Bioenergetics

Brief history & orientation

Cytochrome spectra from the respiratory pigments in animal tissues were originally observed in the
nineteenth century, but dismissed as an artefact by the scientific establishment of the day. Oxygen uptake by
tissue homogenates, and the action of simple respiratory inhibitors were first studied systematically in the
1930s, when it was realised that electrons flowed from substrates to oxygen via a sequence of redox carriers,
several of which showed distinctive spectral changes when oxidised and reduced.

The isolation of intact mitochondria followed improvements in centrifuge technology in the 1940s,
and the introduction of isotonic sucrose preparation media to prevent osmotic lysis. Investigators noted the
phenomenon of latency: the internal mitochondrial enzymes could not be demonstrated until the organelles
had been broken open. The basic structure of mitochondria was established by electron microscopy and it
was shown by controlled disruption that most of the respiratory enzymes responsible for oxygen uptake
were attached to the inner mitochondrial membrane (which is extensively folded to form the
mitochondrial cristae) while the soluble enzymes catalysing the Krebs cycle and fatty acid oxidation
pathways were confined to the internal matrix space.

It was later shown that the inner membrane was a major permeability barrier within the cell, but the
outer membrane contained a protein called porin which rendered it largely largely permeable to molecules
less than about 1500 daltons. An important group of enzymes which metabolise ATP, including myokinase,
creatine kinase, and the nucleoside diphosphate kinases, are trapped in the inter-membrane space, between
the inner and the outer membranes.

myokinase: ATP + AMP <=> ADP + ADP

creatine kinase: ATP + creatine <=> ADP + creatine phosphate

cytidine diphosphate kinase: CDP + ATP <=> CTP + ADP (many similar enzymes exist)

Experimental work accelerated with the introduction of the more convenient Clarke-type oxygen
electrode in the 1950's in place of the older manometric methods. It was shown that the uptake of oxygen by
intact mitochondria depended on the simultaneous conversion of ADP and inorganic phosphate into ATP.
The phenomenon is termed respiratory control. The obligatory dependence of a highly favourable
chemical reaction (substrate oxidation) on the simultaneous execution of an unrelated and extremely
unfavourable reaction (ATP synthesis) was immediately recognised as a novel and important concept by the
biochemists of the day.

Oxygen uptake which is dependent on the presence of ADP and phosphate is termed coupled
respiration. The coupling of respiration and phosphorylation could be broken by mechanical or osmotic
disruption of the mitochondria, or by the addition of uncoupling agents, in which case respiration proceeded
rapidly without any concomitant ATP synthesis. The original uncoupling agent was 2,4-dinitrophenol,
which has now been replaced by more effective compounds such as FCCP (para-trifluoromethoxy carbonyl
cyanide phenylhydrazone) and CCCP (meta-chloro carbonyl cyanide phenylhydrazone).

Two major classes of mitochondrial inhibitor could be distinguished: respiration inhibitors and
phosphorylation inhibitors. Both types were effective against intact mitochondria, but only respiration
inhibitors could prevent oxygen uptake after the addition of an uncoupling agent. These experiments gave
rise to the concept of an energetically favourable electron transport system, which in some way powered an
energetically unfavourable phosphorylation system. The free energy available from the redox reactions was
used to drive ATP synthesis. Respiration inhibitors such as cyanide ions blocked the electron transport chain
and were always effective in suppressing oxygen uptake, whereas phosphorylation inhibitors such as
oligomycin, which prevented ATP manufacture, could only block respiration if the coupling were intact.
 From this point, in the mid 1950s, the subject diverted into a long and occasionally acrimonious
blind alley, as scientists attempted to find a chemical intermediate linking respiration to ATP generation.
This idea derived from the substrate level phosphorylations observed in the glycolytic pathway. This search
was unsuccessful as no such intermediate exists. We now know that the energy from respiration is first
captured in the form of pH and electrical potential gradients across the inner mitochondrial membrane. The
energy stored in these gradients is later exploited to drive the synthesis of ATP. This chemiosmotic theory
was first proposed in 1961 by a British biochemist, Peter Mitchell, who worked for many years without
much official recognition or significant public funding until he was awarded the Nobel Prize for Chemistry
in 1978.

Oxygen electrodes

Much of our knowledge of electron transport in mitochondria and chloroplasts comes from oxygen
electrode recordings. The oxygen concentration in a sealed incubation chamber is continuously monitored,
and the effects of making various additions to the chamber can be observed. A cross section through a
typical apparatus is shown below:

The upper section containing a transparent, thermostatted

sample chamber is secured to the lower electrode
assembly with a screwed ring. A thin teflon membrane is
trapped between the two sections and separates the
isotonic incubation medium from the strong KCl
electrolyte in the electrode compartment. The adjustable
stopper is used to seal the incubation chamber and prevent
room air dissolving during the experiments. The small
hole in the centre of the stopper permits the expulsion of
air bubbles and allows small volumes of reagents to be
added with a microlitre syringe. The contents of the
chamber are stirred continuously with a magnetic flea.

A small polarising voltage (ca. 0.6 volt) is applied between the silver anode (+) and the platinum
cathode (-). Oxygen diffuses through the teflon membrane and is reduced to water at the platinum cathode:

O2 + 4H+ + 4e-1 = 2H2O

The circuit is completed at the silver anode, which is

slowly corroded by the KCl electrolyte:

Ag + Cl- = Ag Cl + e-1

The resulting current is proportional to the oxygen

concentration in the sample chamber. This signal can be
amplified and recorded.

The whole process is totally dependent on the supply of oxygen. The rate of oxygen diffusion to the
cathode (and hence the current) depends on the oxygen concentration in the main incubation chamber. It
also depends on several other factors: temperature, membrane thickness and permeability, sample viscosity
and stirring speed. In contrast to pH electrodes which measure an equilibrium position, oxygen electrodes
measure the velocity of a physico-chemical process that is far from equilibrium. pH electrodes have an
intrinsic thermodynamic response which is relatively insensitive to temperature and sample composition, but
there is no intrinsic calibration to an oxygen electrode - at regular intervals, or if the instrument is dis-
assembled, it must be re-calibrated against a known standard, usually air. It is particularly important to
control the temperature.
Oxygen electrode (oxygraph) recordings

If mitochondria are incubated in an oxygraph

apparatus (oxygen electrode) in an isotonic medium
containing substrate and phosphate, then addition of ADP
causes a sudden burst of oxygen uptake as the ADP is
converted into ATP:

The actively respiring state is sometimes refered

to as "state 3" respiration, while the slower rate after all
the ADP has been phosphorylated to form ATP is refered
to as "state 4".

State 4 respiration is usually faster than the

original rate before the first addition of ADP because
some ATP is broken down by ATPase activities contaminating the preparation, and the resulting ADP is
then re-phosphorylated by the intact mitochondria.

The ratio [state 3 rate] : [state 4 rate] is called the respiratory control index and indicates the
tightness of the coupling between respiration and phosphorylation. With isolated mitochondria the coupling
is not perfect, probably as a result of mechanical damage during the isolation procedure. Typical RCI values
range from 3 to 10, varying with the substrate and the quality of the preparation. Coupling is thought to be
better in vivo, but may still not achieve 100%.

It is possible to calculate a P:O ratio (the relationship between ATP synthesis and oxygen
consumption) by measuring the decrease in oxygen concentration during the rapid burst of state 3 respiration
after adding a known amount of ADP. It is necessary to subtract the basal respiration due to imperfect
coupling and the recycling of ATP, as shown in the graph above. The change in concentration must then be
multiplied by the chamber volume, so that the answer (in micro-atoms of oxygen) can be related to the
quantity of ADP added. The quantity of oxygen in the chamber is calculated from published oxygen
solubility data at the appropriate temperature. Using the figures from the graph above:

decrease in O2 (from graph) = 0.135 mM; chamber volume = 2.5ml

oxygen atoms consumed in state 3 = 0.135 * 2 * 2.5 = 0.68 micro-atoms (note the factor of 2, allowing for 2
atoms in an oxygen molecule)

injected ADP (20 microlitres of a 50mM solution) = ATP formed = 1 micromole in total

P:O = [ATP formed] : [oxygen consumed] = 1.48 (for succinate oxidation)

NAD-linked substrates give consistently higher values for the P:O ratio (about 2.5) compared with
succinate (about 1.5). These results indicate that electrons from relatively poor reducing agents such as
succinate (also acyl CoA and glycerol phosphate) enter part of the way along the respiratory chain, by-
passing the first coupling site where energy is captured for ATP synthesis.

The sharp changes in slope after exhaustion of ADP and again after all the oxygen has been used up
imply that mitochondria must have very high affinities for ADP and oxygen. The concentration of both these
compounds is very low in most healthy cells, since they are efficiently scavenged by the mitochondria.

The addition of an uncoupling agent (such as dinitrophenol or CCCP) leads to a permanently high
rate of respiration in the absence of ADP, until all the oxygen has been consumed.
 Many natural and synthetic poisons block mitochondrial respiration. If mitochondria are incubated in
an oxygraph experiment with substrates and inorganic phosphate, the interactions between inhibitors and
uncouplers allow two major types of inhibition to be distinguished:

Mitochondrial respiration rates

additions / inhibitors none oligomycin cyanide
none low low zero
ADP high low zero
uncoupling agent high high zero

The conclusion to be drawn from this type of experiment is that cyanide prevents respiration by
blocking the respiratory chain itself, so cyanide is effective whether or not ADP or uncoupler are added.
Oligomycin on the other hand inhibits the ADP phosphorylation system, so it only blocks respiration in
coupled mitochondria.

