Chemical thermodynamics

Thermodynamics is the branch of physical chemistry that deals with the energy

changes. That is, it describes the relationships among the various forms of energy

and how energy affects matter. Biochemical Thermodynamics known as

biochemical energetics or bioenergetics, is the field of biochemistry concerned

with the quantitative study of transformation and use of energy by living cells and

of the nature and function of the chemical processes that causes these energy

transformations.

Thermodynamic principles

There are two fundamental laws of thermodynamics, the first and second.

These laws help us to understand:

(i) the direction of a reaction, whether from left to right or vice versa,

(ii) the accomplishment of work, whether useful or not, and

(iii) whether the energy for driving a reaction must be delivered from an external

source.

The First Law: Principle of Conservation of Energy

In thermodynamics, the system and the surroundings constitute the universe. The

principle of conservation of energy was first formulated by V. Mayer in 1841.

The first law of thermodynamics states that the total amount of energy in the

universe (i.e., the system + surroundings) remains constant meaning that energy

cannot be created nor destroyed.

Mathematically expressed as
∆E = EB – EA = Q – W ........................................................... (1)

∆E = change in internal energy

EA = energy of a system at the start of a process

EB = energy of a system at the end of a process

Q = heat absorbed by the system

W = work done by the system.

The change in energy of a system depends only on the initial and the final stages

and not on the path of transformation.

The Second Law

The first law of thermodynamics cannot be used to predict whether a reaction can

occur spontaneously or not. Entropy comes in, which is denoted by S. This is the

energy in a state of randomness or disorder. It is unavailable and useless energy.

Entropy is a measure of the degree of randomness or disorder of a system. The

entropy of a system increases (i.e., ∆S is positive) when it becomes more

disordered. Entropy becomes maximum in a system as it approaches equilibrium.

When equilibrium is attained, no further change can occur spontaneously unless

additional energy is supplied from outside the system.

Ultimately closed systems obey the second law of thermodynamics. Open

systems, such as ecosystems, however, with their constant input and outflow,

maintain a steady state in spite of the second law of thermodynamics. Biological

systems, thus, do not seem to conform to the second law, for the tendency of life

is to produce order out of disorder.

The second law states that physical and chemical processes proceed in such a way

that the randomness or disorder of the universe (the system and its surroundings)

increases to the maximum possible or simply a spontaneous process is always

accompanied by an increase in entropy of the universe. Represented as:

(∆S system + ∆S surroundings) > 0 for a spontaneous process .................... (2)

Thus, the total entropy of a system must increase if a process is to occur

spontaneously. The difference between the first and second laws of

thermodynamics is that the first law is concerned with the accounting of the

various kinds of energy involved in a given process, whereas the second law is

concerned with the availability of the energy of a given system for doing useful

work.

In 1878, Gibbs created the free energy (G) function by combining the first and

second laws of thermodynamics. It is called Gibbs free energy, the theoretically

available energy for useful work

∆G = ∆H – T∆S ....................................................................... (3)

∆G = change in free energy of a reacting system,

∆ H = change in heat content or enthalpy of this system,

T = absolute temperature at which the process is taking place

∆S = change in entropy of this system.

∆H = ∆E + P∆V ...........................................................................(4)

where, ∆E = change in internal energy of a reaction

∆V = change in volume of this reaction.

In all biochemical reactions, volume change, ∆V is small, hence ∆H is nearly

equal to the change in internal energy, ∆E. Equation 3 becomes

∆G = ∆E – T∆S............................................................................(5)

Facts to note about ∆G is that

i). ∆G determines whether a reaction can occur spontaneously or not. Thus,

(a) If ∆G is negative (exergonic), the reaction proceeds spontaneously with loss

of free energy and if the value is of great magnitude, the reaction goes virtually

to completion and is essentially irreversible.

(b) If ∆G is positive (endergonic), the reaction proceeds only if free energy can

be gained and if the value is of high magnitude, the system is stable with little or

no tendency for a reaction to occur.

(c) If ∆G is zero, the reaction system is at equilibrium and no net change takes

place.

ii). ∆G of a reaction is the free energy of the products minus that of the reactants

and independent of the path of transformation or mechanism. ∆G for the oxidation

of glucose to CO2 and H2O is the same irrespective of the pathway.

iii). ∆G has no information about the rate of a reaction. A negative G indicates

that a reaction can occur spontaneously, but not the rate.

Consider the oxidation of glucose to CO2 and H2O at constant temperature and

pressure.

C6H12O6 + 6O2 6CO2 + 6H2O

Assuming at a standard condition of temperature 25°C (or 298 K) and pressure

1.0 atm. (or 760 mm Hg), per one molecule of glucose oxidized, ∆G = – 686,000

cal/mol, ∆H = – 673,000 cal/mol, the entropy can be calculated using equation 3.

∆H ∆G …………………………. .……………… (6)

∆S =
T

- 673,000 – (- 686,000)
∆S =
298

= 44 cal/deg (i.e., the entropy of the universe has increased)

Relationship between standard free energy change and equilibrium constant

In a reaction,

A+B C + D………………………….(7)

The free energy change, ∆G of this reaction is given by

[C] [D]
∆G = ∆G° + RT loge ………………………………….. (8)
∆G° = Standard free energy
[A] [B]
change, when reactants A, B, C and D is present at a

concentration of 1.0 M., R = Gas constant, T = Absolute temperature, and [A],

[B], [C] and [D] = Molar concentrations of the reactants

At equilibrium, ∆G = 0. Equation 8 becomes,

[C] [D]
0 = ∆G° + RT loge ………………………………….. (9)
[A] [B]
 [C] [D]
∆G° = - RT loge ………………………………….. (10)
[A] [B]

A+B C+D
The equilibrium constant, K′eq for the reaction

is given by
[C] [D]
K′eq = ………………………………….. (11)
[A] [B]

However, in reactions in which more than one molecule of any reactant or product

participates, the reaction becomes

aA + bB cC + dD

where a, b, c and d are the number of molecules of A, B, C and D participating in

the reaction, the equilibrium constant is

[C]c [D]d
K′eq = ………………………………….. (12)

[A]a [B]b

Substituting equation 11 into equation 10 gives

∆G° = –RT loge K′eq ......................................................................... (13)

or ∆G° = –2.303 RT log10 K′eq ..................................................... (14)

or K′eq = 10–∆G°/(2.303RT) ............................................................... (15)

Substituting R = 1.98 ×10–3 kcal mol–1 degree–1 and T = 298°K (25°C) in equation

15

K′eq = 10–∆G°/1.36 .................................................................................(16)

∆G° is expressed in kcal/mol. A change in equilibrium constant by a factor of 10

results in a change in standard free energy of –1.36 kcal/mol at 25°C. ∆G° is

defined as the difference between the free energy content of the reactants and that

of the products under standard conditions (i.e., 298 K pressure and 1.0

atmospheric pressure) when the reactants and products are present in their

standard concentrations of 1.0M. When K′eq is greater than 1, ∆G° is negative

the reaction will proceed to form the products under standard conditions (forward

reaction), when K′eq is less than 1, ∆G° is positive the reaction will proceed in a

reverse direction (backward reaction) and when K′eq is 1, ∆G° is zero the reaction

will be at equilibrium.

