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L13 – Mitochondira; introduction, energy metabolism, chemiosmotic
coupling, respiratory chain, ATP synthase
Mitochondria and energy requirements
• Humans require a continuous supply of energy to survive, equivalent to a required
power supply of around 100 watts
...

• The majority of the ATP is regenerated in our mitochondria
...

• Their shapes are very varied, even within the same cell type in different conditions
...

• There are two membranes – outer and inner
...

• The inner membrane is highly invaginated to form cristae
...
The respiratory chain and ATP synthase machinery are located in the
inner mitochondrial membrane and provide most of the energy required for cellular
function in the form of ATP, which is exported into the cytoplasm
...

• The mitochondrial DNA are maternally inherited
...

• Mitochondria still retain many bacterial qualities, for example, the ribosome are
bacterial ribosomes, so inhibitors that are specific to bacterial ribosomes would not
affect the normal eukaryotic ones but will inhibit mitochondrial ribosomes
...

• The majority of the ATP used by eukaryotic cells is formed in mitochondria
...
g
...

•
•
•

Sugars are metabolised by glycolysis to form 2 molecules of pyruvate and 2 NADH
molecules in the cytoplasm
...

Its acetyl group is attached to carbon dioxide to form acetyl CoA (for every glucose
molecule, 2 acetyl CoA is produced in this reaction) with formation of NADH (for
every glucose molecule, 2 NADH is produced in this reaction)
...

More NADH is produced (6 molecules for every glucose)
...

Matrix NADH is oxidized by NADH dehydrogenase (complex I) of the respiratory
electron transfer chain
...

This gives us a net total of 10 NADH, 2 FADH2 and 2 ATP and 2 GTP (or ATP)
...

• The most active NADH shuttle, which functions in liver, kidney and heart
mitochondria is the malate aspartate shuttle
...

• The malate thus formed can pass through the inner mitochondrial membrane via the
malate-alpha-ketoglutarate transporter
...

• About 2
...

• Cytosolic oxaloacetate is regenerated by transamination reactions
...

• The IMM itself is impermeable to protons
...

• Electron transfer results in proton uptake at one side of the IMM and proton release
at the other side
...

• Protons can pass back across the IMM through the membrane part of the ATP
synthase, liberating energy that is used to drive ATP synthesis
...
This proton
motive force is universally used, including by bacteria; bacteria use their outer plasma
membrane to generate a proton motive force and they also synthesise ATP in the same
way
...
The hydrogen carriers have redox-linked pK values such that
reduction causes protonation and oxidation causes deprotonation
...
Since a proton and electron are being
transferred together, this process is electroneutral
...
In this case, an uncompensated charge is moved across the membrane, and so
generates an electric field across the membrane
...
It is this proton motive
force, which is used subsequently to drive energy-requiring processes such as ATP
synthesis
...

The respiratory chain
The respiratory chain has four central enzyme complexes:
• They act as a pathway to channel electrons from the citric acid cycle (via complexes
I and II) through to complex III and onto complex IV
...

• Electron transfer causes proton movement across the membrane
...

The enzymes evolved are highly complicated
...
Complex I oxidises NADH
and reduces ubiquinone in the membrane on the matrix side
...
The ubiquinol diffuses across the membrane and it is oxidised by complex III
releasing its protons into the intermembrane space, passing the electrons to a small
molecule on the intermembrane space called cytochrome c
...
Part of the pumping of the protons is described by the vectorial
redox loop, but there are other processes as well
...
Complex II, the smallest is only comprised of 4
proteins
...

Experiment:
Coupling can be demonstrated under conditions in which O2 and oxidizable substrates are
present, but not ADP
...
At intervals, samples are
removed and assayed for the presence of ATP
...
Conversely, if the ATP synthase was to be inhibited
by for example, oligomycin, both ATP synthase and O2 consumption does not occur
...

This is because when the flow of protons from the intermembrane space to the matrix is
blocked through ATP synthase, there is no path that exists that will return the protons to
the matrix, thereby generating a large proton gradient
...

ATP synthase
ATP synthase has two functional domains, the transmembrane protein FO and the
peripheral membrane protein F1
...
ATP synthase catalyses the
endergonic formation of ATP from ADP and Pi, accompanied by the flow of electrons from
the IMS to the matrix
...