Respiratory chain components

The system of mitochondrial enzymes and redox carrier molecules which ferry reducing equivalents from
substrates to oxygen are collectively known as the electron transport system, or the respiratory chain. This
system captures the free energy available from substrate oxidation so that it may later be applied to the
synthesis of ATP. Many respiratory chain components were first identified in crude homogenates through
their spectral properties, which frequently change when a carrier is oxidised or reduced. Fractionation of
mitochondria in the presence of mild detergents or chaotropic salts dissected the respiratory chain into four
large multi-subunit complexes containing the principal respiratory carriers, named Complex 1 to Complex
4. These substantial protein "icebergs" float in the sheet of inner membrane lipids, often presenting one face
to the mitochondrial matrix and another to the inter - membrane space. Many of their components have now
been isolated in a relatively pure form. Other membrane bound enzymes such as the energy linked
transhydrogenase (ELTH) are also present which fulfil ancillary roles.

The main components participate in the approximate order of their redox potentials, and the bulky
complexes are linked by low molecular weight mobile carriers which ferry the reducing equivalents from
one complex to the next. Except for succinate dehydrogenase (complex 2) all these complexes pump protons
from the matrix space into the cytosol as they transfer reducing equivalents (either hydrogen atoms or
electrons) from one carrier to the next. The diagram above shows the flow of reducing equivalents in purple,
and movement of the positively charged protons in red. Proton pumping is an arduous task which creates
substantial pH and electrical gradients across the mitochondrial inner membrane. These protons eventually
re-enter the matrix space via the F1 ATPase, driving the synthesis of ATP as they return.

The number of protons and the number of positive charges crossing the inner membrane need not
necessarily agree for each individual transmembrane protein, although the accounts must balance for the
whole ensemble. This discrepancy is illustrated on the diagram above, and is explained in greater detail
below.
 The sequence of of the carriers in the respiratory chain was clarified by the use of specific inhibitors
to block the flow of electrons from substrates to oxygen. All components on the substrate side of a block
become more reduced, while those which follow become more oxidised. These changes can often be
observed spectroscopically. If a range of inhibitors are available, acting at different places, then their precise
points of action and the order of the carriers is defined.

Further information was gleaned from the use of artificial redox mediators. Electron donors, such as
ascorbate [vitamin C] plus the synthetic compound tetramethyl phenylene diamine can contribute electrons
to cytochrome c, while artificial acceptors such as ferricyanide would collect electrons from complex 1 or
complex 2 and by-pass the remainder of the chain. This could be particularly informative when used in
conjunction with specific inhibitors, or when ATP yields were measured for particular segments of the
chain.

Respiratory Chain Components Location Prosthetic Function

NADPH / NADP (almost 100% in
the reduced form)
energy-linked transhydrogenase
NADPH + NAD+
=> NADH + NADP+
NADH / NAD (less than 30% in the
reduced form)
NADH dehydrogenase
(complex 1)
succinate dehydrogenase
(complex 2) [see note 1]
ubiquinol / ubiquinone
ubiquinol:cytochrome c reductase
(complex 3)
cytochrome c
(ferrous / ferric)
cytochrome c oxidase (complex 4)
F0 / F1 ATPase
(ATP synthetase)
groups
matrix space (separate - mobile carrier
cytosolic pool)
membrane spanning none proton pump 2H+/2e-1
protein
matrix space (separate - mobile carrier
cytosolic pool)
membrane spanning non-heme iron proton pump 4H+/2e-1
multi-subunit protein & FMN
membrane spanning non-heme iron no proton pumping
multi-subunit protein & FAD
dissolved in the inner - mobile carrier
membrane lipids
membrane spanning non-heme iron, heme b proton pump 4H+/2e-1
multi-subunit protein & heme c1 [see note 2]
inter-membrane space heme c mobile carrier
membrane spanning copper, heme a proton pump 2H+/2e-1
multi-subunit protein & heme a3 [see note 2]
membrane spanning none proton pump
multi-subunit protein 3H+ / ATP

Note 1: In addition to succinate dehydrogenase, several other enzymes contribute electrons to

directly to ubiquinone. Acyl CoA dehydrogenase is a soluble flavoprotein involved in fatty acid oxidation in
the mitochondrial matrix space. It feeds its reducing equivalents to ubiquinone via an intermediate low
molecular weight electron transferring flavoprotein (ETF). Glycerol phosphate "oxidase" is another
flavoprotein which also feeds reducing equivalents to ubiquinone. It is an inner membrane protein, but the
relevant active centre faces outwards and reacts with its substrate in the cytosolic compartment, not in the
matrix space. None of these enzymes contributes to proton pumping, so the P:O ratio from their substrates is
only1.5

Note 2: The proton pumping stoichiometry for complex 3 and complex 4 is difficult because the
electron acceptor for complex 3 (cytochrome c) is located on the outside of the inner membrane, whereas the
oxidant for complex 4 (molecular oxygen) is reduced on the inside face of the inner membrane. It is
necessary to count the negative charges on the electrons traversing the chain as well as the positive charges
on the protons. Moreover, reduction of oxygen consumes two additional "chemical" protons to form water,
in addition to those pumped by complex 4. These "chemical" protons don't count because they exactly
balance the two "chemical" protons produced earlier when the hydrogen atoms were first removed during
substrate oxidation. The net effect of all this is that complex 3 exports four protons but only two positive
charges for each pair of electrons traversing the respiratory chain, while complex 4 exports two protons but
four positive charges.

Liposomes are membranous vesicles which form spontaneously when many phospholipids are
dispersed in aqueous media. If the lipids are simply shaken with water large multi-layered structures are
produced with an "onion skin" arrangement. If the multi-layered liposomes are treated with ultrasonic
vibrations they yield much smaller unilamellar vesicles with a simple limiting membrane composed of a
single phospholipid bilayer. Both single and multi-layered liposomes can be prepared which are
impermeable to the majority of charged ions, although uncharged, hydrophobic molecules have ready access
to the interior. A small volume of the original preparation medium becomes trapped within each liposome as
the membrane seals up.

Reconstitution: If hydrophobic membrane proteins are included during the lipid sonication step,
these are incorporated into the liposome membranes. This has been used to re-constitute the respiratory
chain from purified fragments. Unfortunately the proteins adopt a random orientation as they are
incorporated into the liposome membrane, so half of them finish facing the wrong way round. It is therefore
difficult to re-create a proton gradient. A variety of ingenious techniques have been used to select the
correctly oriented protein population, for example by trapping the electron donor within the liposome and
supplying the acceptor only to the external face.

Liposomes are also being actively researched as a means of delivering cytotoxic drugs to tumour
cells, which have been targeted with monoclonal antibodies.

Uncouplers and Inhibitors

Much of our knowledge of mitochondrial function results from the study of toxic compounds.
Specific inhibitors were used to distinguish the electron transport system from the phosphorylation system
and helped to define the sequence of redox carriers along the respiratory chain. If the chain is blocked then
all the intermediates on the substrate side of the block become more reduced, while all those on the oxygen
side become more oxidised. It is easy to see what has happened because the oxidised and reduced carriers
often differ in their spectral properties. If a variety of different inhibitors are available then many of the
respiratory carriers can be placed in the correct order.

There are six distinct types of poison which may affect mitochondrial function:

1) Respiratory chain inhibitors (e.g. cyanide, antimycin, rotenone & TTFA) block respiration in the
presence of either ADP or uncouplers.

2) Phosphorylation inhibitors (e.g. oligomycin) abolish the burst of oxygen consumption after adding
ADP, but have no effect on uncoupler-stimulated respiration.

3) Uncoupling agents (e.g. dinitrophenol, CCCP, FCCP) abolish the obligatory linkage between the
respiratory chain and the phosphorylation system which is observed with intact mitochondria.

4) Transport inhibitors (e.g. atractyloside, bongkrekic acid, NEM) either prevent the export of ATP, or the
import of raw materials across the the mitochondrial inner membrane.

5) Ionophores (e.g. valinomycin, nigericin) make the inner membrane permeable to compounds which are
ordinarily unable to cross.
6) Krebs cycle inhibitors (e.g. arsenite, aminooxyacetate) which block one or more of the TCA cycle
enzymes, or an ancillary reation.

Some of the best-known compounds are listed below:

Compound Mode of action and effects

Amino- Ancillary enzyme inhibitor. Inhibits all transaminases by reacting covalently with their
oxyacetate pyridoxal phosphate prosthetic group. Blocks the malate / aspartate cycle by inhibiting
glutamate - oxaloacetate transaminase (GOT).
Antimycin A Respiration inhibitor. Blocks the respiratory chain at complex 3 between cytochrome b
and cytochrome c1. It therefore prevents the oxidation of both NADH and succinate, but
has no effect on ascorbate + TMPD.
Arsenite Krebs cycle inhibitor. Reacts with the disulfide linkage in oxidised lipoic acid, forming
a cyclic adduct. Inhibits all the oxo-acid dehydrogenases, including pyruvate
dehydrogenase, oxoglutarate dehydrogenase and the branched chain oxo-acid
dehydrogenase.
Atractyloside Transport inhibitor. Blocks the adenine nucleotide porter by binding to the outward -
facing conformation (contrast with bongkrekic acid). It has no effect on sub-
mitochondrial particles, which re-seal spontaneously after sonication with the
membranes inside-out. This ATP/ADP transport inhibitor resembles oligomycin when
used with intact mitochondria. (See also Bongkrekic acid.)
Bongkrekic acid Transport inhibitor. Blocks the adenine nucleotide porter by binding to the inward-
facing conformation (contrast with atractyloside).
Cyanide Respiration inhibitor. Blocks cytochrome oxidase (complex 4) and prevents both
coupled and uncoupled respiration with all substrates, including NADH, succinate and
ascorbate + TMPD.
Mersalyl Transport inhibitor. Dose dependent inhibition of the phosphate and dicarboxylate
porters. Mersalyl is an outdated mecurial diuretic.
N-ethyl Transport inhibitor. Blocks the phosphate porter by reacting with -SH groups, and
maleimide prevents respiration by coupled mitochondria and phosphate - mediated swelling.
(NEM)
Oligomycin Phosphorylation inhibitor. Binds to a 23kd polypeptide (OSCP) in the F0 baseplate and
blocks ATP synthesis (and degradation) by the F0 /F1 ATPase. It abolishes ADP-
stimulated respiration in intact mitochondria and all ATP-driven functions in sub-
mitochondrial particles.
Rotenone Respiration inhibitor. Blocks NADH dehydrogenase (complex 1) in the respiratory
chain but has no effect on the oxidation of either succinate or ascorbate + TMPD.
(TMPD is tetra methyl phenylene diamine, an artificial redox mediator which assists the
transfer of electrons from ascorbate to cytochrome c.)
Thenoyl trifluoro Blocks succinate dehydrogenase (complex 2). It has no effect on the oxidation of
acetone (TTFA) NADH-linked substrates or ascorbate + TMPD.

