RESPIRATORY CHAIN & OXIDATIVE PHOSPORYLATION AND KREBS CYCLE

Dr. Menorca
1ST SHIFTING

July 21, 2015

I. THE MITOCHONDRIAL MEMBRANE

B.

Electron Flow through the Respiratory Chain

C.

The Complexes of Respiratory Chain

**** Glycerohosphate Acyltransferase

II. THE RESPIRATORY CHAIN

A.

Role of Respiratory Chain of Mitochondria

1. Complex I
Catalyzes two simultaneous and obligately
coupled coupled processes

Note that most energy liberated during the BIOLOGIC

OXIDATION of carbohydrates, fatty acids and amino
acids is made available within the mitochondria as
REDUCING EQUIVALENTS (-H OR e-).

a.

Exergonic transfer Q of A HYDRIDE ION :Hfrom NADH and a proton H+ from the matrix

b.

Endergonic transfer of 4H+from the matrix to

the IMS (i. e. endergonic transfer of 4H+/2e-)

NADH + Q + 5H+matrix NAD + QH2 + 4H+IMS

2. Complex II

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a.

Electrons pass from SUCCINATE TO FAD, then

through the Fe S CENTERS then to Q
(SUCCINATE DH is the only membrane-bound
enzyme in the CAC).

b.

Others pass electrons at LEVEL OF Q but not

through Complex II.
i.

Acyl CoA DH flavoprotein first step in

the B- ox. of fatty acids. So... from
substrate (Fatty Acyl CoA) to FAD to
electron-transferring flavoprotein (ETF) to
Fe-S centers to Q.

Glycerol 3 PO4 DH In glycolysis.

So from substrate (Glycerol3-PO4) to
FAD to Q.
NET RESULT: pool of reduced UBIQUINONE
(QH2).
ii.

c.

3. Complex III REOXIDATION OF REDUCED Q

(QH2)
i.

ii.

Transfer of electron from UBIQUINOL to

CYTOCHROME C, also called b, c1
complex.
The net equation of this redox reaction
can be visualized as the Q CYCLE.

QH2 + 2Cyt cox + 2H+matrix Q + 2Cyt cred + 4H+IMS

iii.

The Q CYCLE accommodate the switch

between the 2e- CARRIER Qand the one
electron carrier Cyt b, c1, and explains the
measured stoich of the 4 protons
translocated per pair of electron.

4. Complex IV FINAL H+ REACTION WITH

OXYGEN
a.

After its single heme accepts and electron from

COMPLEX III, CYTOCHROME C moves to
COMPLEX IV to donate electrons to a
BINUCLEAR COPPER CENTER in the ENZYME
(Cu A and Cu B).

b.

Thus, electron is transferred from CYTOCHROME

C to CuA to HEME A to HEME A3CuB CENTER
to OXYGEN.

c.

Since there are 2 ATOMS of OXYGEN, it requires

4e-. for every 4e-passing through this complex, the
enzyme comsumes 4H+ from the matrix in
covertingO2 TO 2H2O.

d.

It also uses the energy of this redox reaction to

pump one H+into IMS for each e- (TOTAL OF
4H+) 4H+ + 4H+ = 8H+, THEREFORE:

4Cyt creduced +O2+ 8H+matrix 4Cyt coxidized +2H2O+4H+IMS

This transfer of four electrons from Cytochrome C to O2

involves the two heme groups, a and a3 and CU. Of the eight H+
removed from the matrix, four are used to form two water
molecules and four are pumped into the intermembrane space.
Thus, for every pair of electrons passing down the chain from
NADH or FADH2, 2H+ are pumped across the membrane by
Complex IV.

Note: While Q carries two electrons, the

cytochromes carries only one, thus the oxidation
of one QH2 is coupled to the reduction of 2
molecules of Cytochrome C.

