Oxidative Phos - Abali 3/7/16 Flashcards

1
Q

why do we need oxygen?

what types of work are driven using energy from ox phos?

A

required for total oxidation of glucose; oxygen is fully reduced to pick up the electrons given off in that process

  1. mechanical work (muscular contraction)
  2. chemical work
  3. transport work
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2
Q

oxidative phosphorylation

  • parts
  • process
A

two parts

1. electron transport chain :

  • electrons are donated by NADH molecules (generated from metabolism of food)
  • drives Complexes I, III, IV to pump H into intermembrane space

2. chemiosmosis :

  • H gradient generated by etc - proton motive force - that drives Complex V (ATP synthase) to pump H back out and turn ADP into ATP
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3
Q

2 processes that generate ATP

  • % of ATP generated
  • how does the process sustain itself (NAD+)
A

glycolysis

  • 10% of ATP
  • NAD+ regenerated via anaerobic production of lactate [lactate dehydrogenase]

oxidative phosphorylation

  • 90% of ATP
  • NAD+ regenerated via donation of e to electron transport chain by NADH
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4
Q

overall role of cardiopulm system in context of biochemical processes

  • two biochemical scenarios that potentiate O2 delivery in tissues
A

cardiopulm system provides the transport mechanism (blood/Hb as carriers, heart as the motor, lungs as dropoff/pickup vehicles) to move…

  • CO2 waste products of TCA cycle
  • O2 required for oxphos

O2 delivery is enhanced in conditions of…

  • 2,3BPG buildup (intermediate of glycolysis)
  • low pH
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5
Q

functional parts of the mitochondria and what they contain

A

outer membrane

  • contains porins, permeable up to 10kDa

intermembrane space

  • generally low pH (if etc is doing its thing)
  • location of apoptotic molecules (triggered by cyt c)

inner membrane

  • impermeable to everything but H2O, O2, CO2, NH3
    • everything else passes through transport shuttles
  • Complexes I, II, III, IV, V + CoQ making up the respiratory chain needed to generate proton motive force

mitochondrial matrix

  • generally high pH
  • pyruvate dehydrogenase
  • TCA cycle enzymes (all except succinyl dehydrogenase/Complex II - FADH2 generator - which is in inner membrane)
  • FA beta ox enzymes
  • some urea cycle enzymes (protein metab)
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6
Q

redox reactions: players

A

oxidation (LEO, OIL)

  • reductant loses electrons = gets oxidized
    • facilitated by an oxidizing agent

reduction (GER, RIG)

  • oxidant gains electrons = gets reduced
    • facilitated by a reducing agent
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7
Q

how is energy harvested through the etc?

A

NADH/FADH2 act as reductants, deliver electrons to etc

  • high e electrons lose energy in several steps, which allows energy to be siphoned off for use in ATP generation (instead of rapidly dissipated as heat)
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8
Q

practical meaning of high/low ΔEo’

A
  • low ΔEo’ = willing to lose electrons (low affinity for e), be oxidized
  • high ΔEo’ = willing to pick up electrons (high affinity for e), be reduced
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9
Q

relative efficiency of oxphos

A

approx 40% of energy that goes through oxphos is captured to generate ATP

other 60% is lost as heat

relatively efficient!

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10
Q

breakdown of oxphos complexes and their critical components

A

Complex I (aka NADH dehydrogenase)

  • Fe-S center
  • FMN

Complex II (aka succinate dehydrogenase)

  • Fe-S center
  • uses FAD/FADH 2 to pump e to CoQ
    • generates less ATP bc it doesnt contribute to the proton gradient!

Coenzyme Q (aka Q10, CoQ, ubiquinone)

  • hydrophobic/lipophilic molecule in inner membrane

Complex III (aka cytochrome c reductase)

  • Fe-S center
  • Cyt b
  • Cyt c1

cytochrome c

  • water soluble; moves in vicinity of outer face of inner membrane, shuttling electrons one at a time from Complex III to Complex IV

Complex IV (aka cytochrome c oxidase)

  • Fe, Cu
  • Cyt a
  • Cyt a3
  • transfers 2 e to 1/2 O2: catalyzes 1/2O2→H2O

Complex V (aka ATP synthase, F0/F1 ATPase)

  • F0: transmembrane proton pump, inhibited by oligomycin
  • F1: ATP synthesizer
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11
Q

stops on etc for an electron

A

NADH → complex I → CoQ → complex III → cyt c → complex IV → O2

on their journey from NADH to oxygen, e stop at…

  • 4 protein complexes in inner mito membrane: I, II, III, IV
  • 2 mobile carriers
    • lipid soluble CoQ (inner mito mem)
    • water soluble cyt c

overall, releases energy used for ATP synth and generates water

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12
Q

NADH

  • role
  • where it comes from
A

NADH carriers e obtained from metabolism through glycolysis and/or TCA cycle to the etc