Uncouplers & Ionophores: All of these compounds are small amphipathic molecules which
dissolve in phospholipid bilayers and enormously increase their ionic permeability. They shield the electric
charge as the ion passes through the membrane, providing a polar environment for the ion and a
hydrophobic face to the outside world. Ionophores transport a variety of ions, but uncoupling agents
specifically increase the proton permeability, and disconnect the electron transport chain from the formation
of ATP. Some ionophores are natural products isolated from micro-organisms, but others are synthetic
compounds, tailored to a specific application. The uncoupling agents are all synthetic, although there is a
delicately regulated uncoupling protein involved in thermogenesis in brown adipose tissue mitochondria.
 CCCP: (carbonyl cyanide m-chloro phenyl hydrazone) This is a
lipid-soluble weak acid which is a very powerful mitochondrial
uncoupling agent. The compound FCCP (p-trifluoromethoxy
carbonyl cyanide phenyl hydrazone) is similar.

The negative charge is extensively delocalised over about ten

atoms in the ionised form of CCCP, so the electric field
surrounding the CCCP anion is very weak. This allows the anion
to diffuse freely through non-polar media, such as phospholipid
membranes. This behaviour is very unusual: the vast majority of
electrically charged ions are excluded from non-polar
environments.

With intact mitochondria, CCCP enters in the protonated form, discharging the pH gradient, and then
promptly leaves as the anion, destroying the membrane potential. The process can be repeated millions of
times, so that a tiny amount of CCCP can catalyse the movement of huge numbers of protons, and short-
circuit the respiratory chain.

Ionophores can be divided in to channel formers (such as gramicidin) which form a tiny pore through
the membrane, and mobile carriers which diffuse backwards and forwards across the membrane. The
ionophores used to study mitochondria normally belong to the mobile carrier group. They show considerable
ionic specificity because the ion must be accommodated within a confined space inside the carrier: there are
potassium ionophores, calcium ionophores and so forth.

Valinomycin: This mobile carrier catalyses the electrical movement of K+ across phospholipid
bilayers. This implies that the potassium distribution across the membrane obeys the Nernst equation once
equilibrium has been attained.

It is a cyclic amide/ester in which the sequence D-hydroxy- isovalerate, L-valine, L-lactate and D-
valine is repeated three times (-A-B-C-D- on the diagram). The ionophore provides a polar interior to
accommodate the potassium ion, but presents a non-polar lipophilic exterior to the outside world.

Nigericin: This mobile carrier resembles valinomycin but contains a

carboxyl group and forms a potassium salt. It therefore catalyses an
electroneutral potassium/proton exchange across lipid bilayers. This
implies that the potassium distribution will be related to the pH gradient
once equilibrium has been attained:

[K+ ]in / [H+ ]in = [K+ ]out / [H+ ]out

 In some cases (e.g. valinomycin) the ionophore - ion complex has a net electrical charge, whereas the
empty carrier is neutral. Other ionophores (e.g. nigericin) only form electrically neutral complexes. Charge
makes an enormous difference to the behaviour of each carrier, since the charged complexes interact with
any membrane potentials, whereas the neutral complexes are unaffected. We speak of electrical and
electroneutral carrier mechanisms to draw attention to this difference.

This concept can be extended to cover the naturally occurring transport proteins in the inner
mitochondrial membrane. Many of these proteins (and some ionophores) actually catalyse a swop of one
ligand for another, hence the term antiporters. We also speak of symports (two molecules travel together in
the same direction) and uniports (one molecule travels on its own.) Once again, the precise charge
stoichiometry has a huge influence on the results.

Non-shivering thermogenesis: The mitochondria found in brown adipose tissue contain a unique
uncoupling protein called thermogenin, which allows the controlled entry of protons without ATP sythesis
in order to generate heat. The protein is a 33kd dimer, structurally related to the adenine nucleotide porter. It
is inhibited by GTP, and activation is controlled by the sympathetic nervous system. This process is
particularly important in new-born babies, which can lose heat very rapidly to their surroundings, but also
occurs in adults and in hibernating animals. In contrast to the more familiar white adipose tissue, brown fat
has an excellent capillary blood supply and can achieve very high metabolic rates. The major depot in
humans is behind the shoulder blades, with other patches along the spine. The brown colour arises from the
respiratory enzymes.

Sub-mitochondrial particles

Treatment of mitochondria with ultrasonic vibrations under appropriate conditions tears open the
membranes and leads to the formation of tiny inside-out vesicles derived from the inner membrane,
containing trapped cytochrome c originating from the inter-membrane space. Sub-mitochondrial particles
may retain partial coupling between electron transport and ADP phosphorylation. They have been much
used for experimental work because their inverted membrane orientation provides direct access to the
respiratory chain without the complications introduced by the substrate transport systems.

Two activities which are most easily studied in sub-mitochondrial particles (although they also occur
with intact mitochondria) are the energy linked transhydrogenase and reversed electron transport.

The energy-linked transhydrogenase: This bizarre membrane-spanning enzyme catalyses the

reversible transfer of reducing equivalents between the mitochondrial NADPH + NADP pool and the
NADH + NAD pool, while simultaneously pumping 2 protons and 2 charges across the inner mitochondrial
membrane. The energy linkage keeps the NADPH/NADP couple about 500 times more reduced than the
NADH/NAD couple, despite the exact equivalence of their standard redox potentials.

NADPH + NAD+ + 2H+(inside) <=> NADP+ + NADH + 2H+(outside)

It is not clear which direction the transhydrogenase takes in vivo. Enzymes reacting with NADP
generally have a more negative redox potential than those reacting with NAD, except for glutamate
dehydrogenase, which is the only enzyme with a dual coenzyme specificity. This is a problem, since the dual
coenzyme specificity should lead to an energy-wasting futile cycle if the two coenzyme pools are at different
redox potentials.

The arrangement must confer some selective advantage (having persisted unchanged for 2000
million years!) but the biological benefits remain obscure. It may involve the overall regulation of nitrogen
balance, by using the ammonia-fixing reaction with NADPH to partially counteract the ammonia formation
with NAD, but this hypothesis remains highly speculative. Mitchell once suggested that the NADPH-linked
pathways for isocitrate, malate and glutamate were equivalent to fifth gear on a car, giving enhanced ATP
yields and better fuel economy at the expense of snappy performance. When maximum ATP flux was
required the ADP-mediated activation of the NAD-linked pathways would "drop down a gear" to enter the
overtaking lane.

Reversed electron transport: Oxidative phosphorylation is a partially reversible process and in the
presence of an artificially high ATP/ADP ratio electrons from a weak reducing agent like succinate can be
forced backwards through the respiratory chain carriers to yield a stronger reductant such as NADH:

succinate + NAD+ + energy => fumarate + NADH + H+ (overall reaction)

Ethanol is relatively poor reducing agent, and will only reduce a tiny proportion of any added NAD.
However, in an artificial system containing sub-mitochondrial particles, added alcohol dehydrogenase, a
trace of NAD and excess NADP, electrons from ethanol can be forced backwards via a small pool of NADH
through the energy-linked transhydrogenase to form large amounts of the excellent reducing agent, NADPH.
The reaction requires a source of energy: either added ATP, or respiration using another segment of the
respiratory chain.

ethanol + NAD+ => acetaldehyde + NADH + H+

NADH + NADP+ + energy => NAD+ + NADPH

The process has little physiological relevance, but gave enormous insight into mitochondrial
function. Reversed electron transport from ethanol to NADP in rotenone-blocked sub-mitochondrial
particles can be driven either by energy from external ATP (reversing the normal operation of the
F1ATPase) or by the energy from succinate oxidation via complex 2, complex 3 and complex 4. Both routes
are sensitive to uncouplers, but oligomycin only blocks the process when it is driven by ATP. This key
observation showed that there must be a common high energy intermediate between the coupling sites on
the respiratory chain and the manufacture of ATP. The intermediate is known as the high energy pool.

The High-Energy Pool

Reversed electron transport experiments lead to a realisation in the 1950s that there must be a
common, non-phosphorylated high-energy intermediate between the respiratory chain carriers and the
synthesis of ATP. The concept is illustrated below. The arrows show the normal direction of flow, but most
of the reactions are freely reversible, and energy added to the system via one process can be utilised
elsewhere. The high energy pool was originally thought to be a chemical compound, but nowadays we know
that it is actually the pH and electrical gradients developed across the inner mitochondrial membrane.

It is possible to measure the gradients by studying the distribution of permeant molecules across the
mitochondrial inner membrane. This requires a method for the rapid separation of mitochondria from their
incubation medium, either by rapid ultrafiltration, or by centrifugation through a layer of silicone oil. An
impermeable radioactive marker (commonly sucrose) is added to the incubation in order to correct for
external medium contaminating the organelles.
 Lipid soluble weak acids can be used to measure the pH differential. This method exploits the easy
membrane permeability of the electrically neutral free acid, and the impermeability of its ionised salts. It is
vital that the free acid crosses the membrane in the protonated form without net movement of electrical
charge. At equilibrium the concentration of the free acid is the same everywhere, but the ratio of [free acid]
to [ionised salt] differs in the two compartments. In the matrix space the amount of salt is proportional to the
internal pH, but outside the mitochondria the ionised salt concentration depends on the external pH. If the
internal and external salt concentrations can be measured then application of the Henderson Hasselbach
equation yields the internal pH.