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III. THE ELECTRON TRANSPORT CHAIN (ETC) IN

SYNTHESIS OF ATP
A. ATP is generated when electrons flow through the
respiratory chain.
B. The process of generating ATPs through the ETC is
called OXIDATIVE PHOSPHORYLATION.
C. The mechanism of generation of ATPs by the ETC is
described by CHEMIOSMOTIC THEORY.

IV. MECHANISM OF ATP PRODUCTION BY THE

MEMBRANE-LOCATED ATP SYNTHASE
A. ATP Synthase is an Enzyme Complex
i. F0subcomplex
1. A disk of C protein subunits
2. b. Attached to the disk is a subunit in the form of a
bent axle
3. c. These disk and axle rotate when protons pass
through them.
**rotates once hydrogen enters from the outside through
the force of the electrochemical gradient
ii.

F1subcomplex
1. Consists of three and three subunits
2. Where the opposite end of the subunit fits
3. Fixed to the membrane and do not rotate

**Every time this rotates because of the entry of proton, ADP is

phosphorylated and forms ATP. The pumping out of proton
from the matrix triggers the entry.
B. Schematic Diagram

The inner mitochondrial membrane is impermeable for ions,

when proton is pumped out, it cannot go back. So since
ptoton goes out into the intermembrane space (IMS), this
results to an electrical gradient in which the IMS becomes
more positive while the matrix becomes more negative.
The pumping out of proton happens in complexes I, III and
IV. While complex II, increases number of reduced
ubiquinone.
The presence of an electrical gradient will create a
momentary permeability for protons from the IMS to be
pumped into the matrix. When a proton enters, this fuels the
ATP synthase to be activated and converts ADP and Pi to
ATP.

V. ENERGY CAPTURED BY THE ETC DURING THE

CATABOLISM
Glucose undergoes glycolysis first then the citric acid
cycle.
From glycolysis, there is a reaction there that utilizes
NADH (extramitochondrial NADH introduction).
Glycolysis happens outside the mitochondria.

Each of the three complexes will pump out one ATP. If the
reducing equivalent enters in complex I, one mole of the
substrate will produce three ATP. Pero if sa complex II
nawalangganyang mechanism, at diretsosyasa Q, it will only
produce two ATP. That is the chemiosmotic theory.
Perosabagonglibro, hindinasya exactly three ATPs. Pag
NAD, 2.5 ATP at pag FAD from complex II or any substance
that passes Q and bypasses complex I, it produces 1.5 ATP.

NADH needs a shuttle to enter the mitochondria and

into electron transport chain:
a.

Malate shuttle remains as NADH therefore it

produces 2.5 ATP x 2 =5 ATPs; it will pass by the
complex I, III and IV

***from 1B 2017 trans

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VI. RESPIRATORY CONTROL THAT ASSURES CONSTANT

SUPPLY OF ATP, THE ROLE OF AVAILABILITY OF ADP
There is a need to control the cellular respiration since
there will be a cellular burnout.

b.

A.

Oxidation cannot proceed via the ETC without

the concomitant phosphorylation of ADP.

B.

Most cells in the resting state are in state 4 (below),

and respiration is controlled by the availability of ADP.

C.

There are 5 conditions controlling the rate of

respiration in the mitochondria:
a. State 1 Availability of ADP and Substrate
b. State 2 Availability of Substrate ONLY
c. State 3 capacity of the respiratory chain
itself, when all substrates and
components are present in saturating
amounts.
d. Availability of ADP ONLY
e. Availability of Oxygen ONLY

D.

As respiration increases (as in exercise), the

cell approaches state 3 or 5 when either the capacity
of the respiration chain becomes saturated of the PO2
decreases below the Km for heme a3.

E.

There is also the possibility that the ADP/ATP

transporter which facilitates the entry of ADP into and
ATP out of the mitochondrion becomes rate-limiting.

Glycerophosphate shuttle NADH to FAD therefore

it produces 1.5 ATP x 2 = 3 ATPs; bypasses Complex
I

VII. POISONS THAT INHIBIT THE RESPIRATORY CHAIN

A.

