Oxidative Phosphorylation Flashcards
Oxidative phosphorylation definition
The metabolic pathway in which cells use enzymes to oxidise nutrients, thereby releasing energy which is used to produce adenosine triphosphate.
Where does oxidative phosphorylation take place?
Inside the mitochondria
What is the normal concentration of AMP in the cell?
5 nM
What reactions happen during low ATP + the significance?
adenylate kinase reactions, which catalyse the addition of two ADPs to form one ATP and one AMP
adenylate kinase acts as an energy scavanger
the increase in AMP concentration highlights that the cell is in energy distress
cytosolic AMP activates AMP-activated protein kinase which upregulates energy generating pathways and suppresses energy consuming ones
Mitochondria structure
contain a double membrane
outer membrane is relatively permeable, due to large, non specific porin channels
highly impermeable inner membrane with highly specific transporters
internal cristae structures to increase membrane surface area
internal matrix space
What tissue has a lot of mitochondria + why?
cardiomyocyte staining shows densely packed mitochondria = 30% of cardiomyocyte cell volume
heart needs more ATP than any other organ in heart
what percentage of total ATP is produced in the different processes?
5% substrate level phosphorylation in glycolysis and TCA
95% oxidative phosphorylation
Two processes in oxidative phosphorylation
- generation of proton gradient- electron transport
- respiration generates a proton gradient across the inner membrane by oxidising hydrogen carriers and transporting electrons. This causes a charge separation, with the matrix side of the membrane become negatively charged. - utilisation of proton and electrical gradient- ADP phosphorylation
- ATP synthesis
What did Mitchell’s theory propose?
- movement of electrons drives proton pumping
- these protons are pumped from the matrix into the inter membrane space
- this creates an electrical and pH gradient across the highly impermeable inner membrane- called the protein motive force
- protons then move down their gradient through the phosphorylation apparatus to drive ATP synthesis
What is the electron transport chain?
a series of protein complexes that transfer electrons from electron donors to electron acceptors via redox reactions and couples this electron transfer with the transfer of protons across a membrane
4 large complexes in the eukaryotic ETC
Complex I - NADH Q Oxidoreductase
Complex II- Succinate Q reductase
Complex III- Q cytochrome c oxidoreductase
Complex IV- Cytochrome c oxidase
What are the 4 complexes linked by?
smaller, mobile intermediates
Ubiquinone linked Complexes I and II onto III
Cytochrome C links complex III onto IV
The synthesis of ATP via the respiratory chain is the result of which two coupled processes?
electron transport and oxidative phosphorylation
What acts as electron donors + why?
FADH2 and NADH, as they have negative redox potentials thus can act as reducing agents and readily donate electrons
What acts as an electron acceptor + why?
oxygen acts as the terminal electron acceptor, as it has a positive redox potential therefore can act as an oxidising agent and readily accept electrons
What changes along the electron transport chain? + importance
There is an increase in electron affinity across the complexes, and they move from a more negative redox potential to a more positive redox potential, which ensures the pull of electrons in one direction
the oxidation/reduction reactions have an increased redox potential
This results in there being a large potential difference and free energy of -220kj/mol
what does the -220kj/mol power?
drives the pumping of protons across the inner membrane
What else does the ETC need to transfer electrons?
