1.

Define chemiosmotic principle

Chemiosmosis is the movement of ions across a semipermeable membrane, down

their electrochemical gradient.
An example of this would be the generation of ATP by the movement of H+ across
a membrane during cellular respiration or photosynthesis.
The chemiosmotic theory explains the functioning of electron transport chains. According to
this theory, the transfer of electrons down an electron transport system through a series of
oxidation-reduction reactions releases energy. This energy allows certain carriers in the
chain to transport hydrogen ions (H+ or protons) across a membrane. As the hydrogen ions
accumulate on one side of a membrane, the concentration of hydrogen ions creates an
electrochemical gradient or potential difference (voltage) across the membrane. (The fluid
on the side of the membrane where the protons accumulate acquires a positive charge; the
fluid on the opposite side of the membrane is left with a negative charge.) The energized
state of the membrane as a result of this charge separation is called proton motive force or
PMF.
This proton motive force provides the energy necessary for enzymes called ATP synthases,
also located in the membranes mentioned above, to catalyse the synthesis of ATP from ADP
and phosphate. This generation of ATP occurs as the protons cross the membrane through
the ATP synthase complexes and re-enter either the bacterial cytoplasm or the matrix of the
mitochondria. As the protons move down the concentration gradient through the ATP
synthase, the energy released causes the rotor (F0) and stalk of the ATP synthase to rotate.
The mechanical energy from this rotation is converted into chemical energy as phosphate is
added to ADP to form ATP in the catalytic head (F1 domain)

2. Describe the electron transport chain and how this results in ATP Generation

During chemiosmosis, electron carriers like NADH and FADH donate electrons to the
electron transport chain. The electrons cause conformation changes in the shapes of the
proteins to pump H+ across a selectively permeable cell membrane. The uneven
distribution of H+ ions across the membrane establishes both concentration and electrical
gradients (thus, an electrochemical gradient) owing to the hydrogen ions' positive charge
and their aggregation on one side of the membrane.

The Chemiosmotic Theory states that coupling of electron transfer to ATP synthesis is
indirect, via a H+ electrochemical gradient:

1. Respiration: Spontaneous electron transfer through complexes I, III, and IV is

coupled to non-spontaneous H+ ejection from the mitochondrial matrix. H+ ejection
creates a membrane potential (DY, negative in the matrix) and a pH gradient (DpH,
alkaline in the matrix).
2. F1Fo ATP Synthase: Non-spontaneous ATP synthesis is coupled to spontaneous
H+ transport into the matrix compartment. The pH and electrical gradients created
 by respiration are together the driving force for H+ uptake.
Return of protons to the matrix via Fo "uses up" the pH and electrical gradients.

A total of 10 protons are ejected from the mitochondrial matrix per 2

electrons transferred from NADH to oxygen via the respiratory chain.

Complex I (NADH Dehydrogenase) transports 4H+ out of the

mitochondrial matrix per 2e- transferred from NADH to coenzyme Q.

Complex III (bc1 complex): H+ transport in complex III involves coenzyme Q (CoQ). The "Q
cycle" depends on the mobility of CoQwithin the lipid bilayer. There is evidence for one-
electron transfers, with an intermediate semiquinone radical state.

Q Cycle:
Electrons enter complex III via coenzyme QH2, which binds at a site on the positive
side of the inner mitochondrial membrane, adjacent to the intermembrane space.
QH2 gives up one electron to the Rieske iron-sulfur center (Fe-S).
Fe-S is reoxidized by transfer of the electron to cytochrome c1, which passes it out
of the complex to cytochrome c.
The loss of one electron from QH2 would generate a semiquinone radical, shown
here as Q-, although the semiquinone might initially retain a proton as QH.
A second electron is transferred from the semiquinone to cytochrome bL (heme
bL), which passes it across the membrane via cytochrome bH (heme bH) to
another CoQ bound at a site on the matrix side of the membrane.
The fully oxidized CoQ, generated as the second electron is passed to the b
cytochromes, may then dissociate from its binding site adjacent to the
intermembrane space.
Accompanying the two-electron oxidation of bound QH2, 2H+ are released to the
intermembrane space.
 Overall reaction catalyzed by complex III, including net inputs and outputs of the Q
cycle:
QH2 + 2H+(matrix side) + 2 cyt c (Fe3+) Q + 4H+(outside) + 2 cyt c (Fe2+)

Complex III is an obligate homo-dimer. The iron-sulfur center in one half of the
dimer may interact with bound CoQ and heme c1 in the other half of the dimer.

