REDOX POTENTIAL & ETC

DR. RABIA
(DPT)
BIOCHEMISTRY
LECTURE#02
LECTURER: ALLIED COLLEGE OF HEALTH SCIENCES
MULTAN
DATE: 27-05-2021
REDOX POTENTIAL

• Oxidising or reducing agent may exist in two forms, (a) the oxidised form or oxidant
which can accept electrons and (b) the reductant which can donate its electrons to a
substrate. Each oxidising or reducing agent exists as a conjugate pair of electron acceptor
oxidant and electron donor reductant forms
• The pair consisting of the oxidant and reductant forms of an oxidising or reducing agent
is known as a redox couple or conjugate redox pair, e.g. NAD+/NADH, FMN/FMNH2.
• Oxidising agents differ in their electron affinity.
• The standard redox potential Eo is a measure of the tendency of a redox couple to
donate or accept electrons under standard conditions (at 7 pH & 25˚C). Redox potential
of a given system is intimately related to its free energy change. If redox-potential of a
given system is known, the corresponding free energy change which might occur in the
system on oxidation or reduction may be found as follows
• If the sign of free-energy thus calculated is negative, it indicates free energy release and if
positive, it indicates free energy consumption
• The more negative the Eo of a redox pair, the greater the tendency of the reductant
member of that pair to lose electrons. The more positive the Eo, the greater the tendency
of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the
pair with the more negative Eo to that with the more positive Eo.
• In the mitochondria, the hydrogen/electrons pass through different carriers in the
sequence of increasing order of their positive potential.
• ∆G˚ of ATP: The ∆G˚ for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport
of a pair of electrons from NADH to O2 through the ETC releases 52.58 kcal. Therefore,
more than sufficient energy is available to produce 3 ATP from 3 ADP and 3 Pi (3 × 7.3 = 21.9
kcal/mol)from pair of electron, sometimes expressed as a P:O ratio (ATP made per O atom
reduced) of 3:1. The remaining calories are used for ancillary reactions or released as heat.

• The P:O for FADH2 is 2:1 because Complex I is bypassed.

• There is a strong evidence now to suggest a P:O ratio of 2.5 for NADH, and 1.5 for FADH2,
since ten protons (for NADH) and six protons (for FADH2) are pumped across mitochondrial
membrane.
• Synthesis of one ATP requires four protons
ELECTRON TRANSPORT CHAIN

• The energy-rich carbohydrates (particularly glucose), fatty acids and amino acids undergo a series of metabolic reactions
and, finally, get oxidized to CO2 and H2O. The reducing equivlents from various metabolic intermediates are
transferred to coenzymes NAD+ and FAD to produce, respectively, NADH and FADH. The latter two reduced
coenzymes pass through the electron transport chain (ETC) or respiratory chain and, finally, reduce oxygen to water.
• Definition: This is the final common pathway in aerobic cells by which electrons derived from various substrates are
transferred to oxygen. Electron transport chain (ETC) is a series of highly organised oxidation-reduction enzymes
• Localisation: The ETC is localised in the inner mitochondrial membrane.
• Structure of mitochondria: The outer membrane of mitochondria is permeable to most of the small molecules. The
outer membrane contains special channels (formed by the protein porin), making it freely permeable to most ions and
small molecules.
• There is an intermediate space which presents no barrier to passage of intermediates.
• The inner membrane shows a highly selective permeability. It has transport systems only for specific substances such as
ATP, ADP, pyruvate, succinate, α-ketoglutarate, malate and citrate etc. The enzymes of the electron transport chain are
embedded in the inner membrane in association with the enzymes of oxidative phosphorylation. The inner mitochondrial
membrane is unusually rich in protein. This membrane is highly folded to form cristae. The surface area of inner
mitochondrial membrane is greatly increased due to cristae. The inner surface of the inner mitochondrial membrane
possesses specialized particles (that look like lollipops), the phosphorylating subunits which are the centres for ATP
production.
• Mitochondrial matrix The interior gel like ground substance forms the matrix of mitochondria. It is also rich in the
enzymes responsible for the citric acid cycle, β-oxidation of fatty acids and oxidation of amino acids. The synthesis of
glucose, urea, and heme occurs partially in the matrix of mitochondria. In addition, the matrix contains NAD+ and FAD
(the oxidized forms of the two coenzymes that are required as hydrogen acceptors), and ADP and Pi, which are used to
produce ATP. The matrix also contains mitochondrial DNA (mtDNA) and RNA (mtRNA) and ribosomes
• The redox potentials in the ETC are in increasing order except in the case of ubiquinone
• The chain actually consists of a series of redox couples, at each step electrons flow from the reductant of a redox couple
with more negative redox potential to the oxidant of the next redox couple having a more positive redox potential
STRUCTURAL ORGANIZATION OF RESPIRATORY
CHAIN
• The inner mitochondrial membrane can be disrupted into five distinct respiratory or
enzyme complexes, denoted as complex I, II, III, IV and V. The complexes I-IV are
carriers of electrons while complex V is responsible for ATP synthesis. Besides these
enzyme complexes, there are certain mobile electron carriers in the respiratory chain.
These include NADH, coenzyme Q, cytochrome c and oxygen. The enzyme complexes
(I-IV) and the mobile carriers are collectively involved in the transport of electrons
which, ultimately, combine with oxygen to produce water.
• The largest proportion of the oxygen supplied to the body is utilized by the mitochondria
for the operation of electron transport chain.
COMPONENTS AND REACTIONS OF THE
ELECTRON TRANSPORT CHAIN
• There are five distinct carriers that participate in the electron transport chain (ETC). These carriers are
sequentially arranged and are responsible for the transfer of electrons from a given substrate to
ultimately combine with proton and oxygen to form water.
Nicotinamide nucleotides: Of the two coenzymes NAD+ and NADP+ derived from the vitamin
niacin, NAD+ is more actively involved in the ETC. Dehydrogenation of substrate is the first step in
the process of respiratory chain oxidation. Most of the dehydrogenases require NAD which can accept
a hydride ion (H– ) which is formed by one hydrogen atom and an electron. The electron is received
from the second hydrogen atom releasing the second hydrogen atom in the form of a proton (H+). The
second type of dehydrogenase reaction makes use of FAD as the coenzyme. Substrates include
glyceraldehyde-3 phosphate, pyruvate, isocitrate, D-ketoglutarate and malate.
 AH2 + NAD+↔ A + NADH + H+
NADPH + H+ produced by NADP+-dependent dehydrogenase is not usually a substrate for ETC. NADPH is
more effectively utilized for anabolic reactions (e.g. fatty acid synthesis, cholesterol synthesis.