Measurement of the membrane potential ( ) requires a permeant cation, e.g. a radioactive alkali
metal plus valinomycin, or the lipid soluble tetraphenyl phosphonium. In this case it is important that the
cation penetrates the membrane with net movement of charge, so that the cation distribution is influenced by
the membrane potential. If the internal and external concentrations of a permeant cation can be measured,
then the membrane potential can be calculated from the Nernst equation.

Some dyestuffs change their absorbtion or fluorescence properties when they are aligned in a strong
electric field. This provides an alternative method for the continuous measurement of mitochondrial
membrane potential by adding such compounds to a mitochondrial suspension. Suitable compounds include
carbocyanines, phenosafranine and bisoxonols. Although it is very convenient, this technique must be
calibrated using the older, single point methods.

The sum of the membrane potential and the pH gradient are together known as the proton motive
force (PMF). This indicates the total potential energy stored in the transmembrane gradients, which is
available to drive protons back into the matrix space, and provide the power for biologically useful
processes. Using the correct sign for the pH gradient (negative!) and substituting numerical values for the
gas constant, faraday constant and the absolute temperature, it follows that:

PMF (in millivolts) = - 60 pH

at 37oC. The minus sign is confusing: the pH gradient is itself negative, so the two components are normally
additive in their effects.

In mitochondria the electrical gradient, (150mV, inside negative) makes a larger contribution than
the pH gradient (0.5pH units, inside alkaline). In chloroplasts pH is more important. The mitochondrial
pH corresponds to a three-fold difference in hydrogen ion concentration between matrix and cytosol. For
each transmembrane process, the pH and components may act either separately or together, depending
on the enzyme structure and the balance of biological advantage. A proton movement would normally carry
one positive charge, but other ions may move as well, so there is no need for the proton and charge counts to
balance for any individual membrane transport process, although the totals must obviously balance overall.

The best current estimates for the proton and charge counts for the repiratory chain are shown in the
section on redox carriers. For each pair of electrons to traverse the chain, NADH dehydrogenase moves 4
protons and 4 positive charges outwards across the inner membrane from matrix to cytosol, ubiquinol
cytochrome c reductase moves 4 protons and 2 charges, while cytochrome oxidase moves 2 protons and 4
charges. The re-oxidation of NADH to form NAD and water is thus associated with the net export of 10
protons and 10 positve charges from the mitochondrial matrix to the cytosolic compartment.

ATP synthesis by the F1ATPase is thought to require 3 protons carrying 3 positive charges to re-
enter the matrix space. In addition, 1 proton (but no charge) is needed for phosphate uptake and 1 charge
(but no proton) for the ATP/ADP exchange with the cytosol (see the metabolite transport pages for details).
Thus the overall requirement is 4 protons and 4 charges for each ATP delivered to the outside world. This
implies that the P:O ratio is 2.5 for NAD-linked substrates, and 1.5 for succinate, which is rather lower than
some older text-book values, but much closer to the actual experimental results!
 The lipid bilayer in the inner membrane is only about 5nm thick and is therefore subject to enormous
electrical forces:

150 x 10-3 volts across 5 x 10-9 metres = 30,000,000 volts / metre

The inner membrane has an unusual phospholipid composition which includes cardiolipin
(diphosphatidylglycerol) and is particularly "leak proof" to small ions.

Most types of phospholipid will spontaneously form liposomes when agitated with water, and this
has been exploited to re-constitute the respiratory chain from purified components.

The F0F1-ATPase

This substantial enzyme (Mr approximately 500,000) is readily visible in electron micrographs as 8.5
nm spheres attached to the matrix side of the mitochondrial
inner membrane. The spheres can be detached by a variety
of methods, after which they act as an ATPase. The
physiological function of this enzyme is the synthesis of
ATP, using the energy stored in the transmembrane pH and
potential gradients.

The complete assembly contains at least 12 different

types of polypeptide chain, several of which are present in
multiple copies. The catalytic head group is connected by
an oligomycin sensitive stalk to a proton conducting
baseplate in the mitochondrial inner membrane. Three
protons are thought to pass through the membrane from the external P phase to the internal N phase for each
molecule of ATP manufactured by the complex.

The F1 head group contains three nucleotide binding sites, and the enzyme probably performs a
three-phase catalytic cycle. In the first phase, ADP and phosphate bind to one active centre, which catalyses
the formation of bound ATP. This step is energetically possible because the free energy released by tightly
binding the ATP to the active centre compensates for the instability of the new phospho anhydride bond.
The energy from the proton motive force is required to prise the ATP from the active centre.

The F0 base piece embedded in the mitochondrial inner membrane is a molecular turbine driven by
the trans-membrane proton gradient. Proton entry forces a central camshaft to rotate within the F0 baseplate
and the F1 head group, altering the subunit conformation as this movement takes place. A second, off-centre
protein tether connects the head group to the base piece and prevents the head piece spinning uselessly as the
central shaft rotates. Energy is transmitted to the catalytic subunits in the ATP synthase F1 headpiece by the
rotation of the camshaft. The "cam" distorts the protein subunits, destroying their ability to bind ATP. The
energy input is used to drive ATP release, not for bond formation.

It is presumably necessary to disable the catalytic mechanism on the centre which has just formed
ATP (to stop this centre hydrolysing its own product) before destroying its ability to bind ATP. This allows
the product to be released. Meanwhile, the two other active centres are performing their own parts of the
catalytic cycle. The three active centres operate simultaneously, but 120o out of phase. It takes at least 9
protons (possibly as many as 12) to drive one revolution of the camshaft and produce 3 ATP molecules.

Remember that the whole complex is reversible. Normally the energy from the proton gradient is
used to manufacture ATP, but it is equally possible in vitro to do things the other way round, and use the
hydrolysis of ATP to drive the camshaft, and ultimately pump protons back through the turbine and into the
extramitochondrial compartment. If the F0 base piece is not attached to a membrane nothing useful will be
accomplished, and the complex will simply act as an ATPase, as was originally observed.
 It is possible to directly observe the rotation of the F1ATPase cam shaft using a fluorescence
microscope, although considerable ingenuity is required. Noji et al (1997) Nature 386, 299-302 used a
genetically modified F1ATPase from a thermophilic bacterium expressed in E. coli. They discarded the F0
basepiece and tethered the F1 motor head groups to a glass plate using polyhistidine tags attached to the N-
termini of all three beta subunits. The glass plate had been pre-treated with horseradish peroxidase
conjugated with the nickel complex of nitrilotriacetic acid, to which polyhistidine binds with high affinity.
[Nitrilotriacetic acid looks like half an EDTA molecule, so it leaves un-coordinated nickel positions
available for external ligands.]

The motors were glued down by their large catalytic subunits, leaving the motor shafts exposed, and
facing away from the glass. The gamma subunits which form the shaft were modified by site directed
mutagenesis to remove the original Cys193 (which is inconveniently far down the shaft) and replace it with
serine. These workers also replaced Ser107 in the stalk region with cysteine.

This single cysteine residue (the only one in the molecule) could then be biotinylated, and linked
using streptavidin to fluorescently labelled, biotinylated actin filaments. [Streptavidin has four biotin binding
sites.]

The fluorescent actin filaments were many times larger than the tethered motors and could be
visualised in a light microscope.

Addition of 2mM ATP caused a small number of the motor shafts marked by the actin filaments to
rotate in a counter- clockwise direction. The movie shows the results they obtained.

Circular motion also occurs in the proteins which rotate bacterial flagellae, another important
enzyme system which is driven by the proton motive force. It is apparent that the wheel has been in
continuous use for at least 2000 million years.

Bioenergetics

Redox potentials: These provide a quantitative measure of oxidising or reducing power. Strong oxidising
agents (like molecular oxygen) have positve redox potentials, good reducing agents have negative redox
potentials. Electrons move spontaneously towards those compounds with the more positive redox potentials.
The standard for the whole scale is pure hydrogen gas at 1 atmosphere pressure in contact with a platinised
platinum electrode immersed in 1 molar acid. This combination is called a standard hydrogen electrode
(SHE) and is defined to have a redox potential of zero.

Standard Redox Potentials (Eo) are measured with 50% oxidised form and 50% reduced form for the
compound in question. [This is similar to pKa in the Henderson Hasselbach equation, which is equal to the
pH value measured with 50% protonated buffer and 50% deprotonated buffer.] If the oxidation / reduction
reaction involves protons (many do) then you need 1 molar acid present as well. This is a bit inconvenient
for biological systems, so we often work with Eo' which is the redox potential measured at pH 7. This has a
major effect: at pH 7 hydrogen gas has a redox potential of -420 mV.

The effective redox potential depends on the proportion of the oxidised and reduced forms. This can make a
big difference: the NADPH / NADP couple (maintained by cells almost 100% in the reduced form) is a
much better biological reducing agent than NADH / NAD (no more than 30% reduced under normal
conditions) despite the fact that their standard redox potenials are identical.

E = Eo + R T ln( [oxidised form] / [reduced form] ) / z F

(E is measured in volts, R is the gas constant [8.31 joules / degree / mole], T is the absolute temperature, F is
the faraday constant [96,500 coulombs / mole], z is the number of electrons involved in the reaction and ln
denotes the natural logarithm. If natural logs are replaced by base 10 logarithms, these must be multiplied by
a conversion factor of 2.303)
Note that it is meaningless to speak of the redox potential for a single compound in isolation: both the
oxidised and the reduced forms must be defined, although in some cases (e.g. NADH) the oxidised form
(NAD) is obvious. For hydrogen gas the oxidised form could be either protons or water (you must specify
which) while for oxygen the reduced form is usually either hydroxyl ions or water, but it could be
superoxide or peroxide, and this would affect the value of the redox potential.