Glycolysis: 3-5 ATP; depends on the shuttle

Inhibitors of the Respiration Chain

1. Acting on Complex
a.

PATHWAY
Glycolysis

REACTION
Gly-3-PO4DHRxn----->2

ATP/mol
glucose
3-5

b.
2.

CitricAcid
Cycle

NADH
-malateshuttle
-glycerophosphateshuttle
PyruvateDH Rxn------>2
NADH
IsocitrateDH Rxn--->2NADH
-ketoglutarateRxn-->2

2x2.5
2x1.5
5

NADHRxn--->2
SuccinateDH

FADH
2
MalateDH
Rxn------->2

Total NADH

3.

4.

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B.
1.

AntimycinA
Dimecarpol

Acting on Complex IV
a.
b.
c.

Oxidative phosphorylation greatest source of ATP

By competitive inhibition
Malonate

Acting on Complex III

a.
b.

26- 28

Therefore, 90% of the high-energy phosphate (32 x

0.90) produced from the complete oxidation of 1 mole
of glucose is obtained via oxidative phosphorylation
coupled to the respiratory chain (ETC).

Acting on Complex II
a.
b.

5
5

Blocksthe transfer of electron from Fe-S

to Q
Barbiturates such as a mobarbita

H2S
Carbonmonoxide
Cyanide

Inhibitors of Oxidative Phosphorylation

Inhibition of the transporter of ADP into ATP out of the
mitochondrion. e.g. Atractyloside

2.

Blocks the flow of protons through ATP synthase

When protons are pumped, it is the signal for the

ATP synthase to open.
There are protons that block the opening of ATP

synthase
E.g.Oligomycin,aureovertin

C. Uncouplers of Oxidative Phosphorylation

1.
2.

Dissociate oxidation in the respiration chain from

phosphorylation

3. Dicarboxylate Transporter (DT)

Requires inorganic PO4

Cause respiration to become uncontrolled since the

rate is no longer limited by the concentration of ADP
or Pi.
E.g. 2,4-dinitrophenol,dicumarol , thermogenin and
free salicylate

VIII. THE RELATIVE IMPERMEABILITY OF THE INNER

MITOCHONDRIAL MEMBRANE NECESSITATES
EXCHANGE TRANSPORTERS
A.

Exchange Diffusion Systems present in the Inner

Mitochondrial Membrane

4. Tricarboxylate Transporter (TT)

Requires malate
Transports citrate, isocitrate, cis-aconitate

1. Phosphate Transporter (PT)

Transports di- and tricarboxylic anions

Note: monocarboxylic anions and

neutralsubstances are PERMEABLE, thus they do not
needtransporters

Exchange with OH- in their H2PO4- form to

maintainelectrical neutrality.

5. Alpha-ketoglutarate Transporter (AKT)

6. Adenine Nucleotide Transporter (ANT)

2. Pyruvate Symport (PS)

Vital in allowing ATP exit from mitochondria to the

sites of extramitochondrial utilization and in allowing
the return of ADP and ATP production within the
mitochondrion
Four negative charges are removed from the matrix for
every three taken in.

Utilizes H+ gradient

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2 types of enzymes:
a.
b.

Cytosolic Glycerol-3-PO4 Dehydrogenase

uses NADH
Mitochondrion Glycerol-3-PO4
preferably uses FADH

7. Long chain fatty acids are transported in the

mitochondria via the carnitine system

B.

C.

D.

Malate shuttle (kahit late paborito pa rin siya)

Whether inside or outside NADH is used as

coenzyme so theres no reduction in production of ATP
for each extramitochondrial NADH produced.

3.

