Oxidative Phos - Abali 3/7/16 Flashcards
why do we need oxygen?
what types of work are driven using energy from ox phos?
required for total oxidation of glucose; oxygen is fully reduced to pick up the electrons given off in that process
- mechanical work (muscular contraction)
- chemical work
- transport work
oxidative phosphorylation
- parts
- process
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
2 processes that generate ATP
- % of ATP generated
- how does the process sustain itself (NAD+)
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
overall role of cardiopulm system in context of biochemical processes
- two biochemical scenarios that potentiate O2 delivery in tissues
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
functional parts of the mitochondria and what they contain
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)
redox reactions: players
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
how is energy harvested through the etc?
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)
practical meaning of high/low ΔEo’
- 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
relative efficiency of oxphos
approx 40% of energy that goes through oxphos is captured to generate ATP
other 60% is lost as heat
relatively efficient!
breakdown of oxphos complexes and their critical components
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
stops on etc for an electron
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
NADH
- role
- where it comes from
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
bypass reactions
- definition/naming
- 3 rxns
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)
chemiosmosis and complex V
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
prerequisites for ox phos to take place
- availability of reducing agents: NADH, FADH2
* from glycolysis, FA oxidation, TCA cycle - pH gradient (proton motive force, low pH in intermembrane space)
- terminal oxidizing agent (O2)
- high ADP/ATP ratio
- sufficient mitochondria with the enzymes and metab machinery for oxphos to take place
what factors limit ATP synthesis during strenuous exercise
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
where does the ADP for ATP synth come from?
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!
regulation of respiration
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
hypoxia: consequences
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
role of inhibitors of oxphos
bind to a specific etc complex and stop the redox rxns/electron transfer
role of uncouplers on oxphos
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
atractyloside
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 :(
amytal
(amobarbital)
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
rotenone
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
antimycin
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
cyanide (CN)
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
how to reverse cyanide poisoning
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
oligomycin
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
uncouplers: mech of action
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
uncouplers: examples
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
- proton channels
* thermogenin (UCP-1)
physiological means of heat production
- role of proton channels
two ways to generate heat:
- shivering : local contraction, upreg of resp rate
- 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
regulation of UCP-1
cold triggers hypothalamus → norepi released → cAMP pathway → TAG degradation, FA release
- in BAT cell, FA release activates UCP-1
- proton gradient dissipated as heat
getting cytosolic NADH into the mito matrix
no NADH cotransporter!
use transporters/shuttles instead
- malate/aspartate shuttle
* heart, liver, kidneys - glycerol phosphate shuttle
* brain, sk muscle
reversible pathway of getting NADH into mito matrix
- steps
- major advantage
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
irreversible pathway of getting NADH into mito matrix
- steps
- why?
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
summary: malate aspartate shuttle
transfers reducing eq of cytoplasm NADH → mito NADH
- only works if [NADH]/[NAD+] in cyto > mito
- reversible
- 3 ATP/NADH
summary: glycerol phosphate shuttle
transfers reducing eq of cytoplasm NADH → mito FADH2
- works under almost all conditions
- irreversible
- 2 ATP produced
