Krebs cycle ,electron transport chain

Topics to be covered
Oxidative Decarboxylation of Pyruvate.
Krebs cycle & Anaphoretic reactions.
Electron Transport and Oxidative Phosphorylation.
components of respiratory chain,

coupling of electron transport and

oxidative phosphorylation,

energy yield of reduced coenzymes,

inhibitors and un couplers

Introduction
♣ During Cellular respiration
♣ cells consume O2 & produce CO2.
Oxidative Decarboxylation of Pyruvate
Pyruvate from glucose or AAs (i.e. Ala) passes into
mitochondria &
oxidized into acetyl-CoA (i.e., oxidation &
decarboxylation).
by multi enzyme pyruvate dehydrogenase (PDH)
complex.
PDH complex requires sequential action of three d/t
enzymes and five d/t coenzymes.
⇒.PDH complex

 functions as a unit consisting of 3 principal enzymes:

 .Pyruvate decarboxylase(pyruvate dehydrogenase) (E1) .
 .Dihydro lipoyl transacetylase(E2) .
 .Dihydro lipoyl dehydrogenase(E3)

coenzymes
1.thiamine pyrophosphate(TPP),
2.flavin adenine dinucleotide(FAD),
3.coenzymeA(CoA-SH)
4.nicotinamide adenine dinucleotide(NAD),
5.lipoate.
Reaction mechanism of pyruvate dehydrogenase complex.
mechanism
 Mechanism of conversion of pyruvate to acetyl CoA by pyruvate dehydrogenase complex.
Step 1: Pyruvate and thimine pyrophosphate (TPP) combine to form a condensation
product. Step 2: Pyruvate decarboxylase catalyzes release of carbon
 dioxide from the condensation product to form a hydroxyethyl intermediate. The latter is
attached to the reactive carbon of the
 TPP. Step 3: Transfer of acetyl group from the hydroxyethyl intermediate to the lipoic
acid (oxidized) occurs to form acetyl lipoic
 acid. The reaction is catalyzed by dihydrolipoyl transacetylase. Step 4: Transfer of an
acetyl group from the acetyl lipoic acid to
 coenzyme A (CoASH) occurs next, converting the latter to acetyl CoA. The acetyl CoA
then enters the TCA cycle. The other product of this reaction is reduced lipoic acid. Step
5: This step regenerates the oxidized lipoic acid (from the reduced lipoic acid),
 which then participates in the next cycle of reactions. This conversion is catalyzed by the
dihydrolipoyl dehydrogenase component
 of the enzyme complex, which catalyzes transfer of the reducing equivalents fi rst to FAD
and then to NAD_x0003_ .
 Overall: Pyruvate _x0003_ NAD_x0003_ _x0003_ CoASH _x0002_ Acetyl CoA _x0003_
CO2_x0003_ NADH _x0003_ H_x0003_ .
Reaction mechanism of PDH

Where :-E1: decarboxylation & oxidation;

E2: acetylation and
trasacetylation;E3:dehydrogenation
Regulation of PDH

 Protein kinase inactivates the enzyme by phosphorylation.

kinase activated by acetyl-CoA, NADH.H+ and ATP and
inhibitedby increased Ca2+concentration during
muscle exercise, CoASH, pyruvate, NAD+ and ADP.
PDH and glycolysis are inhibited
by high-energy potential, e.g., during FA
oxidation that produces large amount of
energy.
 Insulin stimulates PDH by stimulating protein
phophatase.
Calcium released during muscle contraction
stimulates PDH (by increasing phosphatase
activity) for energy Production.
Arsenic Poisoning and PDH

PDH& a-ketoglutarate DH are inhibited by arsenate.

 Arsenate binds tothiol(SH)groups of lipoic acid & makes it
unavailable to serve as cofactor.

Thiamine Deficiency and PDH-Complex:

•TPP important coenzyme for the reaction.
 thiamine deficiency disease (Beriberi &Wernicke-

Korsakoff syndrome) results in impaired oxidation of

pyruvate.
Tri carboxylic Acid Cycle (TCA Cycle)

(Krebs' cycle, Citric acid Cycle, TCA cycle or common pathway )

Terminal oxidation pathway for most biomolecules.

CHOs, lipids as well as proteins pour their partially

oxidized catabolites to complete catabolism.
Tri carboxylic Acid Cycle (TCA Cycle)

completely oxidized acetyl

CoA into CO2,

electron-containing H-transfer
to FADH2 and NADH.H+.

