr
• Dont physically move NADH
• Get indirect transfers of reducing equivs
• Two transporters in shuttle system
• Malate - a ketoglutarate
• Glutamate aspartate
• Relies on NAD already avail in matrix

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Fig.19-26
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Shuttle contd

• Cytosolic NADH can move into intermembr space
• Reduces oxaloacetate ? malate
• Malate dehydrogenase
• Malate transported into matrix
• Antiport a-ketoglutarate transported from matrix
• In matrix, malate oxd ? oxaloacetate
• Malate dehydrogenase
• Concurrent redn NAD ? NADH
• So NADH avail in matrix

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Shuttle contd

• Oxaloacetate ? aspartate
• Aspartate aminotransferase
• Concurrent deamination glutamate ?
  a-ketoglutarate
• a-Ketoglutarate now avail to antiporter w/ malate
• Aspartate transported into intermembr space
• Antiport glutamate transported from intermembr
  space

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Shuttle contd

• In intermembr space, aspartate ? oxaloacetate
• Concurrent a-ketoglutarate ? glutamate
• Glutamate now avail to antiporter w/ aspartate
• More oxaloacetate now avail

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1 Glucose Molecule Yields

• 12 redns of cofactors ? 26 or 28 ATP
• 1.5-2.5 ATP/NADH
• 1.5 ATP/FADH2
• 4 substr-level phosphns ? 4 ATP (or 2 ATP 2
  GTP)
• Book anaerobic metab of glucose ? 2 ATP
• You should now understand the term oxidative
  phosphorylation

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Regulation of Respiration

• Intracell ADP, not ATP measures cell energy
• Related to ATP ATP / ADPPi
• Normal ratio high
• When ADP?, more avail as substr for oxve
  phosphn
• Happens when ATP used up
• ? ? respn and ? ATP until normal intracell
  ATP reached
• REMEMBER translocase allows 1 ADP (substrate)
  into matrix for 1 ATP (product) out

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Regulation of Respiration

• Regulates ATP formn only as cell uses it up
• ATP / ADP ratio also controls glycolysis,
  Krebs cycle (19-29)
• When ratio ?, metab enzs stimd
• ? e- avail to ets
• ? ? oxve phosphn
• ? ? ATP to normal

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Fig.19-29
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Fig.19-29