The numerical value of 2.303 R T / F is about 60mV at 37oC. Can you use this information to calculate the
change in redox potential for hydrogen gas, going from 1 molar acid in a standard hydrogen electrode to the
biological standard at pH 7.0 ?

A few redox potentials can be measured directly (by inserting a platinum electode into a mixture of the
oxidised and reduced forms, and completing the circuit through a salt bridge connected to a suitable
reference electrode) but biological redox potentials are more commonly obtained indirectly by studying the
equilibrium position of reactions where one participant has a known redox potential. When everything is
present in equimolar amounts, a good reducing couple can reduce a weaker couple. However, a weak
reducing agent could reduce a stonger reducing agent, providing that the weaker couple were largely in the
reduced form, and the stronger couple largely in the oxidised form. Similar considerations apply to oxidising
agents, with the argument appropriately inverted.

When an oxidising reagent interacts with a reducing agent, the difference between their respective redox
potentials E is related to the Gibbs free energy G for the overall reaction:

G= -zF E

(where G is measured in joules, E is measured in volts, F is the faraday constant [96,500 coulombs /
mole] and z is the number of electrons transfered in the reaction). A little care is needed with the
arithmetical signs in deciding which number should be subtracted from what. Remember that G is
negative if the overall reaction is favourable.

The numerous electron carriers that make up the respiratory chain are arranged in the approximate order of
their redox potentials: the best reducing couples at the substrate end and the best oxidising couples at the
oxygen end. At key points along the chain, the difference in redox potential between adjacent carriers
provides the driving force to pump protons out of the matrix space and into the cytosol as part of the overall
energy coupling mechanism.

The redox potentials for some important biological reactions are listed in the table:

Chemical reaction Eo' (mV)

isocitrate => oxoglutarate + CO2 + 2e-1 -380
hydroxybutyrate => acetoacetate + 2e-1 -346
pyruvate + CoASH => acetyl-CoA + CO2 + 2e-1 ?
NADH => NAD+ + H+ + 2e-1 -320
lactate => pyruvate + 2e-1 -190
malate => oxaloacetate + 2e-1 -166
succinate => fumarate + 2e-1 +30
ubiquinol => ubiquinone + 2H+ + 2e-1 +45
cytochrome c2+ => cytochrome c3+ + e-1 +230
H2O => 1/2 O2 + 2H+ + 2e-1 +820
Nernst equation: This equation has various forms: do not be surprised if you find another version. The
form most commonly encountered in biological systems relates the membrane potential to the
concentrations of a diffusible ion in equilibrium with the potential on each side of the membrane:

= 2.303 R T log( [Cin] / [Cout] ) / z F

(where is the membrane potential in volts, R is the gas constant [8.31 joules / degree / mole], T is the
absolute temperature, Cin and Cout are the two ionic concentrations, z is the electric charge on the ion, F is
the faraday constant [96,500 coulombs / mole]. The factor of 2.303 arises from the use of log10 instead of
natural logarithms.)

When the system reaches equilibrium, the tendency for the diffusible ion to escape through the membrane,
down its concentration gradient, is exactly balanced by the electrical force attracting it in the opposite
direction. The membrane potential changes sign if you reverse the orientation of the gradient, or if you
substitute a diffusible anion for a diffusible cation keeping all the concentrations unchanged. Use your
common sense to work this out: at equilibrium, the highest concentration of a positive ion will be on the
negative side of the membrane, and vice versa.

At 37oC the value of 2.303 R T / z F is about 60mV so the membrane potential increases by 60mV for each
tenfold increase in ion gradient. This is the same as the electrical output from a glass pH electrode (60mV
per pH unit) which is not surprising because the voltage arises through the same mechanism.

Note that the ion must be able to cross the membrane with movement of charge for the above equation to
apply, and it must have reached equilibrium with the electrical gradient. The Nernst equation does not apply
to impermeant ions, or those which cross by an electroneutral exchange mechanism. No useful work can be
obtained from an ion gradient subject to the Nernst equation (see below for a detailed discussion) but it is
possible to obtain energy from an ion gradient which has NOT reached equilibrium, for example the sodium
gradient across the plasmalemma, or the proton gradient across the mitochondrial inner membrane.

The Nernst equation can be used to calculate the mitochondrial membrane potential by measuring the
distribution of a lipid soluble cation such as tetraphenylammonium, or rubidium plus valinomycin.

Gibbs free energy: ( G) This is the useful work which can be obtained from a chemical reaction, and
reflects its displacement from equilibrium. No useful work can be obtained from a reaction which has
reached equilibrium, and in this case G = 0.

For a chemical reaction where a series of reactants Rn form a series of products Pm:

R1 + R2 + R3 + ... <=> P1 + P2 + P3 + ...

the precise relationship between G and the extent of reaction is given by the Gibbs equation:

G = G0 + R T ln ( [P1] . [P2] . [P3] ... / [R1] . [R2] . [R3] ...)

(where R is the gas constant [8.31 joules / degree / mole], T is the absolute temperature, and ln( ) is the
natural logarithm of all the product concentrations multiplied together, then divided by all the reactant
concentrations multiplied together). If you prefer to work with base 10 logarithms, you must multiply the
whole of the last term above [starting with R T ln( ....) ] by 2.303

G0 is the standard free energy for the reaction, measured when all the reactants and products are present at
1 molar concentration.
 This graph shows the relationship between G and
reaction progress for two different chemical reactions.

red reaction: G0 is positive

blue reaction: G0 is negative.

In both cases G is zero at equilibrium, but the red

equilibrium position favours the reactants while the blue
equilibrium favours the products.

The horizontal axis for this graph is R T ln ( [P1] . [P2] . [P3] ... / [R1] . [R2] . [R3] ...)

Since G is zero at equibrium, it follows that:

G0 = - R T ln ( [P1E] . [P2E] . [P3E] ... / [R1E] . [R2E] . [R3E] ...)

where the subscript E denotes the concentration of reactant or product present at equilibrium. Under these
conditions, the terms inside the round brackets then represent the equilibrium constant, Keq for the overall
reaction, leading to the important conclusion that:

G0 = - R T ln (Keq)

where Keq = [P1E] . [P2E] . [P3E] ... / [R1E] . [R2E] . [R3E] ...

Free energy of an ion gradient: No useful work can be obtained from ions subject to the Nernst equation
(unless you alter the electrical gradient) because these ions have already reached equilibrium with the
membrane potential, but energy can be obtained from the dissipation of a gradient for non-diffusible ions
across the same membrane. There are two separable components to this free energy, one of which arises
from the electrical gradient across the membrane, and the other from the difference in ionic concentrations.
To calculate the free energy, imagine the ions performing some useful task as they escape across a tiny
membrane separating two enormous reservoirs of effectively infinite capacity. Visualise moving one mole of
the non-permeant ion from one side of the membrane to the other, keeping everything else (ionic
concentrations, voltages) constant

G=zF + 2.303 R T log ( [Cout] / [Cin] )

(where G is the free energy, z is the charge on the ion, F is the faraday constant [96,500 coulombs / mole]
and is the membrane potential, R is the gas constant [8.31 joules / degree / mole], T is the absolute
temperature, Cin and Cout are the concentrations in the two compartments). The first term on the right hand
side is like an electricity bill, basically amps x volts x time. The second term is a slightly modified form of
the Gibbs equation, where the trapped ions are regarded as reactants, and the escaped ions are seen as
products. G0 is zero because no energy is available from the concentration term if the ion has the same
concentration on both sides of the membrane. The factor of 2.303 allows for the use of base 10 logarithms in
place of natural logs.

The above equation allows almost infinite opportunity for getting the signs muddled up. Remember that G
is negative for favourable reactions. You need the correct sign for the membrane potential, and the correct
charge on the ion, and remember that the second concentration term may either add to or subtract from the
first electrical term, depending on whether Cout is greater or less than Cin. Use your common sense to
visualise what is happening, and then insert the correct signs as appropriate.
Henderson Hasselbach equation: This relates the pH of a buffer solution to the ionisation constant for the
buffer, and the proportion of the protonated and non-protonated forms:

pH = pKa + log10 ([deprotonated form] / [protonated form])

It is easy to remember this: the greater the proportion of the protonated form, the more acidic the buffer must
be. For a weak acid buffer system such as acetic acid and sodium acetate, the free acetic acid is the
protonated form and the deprotonated form is the salt. For an amine buffer, the salt would be the protonated
form.

The pKa reflects the intrinsic affinity of the buffer for protons, and is simply log10 of the association
constant, Ka:

BH <=> B- + H+ Ka = [BH] / ( [B-] . [H+] )

Again, it is easy to get this the right way round: strong bases have large pK values, an alkaline pH and a
strong affinity for protons. When a buffer is exactly half-neutralised, the concentrations of the protonated
and deprotonated forms are equal. At this point the system has maximum buffering capacity and the pH =
pKa

Mitochondrial transport systems

Normal operation of the respiratory chain creates both a pH differential and a voltage gradient of 30,000,000
volts/metre across the inner mitochondrial membrane. This demands highly specific transport proteins to
control the movement of small ions across the membrane, and to prevent the dissipation of the various
gradients.

The majority of inner membrane carriers are antiporters which exchange one molecule for another. If the
electrical charges on the two molecules exactly balance the exchange will be an electroneutral process and
the huge membrane potential will have no effect on the result. However, the pH gradient may affect the
equilibrium position for an electroneutral exchange if one or more participants are acidic or basic.

Symports transport two molecules in the same direction. If the molecules have opposite charges this may
still be an electroneutral process. It is experimentally difficult to distinguish between a proton symport and a
hydroxyl antiport.

If the charges differ on the transported molecules for either symports or antiports, then there will be an
electrical (sometimes called electrogenic) transport process. Such carriers can "feel" the membrane
potential, which will exert a major influence on the result. In general the gradients affected by the pH
component of the proton motive force are fairly modest, but those influenced by the membrane potential are
substantial.

