Oxidative Phosphorylation In Health + Disease Wk4

Question 1

Oxidative phosphorylation overview

Answer 1

-electrons transferred through four protein complexes - to oxygen to form water
-transfer of electrons drive proton pumps establishing a proton gradient
-proton gradient drives ATP-synthase to generate ATP from ADP

Question 2

Glycolysis generates Pyruvate

Answer 2

Pyruvate feeds into TCA/Krebs cycle
NADH and FADH2 generation feeds into electron transport chain

Question 3

Electron flow through complexes is achieved via Fe-S or Haem groups

Question 4

Electron carriers in complex proteins

Answer 4

Fe based EC are found in protein complexes + cytochromes
Fe-S (lower affinity) predominate in early complexes
Haem groups (higher affinity) predominate in later complexes
Even if Fe-S centres have multiple Fe atoms, each Fe-S will only carry 1 electron at a time

Question 5

Mobile electron carriers - Ubiquinone

Answer 5

-Coenzyme Q
-hydrophobic quinone molecule, move through lipid bilayer
-carries electrons from complexes | and || to |||

Question 6

MEC - cytochrome c

Answer 6

Carries electrons from complex ||| to |V
-fe groups in Haem c group
-highly conserved through evolution
-water soluble, loosely associated with outer side of IMM
Acts as anti-oxidant

Question 7

Complex I of ETC - NADH-ubiquinone oxidoreductase

Answer 7

-L-shaped membrane bound protein
-peripheral arm responsible for electron transfer
Membrane arm responsible for proton pumping
14 core subunits (7 peripheral, 7 membrane) 30 accessory subunits

Question 8

I structure-function

Answer 8

-electrons donated from NADH to FMN
-^ transferred through 7 Fe-S clusters
-final Fe-S donates to ubiquinone
-negative charge on ubiquinone drives conformational changes in the E-channel
Moves through ‘open’ and ‘closed’ states driving proton transport through the membrane region

Question 9

Complex II - succinct dehydrogenase

Answer 9

Involved in Krebs cycle + ETC
-catalyses conversion of Succinate to Fumarate in Krebs
- passes electrons to ubiquinone for transfer to complex III

Question 10

II - electron flow

Answer 10

-four subunit protein complex
-succinate to Fumarate conversion transfers electrons to FAD to generate FADH2
-electrons are passed through 3 Fe-S groups to ubiquinone

Question 11

Complex III - Cytochrome bc1 oxidoreductase

Answer 11

-11 subunits : 3 respiratory subunits, 2 core proteins + 6 low-molecular weight proteins
-initiates proton flow via the Q cycle

Question 12

III - the Q cycle - step 1

Answer 12

Cytochrome b binds a ubiquinol and a ubiquinone

Ubiquinol releases electron to Fe-S and L-Haem, releasing 2 protons

Fe-S transfers electron to cytochrome C1. Cytochrome C1 passes electron to Cytochrome C

L-Haem transfers electron to H-Haem. H-Haem passes electron to ubiquinone generating semiquinone

The first ubiquinol (now oxidised to ubiquinone) is released

Question 13

Complex III – the Q cycle
Step 2

Answer 13

A second ubiquinol is bound

Ubiquinol releases electron to Fe-S and L-Haem, releasing 2 protons

Fe-S transfers electron to cytochrome C1. Cytochrome C1 passes electron to Cytochrome C

L-Haem transfers electron to H-Haem. H-Haem passes electron to semiquinone along with 2 protons from the matrix forming ubiquinol

The ubiquinone and ubiquinol are released

Question 14

Complex IV - cytochrome c oxidase

Answer 14

-14 subunits
- 22 heme groups, 1 cytochrome a, 1 cytochrome a3, 2 copper centres

Question 15

Complex IV – electron flow

Answer 15

• electrons passed from reduced cytochrome C via Cu and Fe centres to a binuclear centre
• ^ catalyses oxygen reduction to form water, with protons from matrix side
  -Electron transfer linked proton transport shuttles protons to the intermembrane space

Question 16

Chemi osmosis - linking H+ gradient to ATP production

Answer 16

^ describes movement of ions across a membrane following electrochemical gradient
- peter D Mitchell hypothesised that in mitochondria this movement of ions was utilised to drive the synthesis of ATP
-not accepted at the time,
-chemiosmosis essential in photosynthesis

Question 17

Complex V - ATP synthase

Answer 17

Comprised of membrane bound F0 section + a Cytoplasmic F1 section
- F0 formed of ring of C subunits, an a subunit through protons can move + a b subunit which connects to F1
-F1 is formed of a central Y subunit which connects to the F0 c ring, + ring of alternating a and b subunits

Question 18

Complex V – protons drive rotation in F0

Answer 18

Proton mov through F0 drives rotation of the g subunit which drives ATP formation in F1
Protons enter a subunit then C subunit driving rotation of c ring
-following rotation, protons are released on matrix side through a subunit
-rotation of c ring drives rotation of g subunit connecting to F1

Question 19

Complex V – g rotation drives ATP synthesis

Answer 19

In F1, g rotates against static a and b
- g rotation drives conformational changes at three actives sites in a cycle of
1. Binding of ADP and Pi (loose state)
2. Confirmation change brings ADP and Pi together = ATP (tight state)
3. Release of ATP (open state)

Question 20

The P:O ratio

Answer 20

Molecular ratio of amount of |p incorporated into ATP per oxygen
- for NADH ~2.5
-FADH2 `1.5

Question 21

Uncouplers

Answer 21

Uncoupling uncouples proton electron transport and phosphorylation reactions
Inhibit ATP synthesis without affecting respiratory chain and ATP synthase (H(+)-ATPase)

Question 22

2,4-dinitrophenol (DNP)

Answer 22

-used as diet pills + pesticide 1933 1998
-functions as protonophore
-hydrophobic, diffuses across membrane
-carries protons
-removes proton gradient

-ETC still functions but without proton gradient ATP cannot be produced via complex V
-energy is used for ETC but no ATP payout
= metabolic rate increased

Question 23

Uncoupling proteins

Answer 23

Proton transporter proteins
- localised in mitochondrial membrane
-disrupt proton gradient, uncoupling ATP synthesis from electron flow
-UCP1 thermogenin
Expressed in brown fat, controlled by GPCR
Regulated heat generation during hibernation

-UCP2 and UCP3
Expressed in tisssues incl. pancreas + hippocampus
Regulates negative feedback loops in mitochondria or ROS production

Question 24

Inhibitors of specific ETC components

Answer 24

Drugs + toxins can disrupt specific components of ECT

Question 25

Rotenone

Answer 25

Acts on complex I
-inhibits transfer of electrons from Fe-S centres to ubiquinone
-unable to pass electrons to ubiquinone leading to backup of electrons in matrix
-can cause mitochondrial damage due to ROS production
-used as pesticide
- can be used against invasive fish species

Question 26

Antimycin A

Answer 26

Blocks Qi site of complex III = blocking ubiquinol oxidation