Complex IV (Cytochrome Oxidase): As discussed in the section on the respiratory

chain, electrons are donated to complex IV, one at a time, by cytochrome c, which binds
from the intermembrane space. Each electron passes via CuA and heme a to the binuclear
center, buried within the complex, that catalyzes oxygen reduction:
4e- + 4H+ + O2 2H2O.
Protons utilized in this reaction are taken up from the matrix compartment.

H+ pumping by complex IV:

In addition to the protons utilized in the reduction of O2, there is electron transfer-
linked transport of 2H+ per 2e- (4H+ per 4e-) from the matrix to the intermembrane space.

3. What is Dinitrophenol? And what are its mechanisms of action?

Dinitrophenol (DNP) produced by hydrolysis of 1-chloro-2,4-dinitrobenzene.

It is commercially used as an antiseptic and as a pesticide -> chemical intermediate in
production of sulfur dye, wood preservative and some herbicides

DNP is an uncoupler of oxidative phosphorylation and works in the mitochondria. In living

cells DNP acts as a proton ionophore (is a chemical species that reversibly binds ions) that
shuttles protons across the biological membranes -> dissipates proton gradient across
mitochondria and chloroplast membranes
- causes collapse of proton motive force used to produce ATP, and instead of
producing atp the energy is lost as heat
As a result, the protons are able to bypass the action of ATP synthase and simply diffuse
back inside the inner membrane. This heat, incidentally, accounts for the danger posed by
acute overdoses of DNP. The body has no natural feedback cycle by which to counteract it,
and it increases in proportion to the dosage of DNP administered. Therefore, an acute
overdose of DNP can cause dangerous hyperthermia.
Importantly, DNP does not disrupt any other step in the process of respiration and oxygen
consumption. By allowing protons to bypass ATP synthase, DNP makes respiration
much less efficient; it also increases oxygen consumption, in much the same way as would
cardio exercise. But it leaves the respiratory chain intact.

4. Define uncoupler

Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the

electron transport and phosphorylation reactions and thus inhibit ATP synthesis without
affecting the respiratory chain and ATP synthase (H(+)-ATPase).
- An uncoupler is a protonophore (CCCP, FCCP, DNP) which cycles across the inner mt-
membrane with transport of protons and dissipation of the electrochemical proton
gradient
2,4-Dinitrophenol, dicumarol and carbonyl cyanidep-trifluorocarbonyl-cyanide
methoxyphenyl hydrazone (FCCP) all have hydrophobic character making them soluble in
the bilipid membrane.
All of these decouplers also have dissociable protons allowing them to carry protons from
the intermembrane space to the matrix which collapses the pH gradient.
DNP decreases the formation of high-energy phosphate bonds in mitochondria and at the
same time stimulates systemic oxygen consumption. This dissociative effect is known as
uncoupling of oxidative phosphorylation. Adenosine triphosphate (ATP) production is the
final product of the tricarboxylic acid (Krebs) cycle in mitocondria along with CO2 and H2O.
During glycolysis, there is a net production of two ATP molecules, but the majority of
energy-rich phosphate bonds (38 in total) are produced during the final oxidative
phosphorylation process. During this final phase, ATP synthetase converts
adenosinediphosphate to ATP with the addition of an inorganic phosphate molecule. DNP
interferes with the final energy production pathway by preventing the uptake of inorganic
phosphate molecules into the mitochondria. T
his results in the inhibition of all energy-requiring processes and the extra-mitochondrial
accumulation of inorganic phosphate. DNP also acts as a chemical ionophore, stopping the
final energy conversion by exporting the proton ions (H+) needed for ATP production across
the mitochondrial membrane by increasing the basal leak of protons. This shift in the proton
electrochemical gradient then results in potential energy dissipating as heat, instead of
being converted to ATP, with rapid consumption of calories. The heat production represents
a failure in thermoregulatory homeostasis, leading to uncontrolled hyperthermia.
 5. Why was DNP used as a diet pill?