In a very few cases (e.g. for calcium uptake) the carrier simply allows the charged ion to traverse the
membrane. This constitutes an electrical (or electrogenic) uniporter. The membrane potential exerts its full
effect, leading to substantial concentration gradients if the reaction were able to reach equilibrium.

The principal transport systems are listed in the table below, which also shows some of the main inhibitors.

porter stoichiometry inhibitors comments

phosphate electroneutral exchange of NEM all mitochondria
H2PO4- for OH- mersalyl
dicarboxylate random electroneutral exchange of mersalyl all mitochondria
malate2- for succinate2- or HPO42- malonate
tricarboxylate electroneutral exchange of benzene 1,2,3 mammalian adipose tissue
(citrate3- + H+ ) for malate2- tricarboxylate and liver: needed for fatty
acid biosynthesis
oxoglutarate electroneutral exchange of _ most mammalian tissues
malate2- for oxoglutarate2- needed for the malate
aspartate cycle
adenine electrical ATP4-/ADP3- exchange atractyloside all mitochondria
nucleotides bongkrekic
acid
glutamate electroneutral exchange of _ mammalian liver: needed
glutamate- for OH- for the urea cycle
aspartate electrical exchange of _ most mammalian tissues
(glutamate- + H+ ) for aspartate- needed for the malate
aspartate cycle
calcium electrical uniport for Ca++ _ calcium uptake system
calcium electroneutral exchange of _ mitochondrial calcium
Ca++ for 2H+ export system in liver
calcium / electroneutral exchange of _ mitochondrial calcium
sodium Ca++ for 2Na+ export system in heart
sodium / electroneutral exchange of _ widely distributed
proton Na+ for H+

The charge imbalance associated with the adenine nucleotide carrier leads to a large difference in the
ATP/ADP ratio between matrix space and cytosol. ATP is effectively "worth more" in the cytosol, because
the ATP:ADP couple is maintained further away from equilibrium:

Cells do not, however, get something for nothing. The transport of ATP, ADP and phosphate across the
inner mitochondrial membrane costs 33% additional energy, over the minimum required for the synthesis of
ATP within the mitochondrial matrix compartment. This extra energy must be supplied by the respiratory
chain. One additional proton is used to drive both of these transport systems: the positive charge on this
single proton drives adenine nucleotide exchanger, while its acidity drives the phosphate uptake. The
electrical and pH components of the proton motive force are exploited separately.

There has recently been great interest in the ATP:ADP carrier protein as a possible auto-antigen in dilated
cardiomyopathy, a common and debilitating cardiac disease. This same enzyme is involved in the
mitochondrial permeability transition and in apoptosis.

The high cytosolic ATP/ADP implies a very low cytosolic AMP concentration, as a result of the myokinase
equilibrium. This enables 5' AMP to serve as an emergency signal, which indicates a threat to the ATP
supply. Several key enzymes (notably glycogen phosphorylase and phospho- fructokinase) are strongly
activated by low concentrations of 5'AMP. This nucleotide also gives rise to adenosine which stimulates
blood flow to active tissues. Note that 5'AMP differs from 3'5' cyclic AMP produced by adenyl cyclase.

Muscle contraction apparently requires a very high

ATP/ADP ratio, and it is difficult to maintain a low
myofibrillar ADP because it will not diffuse quickly
enough back to the mitochondria at low ADP levels.
(Diffusion rate is directly proportional to metabolite
concentration.) Active muscles contain creatine
phosphokinase, (CPK) and large amounts of creatine and
creatine phosphate. These compounds equilibrate with the
adenine nucleotide pools. It is thought that the high
concentrations of these highly diffusible energy carriers
increase the maximum energy transport rate.

Gene knockout experiments have shown that mice lacking both the mitochondrial and myofibrillar CPK iso-
enzymes exhibit abnormal development, however mutants deficient in only one iso-enzyme are superficially
healthy. It is difficult to see why both CPK genes should be widely retained if they don't do anything. It
remains to be established whether the single mutants can maintain the sustained work outputs achieved by
the wild-type controls.

One of the most important asymmetric transporters is the aspartate carrier, which in mammals plays a key
role in the re-oxidation of glycolytic NADH by the malate-aspartate cycle. This cycle is necessary because
the inner membrane is not permeable to either NAD or NADH. The component reactions must revolve twice
for each molecule of glucose oxidised by the cell. The two enzymes involved, malate dehydrogenase (MDH)
and glutamate oxaloacetate transaminase (GOT) are among the most active in the body. Both enzymes exist
in mitochondrial and cytosolic variants, but all four proteins are coded by nuclear genes.

Aspartate- swops for glutamate plus a proton, so the full proton motive force is applied to the aspartate
porter. The overall effect is to bias this otherwise symmetrical cycle, and maintain the cytosolic
compartment in a relatively "oxidising" state (with a low NADH/NAD ratio) while the mitochondrial
compartment is kept correspondingly reduced. This arrangement suppresses lactate formation during aerobic
glycolysis. Invertebrate species employ alternative shuttles to achieve the same effect, and it is doubtful if
eukaryotic cells could work at all without some such arrangement.

It is important to realise that no porter means no transport. Oxaloacetate and fumarate, for example, bind
very badly to the dicarboxylate carrier, and are not transported at any significant rate. If both malate and
oxaloacetate were transported it would be impossible to maintain the redox differential for NADH / NAD
which exists between the mitochondrial matrix space and the cytosol.

Metabolite porter genes are located in the nucleus and are only expressed in those tissues which require
them. For example, liver mitochondria have porters for ornithine and citrulline, which are required for the
urea cycle, but these are not found in other tissues. The citrate carrier is found only in liver cells and
adipocytes, where it is needed for lipogenesis

The outcome of many substrate oxidation experiments in vitro is determined by the porter specificity. For
example: most animal mitochondria oxidise succinate almost quantitatively to malate, because neither
fumarate nor oxaloacetate can be transported out of the matrix space and there is no source of acetyl-CoA to
produce citrate.
 Even in liver mitochondria, which possess a glutamate / hydroxyl
antiporter, glutamate added alone mainly yields aspartate via
glutamate oxaloacetate transaminase (GOT), oxoglutarate
dehydrogenase (OGDH), succinate thiokinase (STK), succinate
dehydrogenase (SDH), fumarase (FUM), malate dehydrogenase
(MDH) and the aspartate porter.

However, if glutamate and malate are added together, mammalian

mitochondria initially produce equal amounts of aspartate and
oxoglutarate using the mitochondrial half of the malate - aspartate cycle. The aspartate and oxoglutarate
porters, GOT, MDH and the respiratory chain are the only enzymes involved under these conditions.

Self assessment question: If mitochondria were incubated with oxoglutarate alone, which pathways would
be operative and which compound(s) would be formed?

In some cases, e.g. for calcium ions, an electrical carrier is used for uptake and an electroneutral system is
used for export. The two components of the proton motive force are used to drive the ion in opposite
directions. This may be exploited by cells to regulate intra-mitochondrial calcium to a different value to the
cytosolic concentration.

Many important metabolites show an asymmetric distribution across the mitochondrial inner membrane. The
operation of the electroneutral anion carriers leads to a modest accumulation of polybasic acids (especially
citrate) within the matrix space. In calculating the equilibrium distribution, it is only the "bottom line" which
counts - the net number of protons which crossed the membrane by whatever process in order to accumulate
the anion. The detailed mechanism is irrelevant, and any intermediate "swops" are discounted. If the overall
reaction for electroneutral metabolite accumulation is:

anionn-(out) + nH+(out) <=> anionn-(in) + nH+(in)

then simple mass action considerations lead to the conclusion that:

[anionn-in ] / [anionn-out ] = ([H+out] / [H+in])n

This is helpful for the operation of the citric acid cycle, and also has implications for the regulation of
glycolysis and lipogenesis by phosphofructokinase and acetyl CoA carboxylase, where citrate is an allosteric
effector.

Mitochondrial swelling experiments: If it is desired to study membrane transport in isolation it is often

necessary to block further metabolism with cyanide or rotenone. The most accurate studies of transport
kinetics involve the rapid separation of mitochondria from their incubation medium using ultrafiltration or
centrifugation through silicone oil. These complex and time consuming experiments require radioactive
tracers to correct for "carry over" of the incubation medium into the mitochondrial fraction. Large scale
metabolite movements will, however, produce alterations in matrix volume through osmotic mechanisms. A
qualitative estimate of transport rates can thus be obtained by measuring the changes in light scattering from
a turbid mitochondrial suspension.
 These results were obtained from 0.5 mg liver
mitochondria suspended at pH 7 in a spectrophotometer
cuvette with 2.5 ml of 150 mM potassium acetate. Acetate
ions can penetrate rapidly by an electroneutral process as
free acetic acid, but no swelling is observed initially
because the potassium cannot enter. Addition of
valinomycin has no effect because this ionophore
catalyses electrical movement. Nigericin allows an
electroneutral exchange of potassium ions for protons and
rapid swelling takes place.

Passive swelling in isotonic ammonium salt solutions can be used to study the major anion transport systems
in cyanide-blocked mitochondria. The solutes move passively down their electrochemical gradients.
Ammonium ions penetrate rapidly as free ammonia without movement of charge, and swelling is observed if
the anion is also transported by an electroneutral process. It can be shown that phosphate, malate, succinate,
oxoglutarate, citrate and sodium are all taken up by electroneutral mechanisms, and the phosphate /
hydroxyl, phosphate / dicarboxylate, dicarboxylate / tricarboxylate and malate / oxoglutarate exchangers can
be demonstrated.

It is also possible to observe "energised swelling" using intact mitochondria where solutes are accumulated
against a concentration gradient using energy from respiration or from external ATP. There are substantial
differences between the energised and passive processes, but in every case (except for ATP-driven pumps)
net ion movements are always down the electro-chemical gradient.

