ATP Generation Flashcards
the main source of energy for ATP synthesis in oxygen dependent tissues
oxidation of acetyle CoA in the tricarboxylic acid cycle and the accompanying oxidation of reduced coenzyme products
what does Pyruvate Dehydrogenase do?
Pyruvate -> Acetyl CoA
and generates NADH & CO2 in the process
advantages of a multienzyme complex
increase rxn rate & minimize side rxns
enables efficient transfer of intermediates between its different active sites
preventing loss of intermediates to other processes (they are usually covalently linked to the enzyme)
E1 of PDH
E1 decarboxylase
requires the catalytic coenzyme prosthetic grp:
TPP thiamin pyrophosphate (vit B1)
E2 of PDH
E2: dihydrolipoyl transacetylase
- transfers acetyl group to CoA
Lipoamide (catalytic coenzyme)
swinging arm to transfer electrons from E1 to E3
E3 of PDH
E3: dihydrolipoyl dehydrogenase
- regenerates the oxidized form of lipoamide(E3)
FAD (flavin adenine dinucleotide)
uses NAD+
what inhibits PDH? how?
Arsenite
reacts w the two thiol groups of reduced lipoamide, preventing reconversion to oxidized (s-s) form, therefore inhibiting its function
in the case of PDH deficiency, why is lactiacidemia expected?
pyruvate can be converted into lactate by the action of lactate dehydrogenase
inhibition of pyruvate dehydrogenase
products down regulate:
NADH (E3)
acetyl CoA (E2)
*both are comp inhibitors
most imp control: covalent inhibition:
P’lation of E1 serine residue
- the kinase that does this is stimulated by ATP/NADH/acetyle CoA and inhibited by pyruvate
*the PDH kinase and phosphatase are physically associated w/ PDH supramolecular complex
activation of pyruvate dehyrogenase
substrates up-regulate:
Ca2+
Insulin - activates the phosphatase
turn off the kinase (release the covalent inhibition) - ADP & Pyruvate
(up-regulation in response to low energy)
catalytic cofactors used by PDH
coenzyme prosthetic groups:
TPP (E1)
lipoamide (E2)
FAD (E3)
stoichiometric cofactors used by PDH
co-enzyme substrates:
CoA (E2)
NAD+ (E3)
overview of the mitochondiral ETC
4 enzymes in the inner membrane
-co-localization brings redox centers together
Electron flow driving by redox potential of components (most neg to most pos)
Enzymes use coenzymes as electron carriers (flavins, iron sulfur centers, heme)
mitochondrial membrane phospholipid important in ETC organization
cardiolipin
deficit in cardiolipin synthesis leads to ETC dysfunction and mito-based disease (Barth syndrome; myopathy)
cytochrome c location and key features
tethered to the outside of the inner membrane by cardiolipin -> located in the space bet the two mito membranes
unique bc it is soluble
its location gives it considerable mobility -> interacts with Complexes III & IV
How does PDH fit in with the TCA?
It is not part of the tricarboxylic acid cycle
Closely linked in terms if mito location and function
Reaction is a critical control point in utilization of pyruvate in the TCA cycle
Location of TCA enzymes?
Location of ETC enzymes?
TCA - mitochondrial matrix
(expt succinate dehydrogenase - inner mito membrane)
ETC - inner mitochondrial membrane
Initiating rxn of the TCA
Citrate Synthesis
condensation of Acetyl CoA + OAA -> Citrate
Irreversible due to hydrolysis of the thioester bond of Acetyl CoA
Aconitase
dehydrase/hydrase
removes H2O and adds it back, resulting in different location of the OH to prepare it for Isocitrate Dehydrogenase (IDH)
The ox-red rxns of the TCA:
4 ox-red rxns of the TCA:
3 - NAD reduction
1 - FAD reduction (succinate dehydrogenase)
the specific intermediates of the TCA that lead to generation of ATP via oxidative phosphorylation via ETC
FADH2
NADH
Isocitrate dehydrogenase
Alpha-ketoglutarate dehydrogenase
Produce CO2 expired (along w/ PDH)
Alpha-ketoglutarate is analagous to PDH
in structure/function, E3 is same
substrate level phosphorylation of the TCA
Alpha-ketoglutarate DH
Succinyl thiokinase
Generates GTP from succinyl CoA
Succinyl thiokinase
aka Succinyl CoA synthetase
Ex of energy coupling where common intermediate can be found bound to the enzyme
thioester bond is high energy
In the TCA; CO2 is generated by
(PDH - not really part of the TCA)
Isocitrate dehydrogenase
Alpha-ketoglutarate dehydrogenase
In the TCA; NADH is generated by
Isocitrate DH
Alpha-ketoglutarate DH
Malate DH
In the TCA; FADH2 is generated by
Succinate DH
In the TCA; GTP is generated by
Succinyl thiokinase
via the hydrolysis of the thioester bond of Succinyl CoA
Overall the TCA is ___ & ___ , and operates under Anaerobic/Aerobic conditions.
exergonic & irreversible
aerobic
The three irreversible steps of the TCA
Citrate synthase
Isocitrate DH
Alpha-ketoglutarate DH
prevent cycle from reversing direction
Irreversibility may be the result of
highly negative delta G
hydrolytic bond cleavage
Regulation of Citrate synthase
inhibited by citrate
no allosteric regulation
Regulation of Isocitrate DH
Inhibited allosterically by:
NADH
ATP
Activated by
ADP (major)
Ca2+
Primary site of control.