 Liberated much of free

energy from fuel molecules.
Acetyl-CoA + 3NAD++ FAD++ GDP + Pi +
2H2O CoASH+ 3NADH.H++ FADH2 + GTP
+ 2CO2.

Electrons and protons from these coenzymes

converted into H2O by respiratory chain coupled to
TCA…………
TCA cycle is an amphipathic pathway.

Site:-It occurs in mitochondria where enzymes required are located

free in the matrix or attached to inner mitochondrial membrane .
TCA……..
Mitochondrial Enzymes

Matrix
 Pyruvate dehydrogenase Complex. Inner Membrane.
 E1-Citrate synthase. E6-Succinate
 E2-Isocitrate dehydrogenases. dehase.
E8-ATPsynthase.
 E2– Aconitase.
 E3-a-Ketoglutarate dehydrogenases.
 E4-SuccinylCoAsynthase.
 E5-Fumarase.
 E7-Malate dehydrogenases.
 Fg: Reactions of the Krebs' cycle. The cycle is closely linked with electron transport
chain.
 Dysfunctional mitochondria as in cancer cells lead to accumulation
Energy yield
Acetyl-CoA + 3NAD++ FAD++ GDP + Pi
+ 2H2O CoASH+ 3NADH.H++ FADH2 +
GTP + 2CO2.
Each NADH molecule ,yields 3 ATPs =3X3=9ATP

 Single FADH2 :-1X2=2ATP

One GTP from =1 ATP

 oxidation of a single acetyl-coA via citric acid cycle

produces 12 ATP molecules.
 Pathways of aerobic glucose catabolism and their linkage to ATP
formation
Regulation of Krebs' cycle
Regulation occurs at the following sites:
Citrate synthase:
Allosterically:

Inhibited by ATP & Long chain fatty

acyl-coA.
Competitively Inhibited by Succinyl-CoA .

Also inhibited by product Citrate

Isocitrate dehydrogenase:
Allosterically:
Activated by ADP and NAD+.

Inhibited by ATP and NADH.

REGULATION…(CONT’D
-Ketoglutarate dehydrogenase complex
 Regulated by phosphorylation/dephosphorylation
mechanism.
In a manner similar to PDH complex.
 Inhibited by accumulation of
ATP, succinyl-CoA and NADH.H+

Respiratory chain itself exerts an overall control

Through OP being inhibited by excess ATP/ADP

ratio.
This ensures oxidation of reduced coenzymes .
Regulation of TCA cycle:
Rate controlling enzymes:

.Citrate synthatase.

.Isocitrate dehydrogenase.

.a- ketoglutarate dehydrogenase.

.Regulation of activity by:

.Substrate availability.

.Product inhibition

.Allosteric inhibition or
activation by other
intermediates.
In Vitro Inhibitors of Krebs' cycle
several toxic chemical inhibitors block several
reactions in Krebs'cycle.
Fluoroacetate inhibits aconitase enzyme. In the form of
fluoro acetyl CoA condenses with oxaloacetate to form
fluorocitrate that inhibits aconitase leading to
accumulation of citrate.
Arsenate inhibits both pyruvate and a-KG-
dehydrogenases.
Malonate or oxaloacetate inhibits succinate
dehydrogenase(competitive inhibition).
Mercury inhibit ssuccinate dehydrogenase.
Biological importance of Krebs'
cycle
Energy Production GTP FADH2,
NADH,
Oxidation of acetyl-CoA formed from
d/t fuels into CO2+ H2O.
It is a major source of succinyl-CoA
which is used for:
 hemoglobin and other porphyrins.
fatty acid synthesis (citrate).
urea and nucleotides (CO2).
It provides intermediates for synthesis
of nonessential AAs :
 a-KG can give rise to glutamic acid

by transamination.
 Oxaloacetate can give rise to

aspartic acid by transamination.

