Download Complex IV

Bioenergetics and oxidative Phosphorylation
Objectives
-To understand the general concepts of Bioenergetics; Enthalpy, entropy
and free energy change and standard free energy and the mathematical
relation between them
-To understand the concepts of high energy compounds and know the most
important examples, like ATP
- To know the general concept of oxidative phosphorylation and the general
mechanism of ATP production.
Bioenergetics
-Enthalpy, entropy and free energy
- Free energy change and standard free energy change and relationship between
them and the equilibrium constant
- ATP is the universal energy carrier in the biological systems
-Structural basis of the high phosphate group transfer potential
-Phosphorylated compound with high phosphate group transfer potential, PEP,
phosphocreatine
-ATP has an intermediate group-transfer potential
Oxidative Phosphorylation
-Electron carriers, NADH and FADH2
-Mitochondria are the respiratory organelles in the cell
-NADH dehydrogenase has two prosthetic groups FMN and iron-sulfur cluster
-QH2 is the entry for electrons from FADH2
-Cytochrome Reductase
-Cytochrome oxidase catalyses the transfer of electrons from CytC to O2
-Chemiosmaotic hypothesis in the phosphorylation of ADP
Bioenergetics & Thermodynamics
Bioenergetics: is the quantitative study of energy transduction in the living cells and nature and
the chemical process underlying these transductions .
•Bioenergetics concerns only with the initial and final energy states of reaction components, NOT
the mechanism of the reaction, Not, the time needed for the reaction to occur. It allows to predict
the spontaneousity of the reaction, wither a reaction will take place or not
Factors that determine the direction of a reaction
The direction and extent to which a chemical reaction proceeds is determined by two factors
- Enthalpy
- Entropy
- Temp
- Enthalpy: DH, a measure of the change in heat reaction content of the reactant and product.
∆H= H2(product)- H1(reactant)
∆H +ve  Endothermic
∆H -ve  Exothermic
-systems tend to go forward to a lowest-energy state
e.g: fall goes downhill, oxidation of fatty acids produce a lot of energy,
these are spontaneous reactions and ∆H is negative, but the melting of Ice
is a spontaneous reaction even it is endothermic reaction and ∆H is
positive
 ∆H alone is not sufficient to predict the direction of a reaction
-Entropy (∆S): a measure of randomness or disorder of the reactants and
products.
-systems have a natural tendency to randomize and the degree of
randomness of a system is defined as S which is the entropy.
∆S= S2(product)-S1(reactant)
∆S +ve  Increased entropy
∆S -ve  Decreased entropy
Entropy (∆S): a measure of randomness or disorder of the
reactants and products.
-Systems tend to increase the entropy (∆S +ve),
e.g. Homogenization of sucrose solution with water. Entropy of ordered state is
lower than that of the disordered state of the same system.
Neither the entropy nor the enthalpy alone can predict the direction of the
reaction.
•Free Energy: Gibbs free energy that correlates the entropy and the enthalpy mathematically
which allow to predict in which direction a reaction proceeds spontaneously.
•Free Energy change, ∆G, Predicts the change in the free energy and thus direction of reaction
at any specified concentration of products and reactants
∆G = ∆ H - T ∆ S
If ∆G is –ve, the reaction proceeds spontaneously.
•Standard Free energy change: ∆Gº: Free energy change under standard conditions; that
when reactant and product concentration are kept at 1M conc.
•The sign of the ∆G predicts the direction of the reaction.
∆G is –ve  exergonic reaction.
∆G is +ve  endergonic reaction.
∆G is zero  equilibrium.
DG of the forward and back reactions
A B
∆G= -500 cal/mol, spontaneous in this direction
The back reaction
B A 
∆G= 500 cal/mol
non-spontaneous at this direction
∆G depends on the concentration of both reactants and products.