  • if obtained from glycolysis, NADH is in cytosol
    • has to be transported in via _____
  • if obtained through TCA cycle, NADH is already in mito matrix, ready to go
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13
Q

bypass reactions

  • definition/naming
  • 3 rxns
A

reactions that produce FADH2, and therefore allow for insertion of 2e- into the etc at CoQ, effectively bypassing complex I

  • complex II/succinate DH (inner part of inner mito mem)
  • G3P DH (outer part of inner mito mem/intermembrane space)
  • fatty acyl CoA DH (inner part of inner mito mem/mito matrix)
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14
Q

chemiosmosis and complex V

A

ATP synthase aka F1F0 ATPase

  • F0 subunit pumps e from intermembrane space into matrix
  • forces the turning of a shaft that in turn causes F1 subunit to rotate
    • rotational energy allows the synthesis of ATP
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15
Q

prerequisites for ox phos to take place

A
  1. availability of reducing agents: NADH, FADH2
    * from glycolysis, FA oxidation, TCA cycle
  2. pH gradient (proton motive force, low pH in intermembrane space)
  3. terminal oxidizing agent (O2)
  4. high ADP/ATP ratio
  5. sufficient mitochondria with the enzymes and metab machinery for oxphos to take place
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16
Q

what factors limit ATP synthesis during strenuous exercise

A

exercise builds muscle proteins and glycogen, but also increases number of mitochondria available for ATP synth

  • during strenuous exercise, ATP synth is limited by inadequate O2 delivery and/or inadequate rate of O2 utilization [shortfall in capacity of mito machinery to make ATP necessary]
    • _​_leads to fatigue, anaerobic resp
17
Q

where does the ADP for ATP synth come from?

A

most of the ADP generated by ATP dephos are in cytoplasm, need to be transported into mito matrix before ATP synthase can do its thing

  • move through porins outer mito mem → intermembrane space
  • move through ATP/ADP antiport aka ATP/ADP translocase aka adenine nucleotide translocase/carrier
    • need a pH gradient for the pump to work correctly!
18
Q

regulation of respiration

A

coupling of e flow through etc and ATP synthesis means that O2 consumption is dependent on availability of ADP

  • high ATP/ADP leads to slowing oxphos/TCA/glycolysis down
  • low ATP/ADP does the opposite
19
Q

hypoxia: consequences

A

decrease in O2 deliver → less mito etc activity → less ATP made → less Na/K ATPase activity

  • increased Na → cellular swelling, increased membrane permeability
  • effect of Ca and impact on mitochondria? check slides/email Abali
20
Q

role of inhibitors of oxphos

A

bind to a specific etc complex and stop the redox rxns/electron transfer

21
Q

role of uncouplers on oxphos

A

breaks the link between rate of electron transport and ATP synthesis

  • energy dissipated as heat
  • body - sensing high ADP/ATP ratio - will keep pumping e into the uncoupled pathway → overheating
22
Q

atractyloside

A

inhibitor

target: ATP/ADP antiporter = ATP/ADP translocase = adenine nucleotide translocase/carrier

  • blocks transport of ADP into matrix → ATP synthesis stops, H gradient builds up, e transport stop
  • poisoning most common in Mediterranean, N African areas among children
    • flower of Atractylis gummifera L. is mistaken for artichoke + sweet tasting so kids ingest it :(
23
Q

amytal

(amobarbital)

A

reversible inhibitor

target: NADH DH = complex I

can still make some ATP via e transported through bypass rxns, but not nearly as much as before

  • aka truth serum
  • used to treat anxiety, insomnia, epilepsy
  • can limit production of ROS, protect cardiac muscle during ischemia/reperfusion
24
Q

rotenone

A

inhibitor

target: NADH DH = complex I

can still make some ATP via e transported through bypass rxns, but not nearly as much as before

  • naturally occuring pesticide aka fish poison
25
Q

antimycin

A

inhibitor

target: complex III

  • binds to cyt b in reduced state, effectively freezing etc
    • complex I, CoQ, complex II fully reduced
    • cyt c, complex IV fully oxidized
  • antifungal used against plants
26
Q

cyanide (CN)

A

inhibitor

target: oxidized form (Fe+3) of heme iron → oxidized complex IV (cyt oxidase a3)

  • stops ATP production
  • prevents formation of water, i.e. delivery of e to final oxidizing agent
27
Q

how to reverse cyanide poisoning

A

CN likes to bind with MetHb (reversible rxn)

