Oxidative phosphorylation

Dr. Rabia
(DPT)
Biochemistry
Lecture#03
Lecturer: Allied college of health sciences Multan
Date: 28-05-2021
Oxidative phosphorylation
• The transport of electrons through the ETC is linked with the release
of free energy. The process of synthesizing ATP from ADP and Pi
coupled with the electron transport chain is known as oxidative
phosphorylation.
• The complex V of the inner mitochondrial membrane is the site of
oxidative phosphorylation.
• This enzyme complex is called Mitochondrial ATPase or H+-ATPase. It
is also called ATP-synthase (since it can hydrolyse ATP to ADP and Pi).
Mechanism of oxidative phosphorylation
• Several hypotheses have been put forth to explain the process of oxidative
phosphorylation. The most important among them—namely, chemical coupling, and
chemiosmotic—are discussed below.
1. Chemical coupling hypothesis: This hypothesis was put forth by Edward Slater (1953).
According to chemical coupling hypothesis, during the course of electron transfer in
respiratory chain, a series of phosphorylated high-energy intermediates are first
produced which are utilized for the synthesis of ATP. These reactions are believed to
be analogous to the substrate level phosphorylation that occurs in glycolysis or citric
acid cycle. However, this hypothesis lacks experimental evidence, since all attempts,
so far, to isolate any one of the high-energy intermediates have not been successful.
2. Chemiosmotic hypothesis: This mechanism, originally proposed by Peter Mitchell
(1961), is now widely accepted. It explains how the transport of electrons through the
respiratory chain is effectively utilized to produce ATP from ADP + Pi. The concept of
chemiosmotic hypothesis is comparable with energy stored in a battery separated by
positive and negative charges
• Proton gradient: The inner mitochondrial membrane, as such, is impermeable to
protons (H+) and hydroxyl ions (OH–). The transport of electrons through ETC is
coupled with the translocation of protons (H+) across the inner mitochondrial
membrane (coupling membrane) from the matrix to the intermembrane space. The
pumping of protons results in an electrochemical or proton gradient. This is due to
the accumulation of more H+ ions (low pH) on the outer side of the inner
mitochondrial membrane than the inner side. The proton gradient developed due to
the electron flow in the respiratory chain is sufficient to result in the synthesis of ATP
from ADP and Pi.
• Enzyme system for ATP synthesis: ATP synthase, present in the complex V, utilizes the
proton gradient for the synthesis of ATP. ATP synthase is a complex enzyme and
consists of two functional subunits, namely F1 and F0. F0 spans inner mitochondrial
membrane F1 protrudes into mitochondrial matrix. Its structure is comparable with
‘lollipops’
• The protons that accumulate on the intermembrane space re-enter the mitochondrial
matrix leading to the synthesis of ATP.
Rotary motor model for ATP generation
• Paul Boyer in 1964 proposed (Nobel Prize, 1997) that a conformational
change in the mitochondrial membrane proteins leads to the synthesis of ATP.
• The original Boyer hypothesis, now considered as rotary motor/engine driving
model or binding change model, is widely accepted for the generation ATP.
According to this hypothesis 3β subunits (catalytic sites) though structurally
similar, but functionally not same at a particular time.
• The enzyme ATP synthase is F0F1 complex (of complex V). The F0 subcomplex
is composed of channel protein ‘C’ subunits to which F1-ATP synthase is
attached. F1 catalyses the ATP synthesis. F1-ATP synthase consists of a central
γ subunit surrounded by alternating 3 α and 3 β subunits (α3 and β3).
• In response to the proton flux, the γ subunit physically rotates. This induces
conformational changes in the β3 subunits that finally lead to the release of ATP.
• According to the binding change mechanism, the three β subunits of F1-ATP synthase
adopt different conformations. One subunit has open (O) conformation, the second
has loose (L) conformation while the third one has tight (T) conformation.
• By an unknown mechanism, protons induce the rotation of γ subunit, which in turn
induces conformation changes in the fixed β subunits of membrane. The substrates
ADP and Pi bind to β subunit in L-conformation. The L site changes to T conformation,
and this leads to the synthesis of ATP. The O site changes to L conformation which
binds to ADP and Pi. The T site changes to O conformation, and releases ATP.
• This cycle of conformation changes of β subunits is repeated. And three ATP are
generated for each revolution. It may be noted that the ATP release in O
conformation is energy dependent (and not ATP synthesis) and very crucial in rotary
motor model for ATP generation.
• The enzyme ATP synthase acts as a proton driving motor, and is an example of rotary
catalysis. Thus, ATP synthase is the world’s smallest molecular motor.
• Coupling in oxidative phosphorylation: In normal mitochondria, ATP
synthesis is coupled to electron transport through the proton
gradient. Increasing (or decreasing) one process has the same effect
on the other.
• For example, hydrolysis of ATP to ADP and Pi in energy-requiring
reactions increases the availability of substrates for ATP synthase and,
thus, increases proton flow through the enzyme. Electron transport
and proton pumping by the ETC increase to maintain the proton
gradient.
