Evidence for Chemiosmosis

In the 1940s it was known by scientists that ATP was the universal energy
currency of life and it was known that this was generated by respiration in the
mitochondrion which used substrate level phosphorylation to produce ATP from
ADP and a phosphate group. This occurred in the process of glycolysis and
during the citric acid cycle. It was also known that the citric acid cycle produced
reduced coenzymes such as NADH an FADH2. It was then theorised that the
energy from the electrons given to the coenzymes was stored in a high energy
intermediate which could be used again for phosphorylation to produce more
ATP. The issue was that after rigorous searching, this intermediate was nowhere
to be found and there didn’t seem to be any indirect evidence to support it either.
In 1961, Peter Mitchell proposed that the energy from the electrons could be
used to create an electrochemical proton gradient across a membrane, the would
be a source of potential energy. The movement of these protons down the
electrochemical gradient as a result of the proton motive force would be an
entropically favourable process that would do work, thus releasing kinetic
energy that could be used to phosphorylate ADP to ATP. It is important to
understand the origin of the proton motive force. One component of this is the
fact that there would be a proton concentration gradient so it would increase the
entropy of the universe for the protons to move down the gradient. Another
component is the fact that the concentration difference in the number of charged
particles means that the intermembrane space would be more positively charged
than the mitochondrial matrix thus creating a potential difference across the
inner membrane, this is also caused by the repulsion between the protons which
help force the protons across the membrane. The potential difference is the
reason why it is termed an electrochemical gradient and as there is a force of
attraction, energy is released as the proton passes through the potential
difference. This is a similar concept to gravity, as a ball falls its gravitational
potential energy is converted into kinetic energy, also similar is electricity where
an electron is accelerated along a potential difference thus releasing energy
which can be used to power appliances. In fact the similarity was so similar to
electricity, the term proticity was also used to describe the phenomenon.
The theory proposed by Mitchell was initially viewed with much scepticism as it
went against the previous hypothesis of the high energy intermediate. However,
over the following years, a mass of evidence would appear in support of
Mitchell’s theory of chemiosmosis leading to him winning the Nobel Prize for
Chemistry in 1978.
Some researchers isolated mitochondrion and placed them in a solution of low
water potential making the outer membrane burst forming mitoblasts. Detergent
was then added to the mitoblasts which burst the inner membrane allowing
researchers to analyse the Link reaction and Citric acid cycle in the matrix and to
conclude that the electron transport chain proteins are embedded within the
inner mitochondrial membrane. The mitoblasts themselves did not produce
much ATP so the researchers figured that the intermembrane space must be
important in producing ATP. Additionally ATP was not synthesised in large
quantities if the headpieces of the ATP synthase stalked particles were removed.

Osaid Ather
Furthermore, the presence of oligomycin, an antibiotic inhibiting proton
transport through ATP synthase, halted the production of ATP. A key discovery
was that the coenzymes within the electron transport chain complexes could use
the energy fro the electrons in the electron transport chain to actively transport
protons across the inner mitochondrial membrane and into the intermembrane
space which suggested that there was an electrochemical gradient, the electrons
were then accepted by Oxygen, the final electron acceptor which would then
bond with protons to form water. The evidence for the electrochemical gradient
was also shown by the measurement of a -200mV potential difference being
measured between the matrix and the intermembrane space and the discovery
that the pH in the intermembrane space is lower than that of the matrix. Having
shown that there was an electrochemical gradient which could store potential
energy, it was then shown that the ATP synthase enzyme could allow protons
back through the inner mitochondrial membrane and use the kinetic energy to
cause mechanical rotation of the axle of the enzyme which would provide the
energy for oxidation phosphorylation of ADP to ATP. This differs from the
substrate level phosphorylation in glycolysis as there is no substrate to remove
groups from such as 1,6-fructosebisphosphate and another key difference is that
oxygen is required as a final electron acceptor for the electron transport chain in
oxidative phosphorylation. If there was no oxygen to accept the electrons, the
electrons may float into the cytoplasm before reaching the end of the electron
transport chain and form superoxides. These are unstable species that will take
electrons from nearby molecules, if the electrons are taken from DNA then it can
lead to mutations that could lead to either cell apoptosis or cancer. Thankfully
the enzyme superoxide dismutase deals with these free electrons to stop this
from happening.

Osaid Ather