Flavoproteins: The enzyme NADH dehydrogenase (NADH coenzyme Q reductase) is a flavoprotein with
FMN as the prosthetic group. The coenzyme FMN accepts two electrons and a proton to form FMNH2.
NADH dehydrogenase is a complex enzyme closely associated with non-heme iron proteins (NHI) or iron-
sulfur proteins (FeS)
NADH + H+ + FMN → NAD+ + FMNH2
Succinate dehydrogenase (succinate-coenzyme Q reductase) is an enzyme found in the inner mitochondrial
membrane. It is also a flavoprotein with FAD as the coenzyme. This can accept two hydrogen atoms (2H+ +
2e–) from succinate.
Succinate + FAD → Fumarate + FADH2
Iron-sulfur proteins: The iron-sulfur (FeS) proteins exist in the oxidized (Fe3+) or
reduced (Fe2+) state. About half a dozen FeS proteins connected with respiratory chain
have been identified. However, the mechanism of action of iron-sulfur proteins in the
ETC is not clearly understood. One FeS participates in the transfer of electrons from
FMN to coenzyme Q. Other FeS proteins associated with cytochrome b and cytochrome
c1 participate in the transport of electrons.
 Coenzyme Q: Coenzyme Q is also known as ubiquinone since it is ubiquitous in living
system. It is a quinone derivative with a variable isoprenoid side chain. The mammalian
tissues possess a quinone with 10 isoprenoid units which is known as coenzyme Q10
(CoQ10).