For a reaction
A ↔ B
A: the reactants
B: the product
[B]
ΔG  ΔG  RTln
[A]
o
The sign of DG and DGº can be different
∆Gº gives prediction of the direction of the reaction only at the standard
conditions:
At standard conditions the [A]=[B]=1 
∆G = ∆Gº + RTln1  ∆G = ∆Gº at standard conditions
Relation between equilibrium constant (Keq) and ∆Gº
A
↔
K
B
eq
at equilibrium

[B] eq
[A]eq
ΔG  ΔG  RTln
o
[B] eq
[A]eq
at equilibrium DG=0
0  ΔG
o
 RTln
ΔG  RTlnk
[B] eq
[A] eq

o
eq
If Keq=1  DGº=0
If Keq>1  DGº < 0 (-ve)
If Keq<1  DGº > 0 (+ve)
Free Energy change profile
∆G is –ve  exergonic
reaction  spontaneous from
A to B
∆G is +ve  endergonic
reaction non-spontaneous from
B to A
The reaction of Glucose 6-PO4 into Fructose 6-PO4 under different conditions
Glucose 6-PO4 ↔ Fructose 6-PO4
Glucose 6-PO4  Fructose
6-PO4
Fructose 6-PO4  Glucose
6-PO4
Glucose 6-PO4 ↔
Fructose 6-PO4
∆Gº of two consecutive reactions are additives and
also DG of pathways are additives
Reactions or processes with a large +ve ∆Gº as moving
against electrochemical gradient are made possible by
coupling the endergonic process with a large –ve
process as hydrolysis of ATP
Favorable and unfavorable reactions are coupled
through common intermediates
A+BC+D
DF
A + B + DC+ D+ F
A+BC+F
∆Gº1 (non-spontaneous)
∆Gº2 (spontaneous)
∆Gº3= ∆Gº1 + ∆Gº2
(spontaneous)
D is a common intermediate and can serve as energy
carriers for this reaction
•ATP is the universal energy carrier in biological systems
ATP: nucleotide consists of adenine, ribose and triphosphate unit, the active form of ATP is
complex with Mg+2 or Mn+2
ATP is energy rich molecule because of its triphosphate unit that contain 2 phosphanhydrid
bonds, large free energy is released when
ATP is hydrolyzed to ADP + Pi or to AMP and PPi
↔ ADP + Pi + H+
ATP + H2O ↔ AMP + PPi + H+
ATP + H2O
∆Gº= -7.3kcal/mol
∆Gº= -7.3kcal/mol
The free energy liberated in the hydrolysis of ATP is used to drive reactions that
requires an input of free energy
ATP is form from ADP and Pi when fuel molecules are oxidized
ATP-ADP cycle: the energy exchange in biological system
Motion
Biosynthesis
Active transport
Signal amplification
ATP is
continuously
formed and
consumed
ATP
ADP
Photosynthesis
Oxidation of Fuel molecules
-Some biosynthesis reactions are driven by nucleotide analogous to ATP and these are:
Gaunosine triphosphate:
GTP
Cytidine triphosphate:
CTP
Uridine triphosphate:
UTP
ATP + GDP
 ADP + GTP
Structural basis of the high P group transfer potential of ATP
ATP + H2O↔ ADP + Pi + H+
Glycerol 3-phosphate + H2O ↔ Glycerol + Pi
ATP has a stronger
tendency to transfer its
terminal phosphoryl
group to water than dose
the glycerol 3-phosphate
 ATP has high
phosphate group transfer
potentials Why?
1- Electrostatic repulsion
2- Resonance stabilization
∆Gº = -7.3 kcal/mol
∆Gº= -2.2 kcal/mol
Other compounds have high phosphate group transfer potential
-Phosphoenol pyruvate (PEP), phosphocreatine have a higher group transfer potential than dose
ATP.
PEP can donate P to ADP to produce ATP
PEP ↔ pyruvate + Pi
∆Gº = -62 kj/mol
ADP + Pi ↔ ATP
∆Gº = +13 kj/mol
PEP + ADP ↔ Pyrovate +ATP
∆Gº = -49 kj/mol ??????