•

•

•
•
•

•

The crystal structure of the F1 shows alternating alpha and beta subunits of which
there are 3 each arranged around a gamma subunit/stalk, which extends from FO to
F1, where it is surrounded by 10 identical subunits (in yeast; in humans, there are 8)
designated c, which form the c-ring
...

Protons then enter the second half channel, from which it is released into the
cytoplasm
...

As the c-ring rotates, the gamma stalk too rotates, inducing conformational changes
in the alpha/beta spheroid, which in turn is held stationary by the b2 and delta
subunits (connects the alpha/beta spheroid to the a subunit)
...
The subunit then changes conformation, assuming the
beta-ATP form that tightly binds and stabilizes ATP
...
The cycle continues
...

The beta subunits interact in such a way that when one assumes the B-empty
conformation, its neighbour must assume the beta-ADP form and the other
neighbour the beta-ATP form
...


The rotation of gamma subunit in F1 was observed by attaching a long, thin, fluorescent
actin polymer via a strep tag to gamma and watching it move relative to the alpha/beta
spheroid, which was immobilized on a microscopic slide by His tags
...

H+/ATP stoichiometry
• One complete rotation produces 3 ATP
...

• The rotor rotates by one c subunit per proton moved into the matrix
...
33 protons for each ATP produced in the
matrix
...
So, overall stoichiometry of H+/ATPcytoplasm is 4
...

• It is likely that the number of c subunits in the ting is different in ATP synthases in
other systems, allowing flexibility in the size of the required proton-motive force
...
That is 2
...
67 protons for every ATP transported
into the cytoplasm
...

The adenine nucleotide translocase
• The inner mitochondrial membrane is impermeable to virtually all molecules
...
This is achieved by the antiporter, adenine nucleotide translocase
...
This gives rise to a net negative charge in the matrix
...

Therefore, 13 protons are required to synthesize ATP and transport it into the cytoplasm in
yeast, whereas in mammals this number is 11
...
Haem, iron-sulphur (Fe-S) centres, copper centres,
and flavins occur commonly as prosthetic groups in many different respiratory chains
...

• They are electron transfer components in diverse metabolic processes such as
photosynthesis, nitrogen fixation and oxidative phosphorylation
...

• The basic [2Fe-2S] centre is formed by two iron ions bridged by two disulphide
bonds and coordinated to four cysteine residues
...
The oxidized
proteins contain two Fe3+ ions, whereas the reduced proteins contain one Fe3+ and
one Fe2+ ion
...


•

•

The [2Fe-2S] Rieske centre, similarly to the [2Fe-2S] centre is formed by two iron
ions bridged by two disulphide bonds, however, these irons are in turn coordinated
to 2 cysteine and 2 histidine residues
...
These potentials are modulated by the protein environment
...
g
...
g
...

• They consist of a porphyrin ring (four pyrroles joined by methane bridges; known as
protoporphyrin) with a central iron coordinated by the four nitrogens
...

• There are different types of heam
...

• Three types of heam cofactor are found in mitochondria: A, B and C; other variants
e
...
, Haem O, D can occur in bacteria
...
Haem A is present in cytochrome oxidase and has a high
redox potential
...
Haem B is
present in complex III (cytochrome bc1 complex) of the electron transport chain
...
This includes cytochrome c and complex III of the electron
transport chain
...


Flavins
• These are a family of organic compounds derived in vivo from riboflavin
...

• FMN (flavin mononucleotide) is a low potential cofactor and in mitochondria is found
only in complex I
...

• They accept 2H (2 electrons + 2 protons), but can only give the electrons one at a
time
...

Cytochrome c is a small, soluble protein in the intermembrane space
...
Its positively charged surface allows efficient docking with
negatively charged surfaces on complexes III and IV for optimal electron transfer
...

This, together with the matrix NADH2 produced by the citric acid cycle is oxidized to
NAD+ by complex I in the electron transport chain
...

• Electron transfer from NADH2 to ubiqinone is coupled to the translocation of 4
protons from the matrix to the intermembrane space
...

• Bacterial forms of complex I are approximately 500kDa in size; they typically consist
of 14 subunits
...

• X-ray crystallography has identified the positions of known redox cofactors in
complex I of the thermophilic bacterium Thermus thermophiles
...