Biochemical reactions take place near pH 7.0 which is conventionally designated

as the standard pH in biochemical energetics. The standard free energy change at

pH 7.0 in biochemical energetics is designated by the ΔG°′.

To calculate ΔG°′, an example of isomerization of dihydroxyacetone phosphate

(DHAP) to
glyceraldehyde 3-phosphate (G-3-P), a glycolysis reaction.

CH2OH CH2OH

C O H C OH

CH2OPO32- CH2OPO32-
Dihydroxyacetone
phosphate DHAP Glyceraldehyde – 3- phosphate

At equilibrium, the ratio (K′eq) of glyceraldehyde 3-phosphate to

dilydroxyacetone phosphate is 0.0475 at 25°C (298°K) and pH 7.0.

The standard free energy change for this reaction can be calculated from equation

14.

ΔG° = –2.303 RT log10 K′eq

= –2.303 × 1.98 × 10–3 × 298 × log10 (0.0475)

= +1.8 k cal/mol

When the initial concentration of DHAP is 2 × 10–4 M and G-3- P is 3 × 10–6 M,

ΔG can be calculated from equation 8.

[C] [D]
∆G = ∆G° + RT loge
[A] [B]

3 × 10–6 M
∆G = 1.8 Kcal/mol + RT log10
2 × 10–4 M
∆G = 1.8 kcal/mol – 2.5 kcal/mol

∆G = –0.7 kcal/mol

Hydrolysis of ATP

Standard free energy of ATP hydrolysis is -30.5 kJ/mol. The actual free energy

of ATP hydrolysis in the cell can be calculated. For example in human

erythrocytes c(ATP) = 2.25 mM c(ADP) = 0.25 mM, c(P i ) = 1.65 mM

[ADP] [Pi]
∆G = ∆G° + RT loge
[ATP]

0.25 mM × 1.65 mM
∆G = - 30.5 kJ/mol + RT log10
2.25 mM

G = -30.5 kJ/mol – 21 kJ/mol

= -51.5 kJ/mol

The negative value for ΔG shows that at the concentration stated, the

isomerization of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate and

hydrolysis of ATP can occur spontaneously. The magnitude of ΔG for a reaction

(whether smaller, larger or the same as ΔG°′) depends on the concentrations of

the reactants. Spontaneity or not for a reaction largely depends on ΔG and not

ΔG°′.

Standard free energy value of chemical reactions are additive.

The overall free energy change for a series of reactions is equal to the sum of the

free energy changes of the individual steps. For example,

A→B ΔG1°′ = – 8 kcal/mol

B→C ΔG2°′ = + 5 kcal/mol

Since A can go to C through the intermediate B, therefore the free energy of

A→C ΔGs°′ = ΔG1°′ + ΔG2°′

= –8 + (+5)

= –3 kcal/mol

Under standard conditions, A is spontaneously converted into B because ΔG is

negative, B cannot be spontaneously converted to C since ΔG is positive but the

conversion of A to C has a ΔG°′ value of –3 kcal/mol, which means that A can

be converted into C spontaneously under standard conditions.

Consider two sequential steps in glycogen breakdown in muscles.

Phosphoglucomutase
Glucose – 1- phosphate Glucose – 6- phosphate
ΔG1°′ = – 1.74 kcal/mol

Glucose phosphate isomerase

Glucose – 6- phosphate Fructose – 6- phosphate
ΔG2°′ = + 0.40 kcal/mol

Adding these two reactions

Glucose – 1- phosphate Fructose – 6- phosphate

The standard free energy change will be,

ΔGs°′ = ΔG1°′ + ΔG2°′

= –1.74 + (+ 0.40)

= –1.36 kcal/mol

Because ΔGs°′ is negative, glucose 1-phosphate is converted into fructose 6-

phosphate in the

muscles spontaneously.

High-energy compounds

In living things continuous supply of free energy is required for metabolic

(anabolic, catabolic and amphibolic) purposes, which are:

(i) to synthesize macromolecules from simpler and smaller precursors,

(ii) to transport molecules and ions across membranes against gradients,

(iii) to perform mechanical work, as in the muscle contraction, and

(iv) to ensure fidelity of information transfer.

The phototrophs obtain this free energy from the sun light while chemotrophs

obtain its own from the oxidation of foodstuffs. This free energy either from light

or the oxidation of foodstuffs are partly transformed into adenosine triphosphate

(ATP) a “high energy phosphate” compound before it is used for metabolic

purposes.

In the breakdown of energy-rich foodstuffs or fuel molecules, free energy is

released from these fuel molecules and used to make ATP from adenosine
diphosphate (ADP) and inorganic phosphate (Pi), a process that requires input of

free energy. ATP then donates much of its chemical energy to energy-requiring

processes like biosynthesis, transport etc. by undergoing a breakdown to ADP

and Pi.

CO2 ATP

Oxidation of Biosynthesis Muscle Genetic

Active
energy rich food contraction information
transport
stuff transfer

ADP + Pi
O2 Recycling of ATP in cells

High-energy phosphate compounds ATP are energy donor to other systems, they

are not used for storing energy for a long-term but for a short-term to carry energy

from one reaction to another for a metabolic process. In a typical cell, an ATP

molecule is consumed within a minute of its formation. The turnover of ATP is

very high. For example, a resting human consumes about 40 kg ATP in a day.