It is significant that ATP has a group-transfer potential that is intermediate among
the biological important phosphorylated molecules. This intermediate position
enable ATP to function efficiently as a carrier of phosphoryl groups. NO enzyme in
cells that transfer P from high-P donor to low energy acceptor  should first
transfer first to ATP to form ADP
ATP is continuously formed and consumed
ATP is intermediate donor of free energy in biological systems rather than as longterm storage form of energy
ATP molecule is consumed after 1 min of its formation and the turnover of ATP is
high, human consumes about 40 kgs of ATPs in 24 hr
ATP hydrolysis is coupled to reaction to shift the reaction toward product
AB
(non-spontaneous)
∆Gº = +4 kcal/mol
ATP + H2O  ADP + Pi
∆Gº = -7.4 kcal/mol
A + ATP + H2O  B + ADP + Pi + H+
DGº = -3.4 kcal/mol
Spontaneous
* NAD+ is the oxidized form of nicotinamide
adenine dinucleotide, NADH is the reduced form
NADH: generation of ATP
NADPH: reductive biosynthesis
* NAD+ is the major eacceptor in oxidation of
fuel molecules.
Oxidizing agent
reducing agent
NAD+ + 2e- + H+ 
NADH
Oxidized form
reduced form
NAD+ is strong oxidizing agent that can
oxidize secondary alcohol into keton
* electron donor= reducing agent
(reductant)
electron acceptor= oxidizing agent
(oxidant)
* Flavin adenine dinucleotide (electron carrier molecule)
FAD: Oxidized Form
FADH2: Reduced Form
FAD + 2e- + 2H+
Oxidizing agent
Oxidant

FADH2
reducing agent
reductant
FAD is strong oxidizing agent it can
oxidize the alkain into alkene
Oxidative Phosphorylation
Oxidative phosphorylation: is the process in which ATP is formed as a result of transfer of
electrons from NADH or FADH2 to O2 by a series of electron carriers
NADH, FADH2 formed glycolysis, Fatty acid oxidation and citric acid cycle. They have a pair of
electrons with high transfer potential when these electrons are transferred to O2, a large free
energy is librated
The flow of electrons from NADH or FADH2 to O2 through protein complexes in the inner
membrane of the mitochondria leads to pumping of protons out the mitochondrial matrix, this
makes pH and Transmembrane electrical gradient
ATP is synthesized when proton flow to the mitochondrial matrix
O2
H2O
ATP
ADP + Pi
-- - - ++++
H+
H+
•The Respiratory chain consists of three proton pumps, linked by two
mobile electron carriers.
• electrons are transferred from NADH to O2 through a chain of three large
protein complexes
Complex I:NADH dehydrogenase, NADH-Q reductase.
Complex III: Cytochrome reductase.
Complex IV: Cytochrome oxidase.
The above protein complexes are pump protons
Ubiquinone (Q): carries electrons from NADH dehydrogenase (I) to
cytochrome reductase (III)
Cytochrome C: carries electrons from cyt-reductase (III) to cytochrome
oxidase (IV).
Complex II: succinate dehydrogenase, (succinate- Q reductase), doesn't pump
protons, production of FADH2 from succinate
NADH
Complex I
NADH dehydrogenase
Complex II
Q
Ubiquinone (Q),
Cytochrome C are
mobile e-carriers
FADH2
Cytochrome reductase.
FAD
Complex III
Cytochrome C
Cytochrome oxidase.
O2
Complex IV
NADH dehydrogenase.
Complex I
Complex II
succinate dehydrogenase (Succinate-Q oxireductase)
Cytochrome reductase.
Complex III
Cytochrome Oxidase.
Complex IV
Oxidative Phosphorylation
NADH dehydrogenase.(Complex I)
The electrons of NADH enter the chain at the NADH dehydrogenase, the initial step is the binding
of NADH and then the transfer the two electrons to the flavin mono nucleotide (FMN) prosthetic
group of this protein to give the reduced form FMNH2
NADH + H+ + FMN  FMNH2 +NAD+
Electrons transfer from the Fe-S cluster of complex I are shuttled to Coenzyme Q
NADH
NAD+
FMN
FMNH2
The flow of two
electrons from
NADH to QH2
leads to pumping of
four H+ from the
matrix to
intermembrane
space
reduced Fe-S
oxidized Fe-S
Q
QH2
QH2 shuttle electrons
from complex I to
cytochrome reductase
(complex III)
It is hydrophobic
quinone diffuse rapidly
within the inner
membrane of
mitochondria
Oxidized form of Q
Intermediate
Reduced form
Electrons flow from Ubiqinol to cyto. C through Cytochrome reductase
Cytochrome is an electron-transferring proteins that contain a heme prosthetic group.
