Oxidative phosphorylation

Why do we need oxygen?

Oxidative phosphorylation is made up of two closely connected components: the
electron transport chain and chemiosmosis. In the electron transport chain, electrons
are passed from one molecule to another, and energy released in these electron
transfers is used to form an electrochemical gradient. In chemiosmosis, the energy
stored in the gradient is used to make ATP.

So, where does oxygen fit into this picture? Oxygen sits at the end of the electron
transport chain, where it accepts electrons and picks up protons to form water. If oxygen
isn’t there to accept electrons (for instance, because a person is not breathing in
enough oxygen), the electron transport chain will stop running, and ATP will no longer
be produced by chemiosmosis. Without enough ATP, cells can’t carry out the reactions
they need to function, and, after a long enough period of time, may even die.

In this article, we'll examine oxidative phosphorylation in depth, seeing how it provides
most of the ready chemical energy (ATP) used by the cells in your body.

Overview: oxidative phosphorylation

The electron transport chain is a series of proteins and organic molecules found in the
inner membrane of the mitochondria. Electrons are passed from one member of the
transport chain to another in a series of redox reactions. Energy released in these
reactions is captured as a proton gradient, which is then used to make ATP in a process
called chemiosmosis. Together, the electron transport chain and chemiosmosis make
up oxidative phosphorylation. The key steps of this process, shown in simplified form
in the diagram above, include:

● Delivery of electrons by NADH and FADH2. Reduced electron carriers (NADH

and FADH2) from other steps of cellular respiration transfer their electrons to
molecules near the beginning of the transport chain. In the process, they turn
back into NAD+ and FAD, which can be reused in other steps of cellular
respiration.
● Electron transfer and proton pumping. As electrons are passed down the
chain, they move from a higher to a lower energy level, releasing energy. Some
of the energy is used to pump H+ ions, moving them out of the matrix and into
the intermembrane space. This pumping establishes an electrochemical gradient.
● Splitting of oxygen to form water. At the end of the electron transport chain,
electrons are transferred to molecular oxygen, which splits in half and takes up
H+ to form water.
● Gradient-driven synthesis of ATP. As H+ ions flow down their gradient and
back into the matrix, they pass through an enzyme called ATP synthase, which
harnesses the flow of protons to synthesize ATP.
The electron transport chain
The electron transport chain is a collection of membrane-embedded proteins and
organic molecules, most of them organized into four large complexes labeled I to IV. In
eukaryotes, many copies of these molecules are found in the inner mitochondrial
membrane. In prokaryotes, the electron transport chain components are found in the
plasma membrane.

As the electrons travel through the chain, they go from a higher to a lower energy level,
moving from less electron-hungry to more electron-hungry molecules. Energy is
released in these “downhill” electron transfers, and several of the protein complexes use
the released energy to pump protons from the mitochondrial matrix to the
intermembrane space, forming a proton gradient.

All of the electrons that enter the transport chain come from NADH and FADH2
molecules produced during earlier stages of cellular respiration: glycolysis, pyruvate
oxidation, and the citric acid cycle.

● NADH is very good at donating electrons in redox reactions (that is, its electrons
are at a high energy level), so it can transfer its electrons directly to complex I,
turning back into NAD+. As electrons move through complex I in a series of
 redox reactions, energy is released, and the complex uses this energy to pump
protons from the matrix into the intermembrane space.
● FADH2 is not as good at donating electrons as NADH (that is, its electrons are at
a lower energy level), so it cannot transfer its electrons to complex I. Instead, it
feeds them into the transport chain through complex II, which does not pump
protons across the membrane.

Because of this "bypass," each FADH2 molecule causes fewer protons to be pumped
(and contributes less to the proton gradient) than an NADH.

Beyond the first two complexes, electrons from NADH and FADH2 travel exactly the
same route. Both complex I and complex II pass their electrons to a small, mobile
electron carrier called ubiquinone (Q), which is reduced to form QH2 and travels
through the membrane, delivering the electrons to complex III. As electrons move
through complex III, more H+ ions are pumped across the membrane, and the electrons
are ultimately delivered to another mobile carrier called cytochrome C (cyt C). Cyt C
carries the electrons to complex IV, where a final batch of H+ ions is pumped across the
membrane. Complex IV passes the electrons to O2, which splits into two oxygen atoms
and accepts protons from the matrix to form water. Four electrons are required to
reduce each molecule of O2, and two water molecules are formed in the process.