During strenuous labour, the ATP is consumed at the rate of even 0.5 kg per

minute.
 O- 3 O- 2 O- 1
- P O P O P O Adenosine
O CH2
ɣ β α
O O O

ADP AMP
ATP
1= phosphoester bond, 2 & 3 = Phosphoanhydride bond

Hydrolysis of ATP will yield ADP and orthophosphate or inorganic phosphate

(Pi) by losing the terminal γ phosphate group with a standard free energy change,

ΔG°′ of –7.3 kcal/mol.

ATP + H2O ADP + Pi

Standard free energy changes for the hydrolysis of other phosphorylated

compounds or

organophosphates have also been determined, some phosphates yield more and

some yield less free energy than ATP upon hydrolysis, under standard conditions.

Considering the ΔG°′ value of hydrolysis of ATP, there are three classes of

organo phosphates 1. Super high energy phosphates, the enolphosphates (e.g.,

phosphoenolpyruvate), phosphoguanidines (e.g., creatine phosphate and arginine

phosphate) etc., with ΔG°′ values larger than that of ATP, 2. High energy
phosphates the ATP, ADP and 3. low energy phosphates, the ester phosphates

with ΔG°′ values smaller than that of ATP.

The same ΔG°′ values of –7.3 kcal/mol is obtained when ADP is hydrolysed to

AMP and inorganic phosphate so also when ATP is hydrolysed to ADP and

inorganic phosphate, this shows that the two terminal phosphate groups of ATP

(β and ɣ) are both high energy groups. The ΔG°′ value of hydrolysis of AMP to

adenosine and phosphate is –3.4 kcal/mol, meaning that the phosphate group of

AMP (α phosphate group of ATP) is of low energy. The hydrolysis of ATP to

yield AMP and PPi has ΔG°′ value of –7.7 kcal/mol, which is slightly greater than

ΔG°′ value for the hydrolysis of the terminal or ɣ phosphate bond.

ATP + H2O AMP + PPi ΔG°′ = –7.7 kcal/mol,

The inorganic pyrophosphate PPi is then hydrolysed to 2 moles of inorganic

orthophosphate Pi by the enzyme pyrophosphatase with ΔG°′ value of –6.9

kcal/mol.

PPi + H2O 2Pi ΔG°′ = –6.9 kcal/mol,

Then

ATP + H2O AMP + 2Pi ΔG°′ = –14.6 kcal/mol,

The ΔG°′ value of –14.6 kcal/mol is the sum of the ΔG°′ value of the two

sequential component reactions which is twice the ΔG°′ value of the terminal

phosphate groups of ATP and ADP.

Hydrolysis of ATP and coupled reactions

To understand the role of ATP in energy coupling, consider this reaction

A B ΔG°′ = +3 kcal/mol,

Calculating the equilibrium constant,

[B]
K′eq = = 10–∆G°/1.36
[A]

= 6.22 × 10–3

A cannot be converted into B spontaneously when the K′eq is 6.22 × 10–3or above

but if the reaction is coupled to the hydrolysis of ATP with ΔG°′ of –7. 3 kcal/mol,

the new reaction will be

A + ATP + H2O B + ADP + Pi + H+

The ΔG°′ for the reaction is additive i.e ΔG°′ for A goes to B and ΔG°′ for ATP

goes to ADP are added together.

= + 3 + (–7.3)

= –4.3 kcal/mol

The equilibrium constant of the coupled reaction can now be calculated as

 [ADP] [Pi]
[B]
K′eq = ×
[A] [ATP]

= 104.3/1.36

= 1.45 × 103

At equilibrium

[B] [ATP]
= K′eq ×
[A] [ADP] [Pi]

The [ATP]/[ADP] [Pi] ratio in the cells generating ATP is maintained at a high

level usually in the order of 500. Therefore,

[B] = 1.45 × 103 × 500

[A]
= 7.26 × 105

This means that the hydrolysis of ATP enables A to be converted to B until the

[B]/[A] ratio

reaches a value of 7.26 × 105 instead of 6.22 × 10–3 that does not include ATP

hydrolysis. The coupling of hydrolysis of ATP to the reaction has changed the

equilibrium ratio of B to A by a factor of about 108. The hydrolysis of one ATP

molecule has 108 effect on the equilibrium constant of a coupled reaction.

Therefore, the hydrolysis of nATP molecules will have 108n effect on the
equilibrium constant of the coupled reaction. If 3 ATP molecules is coupled to a

reaction the equilibrium ratio will change by a factor of 1024 (108×3). A

thermodynamically unfavourable reaction can be converted into a favourable

reaction by coupling it to the hydrolysis of a sufficient number of ATP molecules.

ATP as high energy phosphates in the cell

ATP is in the mid position in the standard free energies of hydrolysis of

organophosphates. Therefore, it can donate high energy phosphates (~P) to the

compounds having lower free energies below it, ADP can accept high energy

phosphate, in enzyme catalysed reaction, to form ATP from compounds with

higher free energies above ATP. Hence ATP/ADP cycle is the connector between

the process that generate ~P and those that utilize ~P.

Processes that generate ~P into ATP/ADP cycle

a. Oxidative phosphorylations catalyzed by the enzyme ATP synthetase

b. Catabolism of glucose to lactic acid known as Embden-Meyerhof-Parnas

(E.M.P.) pathway of glycolysis catalysed by the enzyme kinase

Phosphoglycerate kinase
1,3 dPG + ADP 3PG + ATP

Pyruvate kinase
2PEP + ADP Pyruvate + ATP
c. Oxidation of pyruvic acid at the succinyl thiokinase step of the Krebs’ Cycle

where succinate is produced from succinyl CoA and the GTP formed can transfer

its terminal P to ADP

GDP + Pi GTP

Succinyl CoA Succinate

d. The muscle compounds. Hydrolysis of phosphocreatine in vertebrate muscle

and arginine phosphate in invertebrate muscle.

Creatine kinase
Phosphocreatine + ADP Creatine + ATP

Arginine kinase
Arginine phosphate + ADP Arginine + ATP

Processes that utilize ~P in ATP/ADP cycle

When adenosine triphosphate (ATP) acts as a phosphate donor, the phosphate

group is invariably converted to one with low energy, producing phosphoric

esters of alcohols. Examples of such reaction are formation of D–glucose 6-

phosphate from D-glucose catalysed by hexokinase, also formation of Glycerol

3-phosphate from Glycerol catalysed by glycerol kinase.