  • force the conversion of 10-15% Hb (Fe+2)→ MetHb (Fe+3)
    • give CN another option for binding, while preventing the inhibition of complex IV

tx: nitrite, followed by thiosulfate

  • nitrite turns Hb → MetHb
  • MetHb binds CN → CN-MetHb
  • S2O3 binds to create soluble molecules that can be excreted → SCN + SO3
    • SCN also toxic, but less so, and excreted
28
Q

oligomycin

A

inhibitor

target: Fo subunit of complex V (ATP synthase)

blocks e transport into matrix through Fo subunit, leads to buildup of H gradient, stops etc

mostly applicable in lab setting

29
Q

uncouplers: mech of action

A

allow H to move from intermembrane space back into matrix independently of ATP synthase

  • uncouples electron transfer (and H gradient produced) from phosphorylation of ADP
    • ATP synthase is defunct

consequences

  • levels of ADP/ATP are no longer reg/control mechanisms for resp rate
    • resp rate accelerates, TCA cycle ramps up, e transfer to O2 picks up
  • dissipation of proton gradient generates a ton of heat
30
Q

uncouplers: examples

A

1. membrane damaging molecules

  • make inner membrane permeable to H, dissipate H gradient
  • AraC (cancer therapeutic agent), AZT (HIV tx)

2. mobile proton carriers

  • lipophilic weak bases: pick H up in intermembrane space, carry to matrix
  • high dose aspirin, DNP
  1. proton channels
    * thermogenin (UCP-1)
31
Q

physiological means of heat production

  • role of proton channels
A

two ways to generate heat:

  1. shivering : local contraction, upreg of resp rate
  2. thermogenesis : uncoupling action in brown adipose tissue (BAT)
  • imp in babies: dont know how to shiver, but have a kite-shaped store of BAT by neck for heat gen
  • adults: residual BAT and upregulation of UCP-1 activity in prolonged exposure to low temp

BAT is well-vascularized, lots of mito, lots of UCP-1

32
Q

regulation of UCP-1

A

cold triggers hypothalamus → norepi released → cAMP pathway → TAG degradation, FA release

  • in BAT cell, FA release activates UCP-1
  • proton gradient dissipated as heat
33
Q

getting cytosolic NADH into the mito matrix

A

no NADH cotransporter!

use transporters/shuttles instead

  1. malate/aspartate shuttle
    * heart, liver, kidneys
  2. glycerol phosphate shuttle
    * brain, sk muscle
34
Q

reversible pathway of getting NADH into mito matrix

  • steps
  • major advantage
A

reversible pathway

  • start with NADH in cytoplasm, generate NADH in mitochondria WITH NO LOSS (can still generate 3ATP/NADH)

in cytoplasm:

  • cytosolic NADH drops off 2 e
    • oxaloacetate → malate [via cytosolic malate dehydrogenase, cMD]
  • malate moves through outer mem porin and through the malate/alpha-ketoglutarate shuttle into mito matrix

in mitochondria:

  • malate drops off 2 e to turn NAD+ → NADH
    • malate → oxaloacetate [via mitochondrial malate dehydrogenase, mMD]
    • OAA → alpha-KG [via AST, which takes Glu → Asp]
  • alpha-KG moves through malate/alpha-ketoglutarate shuttle and porin back into cytoplasm

in cytoplasm:

  • alpha-KG → OAA [via AST, which takes Asp → Glu]

at the end of it all, you have…

regenerated your carriers and “moved” NADH into mito matrix, where it can pump e into complex I

35
Q

irreversible pathway of getting NADH into mito matrix

  • steps
  • why?
A

irreversible pathway

  • start with NADH in cytoplasm, generate FADH2 in mitochondria WITH LOSS
    • go from potential for 3ATP/NADH → 2ATP/FADH2
  • why? used by brain/heart/active muscle because pathway allows e to be transported continuously

in cytoplasm:

  • cytosolic NADH drops off 2 e
    • DHAP → G3P [via cytosolic glycerol3P dehydrogenase, cGPDH]
  • G3P moves through porin into intermembrane space

on outer surface of inner mito mem:

  • G3P drops off 2 e to turn FAD → FADH2
    • G3P → DHAP [via mitochondrial glycerol3P dehydrogenase, mGPDH]
  • FADH2 drops its e off at CoQ/ubiquinone as usual, and they make their way to complex III and beyond
36
Q

summary: malate aspartate shuttle

A

transfers reducing eq of cytoplasm NADH → mito NADH

  • only works if [NADH]/[NAD+] in cyto > mito
  • reversible
  • 3 ATP/NADH
37
Q

summary: glycerol phosphate shuttle

A

transfers reducing eq of cytoplasm NADH → mito FADH2

  • works under almost all conditions
  • irreversible
  • 2 ATP produced