Coenzyme Q is a lipophilic electron carrier. It can accept electrons from FMNH2 produced
in the ETC by NADH dehydrogenase or FADH2 produced outside ETC (e.g. succinate
dehydrogenase, acyl CoA dehydrogenase). Coenzyme Q is not found in mycobacteria.
Vitamin K performs similar function as coenzyme Q in these organisms. Coenzyme Q has no
known vitamin precursor in animals. It is directly synthesized in the body.
Cytochromes: The cytochromes are conjugated proteins containing heme group. The latter
consists of a porphyrin ring with iron atom. The heme group of cytochromes differ from that
found in the structure of hemoglobin and myoglobin. The iron of heme in cytochromes is
alternately oxidized (Fe3+) and reduced (Fe2+), which is essential for the transport of
electrons in the ETC. This is in contrast to the heme iron of hemoglobin and myoglobin which
remains in the ferrous (Fe2+) state. Three cytochromes were initially discovered from the
mammalian mitochondria. They were designated as cytochrome a, b and c depending on the
type of heme present and the respective absorption spectrum. Additional cytochromes such as
c1, b1, b2, a3 etc. were discovered later. The electrons are transported from coenzyme Q to
cytochromes (in the order) b, c1, c, a and a3. The property of reversible oxidation-reduction of
heme iron Fe2+ Fe3+ present in cytochromes allows them to function as effective carriers of
electrons in ETC
Cytochrome c (mol. wt. 13,000) is a small protein containing 104 amino acids and a heme
group. It is a central member of ETC with an intermediate redox potential. It is rather loosely
bound to inner mitochondrial membrane and can be easily extracted.
Cytochrome a and a3 : The term cytochrome oxidase is frequently used to collectively
represent cytochrome a and a3 which is the terminal component of ETC. Cytochrome oxidase
is the only electron carrier, the heme iron of which can directly react with molecular oxygen.
Besides heme (with iron), this oxidase also contains copper that undergoes oxidation
reduction (Cu2+ Cu+) during the transport of electrons. In the final stage of ETC, the
transported electrons, the free protons and the molecular oxygen combine to produce water
 Complex I: NADH-CoQ Reductase This system has two functions: Electron transfer &
Acts as a proton pump. The system catalyses transfer of two electrons from NADH to
small lipid soluble CoQ via FMN and Fes clusters.

From FMN.H2 electrons are transferred to a group of Fes proteins. Fe atoms of FeS protein
oscillate between Fe++ and Fe+++. The electrons are then transferred to CoQ.

The process is accompanied by pumping of protons from mitochondrial matrix into

intermembrane space. Permits one ATP Formation (Site I)
 Complex II: Succinate-CoQ Reductase Flow of electrons from succinate to CoQ occurs
via FAD.H2.

Standard reduction potential for transfer of electrons from FAD.H2 to CoQ is + 0.113V
(much lower than +0.420 V energy change for the reaction of complex I). The small energy
change does not allow “succinate-CoQ reductase” system to pump protons across the
mitochondrial membrane, hence this protein complex does not contribute to proton
gradient. Hence no ATP is formed.
 Complex III: CoQ-Cyt.C Reductase functions as proton pump, and catalyses transfer of
electrons. This system catalyses transfer of electrons from CoQ.H2 to Cyt-c via Cyt-b and
Cyt-c1. The electrons from CoQ.H2 is first accepted by Cyt-b566 and then transferred to
Cyt-b562, which reduces
Co.Q.H2→ CoQ
Fe+++ accepts electron and is oxidised to Fe++. The system also acts as a proton pump. It is
believed that 4 (four) protons are pumped across the mitochondrial membrane during the
oxidation

The energy change permits ATP formation (Site II)

The process is believed to involve cytochromes Cv, b1 and bHh and a Rieske FeS. Rieske FeS is an
unusual FeS in which one of the Fe atoms is linked to two histidine –SH groups rather than two cysteine
–SH goups and is called as the Q cycle. Q may exist in three forms:
Oxidised quinone,
Reduced quinol,
Semiquinone.
The semiquinone is formed transiently during the cycle, one turn of which results in the oxidation of
2Q.H2 to Q, releasing 4H+ into the intermembrane space, and the reduction of one Q to QH2, causing
2H+ to be taken up from the matrix
 Complex IV: Cyt-c Oxidase The system functions as proton pump & catalyses transfer of
electrons to molecular O2 to form H2O. This is the terminal component of ETC. It catalyses the
transfer of electrons from Cyt-c to molecular O2 via Cyt-a, Cu++ ions and Cyt-a3.

The flow of electrons is as follows:

Role of Cu Ions: Cu atom adjacent to cyt. haem a is Cu-A (Sub unit II) and Cyt-haem a3 is close to
CuB (Sub unit I). From Cyt-c, the electrons are transferred to haem-a-CuA cluster and then haem
a3-CuB cluster. The system also acts as a proton pump, it pumps two protons into intermembrane
space. The energy change permits ATP formation (Site III) between cyt a3 and molecular O2.
INHIBITORS OF ELECTRON TRANSPORT CHAIN

• Transfer of electrons is selectively inhibited at various components of the electron

transport chain by a variety of substances. Some of these are used as poisons (e.g.
insecticides) and some of which are used as drugs.
• Mitochondrial Shuttle Systems: Glycolysis produces NADH in the cytoplasm which
cannot enter mitochondria. Shuttles between cytoplasm and mitochondria operate. Two
such shuttles are of considerable importance:
(a) Glycerophosphate Shuttle
(b) Malate Shuttle