Oxidative Phosphorylation

Question 1

Oxidative phosphorylation definition

Answer 1

The metabolic pathway in which cells use enzymes to oxidise nutrients, thereby releasing energy which is used to produce adenosine triphosphate.

Question 2

Where does oxidative phosphorylation take place?

Answer 2

Inside the mitochondria

Question 3

What is the normal concentration of AMP in the cell?

Answer 3

5 nM

Question 4

What reactions happen during low ATP + the significance?

Answer 4

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

Question 5

Mitochondria structure

Answer 5

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

Question 6

What tissue has a lot of mitochondria + why?

Answer 6

cardiomyocyte staining shows densely packed mitochondria = 30% of cardiomyocyte cell volume

heart needs more ATP than any other organ in heart

Question 7

what percentage of total ATP is produced in the different processes?

Answer 7

5% substrate level phosphorylation in glycolysis and TCA

95% oxidative phosphorylation

Question 8

Two processes in oxidative phosphorylation

Answer 8

1. 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.
2. utilisation of proton and electrical gradient- ADP phosphorylation
   - ATP synthesis

Question 9

What did Mitchell’s theory propose?

Answer 9

1. movement of electrons drives proton pumping
2. these protons are pumped from the matrix into the inter membrane space
3. this creates an electrical and pH gradient across the highly impermeable inner membrane- called the protein motive force
4. protons then move down their gradient through the phosphorylation apparatus to drive ATP synthesis

Question 10

What is the electron transport chain?

Answer 10

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

Question 11

4 large complexes in the eukaryotic ETC

Answer 11

Complex I - NADH Q Oxidoreductase

Complex II- Succinate Q reductase

Complex III- Q cytochrome c oxidoreductase

Complex IV- Cytochrome c oxidase

Question 12

What are the 4 complexes linked by?

Answer 12

smaller, mobile intermediates

Ubiquinone linked Complexes I and II onto III

Cytochrome C links complex III onto IV

Question 13

The synthesis of ATP via the respiratory chain is the result of which two coupled processes?

Answer 13

electron transport and oxidative phosphorylation

Question 14

What acts as electron donors + why?

Answer 14

FADH2 and NADH, as they have negative redox potentials thus can act as reducing agents and readily donate electrons

Question 15

What acts as an electron acceptor + why?

Answer 15

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

Question 16

What changes along the electron transport chain? + importance

Answer 16

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

Question 17

what does the -220kj/mol power?

Answer 17

drives the pumping of protons across the inner membrane

Question 18

What else does the ETC need to transfer electrons?

Answer 18

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

Question 19

where are iron-sulphur clusters found ? + importance

Answer 19

complexes I, II, III

the fe2+ can be oxidised to Fe3+ with the release of an electron

Question 20

where are haem groups found? + importance

Answer 20

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

Question 21

where is copper found? + importance

Answer 21

complex IV

final step of the ETC

Cu+ to Cu2+ and an electron

Question 22

First stage of oxidative phosphorylation explained

Answer 22

uses NADH H+ as its substrate, which is freely diffusible in the matrix

1. 2 electrons pass from NADH through complex 1 to Flavin Mono Nucleotide FMN, reducing it to FMNH2
2. secondly, electrons from FMNH2 reduce a series of iron sulphur clusters within complex 1
3. electrons pass onto ubiquinone Q, combining with two hydrogens from the matrix, forming QH2
4. This process provides sufficient power to pump 4H+ from the matrix into the inter membranous space

Question 23

structure of NADH-coenzyme Q oxidoreductase

Answer 23

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

Question 24

second stage of oxidative phosphorylation explained

Answer 24

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

1. FADH2 physically linked to succinate dehydrogenase, forming a complex which tethers the Krebs cycle to the inner membrane (succinate Q reductase)
2. 2 electrons pass from FADH2 to a series of iron sulphur clusters
3. electrons then pass onto ubiquinone Q along with two hydrogens from the matrix, forming QH2

Question 25

structure of succinate Q reductase

Answer 25

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

Question 26

Ubiquinone structure

Answer 26

co-enzyme Q10 long hydrocarbon chain, makes it highly hydrophobic this retains Q within the inner membrane, moving within the hydrophobic phospholipid enviroment

Question 27

ubiquinone function and importance

Answer 27

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

Question 28

Third stage of oxidative phosphorylation explained

Answer 28

ubiquinol used as the substrate role of Complex III is to transfer 1 electron instead of the 2 that are entering 1. substrate QH2 arrives at Complex III and transfers 2 electrons 2. 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

Question 29

What does Complex III contain?