Overall, what does the electron transport chain do for the cell? It has two important
functions:

● Regenerates electron carriers. NADH and FADH2 pass their electrons to the
electron transport chain, turning back into NAD+ and FAD. This is important
because the oxidized forms of these electron carriers are used in glycolysis and
the citric acid cycle and must be available to keep these processes running.
● Makes a proton gradient. The transport chain builds a proton gradient across
the inner mitochondrial membrane, with a higher concentration of H+ in the
intermembrane space and a lower concentration in the matrix. This gradient
represents a stored form of energy, and, as we’ll see, it can be used to make
ATP.
Chemiosmosis
Complexes I, III, and IV of the electron transport chain are proton pumps. As electrons
move energetically downhill, the complexes capture the released energy and use it to
pump H+ ions from the matrix to the intermembrane space. This pumping forms an
electrochemical gradient across the inner mitochondrial membrane. The gradient is
sometimes called the proton-motive force, and you can think of it as a form of stored
energy, kind of like a battery.

Like many other ions, protons can't pass directly through the phospholipid bilayer of the
membrane because its core is too hydrophobic. Instead, H+ ions can move down their
concentration gradient only with the help of channel proteins that form hydrophilic
tunnels across the membrane.

In the inner mitochondrial membrane, H+ ions have just one channel available: a
membrane-spanning protein known as ATP synthase. Conceptually, ATP synthase is a
lot like a turbine in a hydroelectric power plant. Instead of being turned by water, it’s
turned by the flow of H+ ions moving down their electrochemical gradient. As ATP
synthase turns, it catalyzes the addition of a phosphate to ADP, capturing energy from
the proton gradient as ATP.

This process, in which energy from a proton gradient is used to make ATP, is called
chemiosmosis. More broadly, chemiosmosis can refer to any process in which energy
stored in a proton gradient is used to do work. Although chemiosmosis accounts for
over 80% of ATP made during glucose breakdown in cellular respiration, it’s not unique
to cellular respiration. For instance, chemiosmosis is also involved in the light reactions
of photosynthesis.

What would happen to the energy stored in the proton gradient if it weren't used to
synthesize ATP or do other cellular work? It would be released as heat, and
interestingly enough, some types of cells deliberately use the proton gradient for heat
generation rather than ATP synthesis. This might seem wasteful, but it's an important
strategy for animals that need to keep warm. For instance, hibernating mammals (such
as bears) have specialized cells known as brown fat cells. In the brown fat cells,
uncoupling proteins are produced and inserted into the inner mitochondrial
membrane. These proteins are simply channels that allow protons to pass from the
intermembrane space to the matrix without traveling through ATP synthase. By
providing an alternate route for protons to flow back into the matrix, the uncoupling
proteins allow the energy of the gradient to be dissipated as heat.

ATP yield

How many ATP do we get per glucose in cellular respiration? If you look in different
books, or ask different professors, you'll probably get slightly different answers.
However, most current sources estimate that the maximum ATP yield for a molecule of
glucose is around 30-32 ATP2,3,4. This range is lower than previous estimates because
it accounts for the necessary transport of ADP into, and ATP out of, the mitochondrion.

Where does the figure of 30-32 ATP come from? Two net ATP are made in glycolysis,
and another two ATP (or energetically equivalent GTP) are made in the citric acid cycle.
Beyond those four, the remaining ATP all come from oxidative phosphorylation. Based
on a lot of experimental work, it appears that four H+ ions must flow back into the matrix
through ATP synthase to power the synthesis of one ATP molecule. When electrons
from NADH move through the transport chain, about 10 H+ ions are pumped from the
matrix to the intermembrane space, so each NADH yields about 2.5 ATP. Electrons from
FADH2, which enter the chain at a later stage, drive pumping of only 6 H+, leading to
production of about 1.5 ATP.
With this information, we can do a little inventory for the breakdown of one molecule of
glucose:

Stage Direct products (net) Ultimate ATP yield (net)

Glycolysis 2 ATP 2 ATP

2 NADH 3-5 ATP

Pyruvate oxidation 2 NADH 5 ATP

Citric acid cycle 2 ATP/GTP 2 ATP

6 NADH 15 ATP

2 FADH2 3 ATP

Total 30-32 ATP

One number in this table is still not precise: the ATP yield from NADH made in
glycolysis. This is because glycolysis happens in the cytosol, and NADH can't cross the
inner mitochondrial membrane to deliver its electrons to complex I. Instead, it must hand
its electrons off to a molecular “shuttle system” that delivers them, through a series of
steps, to the electron transport chain.
 ● Some cells of your body have a shuttle system that delivers electrons to the
transport chain via FADH2. In this case, only 3 ATP are produced for the two
NADH of glycolysis.
● Other cells of your body have a shuttle system that delivers the electrons via
NADH, resulting in the production of 5 ATP.

In bacteria, both glycolysis and the citric acid cycle happen in the cytosol, so no shuttle
is needed and 5 ATP are produced.

30-32 ATP from the breakdown of one glucose molecule is a high-end estimate, and the
real yield may be lower. For instance, some intermediates from cellular respiration may
be siphoned off by the cell and used in other biosynthetic pathways, reducing the
number of ATP produced. Cellular respiration is a nexus for many different metabolic
pathways in the cell, forming a network that’s larger than the glucose breakdown
pathways alone.