Regulation of Alpha-ketoglutarate DH
Activated by Ca2+ Inhibited by: NADH Succinyl CoA (product) GTP (product)
TCA as source of metabolic substrates:
Citrate ->
Fatty acid and Sterol synthesis
TCA as source of metabolic substrates:
alpha-ketoglutarate ->
Amino acid synthesis -> Neurotransmitters
TCA as source of metabolic substrates:
Succinyl CoA ->
Heme synthesis
TCA as source of metabolic substrates:
Malate ->
Gluconeogenesis
TCA as source of metabolic substrates:
Oxaloacetate ->
Amino acid synthesis
What drives electron flow? How does this apply to the ETC carriers?
Redox potential of the components; most negative to most positive
Lower affinity complex (-E) to Higher affinity complexes (+E)
Concentrations of components allows fine tuning of flow
Protons are pumped across ___ to ___ forming an ____
the inner mitochondrial membrane
the intermembrane
electrochemical gradient
positive redox potential vs. negative redox potential in terms of affinity for electrons
positive - higher affinity
negative - lower affinity
(than a proton)
delta G =
-nFdeltaE
positive reduction potential => favorable delta G
n = # of electrons F = Faraday constant - energy produced by an electron in a 1volt potential
ETC - Location of:
Protein Complexes
CoQ
Cytochrome C
Protein Complexes - intimately assoc w the membrane
CoQ - in the lipid core
Cytochrome C - intermembrane space
Proton Pump Complexes
I, III, IV
NAD vs NADP
NADH: source of electrons from TCA and PDH functions in ox rxns of catabolism most abundant ox-red system in cell cosub coenz for Complex I
NADP:
functions in reductive synthesis
has a phosphate grp (only structural diff)
Both NAD & NADP:
accept 2 electrons and a proton in a form similar to a H- (hydride ion)
weak oxidants
exist free in cell
behave as true co-enzyme co-substrates (bc are weakly bound to enzymes)
Complex I
NADH - ubiquinone oxidoreductase
NADH (substrate) electrons are passed to
FMN (the flavoprotein prosthetic group) then to Ubiquinone via series of FeS centers
4 H+ are pumped
Complex II
Succinate - ubiquinon oxidoreductase, a flavoprotein
Electron flow: succinate via FAD and FeS to Ubiquinone
no H+ are pumped
Which one exists primarily in its reduced form, NADH or NADPH?
NADPH (functions in reductive synthesis)
remember NAD is involved in oxidative phosphorylations so you want it in its oxidized form
Flavin nucleotides
tightly bound coenzyme prosthetic grps
1 or 2 electron transfers
semiquinone intermediates are relatively stable
in ETC do not react w O2 directly; are reox by other components of chain
Complex I
Iron sulfur prosthetic groups
most abundant redox cofactors of ETC
Complexes I,II, III
contain equal amts non-heme iron & sulfide
single electron carriers
Ubiquinone (coenzyme Q)
passes electrons from Complexes I & II to III
ubiquinol = reduced ubiquinone
lives in the hydrophobic lipid core of the mem -> freely diffusable
1 or 2 electron transfers
Complex III
Ubiquinone- cytochrome c oxidoreductase
3 prosthetic groups that = redox centers:
cytochrome b, cytochrome c1, Fe-S protein
Electron flow: UQH2 (ubiquinol) is oxidize as 2 cytochromes are reduced
Electron path is called the Q cycle (within Complex III)
use heme prosthetic groups as electron carriers
Complexes III and IV
Cytochrome C
hemes of the ETC
the cytochromes
a/b/c - based on the structure of the heme
same heme as hemoglobin
1 electron transfers
do not bind O2 -> site occupied by aa side chain (exception; cytochrome a3 - last cytochrome of chain, binds O2 and is ox as a result, binuclear center w Cu in Complex IV)
Complex IV
contains antibody fragment for crystallization
Electron Flow: Cyt c -> Cu (a) (subunit II) -> binuclear center (cyto a3 and Cu b) where O2 is reduced to H2O
4 protons pumped
net: 2 H+ released to cytosol for every 4 taken up from the matrix
ROS forms by incomplete red of O2
cytochrome a3
Complex IV
last cytochrome of chain
Special; binds O2 and is ox as a result, binuclear center w Cu
overview of electron flow in Complex I
NADH -> FMN -> FeS -> CoQ
overview of electron flow in Complex II
FADH2 -> FeS -> CoQ
overview of electron flow in CoQ
CoQH2 -> cytochrome c via Complex III
req ISP, cyto c1, cyto b
overview of electron flow in Cytochrome C
Fe2+ -> Cu a of Complex IV
overview of electron flow in Complex IV
from Cu a -> cyto a1 -> cyto a3-Cub binuclear center -> O2 => water
how many electrons are required to reduce O2 to H2O?