Answer 29

3 cytochromes- C, BL and BH an Fe-S cluster known as the Rieske protein Q cycle

Question 30

Explain 2 stages of Q cycle

Answer 30

1. 1 electron from QH2 reduces the Risk protein, and then reduces Cytochrome C1 2. The other electron is parked within Complex III 3. The second electron reduces Cytochrome B and is passed onto Q which becomes reduced to Q- which is known as the ubisemiquinone free radical 4. the ubisemiquinone free radical remains bound 5. A second molecule of QH2 then moves into the complex and again passes its first electron to a cytochrome C acceptor 6. The second electron is passed to the bound ubisemiquinone, reducing it to QH2, as it gains two protons from the mitochondrial matrix 7. The QH2 is then released from the enzyme

Question 31

How does the Q cycle add to the proton gradient?

Answer 31

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

Question 32

structure of cytochrome C

Answer 32

hydrophilic, sits on the periphery of membrane, closer to the inter membranous space contains a haem prosthetic group

Question 33

structure + function of complex IV

Answer 33

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

Question 34

explain fourth stage of electron transport chain

Answer 34

1. two cytochrome Cs arrive, each carrying one electron 2. 1st electron goes along the chain to CuB 3. 2nd electron goes along the chain to Haem a3 4. reduced copper and haem bind oxygen, which forms a peroxide bridge 5. another 2 cytochrome C arrives with 2 more electrons 6. each donates an electron to the haem A3-CuB peroxide bridge 7. addition of 2 H+ turns the peroxide bridge into 2 Oh groups 8. addition of two more hydrogen ions turns the 2 OH groups into 2 molecules of water

Question 35

How many cytochromes require to produce 2 waters?

Answer 35

4 cytochrome Cs, so 4 electrons needed for each oxygen a process of doubling up occurs at complex 4

Question 36

How many hydrogens pumped across ETC for one NADH?

Answer 36

10 H+

Question 37

How many hydrogens pumped across ETC for one FADH2?

Answer 37

6H+

Question 38

How does ATP synthesis take place?

Answer 38

1. H+ ions flow through the F0 subunit of the ATP Synthase channel 2. This drives rotation of the Y subunit by binding to aspartate residues in c subunit, which neutralises the amino acid charges enabling rotation 3. the rotation of C enables the Y subunit rotation which drives F1 subunit conformational changes 4. conformational changes of F1 drives phosphorylation of ADP

Question 39

Explain importance of F1

Answer 39

ATP synthesis reaction is called the binding change mechanism and involves the active site of beta subunit cycling between three states 1. in the open state, ADP and phosphate anger the active site 2. the protein then closes up around the molecules and binds them loosely- the loose state 3. the enzyme then changes shape again and forces the molecules together, forming the tight state, binding the newly produced ATP molecules with high affinity 4. finally the active site cycles back to the open Tate, releasing ATP and binding more ADP and Pi

Question 40

Evidence for ATP synthesis

Answer 40

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

Question 41

2 more important pumps + function

Answer 41

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

Question 42

experimental evidence adding different substrates to mitochondria

Answer 42

1. mitochondria alone consume minimal oxygen 2. mitochondria with substrate consume minimal oxgen 3. mitochondria only consume oxygen when stimulated by ADP- increased ADP drives oxidative phosphorylation, not decreased ATP 4. 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

Question 43

Thermogenesis definition

Answer 43

The process of heat production in animals, occurring mainly in brown adipose tissue in newborns and hibernating animals

Question 44

What is expressed in these tissues? + function

Answer 44

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

Question 45

What is the process of uncoupling substrate oxidation?

Answer 45

Protons are allowed to leak across the inner mitochondrial membrane and thus bypass ATP Synthase, which makes ATP energy production less efficient.

Question 46

What drug can cause uncoupling + mechanism?

Answer 46

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.

Question 47

neonate definition

Answer 47

new born baby

Question 48

importance of thermogenesis in neonates

Answer 48

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