Creatine Phosphate Shuttle

ATP is used by the sets outside the mitochondria for

the energy requiring processes. ATP is converted to
ADP (less energy). To replace ATP,the creatine
phosphate gives its phosphate to ADPso that ADP is
again converted to ATP.
Outer mitochondrial membrane is permeable to
creatinine. Creatinine needs to be phosphorylated
again. The ATP in the inner membrane space coming
from the oxidative phosphorylation then
rephosphorylatecreatine so that creatine phosphate is
replenished.

Free fatty acids (FFAs) will be converted to AcylCoAvia the enzyme acyl-CoA synthetase.
Acyl-CoA will now enter the OUTER mitochondrial
membrane. It is unable to enter the inner
mitochondrial membrane without the
carnitinetransporter.
Carnitinepalmitoyl-transferase I will now convert
acyl-CoA to acylcarnitine, which can now enter the
inner mitochondrial membrane (Acyl of acylcarnitine
from acyl-CoA and carnitine from carnitine)

The inner mitochondrial membrane is freely permeable to

uncharged small molecules such as 02, H20, CO2, NH3,
and monocarboxylic acids (3-OH butyric acid, acetoacetic
acid, acetic acid). Monocarboxylic acids penetrate more
readily in their undissociated and more lipid soluble form.
A Proton-TranslocatingTranshydrogenase is a source of
intramitochondrial NADPH

2.

Energy-linked transhydrogenase
Passage of protons down the electrochemical gradient
from outside to inside is coupled with transfer of H+
from intramitochondrial NADH to NADPH. NADH is a
factor for the transfer of NADPH.

Shuttle transport for Transport of Extramitochondrial NADH

inside the Mitochondria for Oxidation
1.

Glycerophosphate shuttle NADH produced

extramitochondrially; produces lower ATP output

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THE CYCLE

Oxidative phosphorylation ultimate source of ATP

THE KREBS CYCLE (A.K.A. CITRIC ACID CYCLE OR CAC;
TRICARBOXYLIC ACID OR TCA)

CHEMICAL DETAILS OF KREBS (CAC/TCA) CYCLE

Comparison with Glycolysis

DEFINITION AND IMPORTANCE

A.

B.
C.

The CAC is a sequence of reactions in the

mitochondria that oxidizes the acetyl moiety of acetyl
CoA and reduces coenzymes that are reoxidized
through the ETC, linked to the formation of ATP.
The CAC is the final common pathway for the
oxidation of carbohydrates, lipids and proteins.
Reactions of the CAC liberate reducing equivalents
and CO2

Glycolysis
Linear Pathway
Enzymes are cytoplasmic
Most Intermediates are
activated by phosporylation
Ends with Pyruvate that forms
Acetyl-Coa

8 STEPS REACTIONS OF KREBS CYCLE

Reaction
Number / Type

BIRDS EYE VIEW

1. Hydrolysis
2. N-H
Cleavage
3. Redox
N-H Cleavage
4. Redox
N-H Cleavage

In the above figure we can see a general overview of how the

CAC works. Breakdown or the oxidation of macromolecules
generate Acetyl CoA goes to the cycle liberation of 2
electrons/2H goes in to the ETC/oxidative phosphorylation

5. Phosphoryl
Group Transfer

6. Redox
7. Hydrolysis
8. REDOX

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Krebs Cycle
Cyclic Pathway
Enzymes are mitochondrial
Few Intermediates are
activated by phosphorylation
Begins with Acetyl-Coa that
combines with OAA

Reaction
ACETYL CoA + OAA
+ H2O==
CITRATE + CoA
CITRATECISACONITATE ==
ISOCITRATE
ISOCITRATE + NAD
AKG + CO2 +
NADH + H+
AKG + NAD+ + CoA
SUCC-CoA +
CO2 + NADH+ H+
SUCC-CoA + Pi +
ADP
SUCCINATE +
ATP + CoA
SUCCINATE +
FAD(prosthetic)
FUMARATE + FADH2
FUMARATE + H2O
MALATE
MALATE + NAD+
OAA + NADH +
H+

Enzyme

Cofactor

Citrate
Synthase

None

ACONITASE

Fe-S

ISOCITRATE
DEHYDROGE
NASE

NAD

AKG DH
COMPLEX

LIPOAMI
DE NAD

SUCC-CoA
THIOKINASE

CoA

SUCCINATE
DEHYDROGE
NASE

FAD, FeS

FUMARASE

NONE

MALATE
DEHYDROGE
NASE

NAD

STEP 1 COMBINATION OF ACETYL COA & OAA

a.

b.