• Centre N1a, although close to FMN, but is separate from the main electron chain;
function is not clear
...

The location of the ubiquinone binding site has not yet been determined, but is
thought to be close to N2
...
This is quite a problem because the redox cofactors are located
within the hydrophilic arm projecting into the matrix and it is hard therefore to identify
how the protons traverse the hydrophobic arm
...
It turns out that the hydrophobic arm is comprised of three homologous subunits
(Nqo12,13,14), which have structures similar to Na+/H+ antiporters
...
In addition, a long alpha-helix of Nqu12 runs back towards the
hydrophilic arm
...
Possibly, these are mediated by the structural
alterations of the long alpha helix and other links between subunits, but at present this
remains speculative
...

• Complex II is a component of the citric acid cycle, which catalyses the oxidation of
succinate to fumarate
...

Succinate + ubiquinone = fumarate + ubiquinol
• Complex II is the only complex in the electron transport chain that does not have a
proton translocation mechanism
...

• Subunits A and B are hydrophilic and project into the matrix
...

• Subunit A contains the succinate-binding site and has a covalently bound flavin
cofactor, FAD
...

These transfer electrons over a distance of 40 angstroms from FAD towards the
ubiquinone binding site
...
Four of these
helices are organized into a four-helix bundle that co-ordinates a low spin haem B
via one histidine ligand from each of subunit C and D
...
6 angstroms from the terminal [3Fe-4S] centre
...
The hydrogen on its C2 is in close
proximity to the N5 nitrogen of the FAD isoalloxazine ring
...

• Succinate is further deprotonated to give rise to FADH2
...

• The two protons lost from succinate are released into the matrix
...

Ubiquinone has a C50 side chain and an enormous hydrophobic tail, which allows it to
diffuse along in the lipid bilayer, efficiently connecting complexes I and II to complex III
...

• But there are bacterial homologs of complex III which have got only 3 subunits
...

• These subunits contain a [2Fe-2S] Rieske iron sulphur centre, heam C and 2 heam
B prosthetic groups
...

Structure
• The largest of the core subunits is cytochrome b
...
These two haems are located towards
opposite sides of the membrane
...

• The Fe-S protein also has a globular domain projecting into the intermembrane
space that contains a [2Fe-2S] Rieske iron sulphur centre
...

Mechanism
• UQH2 binds at the UQ-binding site (QO) in the iron sulphur subunit, which folds in
and locks the ubiquinol into this site where one of the hydroxide group is pointing
towards the low potential heam B (bL) in cytochrome c1 the other is pointing towards
the [2Fe-2S] Rieske centre within the iron sulphur subunit
...
The 2 protons are released into the
intermembrane space
...

A second ubiquinol is then reduced in the same way
...
UQ is then reduced in two
steps to produce UQH2 and two protons are taken up from the matrix to facilitate this
step
...
Two electrons, one from
each UQH2 reduces two cytochrome c
...
Therefore, the overall reaction is:
Ubiquinol + 2 cyt c3+ + 2 H+matrix = ubiquinone + 2 cyt c2+ + 4H+cytosol

Complex IV (Cytochrome c oxidase)
• Catalyses the transfer of electron from reduced cytochrome c produced by complex
III to molecular oxygen
...

Structure
• Mammalian complex IV is a homodimer with each half composed of 13 different
polypeptides
...
Subunits I and II catalyse the electron and proton transfer reactions
...

• Subunit II has two membrane spanning alpha helices and a large globular
hydrophilic domain that provides a docking site for cytochrome c
...

• Subunit I, the largest, is composed of 12 membrane spanning alpha helices
arranged in groups of four to form an array around three cavities
...
The heam a iron is
ligated by a histidine residue on its proximal side, but on the distal side faces CuB,
with water or hydroxide ligands between them
...
A non-catalytic magnesium ion is
also present in mammalian subunit I located pretty much halfway between CuA and
the binuclear centre
...

• Oxygen binds in the BNC and is reduced with four electrons to form water, with
consumption of four protons from the matrix
...

This increases the energy-conserving efficiency of the cytochrome c oxidase
reaction because more of the energy released by oxygen reduction is conserved in
the proton motive force across the membrane
...

The pH gradient and charge gradient form the PMF
...