2,4-Dinitrophenol (DNP) is reported to cause rapid loss of weight, but unfortunately is

associated with an unacceptably high rate of significant adverse effects. DNP is sold mostly
over the internet under a number of different names as a weight loss/slimming aid. It causes
uncoupling of oxidative phosphorylation; the classic symptom complex associated with
toxicity of phenol-based products such as DNP is a combination of hyperthermia,
tachycardia, diaphoresis and tachypnoea, eventually leading to death.
Nowadays, DNP is sold mostly over the internet under a number of different names such as
DNP, Dinosan, Dnoc, Solfo Black, Nitrophen, Aldifen and Chemox. The chemical is a
yellow crystalline powder that has a sweet, musty odour and is soluble in water. The dose of
DNP per capsule varies from website to website but it is most commonly sold as either 100-
or 200-mg capsules. Some internet sites have DNP available in bulk quantities, allowing
users to purchase kilograms of DNP powder or hundreds/thousands of DNP-containing
tablets, as well as offering free anabolic steroids and thyroxine to use in combination with
the DNP.

6. Explain nonshivering thermogenesis in humans

The word thermogenesis means the process of heat production in organisms. When adults
are cold, they shiver. Shivering results to increased muscle activity, thus, producing heat. In
newborns these mechanisms of heat production do not occur. Newborns rarely shiver
except at very low temperatures. Shivering is not an effective way of producing heat in the
youngest population.

Nonshivering thermogenesis is the primary method of heat production in infants. It is NOT

shivering that produces heat but the metabolism of brown fat increases body temperature
when the thermal receptors in the skin detect a skin temperature of 35 to 36 degree Celsius
(95 to 96.8 degree Fahrenheit).

Nonshivering thermogenesis is the primary method of heat production in infants. It is NOT

shivering that produces heat but the metabolism of brown fat increases body temperature
when the thermal receptors in the skin detect a skin temperature of 35 to 36 degree Celsius
(95 to 96.8 degree Fahrenheit).
Brown fat, also called adipose tissue or brown adipose tissue (BAT), is a special kind of highly
vascular fat found in newborns. It contains an ample supply of blood vessels which cause
the brown color. Brown fats are located primarily in the following areas:

1. Back of the neck

2. In the axillae
3. Around the kidneys
4. Adrenals
5. Sternum
6. Between the scapulae
7. Along the abdominal aorta

Some infants have insufficient brown fat stores. Preterm infants may be born before the
stores of brown fat have accumulated. Aside from the said case, intrauterine growth
restriction also deplete brown fat stores before birth occurs. Newborns that are exposed to
prolonged cold stress may have insufficient brown fat stores as large amount brown fat is
consumed for heat production in this situation. Thus, these infants will not be able to raise
their body temperature if they are subjected to further episodes of cold stress. Without
brown fat to be metabolized, no heat production will counteract the cold stress. Hence, the
infant is at risk to serious complications.

Processes Involved in Nonshivering Thermogenesis

1. Nonshivering thermogenesis begins when the thermal receptors in the skin detect a
skin temperature of 35 to 36 degrees Celsius (95 to 96.8 degrees Fahrenheit).
2. The thermal receptors stimulation is then transmitted to the hypothalamus thermal
center.
3. In response to the hypothalamic stimulation, norepinephrine is released in brown fat.
4. Presence of norepinephrine in the brown fat initiates its metabolism.
5. As brown fat is metabolized, it generates more heat than other fats.
6. Thus, blood passing through the brown fats is warmed and carries heat to the systemic
circulation or to the rest of the body.
Brown adipose
Brown adipose dissipates stored energy (fat) as HEAT
Maintains CORE BODY TEMPERATURE
It is of particularly important
Neonates
Small mammals in cold environments
Animals awakening from hibernation to warm
Brown adipose, but NOT white adipocytes contain many MITOCHONDRIA
The mitochondria are rounded with CRISTAE across their entire width
Brown adipocytes, but NOT white adipocytes, express an UNCOUPLING PROTEIN in
the mitochondria
This protein UNCOUPLES oxidative phosphorylation so substrate oxidation generates
heat rather than ATP.
In brown adipocytes, most fatty acids are the substrates (fuels) not storage
molecules
The fatty acids are immediately oxidised in mitochondria by b-oxidation
Uncoupling proteins prevents ATP production instead, large amounts of HEAT are
produced form the electron transfer chain
This process compromises NON SHIVERING THERMOGENESIS
Brown fat deposits are in the NECK and INTERSCAPULAR area
Brown fat may also play a non-trivial role in the regulation of BODY-WEIGHT
Dysfunction of the mitochondrial uncoupling proteins may be one of many factors
involved in the development of OBESITY