Addition of the C2 unit (Acetyl) to the keto double bond

of the C4 acid (OAA) to produce C6 compound
(Citrate).
The enz. is a synthase (not a synthethase)

ENERGETICS OF THE CAC

A. 3 MOLECULES OF NAD (3 X 2.5)

7.5 ATPs

B. ONE MOLECULE OF FAD (1 x 1.5)

1.5 ATP

C. SUCCINATE THIOKINASE RXN (1 X 1) 1.0 ATP

10.0 ATPs

STEP 2 NON-HYDROLYTIC CLEAVAGE

a.

b.

TEN ATPs ARE FORMED PER TURN OF CAC

The CAC is oxidative, but Citrate, a tertiary alcohol,

cannot be oxidized.
Citrate therefore is converted to Isocitrate (a
secondary alcohol) making oxidation possible.

THE PIVOTAL ROLE OF CAC IN METABOLISM

AMPHIBOLIC PATHWAY
1.

Oxidative Pathway

STEP 3 OXIDATION & NE DECARBOXYLATION

a. Catabolism
a.

Isocitrate is oxidized.
b. Generation of energy

b.

The oxidation results in an unstable intermediate that

decarboxylates to C5 acid (AKG).

STEP 4 OXIDATIVE DECARBOXYLATION

a.

A complex reaction that requires a complex enzyme

(AKG DH COMPLEX).

b.

Oxidative decarboxylation of AKG requires 5 chemical

steps, 3 enzymes and cofactors.

c.

The purpose of this step is to collect the energy from

AKG to high energy compound SUCCINYL COA.

2.

Synthetic Pathway
a. Transamination and Gluconeogenesis

STEP 5 SUBSTRATE LEVEL PHOSPHORYLATION

a.

The energy from thioester bond of Succinyl CoA is

used to form ATP.

b.

Succinate is produced.

STEP 6 FORMATION OF UNSATURATED ACID

a.

Succinate is oxidized to Fumarate.

b.

Interestingly, C=C is formed by oxidation.

b. Fatty Acid Synthesis

STEP 7 HYDROLYSIS OF DOUBLE BOND

a.

The unsaturated acid Fumarate becomes a substrate

for addition of water.

b.

Fumarate is converted to Malate by Fumarate

hydratase.

STEP 8 OXIDATION OF THE HYDROXYL GROUP OF

MALATE FORMS OXALOACETATE. THE CYCLE IS
REPEATED.....

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REGULATION OF CITRIC ACID CYCLE

A.

Respiratory Control VIA the ETC and Oxidative

Phosphorylation
a.
b.
c.

B.

Supply of NAD
Availability of ADP
Rate of Utilization of ATP

Regulation VIA the Individual Enzymes of the CAC

1.

Enzymes on non-equilibrium reactions:

a. Pyruvate DH
b. Citrate Synthase
c. Isocitrate DH
d. AKG DH

2.

Enzymes responsive to energy status [(ATP/ADP)

& (NADH/NAD) status).
a.
b.
c.

3.

Allosteric inhibition of citrate synthase by

ATP and long chain fatty acyl CoA.
Allosteric activation of mitochondrial
isocitrate DH by ATP and NADH.
Inhibition of succinate DH by
oxaloacetate which level is dependent
on [NADH]/[NAD] ratio.

The alpha-ketoglutarate dehydrogenase complex

is regulated in the same way as pyruvate
dehydrogenase.
a.
b.

End product inhibition

Interconversion to active and inactive
form.

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