Mammalian CcO – the subunits (I, II and III) present in mammalian cytochrome oxidases
and their structures are almost identical to bacterial counterparts
...
However, they are not
involved in the core electron/proton transfer reactions and probably have roles in
assembly, stability and/or control
...
the glycerol 3
phosphate then directly donates electrons to the quinone from the outer side of the inner
mitochondrial membrane, so instead of importing NADH into the mitochondria for it to be
oxidised to complex I, in some types of mitochondria this alternative dominates instead and
electrons are donated directly to the ubiquinone pool from glycolysis
...
this
means that you can have a faster throughput of glycolysis without being limited by ATP
phosphate potential
...
Another one is choline
dehydrogenase, which is involved in cholesterol metabolism
...
So even in our mitochondria, besides those 4 main
respiratory complexes, there’s a whole lot of other components plugged into the respiratory
chain as well
...
for example, they
have sevral types of NADH dehydrogenases which are small, single subunit enzymes both
facing the external medium and the matrix, which can oxidise NADH without being coupled
to proton translocation
...
These allow oxidation of
NADH without being limited by entry by too much build up of proton motive force and too
much ATP
...
There is also
another enzyme which can actually oxudise the ubiquinone pool directly and bypass
complex III and complex IV, this again is a signle subunit enzyme found in the inner side of
the mitochondrial membrane and so for some organsims, they can oxidise the NADH via
the ubiquinol oxidise with no energy coupling at all
...
There are also plants which
flower when there is snow on the ground and they use a process to heat up the snow
...
The ubiquinol oxidase is a protein stuck on the
inside of the memrbaen and it has 2 iron atoms which allows the ubiquinol to come in and
reduce these iron atoms and oxygen is reduced to water
...


One example of that typanasoma brucei which is the cause of slepping sickness which
causes 100s and 1000s of deaths a year in Africa depends entirely on a ubiquinol oxidase
pathway for cellular respiration
...
to make
a proton motive force acroos the mitochondrial membrane, it actually runs the ATP
synthase backwards
...
In many organisms including in higher plant chloroplasts,
there are additional enzymes called chlororespiratory enzymes which can act as
reductants and oxidants for the plastoquinol pool
...
If you sequence the
chloroplast genome, there is 13 of the 14 core subunits of a complex I like structure
...
We think that there is a different module
attached to it possible a ferredoxin oxidase module that plugs in a complex I like modle
which then reduces plastoquinol
...
So there is essentially photosynthetic and
chloroprespiratory electron transfer
...
It was thought that around 2
billion years ago, an ancestral cell in an anaerobic environment, when oxygenic
photosynthesis evolved and increased the amount of oxygen in the atmosphere, some
bacteria evolved respiratory chains
...
The bacterium was protected by the cell and the cell was given a lot of ATP by the
bacterium
...
In
different organisms, the number of genes transferred is variable, it is presumed in us that
there were 13 genes transferred
...
plants have retained many more
...
So a
modern plant cell has three genomes, the nuclear genome, the mitochondrial genome
which is very alrge in plants, and a chloroplast genome as well
...
When this
became incorporated into a eukaryotic cell, the plasma membrane essentially became the
inner mitochondrial membrane of the mitochondrion and the host presumable put the outer
membrane around it to protect it
...
This chemiosmotic process is a universal
process which is used by all bacterial
...
The only exception is the ubiquinol oxidase
...
In genera, the bacterial homologues appear to have identical mechanisms and
very similar structures to their mitochondrial counterparts, the difference is that the
mitochondrial enzymes have a lot of extra subunits of indetermined function
...
In contrast, bacterial ones have only 3 subunits and if you take the 3 functional
subunits in electron transfer from the mitochondrial enzyme, you can virtually overlay them
almost identically on the bacterial structure
...
This is true for all 4 of the respiratory enzyme complexes and fro the ATP
synthase itself, except for the difference in the number of c subunits
...

