Oxidative Phosphorylation

The ATP synthase complex is now known to represent one of the quintessential
protein machines found in living cells, the mitochondrial ATP synthase complex
consists of two large structural components called F1 which encodes the catalytic
activity, and Fo which functions as the proton channel crossing the inner
mitochondrial membrane. The subscript "o" in Fo , written here in lower case to
avoid confusion with zero "0," refers to the finding that the antibiotic oligomycin
inhibits the activity of the Fo proton channel.
Although the F1 and Fo components represent the largest biochemical entities
identified by protein purification, it is useful to organize the F1Fo ATP synthase
complex into three functional units. These three functional units can be described
as 1) the rotor made up of and subunits which sit on top of the c subunit ring
that rotates as protons enter and exit the ring, 2) the catalytic head piece
containing the hexameric 33 unit that contains an enzyme active site in each of
the three subunits and is responsible for ATP synthesis in the intact complex (or
ATP hydrolysis in the isolated F1 component), and 3) the stator consisting of the a
subunit imbedded in the membrane which contains two half channels for protons to
enter and exit the Fo component, and a stabilizing arm made up of the b dimer and
subunits (a-b2-). The term stator refers to immobile parts of a rotary engine
which describes the role of the - stabilizing arm in preventing the 33 headpiece
from rotating along with the subunit.
The binding change mechanism incorporates three basic principles;
1. The subunit directly contacts all three subunits, however, each of these
interactions are distinct giving rise to three different subunit conformations.
2. The ATP binding affinities of the three subunit conformations are defined as T;
tight, L; loose, and O; open, in which ADP and Pi bind to the O and L conformations,
and ATP binds tightly to the T conformation but is released from the enzyme when
the subunit is in the O conformation.
3. As protons flow through Fo the subunit rotates in a counter-clockwise circle
(looking at F1 from the matrix side) such that with each 120 rotation the subunits
sequentially undergo a conformational change from L --> T --> O --> L.
A key element of the Chemiosmotic Theory is that the inner mitochondrial
membrane must be impermeable to ions in order to establish the proton gradient.
Therefore, biomolecules required for the electron transport system and oxidative
phosphorylation must be transported, or "shuttled," back and forth across the inner
mitochondrial membrane by specialized proteins. The most important of these
biomolecules are ATP, ADP and Pi which are products and reactants in the ATP
synthase reaction, and cytosolic NADH, a product of the glyceraldehyde-3phosphate dehydrogenase reaction in glycolysis. Not only does newly synthesized
ATP need to be exported from the matrix to replenish cytosolic ATP pools, but the
substrates for the reaction, ADP and Pi, need to be continually imported into the
matrix to maintain high rates of ATP synthesis. This is accomplished by two
translocase proteins located in the inner mitochondrial membrane. One is the

ATP/ADP translocase, also called the adenine nucleotide translocase, which

functions to export one ATP for every ADP that is imported, and the other is the
phosphate translocase which translocates one Pi and one H+ into the matrix by an
electroneutral import mechanism. The ATP/ADP is called an antiporter because it
translocates molecules in opposite directions across the membrane; for every ADP
molecule that is imported from the cytosol, an ATP molecule is exported from the
matrix.
Cytosolic NADH transfers electrons to the matrix via shuttle systems
Numerous dehydrogenase reactions in the cytosol generate NADH, one of which is
the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. However,
cytosolic NADH cannot cross the inner mitochondrial membrane, instead the cell
uses an indirect mechanism that only transfers the electron pair (2 e-), or two
reducing equivalents, from the cytosol to the matrix using two different "shuttle"
systems. The most widely used shuttle is the malate-aspartate shuttle that has
been found to operate in liver, kidney and heart cells and functions as a reversible
pathway. The key enzymes in this shuttle pathway are cytosolic malate
dehydrogenase which reduces oxaloacetate to malate, and mitochondrial malate
dehydrogenase, the citrate cycle enzyme that oxidizes malate to form oxaloacetate.
NADH reducing equivalents transported to the mitochondrial matrix by the malate
aspartate shuttle are used to generate 2.5 ATP as a result of 10 H+ being pumped
across the inner mitochondrial membrane. The malate-aspartate shuttle is a bit
more complicated than simply reoxidizing malate in the mitochondria to form
oxaloacetate because sooner or later cytosolic oxaloacetate levels will be
insufficient to keep the shuttle going. Therefore, cytosolic oxaloacetate needs to be
replenished which is accomplished by two additional enzymes, mitochondrial
aspartate aminotransferase and cytosolic aspartate aminotransferase, as well as, a
second transporter that exchanges aspartate for glutamate.
Oxidative phosphorylation is regulated by the need for ATP which is reflected in the
energy charge of the cell as a function of [ATP], [ADP] and [AMP]. When ATP is
being consumed at high rates to support metabolic functions, the level of ADP in the
cell rises as a result of increased ATP hydrolysis. The cellular concentration of ADP
is in fact a key control factor of oxidative phosphorylation because it determines not
only the rate of ATP synthesis, but also the rate of NADH oxidation and reduction of
O2 to form H2O. The regulatory function of ADP and ATP in controlling aerobic
respiration extends to the citrate cycle and glycolysis, both of which are activated
by a low energy charge (high [ADP] and low [ATP]. In addition, the ratio of
NADH/NAD+ in the mitochondrial matrix controls multiple steps in the citrate cycle,
which in turn determines the flow of electrons through the electron transport system
and ultimately rates of ATP synthesis The role of the electrochemical proton
gradient in linking substrate oxidation to ATP synthesis can be demonstrated by
experiments using isolated mitochondria that are suspended in buffer containing
O2, but lacking ADP + Pi and an oxidizable substrate such as succinate which
donates a pair of electrons to FAD in complex II of the electron transport system.
When ADP + Pi are added to a mitochondrial suspension lacking succinate, O2
reduction (as measured by O2 consumption) and ATP synthesis increase only

slightly over time. However, when succinate is added then both the rates of O2
reduction and ATP synthesis increase dramatically until substrates become limiting.
Both O2 consumption and ATP synthesis are blocked when cyanide (CN-) is added to
the suspension since proton pumping by the electron transport system stops
resulting in a shut down of the ATP synthase complex.