4
superoxide dismutase rxn
2O2- -> H2O2
catalase rxn
H2O2 -> 2H2O + O2
Complexes of the ETC that pump electrons
Complexes I, III, IV
due to great enough energy diff bet donor of elec and receiver
Complex II doesnt pump bc not enough energy
Oxidative phosphorylation
ATP synthesis linked to ETC and O2 reduction
the proton gradient drives synthesis instead of P’lated intermediates donate electrons
O2 consumption dependent on ADP availability
Relative pH; Matrix, Intermembrane space, Cytosol
Intermembrane Space equivalent to Cytosol
Matrix - more basic (bc pumping H out)
= Chemiosmotic Hypothesis - proton-motive force drives ATP synthase
Chemiosmotic Hypo Rational
- no chemical intermediates
- proton gradient = primary energy source for ATP synthesis
- one directional proton pumps exists for producing and using said electrochem gradient (anisotropically oriented)
ATP synthase
is an ATPase as well
F1 - produces ATP, alpha3 beta3 dont rotate
beta binds ADP - O=open state, L=loose stabilizes ADP - P, T=tight ATP formed
Fo - protons spin the c ring
gamma subunit - rotar, not symmetric
Respiratory control
rate of respiration determined by the rate of ATP synthesis
ATP synthesis limited by availability of ADP or Pi
Reduction potential difference required for ATP synthesis
0.3 V
only Complexes I, III, IV
How many protons per 1 ATP synthesized
3H+
Reversal of the ATP-linked pump
when ATP levels in mito drop
decrease, then reversal of the proton-pumping by the F1Fo ATPase
reversal -> ATP synthesis
Oxphos; Respiratory chain inhibitors
block electron transport
CN, CO
Oxphos: Phosphorylation ihibitors
inhibit the F1Fo ATPase
Oligomycin - prevents mvmt of protons through ATP synthase => reduced O2 consumption
Osphos:Uncouplers
allow protons to leak, depletes gradient, need to make more gradient so O2 consumption increases
Dinitrophenol:
uncouples ATP syn and proton pump, equilibriates pH across inner mem
UCP1 / thermogenin:
brown adipose, mem spanning protein, allows Hs to enter mito w/o ATP formation -> heat generation
(short-circuits proton battery of mito)
electrogenic transporters
affected by the membrane potential
so requires the proton gradient
ex. Ca2+ uniport uptake, ATP/ADP exchange
how is ADP taken into the mito, and ATP released?
electrogenic antiporter
1:1
homodimer but 1 binding site -> alternates how it faces (matrix/intermem)
pH gradient drives ATP out ADP in
how is Ca2+ taken into the mito?
electrogenic uniporter
dependent on proton gradient
high conc -> pore formation, cyto c release, and apoptosis
how is phosphate taken into the mito?
phosphate translocase (electrogenic symporter) 1 H : 1 Pi
Malate-Aspartate Shuttle
transport shuttle for NADH -> get electrons into mito via NADH req; 2 carriers, 4 enzymes alpha-ketoglutarate -> OAA Malate Aspartate -> Glutamate
Oligomycin
Dinitrophenol
reflect coupling of O2 consumption w ATP synthesis
Dinitrophenol:
Uncoupler
uncouples ATP syn and proton pump, equilibriates pH across inner mem by freely crossing the membrane
hydrophobic weak acid
UCP1 / thermogenin:
Uncoupler
brown adipose, mem spanning protein, allows Hs to enter mito w/o ATP formation -> heat generation
(short-circuits proton battery of mito)
Ancillary energy-coupled reactions of mitochondria
do not req ATP but consume energy using the proton gradient
relieve resp control and stimulates e transport
inhibited by respiratory inhibitors & uncouplers
unaffected by phosphorlyation inhibitors
ex:
-Ca uptake, upregs PDH, uniporter
-ATP & Aspartate transport to cytosol, reduces pH gradient
-Energy coupled transhydrogenation (need NADPH to keep glutathione in its functional reduced state GSH)
Leber’s Hereditary Optic neuropathy
lowered activity Complex I due to single bp mutation in 3 mitochondrial disease
Esercise intolerance
mutations in cyt b of complex III
not maternally inheritied
mutations seem somatic in muscle tissue
Mitchondria and Apoptosis
Pore formation -> release of cyto c
cyto c complexes with pro-apoptotic factors (Apaf1) & protease precursors (pro-caspase 9) = Apoptosome
Apoptosome -> act of caspases
Pore formation & protein loss => Bioenergetic Crisis = inability to produce ATP, prelude to apoptosis
Parkinson’s disease
mito ETC dysfuntion, including Complex I, increasinly viewed as having an important link