Some types of oxidases in bacteria – if you map the atomic cooridnates of the metal
centres in CcO, they are virtually identical (a few angstroms away from each other)
...
denitrificans uses an a and a b type heme
...
coli utilises a b heme and an
o heme but it still has the same 3 core subunits, but in this case, it is missing the copper
centre and instead it has a ubiquinol site on it
...
coli, complex I and II reduce ubiquinone
and then the ubiquinol is oxidised directly by an equivalent of cytochrome c oxidase, but is
missing complex III
...
Cytochrome bd oxidises quinol and just has 3 heme groups and
the oxygen site is between two heme groups rather than a heme and a copper centre
...
As a result, besides the variations seen in the homologues and
isozymes of respiratory enzymes found in mitochondria, they have also evolved a vast
array of diverse enzymes to allow many different materials to be used as electron sources
and electron sinks for their electron transfer chains
...
For
example, we might have NADH as the reducing substrate and if we are using oxygen, we
really have a big energy gap which is how we get protons across the membrane
...
The key thing is that
there has to be a redox potential difference between whatever the elctron source is and
whatever the electron sink is
...
For
example, some bacteria can use nitrite and make nitrate or hydrogen sulphide being

oxidised to sulphate or even hydrogen gas can be an electron source, provided that there
is a redox potential between them
...
Almost always these
enzymes donate to a quinone and even the quinone can be varied in bacteria; some of
them use ubiquinone and some use menaquinone, which has a lower redox potential and
then the quinone pool can donate to a whole load of things, such as cytochrome bo or bd,
nitrate or nitrite reductase, fumarate reductase, e
...
c
...
for example, methanol
can be oxidised by some bacteria by an enzyme that reduces cytochrome c, which then
goes to cytochrome oxidase
...
Some bacteria actually
use crude metals, ferric ion or manganese for example which act as an electron sink
...
For example, with Paraccocus denitrificans, which is a soil bacterium, one of
the closest to mitochondria actually, if it is in an aerobic environment, it has a completely
normal respiratory chain identical to our own
...
This is part of soil nitrification
...
One example is paracoccus denitrificans
...
The enzymes are spatially arranged in the
plasma membranes
...
in different bacteria, these enzymes are in different
places, some are insdie the cell and some are out and they don’t necessarily use
cytochrome c, some use other electron sources
...
Hence, it is
possible that cytochrome ocidase evolved from pre-exisitign enzymes for nitrogen
metabolism, probably after oxygenic photosynthesis has increases the atmospheric oxygen
concentration
...
The reduction of sulphate is also a final sink for some bacteria and many bacteria,
strictly in anaerobic environmetns because the sulphate utlilising enzymes are oxygen
sensitive and their often at the bottom of lakes and these lakes become black becayse they
reduce sulphate to sulphide, which then reacts with iron and makes ferrous sulphide
...

Suplahte can be reduce to sulphide in the form of hydrogen sulphide which requires 8
electrons
...
They use these as an electron source and the sulphate acts as the final sink
for the respiratory chain to make a proton motive force and therfroe ATP
...
Some bacteria use sulphur as the electron source
instead and converts it to sulphite and then sulphide
...
Howver, Co2 has a low redox potential and the enrrgy yields are low, so you
need something more reducing in order to be able to reduce Co2
...
Cattle, for example, have methanogenic bacteria in their guts and hydrogen
producing bacteria and basically, co2 in the form of bicarbanote is reduced to methan with
hydrogen gas and they are using this as an electron transfer chain to make a proton
motive force
...
It has been found that there
are a lot of hydrogen generating micorbes in our gut flora
...
The metals are ligated by
cysteine residues and have carbon monoxides and cyanide ligands attached
...

Metal eating bacteria – ferrous has a high redox potential so at normal pHs, you can’t use
oxygen to oxidise Fe2+ to Fe3+ as they are too close in potential, there is not much energy
difference between them, but at very low pH, the oxygen potential is pH dependent and
rises 60mV per pH unit and so acidophilic bacteria are able to use ferrous iron oxidation by
molecular oxygen
...
Interestingly these ones, in order
to get NADH which they cannot generate, they actually utilise complex I backwards and
forces protons to move backwards causing NAD to form NADH
...


The sodium motive circuit – there is a whole range of bacteria that instead of using a
proton motive force use a sodium motive force, so they have enzymes which pump sodium
across the membrane and they use that sodium circuit in exactly the same way as a proton
circuit to get energy
...
An example is Vibrio alginolyticus, which is a marine bacterium and is
the single most greatest casue of sea food poisoning
...
So the sodium
ions go back through the c ring, which drive the rotor, which drives ATP synthesis
...
Rather than
containing lots of iron sulphur centres and one flavin, it contains lots of flavin and one iron
sulphur centre
...

 
 



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