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. DH= H2(product)- H1(reactant) DH +ve Endothermic DH -ve Exothermic

-systems tend to go forward to a lowest-energy statee.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, DG, Predicts the change in the free energy and thus

direction of reaction at any specified concentration of products and reactants DG = D H - T D SIf DG is –ve, the reaction proceeds spontaneously.

•Standard Free energy change: DGº: Free energy change under standard

conditions; that when reactant and product concentration are kept at 1M conc. •The sign of the DG predicts the direction of the reaction.

DG is –ve exergonic reaction.DG is +ve endergonic reaction.DG is zero equilibrium.

DG of the forward and back reactions

A B DG= -500 cal/mol, spontaneous in this directionThe back reaction B A DG= 500 cal/mol non-spontaneous at this direction

DG depends on the concentration of both reactants and products.For a reaction A ↔ BA: the reactants B: the product

[A]

[B]RTlnΔGΔG o

The sign of DG and DGº can be different

DGº gives prediction of the direction of the reaction only at the standard conditions:

At standard conditions the [A]=[B]=1

DG = DGº + RTln1 DG = DGº at standard conditions

Relation between equilibrium constant (Keq) and DGº

at equilibrium DG=0

A ↔ B at equilibrium

eq[A]

eq[B]

eqK

eq

eqo

[A]

[B]RTlnΔGΔG

eq

eqo

[A]

[B]RTlnΔG0

eqo RTlnkΔG

If Keq=1 DGº=0

If Keq>1 DGº < 0 (-ve)

If Keq<1 DGº > 0 (+ve)

DG is –ve exergonic reaction spontaneous from A to B

DG is +ve endergonic reaction non-spontaneous from B to A

Free Energy change profile

Glucose 6-PO4 ↔ Fructose 6-PO4

The reaction of Glucose 6-PO4 into Fructose 6-PO4 under different conditions

Glucose 6-PO4 Fructose 6-PO4

Fructose 6-PO4 Glucose 6-PO4

Glucose 6-PO4 ↔ Fructose 6-PO4

DGº of two consecutive reactions are

additives and also DG of pathways are

additives

Reactions or processes with a large +ve DGº 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 DGº1 (non-spontaneous)

D F DGº2 (spontaneous)

A + B + DC+ D+ F

A + B C + F DGº3= DGº1 + DGº2

(spontaneous)

D is a common intermediate and can serve as

energy carriers for this reaction

•ATP is the universal energy carrier in biological systemsATP: 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

ATP + H2O ↔ ADP + Pi + H+ DGº= -7.3kcal/mol

ATP + H2O ↔ AMP + PPi + H+ DGº= -7.3kcal/mol

ATP-ADP cycle: the energy exchange in biological system

ATP ADP

Motion Biosynthesis

Active transportSignal amplification

PhotosynthesisOxidation 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

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 is continuously formed and consumed

Structural basis of the high P group transfer potential of ATP

ATP + H2O↔ ADP + Pi + H+ DGº = -7.3 kcal/mol

Glycerol 3-phosphate + H2O ↔ Glycerol + Pi DGº= -2.2 kcal/mol

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

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 DGº = -62 kj/mol

ADP + Pi ↔ ATP DGº = +13 kj/mol

PEP + ADP ↔ Pyrovate +ATP DGº = -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 consumedATP is intermediate donor of free energy in biological systems rather than as long-term 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) DGº = +4 kcal/molATP + H2O ADP + Pi DGº = -7.4 kcal/molA + ATP + H2O B + ADP + Pi + H+DGº = -3.4 kcal/molSpontaneous

* NAD+ is the oxidized form of nicotinamide adenine dinucleotide, NADH is the reduced form* NAD+ is the major e- acceptor in oxidation of fuel molecules.

* electron donor= reducing agent (reductant) electron acceptor= oxidizing agent (oxidant)

NADH: generation of ATPNADPH: reductive biosynthesis

NAD+ + 2e- + H+ NADHOxidizing agent reducing agent

Oxidized form reduced form

NAD+ is strong oxidizing agent that can oxidize secondary alcohol into keton

* Flavin adenine dinucleotide (electron carrier molecule)FAD: Oxidized FormFADH2: Reduced Form

FAD + 2e- + 2H+ FADH2

Oxidizing agent reducing agentOxidant 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

H+

-- - - - + + + +

ADP + Pi

H+

ATPO2

H2O

•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 dehydrogenase

NADH

Q

Cytochrome reductase.

Cytochrome C

Cytochrome oxidase.

O2

FADH2 FAD

Ubiquinone (Q), Cytochrome C are mobile e-carriers

Complex II

Complex I

Complex III

Complex IV

NADH dehydrogenase.Complex I

Complex IIsuccinate 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 FMN reduced Fe-S QNAD+ FMNH2 oxidized Fe-S QH2

The flow of two electrons from NADH to QH2 leads to pumping of four H+ from the matrix to intermembrane space

Oxidized form of Q

Intermediate

Reduced form

QH2 shuttle electrons from complex I to cytochrome reductase (complex III)It is hydrophobic quinone diffuse rapidly within the inner membrane of mitochondria

Electrons flow from Ubiqinol to cyto. C through Cytochrome reductaseCytochrome 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 iron-protoporphyrine1X 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 O2In 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

O2 is reduced into water

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

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

Complex II: succinate dehydrogenase (Succinate-Q oxireductase) QH2 is the entry for electrons from FADH2 of FlavoproteinsFADH2 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,

* Oxidation and Phosphorylation are coupled by a proton-motive force

NADH + ½ O2 + H+ H2O + NAD+ DG0= -52.6 kcal/mol

ADP + Pi + H+ ATP + H2O DG0= +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 ATP-synthase 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 )