Hexokinase
D-Glucose + ATP D-Glucose 6-P + ADP
 Glycerol kinase
Glycerol + ATP Glycerol 3-P + ADP

The hydroxyl group of glucose and glycerol (acceptor molecule) is

phosphorylated to give phosphoric ester. Glucose 6-p and glycerol 3-p are the

energized forms of glucose and glycerol respectively which can pass through

further enzymatic reactions for the synthesis of larger molecules like glycogen

and lipid respectively.

Electron transport system and oxidative phosphorylation

In aerobic cells, every enzyme-catalysed reaction of the oxidative degradation of

carbohydrates, fats and amino acids ends up in electron transport and oxidative

phosphorylation, which is the final stage of cell respiration. This is a stage where

electrons flow from organic substrates to oxygen thereby releasing energy to

generate ATP molecules.

This process is very important because the total amount of ATP in the body is

about 50 grams and an adult weighing 70 kg required 190 kilograms of ATP for

his daily work, therefore the 50 g ATP must be broken down into ADP and P and

resynthesized many times in a day so as to meet up with the chemical energy

requirement for the body need.

Electron flow as source of ATP energy

In every round of Kreb cycle, 4 pairs of hydrogen atoms are eliminated, they are

from isocitrate, a ketoglutarate, succinate and malate, catalysed by specific

dehydrogenases. These hydrogen atoms donate their electrons to the electron

transport chain and become H+ ions which escape into the aqueous medium., the

electrons are transported along a chain of electron – carrying molecules until it

gets to cytochrome aa3 (cytochrome oxidase), which takes the electrons to

oxygen, the final electron acceptor in aerobic organisms. For every two electrons

accepted by oxygen atom two H+ are taken up from the aqueous medium to form

water.

Other pairs of hydrogen atoms released from the degradation of pyruvate, fatty

acid and amino acids to Acetyl-CoA and other products also donate their

electrons to the respiratory chain for onward transfer to oxygen.

In the respiratory chain is a series of proteins tightly bound with prosthetic groups

which are capable of accepting and donating electrons to each other in a specific

sequence. The energy-rich electrons entering the electron-transport chain lose

free energy as they pass down the chain step wisely to oxygen. These lost energy

is conserved in the form of ATP in the inner mitochondrial membrane.

As the electrons passes through the respiratory chain from NADH or FADH 2 to

oxygen, 3 moles of ATP are synthesised from ADP and P. The sites of the

respiratory chain at which energy is generated to produce ATP by oxidative

phosphorylation are called energy conserving sites.

 Pyruvate Amino acid Fatty acid

2H 2H
Acetyl CoA

Citrate

Malate

Isocitrate
Fumarate

a ketoglutarate
Succinate
Succinyl CoA
 2e- 2e- NADH
2H NADH
dehydrogenase

Succinate Fumarate
Cytochrome aa3 2e-
2H + ⅟2 O2 H2O
Electron transport and oxidative phosphorylation
Therefore, Oxidative phosphorylation is the process in which ATP is formed as a

result of the transfer of electrons from NADH or FADH 2 to O 2 by a series of

electron carriers. This process takes place in the mitochondria.

The mitochondria.

This is the site of oxidative phosphorylation. It is an oval shaped organelle, made

up of two distinct membranes the outer membrane and inner membrane.

The outer membrane, made up of 50% lipids and 50% proteins, which is

responsible for maintaining the shape of the mitochondria, it contains a protein

called porin with pores that are permeable to smallest molecules of about 10

kilodaltons (10 kDa) or less ions, nutrient molecules, ATP, ADP, etc.

The space between the outer and inner membranes is called the intermembrane

space. It is occupied by soluble proteins large enough that they cannot pass

through porin. It also contains adenylate kinase and some other enzymes.

The inner membrane is made up of 20% lipids and 80% proteins, acts as a barrier

to prevent the movement of most molecules, freely permeable only to O2 and CO2

it is impermeable to nearly all ions and most uncharged molecules and contains

the electron-transport chains, succinate dehydrogenase, ATP-synthesizing

enzymes (ATP synthetase complex) and transport proteins. A few molecules have

specific transporters that allow them to enter or exit the mitochondrion. The inner

membrane contains cristae, which are involutions in inner membrane.

Finally, in the inner membrane is the matrix compartment. This is a very dense

protein solution that contains the TCA cycle enzymes, the pyruvate

dehydrogenase system and the fatty acid oxidation system. It also contains ATP,

ADP, AMP, phosphate, NAD, NADP, coenzyme A and various ions such as K+,

Mg2+ and Ca2+.

Electron transport chain

The process where NADH and FADH2 donate electron pairs to a specialized set

of proteins and act as an electron conduit to oxygen is called the electron transport

chain. In the process of the electrons passing down the chain, they lose much of

their free energy some are captured and stored in the form of a proton gradient

that are used to synthesize ATP from ADP; the remaining energy is lost as heat.

The proton gradient is created because protons are pumped from the matrix to the

intermembrane space across inner mitochondria membrane (IMM) and make the

intermembrane space side of the mitochondrial inner membrane have a higher

concentration of protons than does the other side. The proton gradient generated

by the electron transport chain in the mitochondria has both concentration and

electrical potential of which they contain energy.

A total of five large multi-protein complexes in the inner mitochondrial

membrane is involved in the electron transport and oxidative phosphorylation

pathways. They are complexes I, II, III, IV and V, complexes I, II, III and IV with

varieties of prosthetic groups like metal ions, iron-sulphur centres, hemes, and

flavins are part of the electron transport chain while complex V is the enzyme

complex that carries out the oxidative phosphorylation reaction which is the

actual conversion of ADP to ATP.

Also required in the electron transport chain apart from these large multi-protein

complexes are cytochrome c and Coenzyme Q. Coenzyme Q is not a protein, but

a membrane bound cofactor. Cytochrome c is a small soluble protein in the

intermembrane space. The overall reaction is the oxidation of NADH or FADH2

cofactors which resulted in the reduction of oxygen to water. The electron

transport chain can also be called the respiratory chain, since it is the major reason

for respiration.

Electron carriers

Electrons are transferred from substrates to oxygen via series of electron carriers

(flavins, iron-sulfur complexes, quinones and hemes). It is to be noted that all

electron carriers, except quinones, are prosthetic groups of proteins and almost
all the electron-carrying proteins are insoluble in water and they are situated in

the inner mitochondrial membrane.

1. Pyridine Nucleotides

Most of the electron pairs entering the respiratory chains come from the reaction

of dehydrogenases with the coenzymes NAD+ or NADP+ as electron acceptors.