 oxaloacetate, malate and α-KG are utilized

in gluconeogenesis.
 Regulation of metabolic pathways.
Citrate (PFK1 of glycolysis).
Efflux of Intermediates from the TCA Cycle
Anaplerotic Reactions:
Continuous usage/withdrawal of TCA cycle
intermediates for anabolic or other uses render
the cycle depletion of its resources.
The intermediates can be replenished by a
number of reactions commonly called as‘
Anaplerotic(feeding)reactions’.
Major of these are: …"filling up" reactions...
E) Propionyl-CoA conversion into succinyl-CoA takes
place in liver.
F) Transaminated AAs into α-KG, fumarate &
oxaloacetate.
Most significant is the formation of oxaloacetate by
pyruvate carboxylase:
Pyruvate carboxylase inactive in absence of acetyl-CoA,
its positive allosteric activator.
Whenever acetyl-CoA, is in excess, it stimulates pyruvate
carboxylase to make more oxaloacetate, enabling cycle to
proceed.
…"filling up" reactions...
Electron-Transport chain & Oxidative phosphorylation
Contents:
The components of the respiratory chain.
The ATP synthase and oxidative

phosphorylation.
The mechanisms of coupling electron

transport & oxidative phosphorylation.

Uncouplers and inhibitors electron-

transport.
ETC & Oxidative phosphorylation
 OdP:A process that links oxidation of reduced Coenzymes to
phosphorylation of ADP.

 Energy-rich molecules and metabolic intermediates donate

e-s to specific coenzymes, to form their energy-rich
reduced coenzymes.

 These reduced coenzymes (NADH or FAD(2H)) can in turn,

each donate a pair of e-s to a specialized set of electron
carriers, collectively called the ETC.
ETC & OdP…
 As e-s passed down ETC, they lose much of their free
energy.

 Part of this energy can be captured and stored by

production of ATP from ADP and Pi called oxidative
phosphorylation.

 The remaining free energy is used for heat generation.

ETS & OdP…
Respiratory Chain or Electron Transfer Chain
 sequenceof enzymes & carriers responsible for
transfer of reducing equivalents from substrates to
molecular oxygen.
located within the mitochondria.

arranged in order of increasing redox potential i.e.

from NAD+ / NADH to O2/H2O redox couples.

ETC :a set of complexes of inner Mitochondrial
membrane that transfers e-’s from Reduced Coenzymes to
O 2.
Structural organization of respiratory chain

IMM. five distinct respiratory or enzyme

complexes, denoted as complex I, II, III, IV and

V.
 complexes I-IV carriers of electrons
 complex V responsible for ATP synthesis.
mobile electron carriers
NADH, coenzyme Q, cytochrome C and
oxygen.
 enzyme complexes (I-IV) and mobile carriers
are collectively involved in transport of
electrons,

 ultimately, combine with oxygen to

produce water.

 largest proportion of oxygen supplied to body

utilized by mitochondria for operation of
ETC.
ETS & OdP…
Electron transport components of respiratory chain:
 Complex I : NADH : ubiquinone oxido reductase.
 Complex II : succinate :ubiquinone oxido reductase.

 Complex III : Ubiquinol: cytochrome C oxido reductase.

 Complex IV : cytochrome a,a3:oxygen oxido reductase.

Respiratory Chain or Electron
Transfer Chain
Electrons transferred from NADH / FADH2 to oxygen
along a chain of electron carriers.
arranged in sequence/chain/ in the inner mitochondrial
membrane.
Respiratory Chain or Electron
Transfer Chain
Electron transport from NADH
NADH-Q reductase (Complex I),
Electrons flow from NADH to oxygen through three of
complexes.
 two electrons and two H+ from NADH to
 FMN (flavin mononucleotide).

to iron–sulfur proteins(FeS).

to ubiquinone (CoQ)-a small lipid-soluble

molecule in the inner mitochondrial membrane.

Electron transport from NADH
CoQ-cytochrome c oxido reductase (Complex III)
 cytochrome b and cytochrome c1, as well as FeS.
 Electrons pass from ubiquinol (CoQH2) through
cytochrome b, FeS and cytochrome c1
 to cytochrome c.
Electron transport from NADH
Cytochrome c
small water soluble heme protein.
loosely bound to outer surface of IMM.
binds to CoQ-cytochrome c oxido reductase complex
and accepts an electron via Fe3 to Fe2 transition.
Then binds to cytochrome c oxidase complex
(Complex IV) and donates the electron.
Electron transport from NADH
Cytochrome c oxidase (Complex IV)

contains cytochrome a (paired with, CuA)&

Cytochrome a3 (paired with copper atom, CuB).
from cytochrome c to oxygen .
Electron transport from FADH2
Re -oxidation of FADH2
 via succinate–CoQ reductase (Complex II),
 integral protein of IMM.
 link b/n TCA cycle and ETC.
 Contain :-Succinate dehydrogenase and FeS clusters.

two electrons pass from FADH2 to FeS and to

ubiquinone (CoQ).
 enter main ETC and cause H ions to be pumped out.
 But Complex II itself not H –pump.
Mechanism of ATP formation and ATP
synthase
oxidative Phosphorylation involves
Electron Transport system.