Origin & evolution of mitochondria

The earth was formed about 4,500,000,000 years ago. The original atmosphere was a reducing one,
containing (among other constituents) nitrogen, methane, carbon dioxide, hydrogen sulphide and water
vapour. There was no free oxygen, so oxidative phosphorylation was not an option for the earliest
organisms, which relied instead on a fermentative metabolism. Pre-biotic synthesis in thunderstorms and
volcanic vents is believed to have left a rich legacy of the most common stable metabolites, which were
scavenged by our distant ancestors.

The earliest living organisms were subject from the start to a relentless selection pressure for better substrate
uptake systems. It is conceivable that trans-membrane proton gradients were originally used to drive
substrate uptake. The F1ATPase may at first have fulfilled this important function, as the sodium gradient
and the Na/K-ATPase still do today. Selection pressures would favour efficient ion pumping, leakproof
membranes and steadily increasing gradients in order to scour the remaining metabolites from a depleted
environment.

The importance of substrate uptake is that even marginal improvements yield an immediate selective
advantage. In this situation the evolution of the earliest "photosynthesis" does not appear an unduly difficult
step, serving initially to supplement pre-existing ion gradients from a widely available free power supply.

The earliest photosynthetic organisms were unable to produce oxygen from water, and relied instead on a
variety of easier electron donors such as hydrogen sulfide and ferrous iron. They titrated the earth's crust,
exhausting one reductant after another, subject always to the constant pressure to handle stronger oxidants
and bigger gradients. It is likely that photosynthetic electron transport and oxidative electron transport
systems evolved together, starting in each case from the most highly reducing end of their respective
electron transport chains.

The transhydrogenase may be the most ancient part of the respiratory chain, capable of energising the outer
membrane in a primitive fermentative bacterium long before any oxygen was present in the earth's
atmosphere. The other respiratory chain components were probably added sequentially, as more effective
oxidants slowly became available over hundreds of millions of years through the photosynthetic activities of
the cyanobacteria and, very much later, the green plants. Meanwhile the selection pressure continued for
larger and larger ion gradients. Eventually the trans-membrane pH gradient was sufficient to force the
F1ATPase backwards (in its modern physiological direction) and modern photosynthesis and oxidative
phosphorylation became possible.

All the gaseous oxygen in the present atmosphere is believed to have had a biological origin, and was mostly
formed sometime between 3,000,000,000 and 1,000,000,000 years ago, as a result of photosynthesis by
cyanobacteria and the earliest green plants. Electron transport was a prokaryotic invention, and its
practitioners must have enjoyed a tremendous selective advantage from efficient ATP synthesis. Moreover,
the rising atmospheric concentration of highly toxic and reactive oxygen was a serious threat to our
eukaryotic ancestors. Even today, despite the evolution of effective anti-oxidants, most of our tissues
maintain their intracellular oxygen concentration in the micromolar range,1000 times below the bloodstream
value.

Primitive eukaryotes, however, had evolved one technique which no prokaryote could perform: they were
sufficiently large and flexible to swallow other organisms whole, probably digesting them for food. How
much more efficient, though, to keep a few aerobic bacteria as guests, supplying them with substrates in
return for an endless supply of ATP? The original bacterial ion porters could continue to operate, and a
single host cell mutation might be sufficient to insert an adenine nucleotide carrier through the bacterial cell
wall.

Mitochondria are thought to have evolved at least 2000 million years ago from primitive bacteria which
enjoyed such a symbiotic relationship with early eukaryotic cells.

It must have been difficult initially to synchronise the activities of the two genomes, and there followed a
gradual transfer of mitochondrial genetic functions to the eukaryotic cell nucleus, where they were better
integrated with the other cellular controls. This process has progressed to varying extents in different
species, so that yeast and mammalian mitochondria differ slightly in the functions which they have retained.

Mitochondria still show some signs of their ancient origin. Mitochondrial ribosomes are the 70S (bacterial)
type, in contrast to the 80S ribosomes found elsewhere in the cell. As in prokaryotes there is a very high
proportion of coding DNA, and an absence of repeats. Mitochondrial genes are transcribed as multigenic
transcripts which are cleaved and polyadenylated to yield mature mRNAs. Unlike their nuclear cousins,
mitochondrial genes are small, generally lacking introns, and the chromosomes are circular, conforming to
the bacterial pattern.

In addition to the mitochondrial ribosomes and transfer RNA, the small amount of mitochondrial DNA
codes for only 13 polypeptides in humans. These are mainly the hydrophobic cores of the major trans-
membrane proton pumps, which are sticky and insoluble and difficult to move around the cell. All the
remaining mitochondrial genes have migrated to the nucleus, and the hundreds of other mitochondrial
proteins are now imported from the cytosol.

Maternal inheritance: The vast majority of mitochondria in a fertilised egg derive from the hundreds of
thousands of mitochondria in the large maternal egg rather than the small number in the tiny sperm. As a
result most mitochondrial traits appear to be transmitted exclusively through the maternal line.

Nevertheless, there are obvious difficulties with this hypothesis. If maternal transmission were the only
mechanism, and there were no device within each cell to eliminate defects and synchronise the multiple
copies of the mitochondrial DNA, within a relatively short period there would be thousands of mitochondrial
variants. These are not observed in practice, despite the fact that mitochondrial DNA is less well protected
than nuclear DNA and mitochondrial DNA replication lacks some of the error detection systems employed
in the cell nucleus. Single-parent inheritance also denies to a species the evolutionary advantages of
recombination and sexual reproduction.
Electron micrographs show that sperm contain two types of mitochondria: the large spiral structure which
provides the power for flagellar movements, and two smaller, less specialised mitochondria packaged next
to the male pronucleus with the paternal DNA. Whatever the function of this curious arrangement, its effects
are not apparently manifest in the immediately succeeding generation.

Mitochondrial Eve: Assuming a largely maternal inheritance, analysis of human mitochondrial DNA is
consistent with the hypothesis that the entire modern human race is descended from a single female or a
group of closely related sisters, who lived in Africa about 200,000 years ago.

There are numerous objectors to this hypothesis, nevertheless the limited variability of our nuclear DNA
also suggests that our ancestors recently went through an "evolutionary bottleneck" with a very small
number of survivors. Our presence on the planet appears to have been a close run thing.

Biogenesis of mitochondria

The human mitochondrial genome is only 16.6kb and among the smallest in the animal kingdom. The
circular chromosome was completely sequenced by Sanger's team in 1981 and the map is shown below:

The heavy H strand has a higher guanine content, and in this diagram is transcribed in a clockwise direction
as a single RNA molecule starting from the PH promoter. The light L strand is transcribed anticlockwise
from the PL promoter. Both RNA transcripts are later cleaved to yield functional RNA molecules.
Replication of the heavy strand by DNA polymerase commences anticlockwise from the OH replication
origin; this eventually exposes the OL origin allowing replication of the light strand to be completed. The
red-coloured region near the promoters is known as the D-loop and contains a short length of triple stranded
DNA.
The mitochondrial genetic information is very densely packed. There are no introns and only tiny gaps
between the genes. [In the diagram above, N1 - N6 are NADH dehydrogenase subunits, cyt b is cytochrome
b, OX1 - OX3 are cytochrome oxidase subunits, and the genes for two ATPase subunits partly overlap.]
There are 37 mitochondrial genes in total, but only 13 of these code for polypeptides, the remainder being
the 2 ribosomal subunits and 22 types of transfer RNA. All the hundreds of other mitochondrial proteins,
including DNA polymerase, RNA polymerase, amino acid activating enzymes and all the ribosomal proteins
are coded by nuclear genes and imported from the cytosol.

There are about 2000 copies of the mitochondrial genome in a typical cell, so that despite its small size it
comprises about 0.5% of the DNA mass. Mitochondrial DNA replication appears to be less accurate than
nuclear copying, and it is far from clear how consistency is maintained between these multiple copies, or
how their replication is synchronised with the remainder of the cell.

There are substantial differences between the proteins present in mitochondria from different tissues,
reflecting the tissue specific patterns of nuclear gene expression. Protein turnover, however, seems to be
fairly slow and mitochondrial protein composition does not respond very quickly to dietary or hormonal
stimuli. The total number of mitochondria per cell can be changed (for example, through muscle activity)
over the course of several weeks.

The import of cytosolic proteins to the mitochondrial matrix space takes place at specialised sites where the
inner and outer membranes are in close contact. Cytosolic precursors are marked for import with an N-
terminal leader sequence, bearing a substantial positive charge. The leader folds to form an amphipathic
alpha helix where the hydrophobic amino acids are concentrated along one face. Receptor proteins in the
outer membrane recognise sub-sets of these import signals, and help to direct the precurors towards the
import channel. Mitochondrial proteins are usually more basic than their cytosolic counterparts. They
probably have a net positive charge at physiological pH, and are strongly influenced by the membrane
potential. It appears that the proton motive force provides part of the energy for protein uptake.

Proteins pass through the membranes in an extended linear configuration. It is important that they should not
fold fully before import, since the correctly folded enzymes could not pass through the hole. Folding is
temporarily prevented in the cytosol by binding to the chaperone protein hsp70, while two further
mitochondrial chaperones hsp60 and hsp70 supervise re-folding of the imported proteins after they have
entered the matrix space. ATP hydrolysis is required in the cytosol and in the mitochondria for successful
import of functional proteins, in addition to the membrane potential and pH gradient. This ATP requirement
is probably associated with protein folding.

The matrix targeting sequence is cleaved from the imported proteins by a matrix protease. If the imported
protein is destined for the intermembrane space (e.g. myokinase) cleavage of the first signal sequence is
believed to expose a second signal directing re-export of the protein through the inner membrane. (The
unusually small protein apo-cytochrome c apparently uses the non-specific outer membrane channels to
reach the inter-membrane space. Chelation of heme, and consequent adoption of the native protein
conformation locks this particular protein into its final position.)