[Note: Increased oxidation of NADH at Complex I and, consequently, an
increase in NADH-producing pathways of metabolism, such as the TCA
cycle, results.]
Uncoupling proteins
• Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane of
mammals, including humans. These proteins form channels that allow protons to
reenter the mitochondrial matrix without energy being captured as ATP. The energy
is released as heat, and the process is called non-shivering thermogenesis.
• UCP1, also called thermogenin, is responsible for heat production in the brown
adipocytes of mammals. In brown fat, unlike the more abundant white fat, almost
90% of its respiratory energy is used for thermogenesis in response to cold in the
neonate and during arousal in hibernating animals.
• However, humans appear to have few concentrated deposits of brown fat (except
in the newborn), and UCP1 does not appear to play a major role in energy balance.
• Uncoupling proteins UCP2–UCP5 have been found in other tissues, but their full
significance remains unclear.
Synthetic uncouplers
• Electron transport and phosphorylation can also be uncoupled by
compounds that pick up protons in the intermembrane space and release
them in the matrix, dissipating the gradient.
• The classic example is 2,4- dinitrophenol (DNP), a lipophilic proton carrier
that readily diffuses through the mitochondrial membrane. This
uncoupler causes electron transport to proceed at a rapid rate without
establishing a proton gradient, much as do the UCPs. Again, energy is
released as heat rather than being used to synthesize ATP.
• In high doses, aspirin and other salicylates uncouple oxidative
phosphorylation. This explains the fever that accompanies toxic
overdoses of these drugs.
Membrane transport systems
• The inner mitochondrial membrane is impermeable to most charged or
hydrophilic substances. However, it contains numerous transport proteins
that permit passage of specific molecules from the cytosol (or more
correctly, the intermembrane space) to the mitochondrial matrix.
1. ATP and ADP transport: The inner membrane requires specialized carriers
to transport ADP and Pi from the cytosol (where ATP is hydrolyzed to ADP in
many energy-requiring reactions) into mitochondria, where ATP can be
resynthesized. An adenine nucleotide antiporter imports one ADP from the
cytosol into the matrix, while exporting one ATP from the matrix into the
cytosol. A transporter moves Pi from the cytosol into mitochondria.
2. Transport of reducing equivalents: The inner mitochondrial membrane lacks
an NADH transporter, and NADH produced in the cytosol (for example, in
glycolysis) cannot directly enter the mitochondrial matrix. However, two
electrons (reducing equivalents) of NADH are transported from the cytosol
into the matrix using substrate shuttles.
In the glycerophosphate shuttle, two electrons are transferred from NADH to
dihydroxyacetone phosphate by cytosolic glycerophosphate dehydrogenase.
The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme
as FAD is reduced to FADH2. CoQ of the ETC oxidizes the FADH2. The
glycerophosphate shuttle, therefore, results in the synthesis of two ATPs for
each cytosolic NADH oxidized.
This contrasts with the malate-aspartate shuttle, which produces NADH (rather
than FADH2) in the mitochondrial matrix and, therefore, yields three ATPs for
each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is
reduced to malate. A transport protein moves malate into the matrix.
Inherited disorders of oxidative phosphorylation
• It is estimated that about 100 polypeptides are required for oxidative phosphorylation. Of
these, 13 are coded by mitochondrial DNA (mtDNA) and synthesized in the mitochondria,
while the rest are produced in the cytosol (coded by nuclear DNA) and transported. mtDNA
is maternally inherited since mitochondria from the sperm do not enter the fertilized ovum.
• Mitochondrial DNA is about 10 times more susceptible to mutations than nuclear DNA.
mtDNA mutations are more commonly seen in tissues with high rate of oxidative
phosphorylation (e.g. central nervous system, skeletal and heart muscle, liver).
Leber’s hereditary optic neuropathy is an example for mutations in mtDNA. This disorder is
characterized by loss of bilateral vision due to neuroretinal degeneration.
Infantile mitochondrial myopathy associated with renal dysfunction: The condition is fatal.
There is severe diminution or absence of most of the oxidoreductases of the electron
transport chain.
MELAS: An inherited disorder associated with: Mitochondrial myopathy, Lactic acidosis,
Encephalopathy and Stroke Enzyme deficiency: NADH: Ubiquinone oxidoreductase
(complex 1) or deficiency of cytochrome oxidase.
Mitochondria and apoptosis
• The process of apoptosis, or programmed cell death, may be initiated
through the intrinsic (mitochondrial-mediated) pathway by the
formation of pores in the outer mitochondrial membrane. These
pores allow cytochrome c to leave the intermembrane space and
enter the cytosol. There, cytochrome c, in association with
proapoptotic factors, activates a family of proteolytic enzymes (the
caspases), causing cleavage of key proteins and resulting in the
morphologic and biochemical changes characteristic of apoptosis