The reversible reactions follow this general types:

Reduced substrate + NAD+ Oxidized substrate + NADH + H+

Reduced substrate + NADP+ Oxidized substrate + NADPH + H+

The pyridine-linked dehydrogenases take away 2 hydrogen atoms from their

substrates. One is transferred as a hydride ion (:H–) to the NAD+ or NADP+; the

other appears as H+ in the medium. Each hydride ion carries two reducing

Equivalents (e-): one is transferred as a hydrogen atom to C4 of the nicotinamide

ring, the other as an electron to the ring nitrogen.

NH2 NH2

Substrate
Substrate (Oxidised)
(Reduced)
+
NAD+ H+
NADH
NAD+ can also collect reducing equivalent from substrates acted upon by NADP-

linked dehydrogenases in a reaction catalysed by pyridine nucleotide

transhydrogenase.

NADPH + NAD+ NADP+ + NADH

2. NADH Dehydrogenase (=NADH-Q Reductase)

The transfer of electrons from NADH in the mitochondrial matrix to ubiquinone

in the membrane core, coupling with pumping of protons, is catalysed by a highly

organized enzyme complex, NADH dehydrogenase. This complex is made up of

flavoprotein and iron-sulphide proteins as electron carriers. In this step of electron

transfer, a pair of reducing equivalents is transferred from NADH to NADH

dehydrogenase. In NADH dehydrogenase the first type of prosthetic group flavin

mononucleotide (FMN) is reduced to FMNH2, the electrons then moved from

FMNH2 to the second type of prosthetic group in NADH dehydrogenase the iron-

sulphur complexes (Fe—S), known as non-heme iron proteins (NHI proteins).

NAD + H+ + E FMN NAD+ + E FMNH2

There are 3 principal types of Fe—S complexes, in which the number of iron and

sulphur atoms are always equal, the sulphur atoms are partly supplied by cysteine

residues in the associated protein and partly by inorganic sulphide ions. The iron

atoms in these complexes can be in the reduced (Fe2+) or oxidized (Fe3+) state.

The Fe—S complexes are a. FeCys4 (FeS) type b. Fe2S2Cys4 (Fe2S2) type and c.

Fe4S4Cys4 (Fe4S4) type. NADH dehydrogenase contains Fe2S2Cys4 and Fe4S4Cys4

types of complexes.

3. Ubiquinone (=Coenzyme Q)

Ubiquinone (UQ) is the next carrier of reducing equivalents in the respiratory

chain, it can be called coenzyme Q and abbreviated as CoQ or simply Q. This is

a group of compounds that contain the same quinone structure but substituted

with a long side chain of isoprene units (prenyl groups) usually from 6 to 10

linked head to tail. For example, microorganisms that the ubiquinone contain 6

isoprene units, the compound is referred to as Q6 or CoQ6 or UQ30, where 6 is the

number of isoprene units and 30 the total number of carbon atoms in the side

chain. Mammals usually contains ubiquinone with 10 isoprene units, designated

as Q10 or CoQ10 or UQ50.

The isoprenoid tail makes ubiquinone highly nonpolar which enables it to diffuse

readily in the hydrocarbon phase of the inner mitochondrial membrane.

Ubiquinone is the only electron carrier in the respiratory chain that is not tightly

bound or covalently attached to a protein. It serves as a highly mobile carrier of

E - FMNH2 + Q E – FMN + QH2
electrons between the flavoproteins and the cytochromes of the electron-transport

chain. The function of ubiqunione is to collect reducing equivalents from NADH

dehydrogenase and also from other flavin-linked dehydrogenases of

mitochondria.

Ubiquinones behaves like other quinones in that they may be reduced by one

electron at a time forming the semiquinone free radical or may be reduced directly

to the quinol by two electrons.

O
CH3

CH3 O CH2-C=C- CH2 H

n
CH3 O H
CH3
e- + H+ O
Ubiquinone Q OH CH3

O● CH3 O CH2-C=C- CH2 H

CH3
n
CH3 O CH2-C=C- CH2 H CH3 O H
CH3
n OH
CH3 O H
CH3
OH Ubiquinol QH2
(Dihydroubiquinone)
Ubisemiquinone QH●
(free radical)
4. Cytochromes

The enzyme complex catalysing the oxidation of ubiquinol (QH2) contains an

iron-sulphide protein of Fe2S2 Cys4 type and 2 types of cytochromes.

The cytochromes are the electron-transferring, red or brown proteins that contain

a heme prosthetic group, they are heme proteins or hemoproteins like

haemoglobin, their iron atoms are in oxidized and reduced state to transfer

electrons between other compounds, the iron atom in cytochromes alternates

between a reduced ferrous (Fe2+) state and an oxidized ferric (Fe3+) state during

electron transport. The cytochromes act in sequence to carry electrons from

ubiquinone to molecular oxygen. A heme group, like an Fe – S centre, is one-

electron carrier, in contrast with NADH, flavins and ubiquinone, which are two

electrons carriers.

There are 5 types of cytochromes between ubiquinol (QH2) and oxygen in the
b c1 c a a3
electron-transport chain, they are cytochromes b, c1, c, a and a3.

These cytochromes differ from one other in their heme prosthetic group and their

mode of attachment to the apoprotein part. In cytochrome b, the heme is not

covalently bonded to the protein, while in cytochromes c and c1, the heme is

covalently bonded to the protein by thioether linkages which is formed by the

addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of

the heme. In cytochromes a and a3 the heme is like that of c and c1 but a

hydrophobic polyprenyl chain replaces one of the vinyl groups and one of the
methyl groups of the heme is replaced by a formyl group. The type of heme

present in cytochrome b is called heme B, cytochromes c and c1 is heme C and

cytochromes a and a3 is heme A. Cytochromes a and a3 are the terminal members

of the respiratory chain. They are a complex called cytochrome oxidase. The

cytochrome aa3 complex, differs from other cytochromes because it contains 2

moles of highly-bound heme A and 2 essential copper atoms.

Heme B Heme C

Heme A
Electron transport complexes

NADH dehydrogenase (Complex I)

This contain iron-sulphur centre and Flavin mononucleotide (FMN). It accepts

electrons from NADH to regenerate NAD. It pumps protons, in this process, each

pair of electrons accepted results in the movement of 4 H+ from the matrix to the

intermembrane space. The electrons generated in the breakdown of NADH to

NAD is donated to Coenzyme Q.