Electrochemical gradient.

ATP synthesis.
 Electron Transport system.
Mechanism of ATP formation and ATP synthase

 Arranged from least to highest standard redox potential (E’o) i.e. most
reduced to most oxidized
 from NADH (most negative redox potential, least affinity)
 to oxygen (most positive redox potential, highest affinity for electrons).
 Formation of H gradient
Protons /H+/ translocated from matrix
into intermembrane space as e-s move
from reduced coenzymes to O2.
generation of electrochemical
gradient.
Formation of H gradient
Formation of H gradient
Mechanism of ATP formation and ATP synthase
 ATP synthesis.
oxidative phosphorylation

 synthesizing ATP from ADP and Pi coupled with

electron transport chain.

 complex V of inner mitochondrial membrane.

 synthesis of ATP (phosphorylation) occurs when NADH and
FADH2 oxidized.

 energy liberated by ETC create a proton gradient.

 proton gradient couples ETC and ATP synthesis.

ATP synthase(Complex V) -a rotatory engine
 located in the inner mitochondrial membrane
 Carried out actual synthesis of ATP.
 driven by proton-motive force :-pH gradient and membrane
potential.

 Two subunits F1 and Fo; F stands for coupling factor.

1) F1 :matrix side:-catalyzing actual production of ATP from ADP

and Pi.
2) Fo:-Proton conducting unit :-protons pumped out during electron
flow, return back to mitochondrial matrix.
Energy for ATP synthesis released when protons enter back in to
matrix through Fo .
ATP synthase(Complex V)-a rotatory engine

 Proton pump: across inner mitochondrial membrane from matrix to

intermembrane space at Complexes I, III, and IV.
 creates electrical gradient (more positive charges outside ) and

 pH gradient (lower pH on outside membrane).

 protons reenter matrix through a channel in membrane-spanning
domain (Fo ) of Complex V,
 driving rotation of Fo and, at the same time, dissipating pH and
electrical gradients.
Fo rotation causes conformational changes in the extra-
membranous F1 domain that allow it to bind ADP + Pi,
phosphorylate ADP to ATP, and release ATP.
Stoichiometry or energy production from reducing equivalents

how many protons are pumped outward by

electron transfer from one NADH to O2??????
How many protons must flow inward
through Fo of ATPase to drive synthesis of
one ATP?????

Overall, each NADH donates 2e-s, equivalent to reduction of 1⁄2O2 .

A general accepted estimate of stoichiometry of ATP synthesis is:-
 4H+ are pumped at complex I, III, & 2H+at complex IV (a total of 10)
and (6 for succinate).
 4H+s are translocated for each ATP synthesized.
Stoichiometry
 NADH oxidation :-H pumps; Complex I, III and IV.
 FADH2 oxidation:-H Pumped out only by Complex III and IV.
 ATP from FADH2 < NADH.
 ATP production by complete oxidation of reduced
coenzymes
Coenzyme ATP/Mole
NADH 10/4 2.5
(3)
FADH2 6/4 1.5
(2)
Coupling and respiratory control
Normally Electron transport tightly coupled to ATP synthesis.

 i.e.
electrons do not flow through ETC to oxygen unless
ADP simultaneously phosphorylated to ATP.

 ATP not synthesized unless electron transport provide

proton gradient.

 Thus oxidative phosphorylation needs NADH or FADH2,

oxygen, ADP and inorganic phosphate.
Coupling and respiratory control
The actual rate of oxidative phosphorylation set by
availability of ADP.
 If ADP added to mitochondria, rate of oxygen

consumption rises as electrons flow. Or electrons flow

down the chain only when ATP synthesis needed.

 whenall ADP phosphorylated to ATP(high ATP +low

ADP);-
rate of oxygen utilization falls.
No electron transport occurs,
NADH and FADH2 build up.
Uncouplers of ETC
 Uncouple phosphorylation of ADP from electron
transfer.
Electron transport continue to function,
leading to oxygen consumption,
but phosphorylation of ADP inhibited.
 disrupt permeability of inner membrane (either physically
or chemically) and
 proton gradient/proton motive force/ across

the IMM is dissipated.

 release of protons across membrane coupled with
release of heat ,rather than ATP formation.