a way of facilitating single and double electron transfer:
- iron in iron sulphur clusers
- iron in ham in cytochromes
- copper
these groups allow electron movement through oxidation/reduction reactions
different complexes have different oxidation/reduction centres
the protein environment also influences redox potential
where are iron-sulphur clusters found ? + importance
complexes I, II, III
the fe2+ can be oxidised to Fe3+ with the release of an electron
where are haem groups found? + importance
haem is a porphyrin ring structure with Fe chelated at the centre
found in complexes III and IV, also cytochrome C
Fe2+ oxidised to FE3+ with the release of an electron
where is copper found? + importance
complex IV
final step of the ETC
Cu+ to Cu2+ and an electron
First stage of oxidative phosphorylation explained
uses NADH H+ as its substrate, which is freely diffusible in the matrix
- 2 electrons pass from NADH through complex 1 to Flavin Mono Nucleotide FMN, reducing it to FMNH2
- secondly, electrons from FMNH2 reduce a series of iron sulphur clusters within complex 1
- electrons pass onto ubiquinone Q, combining with two hydrogens from the matrix, forming QH2
- This process provides sufficient power to pump 4H+ from the matrix into the inter membranous space
structure of NADH-coenzyme Q oxidoreductase
46 subunits
molecular mass of about 1,000 kilo daltons
in most organisms, the complex resembles a boot with a large ball poking out from the membrane into the mitochondrion
region embedded in membrane responsible for proton pumping
region protruding is responsible for the redox reactions using NADH2, FMN and iron sulphur clusters
second stage of oxidative phosphorylation explained
uses FADH2 as its substrate, which is the molecules prosthetic group as it cannot freely flow
complex II also acts as another entry site into the electron transport chain, for electrons contained within FADH2
- FADH2 physically linked to succinate dehydrogenase, forming a complex which tethers the Krebs cycle to the inner membrane (succinate Q reductase)
- 2 electrons pass from FADH2 to a series of iron sulphur clusters
- electrons then pass onto ubiquinone Q along with two hydrogens from the matrix, forming QH2
structure of succinate Q reductase
complex II does not fully span the membrane like Complex I does- sits in part of the membrane, predominantly the matrix side
no proton pumping takes place, thus complex II does not contribute to the proton gradient
no communication between complex I and II
Ubiquinone structure
co-enzyme Q10
long hydrocarbon chain, makes it highly hydrophobic
this retains Q within the inner membrane, moving within the hydrophobic phospholipid enviroment
ubiquinone function and importance
has three redox states- fully oxidised ubiquinone, semiquinone and fully reduced ubiquinol
therefore the molecule has the capacity to act as a two electron carrier and a one electron carrier
central role in the ETC, as iron sulphur clusters can only accept one electron at a time
Third stage of oxidative phosphorylation explained
ubiquinol used as the substrate
role of Complex III is to transfer 1 electron instead of the 2 that are entering
- substrate QH2 arrives at Complex III and transfers 2 electrons
- complex IV requires electrons to be transferred to it one at a time from cytochrome C, therefore Complex III must accept 2 electrons from QH2 but only release 1- this requires the Q cycle
What does Complex III contain?
3 cytochromes- C, BL and BH
an Fe-S cluster known as the Rieske protein
Q cycle
Explain 2 stages of Q cycle
- 1 electron from QH2 reduces the Risk protein, and then reduces Cytochrome C1
- The other electron is parked within Complex III
- The second electron reduces Cytochrome B and is passed onto Q which becomes reduced to Q- which is known as the ubisemiquinone free radical
- the ubisemiquinone free radical remains bound
- A second molecule of QH2 then moves into the complex and again passes its first electron to a cytochrome C acceptor
- The second electron is passed to the bound ubisemiquinone, reducing it to QH2, as it gains two protons from the mitochondrial matrix
- The QH2 is then released from the enzyme
How does the Q cycle add to the proton gradient?
Coenzyme Q is reduced to ubiquinol on the inner side of the membrane and oxidised to ubiquinone on the other so there is a net transfer of protons across the membrane
structure of cytochrome C
hydrophilic, sits on the periphery of membrane, closer to the inter membranous space
contains a haem prosthetic group
structure + function of complex IV
cytochrome C oxidase
uses cytochrome C and oxygen as its substrates
generates water as product
oxygen is the terminal electron acceptor
electrons flow through haem copper sandwich
copper-haem-copper
oxygen forms a peroxide bridge between terminal haem and copper
explain fourth stage of electron transport chain
- two cytochrome Cs arrive, each carrying one electron
- 1st electron goes along the chain to CuB
- 2nd electron goes along the chain to Haem a3
- reduced copper and haem bind oxygen, which forms a peroxide bridge
- another 2 cytochrome C arrives with 2 more electrons
- each donates an electron to the haem A3-CuB peroxide bridge
- addition of 2 H+ turns the peroxide bridge into 2 Oh groups
- addition of two more hydrogen ions turns the 2 OH groups into 2 molecules of water
How many cytochromes require to produce 2 waters?