NADH + H+ NAD+ + 2H+ + 2e-

Coenzyme Q

It is a non-protein electron carrier found in IMM. It can transfer one or two

electrons, can accept electrons from Complex I and II or other proteins and

donates the electrons to Complex III.

CH3 CH3

[CH2CH = CCH2]10H [CH2CH = CCH2]10H

CH3O CH3O
O

Oxidised Quinone Semi Quinone

CH3

[CH2CH = CCH2]10H
CH3O

Reduced Quinone (Hydroquinone)

Succinate Dehydrogenase (Complex II)

Succinate dehydrogenase is one of the enzymes in the TCA cycle that is

embedded in IMM. When fumarate is formed from succinate the enzyme-bound

FAD is reduced to FADH.

FAD+ + 2H+ + 2e- FADH2

The oxidation of FADH2 requires the donation of electrons to Coenzyme Q which

means the enzyme cannot carry out the reaction if the electron transport is non-

functional. Succinate dehydrogenase does not pump protons.

Coenzyme Q-dependent cytochrome c reductase (Complex III)

This accepts electrons from Coenzyme Q i.e indirectly, from both Complex I and

Complex II. It is a proton pump and contains several heme prosthetic groups. The

electrons that Complex III receives are donated one at a time to a soluble heme

containing electron carrier protein called cytochrome c. Cytochrome c is located

in the intermembrane space. Although it is a relatively small protein (~12 kDa)

but too large to pass through the porin molecules in the outer membrane.

Cytochrome c oxidase (Complex IV)

Cytochrome c oxidase, accepts electrons from cytochrome c and transfers the

electrons directly to oxygen. It is a proton pump, it is sometimes referred to as the

cytochrome a-a3 complex and contains several heme prosthetic groups. There are

many heme prosthetic groups in the electron transport chain but cytochrome c
oxidase is the only one that is capable of binding oxygen because all others have

their axial heme-iron binding sites occupied by amino acid side-chains except the

ones of cytochrome c.

F1F0-ATPase (ATP Synthase or Complex V)

The final complex required for the synthesis of ATP is the ATP synthase enzyme

complex itself known as complex V. It uses the proton gradient generated by the

proton pumps to synthesize ATP. The ATP synthase is a complicated protein

complex, comprised of two separate multi subunit proteins F0 and F1. The

transmembrane part of the complex which is embedded in the inner membrane

and extends across it which allows protons to move down their gradient (the

proton channel of the enzyme complex) is the F0 while the part of the complex

that extends into the matrix from the inner membrane and acts as the ATP

synthesis enzyme from which ATP is formed from ADP + Pi is the F1.
Mechanisms of oxidative phosphorylation

There are 3 principal hypotheses that gave account for the coupling of oxidation

and phosphorylation. These hypotheses explain how the energy transfer between

electron transport and ATP synthesis occur. They are 1. Chemical Coupling

Hypothesis, 2. Conformational Coupling Hypothesis and 3. Chemiosmotic

Coupling Hypothesis. The simplest and novel mechanism was the chemiosmotic

coupling hypothesis

Chemiosmotic Coupling Hypothesis

This hypothesis was postulated by a British biochemist named Peter Mitchell, in

1961. He proposed that electron transport and ATP synthesis is coupled by a

proton gradient, and not by a covalent high-energy intermediate or an activated

protein. There is transfer of electrons through the respiratory chain which caused

the pumping of protons (H+) from the matrix side to the cytosol or cytoplasmic

side of the inner mitochondrial membrane resulting in higher concentration of H+

on the cytoplasmic side and thus creating an electrochemical potential difference

with more positive ions on the cytoplasmic side. The hypothesis further proposes

that the H+ ions, ejected by electron transport into the cytoplasmic side, flow back

into the matrix through a specific H+ channel or ‘pore’ in the FoF1 ATPase

molecule, which is driven by the concentration gradient of H+.

Free energy is released, as proton (H+) flows back through the ATPase, this causes

the coupled synthesis of ATP from ADP and phosphate by ATP synthetase. The

inner mitochondrial membrane must be intact, completely closed vesicle

otherwise an H+ gradient across the inner membrane will not exist. If, however, a

‘leak’ of proton across the membrane is induced by uncouplers, the proton

gradient would be discharged and consequently, energy-coupling would fail.

In summary, as the high-energy electrons from the hydrogens of NADH and

FADH2 are transported down the respiratory chain in the mitochondrial inner

membrane, the energy released as they pass from one carrier molecule to the next

is used to pump protons (H+) across the inner membrane from the mitochondrial

matrix into the inner membrane space. This creates an electrochemical proton
gradient across the mitochondrial inner membrane, and the backflow of H+ down

this gradient is, in turn, used to drive the membrane-bound enzyme ATP synthase,

which catalyses the conversion of oxidative phosphorylation. The high energy

chemical intermediates are replaced by a link between chemical processes

(“chemi”) and transport process (“osmotic”) hence chemiosmotic coupling.

INNER MITOCHONDRIA MEMBRANE

+
substrate
+

CYTOSOL MATRIX
+

+
Proton
H+ H+
Translocation
+

+
O2
+

ADP + Pi
+
H+ H+
ATP
+

ATP synthetase
Electron-transferring reactions (oxidation - reduction reaction)

The chemical reactions which involve the transferring of electrons from one

molecule to another are called oxidation-reduction reactions or oxidoreductions

or redox reactions. The molecule that donate electron is the reducing agent

(reductant) and the one that accept electron is the oxidizing agent (oxidant). The

reducing or oxidizing agents function as conjugate reductant-oxidant pairs (redox

pairs) with the general equation

Electron donor e- + Electron acceptor

Fe2+ e- + Fe3+

Ferrous ion (Fe2+) = electron donor and ferric ion (Fe3+) = electron acceptor. Fe2+

and Fe3+ = conjugate redox pair.

Four different ways of transferring electrons and they all occur in cells.

a. Direct transfer of electrons

Fe2+ + Cu2+ Fe3+ + Cu+

b. Transferring of hydrogen atoms

AH2 A + 2e– + 2H+
AH2 = hydrogen (or electron) donor, A = hydrogen acceptor and AH2 and A =

conjugate redox
AHpair.
+B A + BH2
2

c. In the form of a hydride ion— The hydride ion (: H–) bears two electrons, as in

the case of NAD-linked dehydrogenases.