 NADH oxidation continues rapidly, oxygen consumption is

increased, and ATP production decreases.
Heat Generation and Uncouples of oxidative Phsph...
When protons leak back into matrix without going
through ATP synthase pore, they dissipate
electrochemical gradient across membrane without
generating ATP.
…………..called “uncoupling” oxidative
phosphorylation.
May with some chemical cpds, “uncouplers” or
“Ionophores”.
Or it may occurs physiologically with uncoupling
proteins(UCPs):that form proton conductance channels
through membrane.
Uncoupling agents create pores in the IMM,

Uncoupling of OdP results in increased O2consumption

and heat production as e-flow and H+ pumping attempt to
maintain the ECG.[electrocardiogram]
Uncoupling proteins (UCPs)
 Mitochondrial uncoupling is mediated mainly by uncoupling
proteins (UCPs), :-
brown adipose tissue
uses almost 90 % of its respiratory energy for
thermogenesis.
in response to cold, newborns, & hibernating
animals.
Their mitochondria contain a uncoupling protein 1,
UCP1(thermogenin).
 Use hormonal stimulus.

initiated by release of free fatty acids.

activation of fatty acid oxidation and heat
generation.
 acts as a channel in the IMM.

control permeability of membrane to protons.

Normal fat is white ,fat conjugated with protein such as thermogenin forms brown .
 Cold or excessive food intake stimulates release of NE inturn it activates TAG-
lipase that releases FAs for oxidation. released free fatty acids bind to UCP1
triggering an uncoupling of the proton gradient.

 The proton conductance protein, thermogenin, activated, and protons are

brought into matrix. This stimulates electron transport chain, which increases
its rate of NADH and FAD(2H) oxidation and produces more heat.
Uncouples of oxidative Phsph...
 Synthetic uncouplers :- oligomycin , dinitrophenol, dnp, 2,4-
dinitrophenol ,high does, aspirin.
Oligomycin a natural antibiotic from Streptomyces, inhibits
mitochondrial ATP synthase.
 binding to oligomycin sensitivity-conferring protein

(OSCP) of F0 portion.
 closing H+channel &prevents reentry of proton into

mitochondrial matrix.
Uncouples of oxidative Phsph...
Dinitrophenol, DNP,
small lipophilic molecule.
DNP is a proton-carrier and can easily diffuse through IMM.
allowing protons to flow back into matrix of the
mitochondria.
In high doses, drug aspirin and other salicylates
uncouple oxidative phosphorylation.
fever :toxic overdoses of these drugs.
Inhibitors of ETC

Inhibitors
 bind
to one of components of ETC & block
transport of electrons.
 accumulation of reduced components

before inhibitor &

oxidized components after that step.
 reduce both ATP generation and oxygen

consumption.
examples include
carbon monoxide(Complex IV ),cyanide (Complex IV),
rotenone (Complex I), antimycin C (Complex III), &
oligomycin, (Complex V inhibitor)
Carbon monoxide;-a common inhibitor of ETC
binds with reduced form of iron in hem groups (Fe++) in

Cytochrome Oxidase (Complex IV ).

blocks transfer of electrons to oxygen.
 bindComplex IV & halt passing of electrons.
 pumping of protons diminished & ATP not
produced.
Rotenone :insecticide, extracted from roots of plant

strong inhibitor of complex I(NADH-CoQ
reductase complex)
Inhibits transport of electron from Fe-S to
ubiquinone.
Certain tribes use it as a fish poison which
paralyse fish.
Oligomycin:antibiotic
Potent inhibitor of ATP synthase complex.

Binds Fo domain closing H- chanal

Prevents phospn. Of ADP.

Oligomycin Sensitivity-conferring Protein[OSCP]

Inhibitors/ Un couplers
Reference

Lehninger: Biochemistry4ed.

Marks, M. L. Marks ’ Basic Medical

Biochemistry: A Clinical Approach , 2nd

Edition(second.).
Harvey, R. A. . Lippincott’s Illustrated Reviews.

Murray, R. K., & Davis, J. C. Harper ’ s Illustrated

Biochemistry.