Mitochondrial diseases

Primary mitochondrial diseases are relatively rare, probably because major defects in the Krebs cycle or the
respiratory chain are incompatible with life and many affected embryos die at an early stage. Nevertheless,
about 150 different types of hereditary mitochondrial defect have been reported, and mitochondria play an
important part in several common conditions. Mitochondrial DNA is maternally inherited through the egg
cell cytoplasm, but many of the inherited defects map to the nuclear genome since the majority of
mitochondrial proteins are imported from the cytosol. Heteroplasmy is a complicating factor: affected cells
may contain a mixed mitochondrial population, and the defect may only involve a proportion of the
mitochondria. (In healthy people all the copies of the mitochondrial DNA are identical.) Mitochondrial
defects may be confined to a limited range of tissues, and, unexpectedly, they may change substantially as
the patient ages. There has recently been great interest in mitochondrial diseases with an autoimmune
component, and in those involving apoptosis (programmed cell death).

Respiratory defects: A huge variety of individual defects have been described, affecting one or more of the
respiratory chain redox carriers. (A defect in mitochondrial protein import, for example, might affect dozens
of enzymes to a greater or lesser extent.) The tissues which rely most extensively on aerobic metabolism are
most severely affected, so patients commonly present with a myopathy or encephalopathy, or both. Lactic
acidosis, muscular weakness, deafness, blindness, ataxia and dementia are common findings. These
mitopathies are often fresh mutations, so there is no family history. In many cases the diseases are obvious
from birth, but may develop in later life if the number of defective mitochondria increases with age.

Light microscopy of muscle biopsies frequently reveals a proportion of "ragged red" fibres or the absence of
key respiratory enzymes from particular cell types after histochemical staining. Abnormal mitochondria may
be visible in the electron microscope: for example "parking lot" mitochondria where the normal cristae are
replaced by a rectangular grid. Even where the nature of the mutation has been identified by DNA
sequencing, there is only limited correlation between the genetic defect and the time course of the disease or
the severity of the symptoms.

The classification of this heterogeneous group of diseases leaves much to be desired, but the following are
typical examples: myoclonic epilepsy with ragged red fibres (MERRF), mitochondrial encephalopathy with
lactic acidosis and stroke (MELAS), neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP).

Auto-immune diseases: There are some relatively common diseases where patients have been reported to
produce auto-antibodies against mitochondrial proteins. These include primary biliary cirrhosis (pyruvate
dehydrogenase complex), dilated cardiomyopathy (adenine nucleotide porter) and Leber's heriditary optic
neuropathy (LHON).

LHON is the most curious, because the antigen displayed on the plasmalemma is derived from a
mitochondrially-encoded subunit of NADH dehydrogenase (complex 1). LHON is closely related to
multiple sclerosis, and is the most common cause of blindness in otherwise healthy young men. It mainly
affects young adult males, but it is maternally transmitted and female relatives are often carriers. It can be
distinguished from an X-linked recessive condition, because male patients are normally unable to transmit
the mutation to their children.

The permeability transition: Under conditions of extreme stress (e.g. pathological calcium ion
concentrations, free radical mediated oxygen damage) mitochondria undergo an autocatalytic collapse,
associated with the loss of the normal membrane potential and a complete failure of ATP production. The
transition is irreversible and is normally a prelude to cell death. It is routinely prevented during the
laboratory isolation of mitochondria by including a calcium ion chelator (EDTA or EGTA) in the isotonic
preparation medium.

The transition involves the incorporation of subunits from the adenine nucleotide porter, and other proteins
derived from the outer membrane, into a large pore which allows unrestricted access of small ions to the
mitochondrial interior. Pore formation is favoured by atractyloside and inhibited by bongkrekic acid, and
can also be blocked by the fungal toxin cyclosporin A which binds to a protein called cyclophillin in the
mitochondrial matrix. [Cyclosporin A is a cyclic peptide which is widely used as an immuno - suppressant
after transplant surgery, but this may be unconnected with its effects on mitochondria.]

Apoptosis: The mitochondrial permeability transition is believed to be involved in the suicidal process of
apoptosis, or programmed cell death. This elaborate self-destruction cascade is responsible for the
programmed loss of cells during tissue differentiation, the self-destruction of tumours and virally - infected
cells, and the unwanted cell damage which follows loss of tissue perfusion in cardiovascular disease. It is
proposed that mitochondria which undergo the permeability transition release a protease from the inter-
membrane space which then activates the subsequent nuclear stages of the apoptotic cascade.
Apoptosis is closely regulated at numerous points along the cascade. Key effectors include (1) the tumour
suppressor protein p53, which induces cells to undergo apoptosis when irreparable DNA damage is detected,
and (2) the proto-oncogene bcl-2 which prevents the permeability transition, suppresses apoptosis and
potentially allows the survival of damaged or cancerous cells. Bcl-2 is concentrated in the mitochondrial
outer membrane, where it is closely involved in regulating the permeability transition.

Some learning objectives

Bioenergetics has traditionally been a difficult area for students, partly because of its inherent complexity,
and partly because some of the theories were fiercely contested. This is less of problem today, but many
people still find particular difficulty with the idea of an ion gradient as a convertible form of energy, with the
resolution of the proton gradient into electrical and pH components, and with the notion that the free energy
available from a chemical reaction depends on the distance from equilibrium. Make sure that you understand
these key topics.

Read the bio-energetics chapters in your text-book, which should be the most recent edition. The relevant
chapters from Molecular Cell Biology by Darnell et al and Molecular Biology of the Cell by Alberts et al are
very good, as are Devlin, Stryer, Voet, Wood and other offerings, provided they are up to date. Revise the
sections on bioenergetics, mitochondria, electron transport and oxidative phosphorylation, and the material
on membrane transport systems. A good specialist account is Bioenergetics 2 by Nicholls & Ferguson
(Academic Press, 1992). There are multiple copies in Leeds in the Medical School Library and in the
Edward Boyle Library. Some of these copies are kept in the student loan or counter collections.

The key points are as follows:

1) Mitochondria are subcellular organelles containing the Krebs cycle, fat oxidation pathway and the
respiratory chain, which produce almost all of the 70kg of ATP used each day in the human body. They can
be purified from tissue homogenates by differential centrifugation, using non-penetrant isotonic media to
provide osmotic support. A few tissues (e.g. red blood cells, eye lens) have no mitochondria, and cannot
respire aerobically.

2) Mitochondrial respiration can be measured with an oxygen electrode. Similar equipment is used in blood
gas analysers, pollution monitoring and industrial process control. How does it work, and what are the
principal sources of error?

3) Understand precisely what is meant by the respiratory control index, P:O ratio and uncoupling. Calculate
P:O ratios for succinate, and for glutamate + malate using intact, coupled mitochondria.

4) Several of the mitochondrial components undergo spectral changes on oxidation and reduction. These
observations suggest that a series of redox carriers (arranged approximately in the order of their
oxidation/reduction potentials) transport reducing equivalents (electrons or hydrogen atoms) from substrates
to oxygen. This sequence of carriers is known as the respiratory chain.

5) Understand the use of specific inhibitors to delineate biochemical mechanisms. Appreciate the differences
between respiratory chain inhibitors, phosphorylation inhibitors and transport inhibitors using cyanide,
antimycin, rotenone, TTFA, oligomycin, mersalyl and atractyloside as examples.

6) Energy captured by the respiratory chain is not converted directly into ATP. There is instead a temporary
"clearing house" known as the high energy pool. Its chemical nature was a mystery for many years, but now
we know that it is an electrochemical gradient for hydrogen ions across the inner mitochondrial membrane.
In human mitochondria this ion gradient or proton motive force has a large electrical component and a small
pH component.

7) Understand the operation of the major transmembrane ion pumps and the ATP synthetase.
8) Lipid bilayers are exceedingly impermeable to small ions, including protons and hydroxyl ions.
Transmembrane ion gradients are another form of energy, interconvertible with chemical, electrical and
mechanical energy. The chemiosmotic mechanism is universal in living organisms and is also exploited by
chloroplasts for photosynthesis, and by bacteria.

9) Understand the mechanism of ionophores, the distinction between electroneutral and electrical carrier
mechanisms, and the importance of trans-membrane ion and potential gradients. Know the meaning of
symports, antiports and electrogenic uniports. Understand where the Nernst equation applies.

10) Appreciate the use of light scattering as a crude measure of particle size and the use of passive swelling
in isotonic ammonium salt solutions to demonstrate the principal transport sytems. Study the interactions
between the carriers and explain the effect of phosphate on malate uptake. Why does passive swelling in
sodium (but not potassium) phosphate indicate the existence of a sodium/proton anti-porter?

11) Understand why the oxidation products from individual substrates are constrained by the transport
systems in the inner mitochondrial membrane, and by the availability of other co-substrates, such as acetyl-
CoA.

12) The electroneutral uptake systems for small anions and cations produce relatively modest concentration
gradients since they exploit only the pH component from the proton motive force, but the electrical
adenine nucleotide carrier produces a large gradient for ATP since it can exploit the much larger electrical
differential across the mitochondrial inner membrane.

13) The Gibbs free energy G available from a physical process or chemical reaction depends on the
distance away from equilibrium. This means that ATP has a higher free energy in the cytosol than it does in
the mitochondria, and this is important for many cellular activities.

14) Many metabolites are unevenly distributed across the mitochondrial membrane. Energy driven
aspartate / glutamate exchange is important for keeping the cytosol highly oxidising, and preventing the
aerobic synthesis of lactic acid.

15) Mitochondria probably evolved from symbiotic bacteria. They still contain a small amount of DNA,
which codes for 70S (bacterial type) ribosomes, transfer RNAs and thirteen polypeptides. These are mostly
the intensely hydrophobic cores of the major proton pumps. Understand the mechanism whereby hundreds
of other mitochondrial proteins are imported from the cytosol. Mitochondrial DNA appears to be maternally
inherited.

16) Mitochondria are involved in many disease processes, although some defects may not be detected as a
result of heteroplasmy, or because the affected embryos are not viable. There is good evidence for auto-
immune effects, and the mitochondrial permeability transition is thought to play an important part in
apoptosis.