NADH + H+ NAD+ + 2H+ + 2e-

d. Direct combination of an organic reductant with oxygen

R CH3 + 12 O2 R CH2 OH

STANDARD OXIDATION-REDUCTION POTENTIAL

The likelihood of a given conjugate redox pair to lose an electron is specified by

a constant called standard oxidation-reduction potential, standard redox potential

or standard potential E′o. This is the electromotive force (e.m.f.) in volts given by

a responsive electrode placed in a solution containing the electron donor and its

conjugate electron acceptor at 0.1 M concentration, 25°C and pH 7.0. Standard

potentials are in volts; for convenience in millivolts. Each conjugate redox pair

has a characteristic standard potential.

The higher the negative values of a standard potential of a system the higher the

tendency of losing electrons, and the higher the positive values of a standard

potential of a system the higher the tendency of accepting electrons. This means
that the more negative the E′o, the lower is the affinity of the system for electrons

and conversely, the more positive the E′o of a system, the greater is its electron

affinity. Thus, electrons tend to flow from one redox couple to another in the

direction of the more positive system. For example, isocitrate/a-ketoglutarate +

CO2 couple with E′o = –0.38 V tends to pass electrons to the redox couple

NADH/NAD+ with E′o = –0.32 V which has a relatively more positive standard

potential. Conversely, H2O/O2 couple with E′o = + 0.82 V, shows that water

molecule has very little tendency to lose electrons to form molecular oxygen but

molecular oxygen has a very high affinity for electrons or hydrogen atoms.

In oxidation systems, the electrons tend to flow from a relatively electronegative

conjugate redox pair, e.g NADH/NAD+ (E′o = – 0.32 V), to the electropositive

pair, e.g reduced cytochrome c/oxidized cytochrome c (E′o = + 0.25 V) and to the

more electropositive pair of water/oxygen pair (E′o = + 0.82 V).

The greater the difference in the E′o between two redox pairs, the greater is the

free-energy loss as electrons pass from the electronegative to the electropositive

pair. Therefore, when electrons flow down the complete electron-transport chain

from NADH to oxygen through several electron-carrying molecules, they lose a

large amount of free energy.

The amount of available free-energy can be calculated as a pair of electrons

passes from NADH to O2 using the equation,

ΔG°′ = –nFΔE′o
ΔG°′ = standard-free-energy in calories, n = number of electrons transferred, F =

faraday constant (23.062 kcal/V. mol) and ΔE′o = difference between E′o of the

electron-donor system and that of the electron-acceptor system.

The standard-free-energy change as a pair of electron (2e-) passes from

NADH/NAD+ pair (E′o = – 0.32 V) to the H2O/O2 pair (E′o = + 0.82 V) can be

calculated from

ΔG°′ = –nFΔE′o

ΔG°′ = – 2 (23.062) [0.82 – (– 0.32)]

= – 2 × 23.062 × 1.14

= –52.58 kcal/mol

This amount of free energy (i.e., –52.58 kcal) is more than sufficient to bring

about the synthesis of 3 moles of ATP, which requires an input of 3 (7.3) = 21.9

kcal under standard conditions. Using this expression ΔG°′ = –nFΔE′o, the free-

energy changes for individual segments of the electron-transport chains can be

calculated from the differences in the standard potentials of the electron-donating

redox pair and the electron-accepting pair.

Importance of electron transport energy

The main function of electron transport in mitochondria is the provision of energy

for the synthesis of ATP during oxidative phosphorylation.

The energy generated can also be used for other biological purposes.
1. The proton gradient generated by electron transport can be used to generate

heat for the maintenance of body temperature especially in hairless mammals and

some hibernating animals.

2. The electron transport energy is used to transport Ca 2+ from the cytosol into

the matrix of mitochondria.

3. Rotation of bacterial flagella is controlled by the proton gradient generated

across the membrane.

4. Transfer of electrons from NADH to NADPH is powered by the proton

gradient.

5. The entry of some amino acids and sugars is governed by the energy generated

during electron transport.

Respiratory inhibitors

A. Inhibitors of Electron Transport chain

These are the inhibitors that stop respiration by combining with members of the

respiratory chain, they act at 3 loci that may be identical to the energy transfer

sites I, II and III, some of these compounds are well-known drugs or poisons.

Rotenone

It complexes with NADH dehydrogenase and acts between the Fe—S proteins

and ubiquinone thereby inhibiting transfer of electrons to Coenzyme Q from

Complex I. Rotenone is relatively nontoxic to mammals because it is absorbed

poorly but the compound is intensely toxic to the fishes and insects as it readily

passes into their gills and breathing tubes respectively.

Piericidin A

It is an antibiotic produced by Streptomyces that have similar action like that of

rotenone.

Barbiturates (Amytal, seconal).

A sedative that also block NADH dehydrogenase, but are required in much higher

concentrations for the purpose. In contrast, barbiturates, rotenone and piericidin

A do not interfere with the oxidation of succinate, because the electrons of these

substrates enter the electron-transport chain beyond the block of coenzyme Q.

Antimycins

These are also antibiotics, produced by Streptomyces. They inhibit the respiratory

chain at or around site II and block electron flow between cytochromes b and c1

inhibiting electron transfer from Complex III to cytochrome c.

Dimercaprol

It is identical in action to the antimycins.

Cyanides

The cyanide ion (CN–) combines tightly with cytochrome oxidase, they bind to

the oxygen binding site on the heme (Fe3+) prosthetic group of cytochrome c

oxidase leading to the cessation of transfer of electrons to oxygen. The effect of

cyanide is as fundamental as deprivation of oxygen, and like the latter causes

rapid damage to the brain.

Azide

It blocks the electron flow between the cytochrome oxidase complex and oxygen.

Azide (N3–), as also cyanides, react with the ferric form (Fe3+) of this carrier.

Hydrogen sulphide (H2S)

This also acts like the HCN but its disagreeing odour gives more warning.

Carbon monoxide

It also attacks between cytochrome oxidase and O2 but, unlike cyanide and azide,

CO inhibits the ferrous form (Fe2+) of the electron carrier. In contrast, cyanide

CN- is much more effective cytochrome c oxidase inhibitor because it has a high

affinity for cytochrome c oxidase, and a relatively low affinity for hemoglobin.

B. Inhibitors of Oxidative Phosphorylation

These compounds inhibit electron transport in cells where such transport is

coupled with ATP synthesis, but has no effect on electron transport that is not

coupled with phosphorylation. Thus, these compounds inhibit both electron

transport and oxidative phosphorylation.