Their iron atoms alternate between a educed ferrous(+2) state and an oxidized ferric(+3)
state during electron transport.
Cyt. Reductase catalyzes the transfer of 2 e- from QH2 to Cyt. C (water soluble protein)
and this is coupled to pumping of 4 H+ to the inter-membrane space
* Cyt. Reductase has two
types of cytochromes; b
and C1
Cyt. C1 and Cyt. C have ironprotoporphyrine1X the same
as heme of myoglobin,
hemoglobin and these hemes
are covalently linked to
protein
Cytochrome reductase.
Complex III
QH2 transfer one of its electrons to Fe-S cluster in the reductase. Then this electron is
shuttled to Cyt. C1 then to Cyt. C which carries it away from the complex
Complex IV: Cytochrome Oxidase.
Cytochrome Oxidase catalyses the transfer of electrons from Cyt C to O2
In this reaction
2Cyt C(+2) + 2H+ +1/2 O2  2Cyt C(+3) +H2O
This process accomplished by pumping 2 protons from matrix to intermembrane space
Cytochrome Oxidase
contains two heme A
groups called heme a
and heme a3 , they are
different because they
differ in their location in
the location.
Cyt. Oxidase contains
also two copper ions
called CuA and CuB
as prosthetic group
O2 is reduced into water
Complex II: succinate dehydrogenase (Succinate-Q oxireductase)
QH2 is the entry for electrons from FADH2 of Flavoproteins
FADH2 is formed in citric acid cycle by the oxidation of the succinate to fumarate by succinate
dehydrogenase (complex II) which is integral protein in the mitochondrial inner membrane,
FADH2 doesn't leave the
complex, but its electrons are
transferred to Fe-S cluster then
to Q for the entry to the
electron transport chain, the
same thing for the FADH2
moieties of glycerol
dehydrogenase, and Fatty acyl
Co dehydrogenase transfer their
high potential electrons to Q
to from QH2, these enzymes
are not proton pumps
* Oxidation and Phosphorylation are coupled by a proton-motive force
NADH + ½ O2 + H+  H2O + NAD+
∆G0= -52.6 kcal/mol
ADP + Pi + H+ ATP + H2O
∆G0= +7.3 kcal/mol
* ATP synthesis is mediated by mitochondrial ATPase (ATP synthase in the inner
membrane of mitochondria)
* Oxidation of NADH is coupled to Phosphorylation of ADP into ATP
The chemiosmotic hypothesis
The transfer of electrons through the respiratory chain leads to pumping of protons
from the matrix to the other side of the inner mitochondrial membrane. The H+
concentration becomes higher on the systolic side and the electrical potential is
generated and this proton motive force drives the synthesis of ATP by the ATPsynthase complex
* Oxidation of 1 NADH  3ATP
1 FADH2  2ATP
Oxidative Phosphorylation
* Electrons transfer in the respiratory chain can be blocked by specific inhibitors
* Oligomycin: drug bind to ATP synthase that prevents the rentry of H+  prevents
ATP synthesis  prevent electron transport so electron transport and phosphorylation
are coupled
The End
iron-sulfur cluster (Fe-S) in iron-sulfur proteins (non-hem proteins) play a critical role in a
wide range of reduction reactions in biological systems, three types of Fe-S cluster:
The simplest one is consisting of one iron
atom coordinated to 4 sulfhydryl group of
four cysteine molecules
A second type [2Fe-2S]
Third type [4Fe-4S]
NADH Dehydrogenase contain the [2Fe-2S]
and [4Fe-4S]
Iron atoms in these clusters cycle between Fe
(reduced ) and Fe (oxidized )