4 cytochrome Cs, so 4 electrons
needed for each oxygen
a process of doubling up occurs at complex 4
How many hydrogens pumped across ETC for one NADH?
10 H+
How many hydrogens pumped across ETC for one FADH2?
6H+
How does ATP synthesis take place?
- H+ ions flow through the F0 subunit of the ATP Synthase channel
- This drives rotation of the Y subunit by binding to aspartate residues in c subunit, which neutralises the amino acid charges enabling rotation
- the rotation of C enables the Y subunit rotation which drives F1 subunit conformational changes
- conformational changes of F1 drives phosphorylation of ADP
Explain importance of F1
ATP synthesis reaction is called the binding change mechanism and involves the active site of beta subunit cycling between three states
- in the open state, ADP and phosphate anger the active site
- the protein then closes up around the molecules and binds them loosely- the loose state
- the enzyme then changes shape again and forces the molecules together, forming the tight state, binding the newly produced ATP molecules with high affinity
- finally the active site cycles back to the open Tate, releasing ATP and binding more ADP and Pi
Evidence for ATP synthesis
Paul Boyer and John Walker used X-ray crystallography to examine the structures
attached fluorescent actin filaments to moving gamma subunit
when proton gradient was present the actin was shown to move- rotate in a way that represent that of the gamma subunit
2 more important pumps + function
adenine nucleotide translocase- exchange ADP3- for ATP4-, making the inside more positive, discharging an electrochemical gradient
phosphate carrier- takes a H+ away from ATP Synthase along with H2PO4-, discharging an electrochemical gradient
experimental evidence adding different substrates to mitochondria
- mitochondria alone consume minimal oxygen
- mitochondria with substrate consume minimal oxgen
- mitochondria only consume oxygen when stimulated by ADP- increased ADP drives oxidative phosphorylation, not decreased ATP
- mitochondria stop consuming oxygen when all the ADP is phosphorylated to ATP
Add chemical inhibitors to any stage of the ETC and ATP synthesis will stop
Thermogenesis definition
The process of heat production in animals, occurring mainly in brown adipose tissue in newborns and hibernating animals
What is expressed in these tissues? + function
uncoupling protein 1- thermogenin
dissipates the H+ gradient independently of ATP synthase, resulting in non shivering heat generation
diverted capacity to synthesise ATP, more used to generate heat
What is the process of uncoupling substrate oxidation?
Protons are allowed to leak across the inner mitochondrial membrane and thus bypass ATP Synthase, which makes ATP energy production less efficient.
What drug can cause uncoupling + mechanism?
2,4-dinitrophenol, a lipophilic weak acid
able to cross the inner membrane and drag a proton with it, chemically uncoupling oxidative phosphorylation
acts as a protonophoe
was used as a diet drug, as due to more of the protons being lost, less ATP was being generated from the available glucose, thus the metabolic rate increases and more fat is burned in order to compensate for the inefficiency and to meet the energy demands.
neonate definition
new born baby
importance of thermogenesis in neonates
foetuses have relatively high metabolic rates compared to that of adults.
heat is transferred to the foetus via the placenta and uterus, thus fetal temperature is maternally dependent until birth
at birth, the neonate rapidly cools in response to the relatively cold extrauterine environment
in order to survive, ATP synthesis must be uncoupled to accelerate non-shivering thermogenesis, which is coupled to the lipolysis of brown adipose tissue