Oligomycins

These polypeptide antibiotics are obtained from various species of Streptomyces.

They inhibit the transfer of high-energy phosphate to ADP and, therefore, inhibit

electron transfers coupled to phosphorylation. However, they do not affect those

redox reactions that are not coupled. They are widely used as experimental tools
to differentiate between the two kinds of reactions. Oligomycin binds the Fo

component of ATP synthetase and it is a potent inhibitor of this enzyme and, thus,

of oxidative phosphorylation.

Rutamycin

This antibiotic also inhibits both electron transport and oxidative phosphorylation

and acts like oligomycin.

Atractylate (Atractyloside)

This is a toxic glycoside extract of rhizomes Atractylis gummifera. It affects

oxidative phosphorylation by competing with ATP and ADP for a site on the

ADP/ATP antiport of the inner mitochondrial membrane. Atractyloside indirectly

inhibits Complex V because it reduces the amount of free ADP substrate

available; the effect is an inhibition both of ATP synthesis and of electron

transport.

Bongkrekate

Toxin formed by Pseudomonas. It also blocks the ADP/ATP antiport.

Rotenone
Piericidin A Antimycin CN, N3,
Amytal dimercaprol H2S, CO
dimercapr
NADH [FMN] [FeS] [Cyt b] [Cyt c1] O2

Site I Site II Site III

C. Uncouplers of Oxidative Phosphorylation

Uncoupling agents are compounds which disconnect (uncouple) the synthesis of

ATP from the transport of electrons through the cytochrome system. In this

situation, electron transport is performed, oxygen is consumed but does not lead

to phosphorylation of ADP. When uncouplers are present, the free energy

released by electron transport appears as heat, rather than as newly-made ATP.

Uncoupling agents causes the inner membrane to be permeable to H+. They are

lipophilic and bind H+ in the region of the membrane with higher H+

concentration and carry it through the membrane to the side with the lower H +

concentration. They are called protonophores because they bind and carry

protons.

The difference between uncoupling and inhibition process is that in uncoupling

there is increase in oxygen consumption and ATP is not utilised whereas in

inhibition of phosphorylation (or inhibition of ADP/ATP antiport) oxygen

consumption diminishes and ATP is utilised. Examples of some uncouplers.

2, 4-dinitrophenol (DNP)

The uncoupling action of DNP is carried out by both the phenol and the

corresponding phenolate ion that are significantly soluble in the lipid core of the

inner mitochondrial membrane, hence it can pass through inner membrane. The

phenol diffuses through the core to the matrix, where it loses a proton forming

the phenolate ion which in turn diffuses back to the cytosol side, where it picks
up a proton to repeat the process. In this process, uncouplers prevent formation

of H+ gradient across the membrane. Dinitrophenol also stimulates the activity of

the enzyme ATPase, which is normally inactive as a hydrolytic enzyme in

mitochondria.

Cytosol Matrix
IMM

H+ H+

Phenolate ion 2,4-dinitrophenol 2,4-dinitrophenol Phenolate ion

Dicoumarol

Dicoumarol arises from the action of microorganisms on coumarin. Its action is

identical to that of 2,4-dinitrophenol.

m-chlocarbonyl cyanide phenylhydrazone (CCCP)

Its action is also similar to that of 2, 4-dinitrophenol but it is about 100 times more

active than DNP.

D. Ionophores (ion carriers) of Oxidative Phosphorylation

They are lipophilic substances, that bind and carry specific cations through the

biologic membranes. They are different from uncouplers in that they cause the

movement of other cations different from H+ through the membrane.

Valinomycin

This is a toxic antibiotic synthesized by Streptomyces. The antibiotic forms a

lipid-soluble complex with K+ which readily passes through the inner

mitochondrial membrane, which normally slowly allowed K+ alone in the

absence of valinomycin. It has a high degree of selectivity for K+. Valinomycin

interferes with oxidative phosphorylation in mitochondria by making them

permeable to K+. This make the mitochondria to use the energy generated by

electron transport to accumulate K+ instead of making ATP.

Nigericin

This acts as an ionophore for K+ but in exchange for H+ thereby preventing the

build-up of pH gradient across the membrane. In the presence of both

valinomycin and nigericin, the membrane potential and the pH gradient are

eliminated, and phosphorylation is completely inhibited.

Regulation of the electron transport pathway

The synthesis of ATP by electron transport and oxidative phosphorylation are

regulated essentially by the availability substrate. The 3 main energy (ATP)-

yielding stages in carbohydrate metabolism are glycolysis, TCA cycle and

oxidative phosphorylation. Each of this stages are regulated as to satisfy the time-

to-time need of the cell for its products and they are self-regulated way. They
produce ATP and certain specific intermediates such as pyruvate and citrate, that

act as precursors for the biosynthesis of other cell components. The relative

concentrations of ATP and ADP controls the rate of electron transport and

oxidative phosphorylation also control the rates of glycolysis, pyruvate oxidation

and the citric acid cycle.

When ATP concentration is high and ADP and AMP concentration is low ([ATP]

: [ADP][Pi] ratio is high), the rates of glycolysis, pyruvate oxidation, the citric

acid cycle and oxidative phosphorylation will be reduced i.e at minimum. When

the rate at which ATP utilization is very high by the cell, ADP, AMP and Pi

concentration will increase, this will result in immediate increase in the rate at

which electron transport and oxidative phosphorylation occurs. Rate of pyruvate

formation via glycolysis, pyruvate oxidation through citric acid cycle will also

increase, thereby increasing the flow of electrons into the respiratory chain. The

pathway to ATP synthesis cannot proceed without ADP + Pi or NADH; when

they are available, then the pathway will result in ATP synthesis.

The regulatory controls are both inhibitory and stimulatory. When ATP and

citrate increases, the two have a concerted allosteric inhibition on

phosphofructokinase (PFK) leading to reduction in glycolysis (production of

pyruvate). When ATP increases, NADH and Acetyl-CoA levels also increases,

which will cause the inhibition of the oxidation of pyruvate to Acetyl-CoA.

 Glucose

Glucose 6 P

Glycolysis
Fructose 6 P

AMP

Fructose 1,6 dP

Pyruvate
NADH & Acetyl CoA

Acetyl CoA

Citrate
TCA cycle
Isocitrate
ATP
a ketoglutatrate
ATP Electron transport chain O2
Succinate

Fumarate
ADP + Pi
Malate

Oxaloacetate