oxidative phosphorylation

what is substrate level phosphorylation

• Phosphorylation of ADP or some other nucleoside 5′-diphosphate coupled to dehydrogenation of an organic substrate (reactive compound)

• indepedent of the ETC/respiratory chain

• most straightforward way to make ATP

where does substrate phosphorylation happen in glycolysis process (e.g. what step)?

• 1,3-bisphosphoglycerate → 3-phosphoglycerate

• phosphoenolpyruvate → pyruvate

• phosphate is donated to ADP

where does substrate phosphorylation happen in citric acid cycle (e.g. what step)?

• succinyl CoA → succinate

• hydrolysis of phosphodiester bond between coenzyme A and succinyl group is very exothermic so can drive phosphorylation of ADP/GDP

what is oxidative phosphorylation?

• final stage of aerobic cellular respiration where cells make energy in the form of ATP

• consists of ETC and chemiosmosis

• reduced coenzymes get oxidised (linked to ATP production)

what are the two reduced coenzymes involved in oxidative phosphorylation?

NADH and FADH₂

FADH₂ is always bound to its enzyme

what are the main structural elements of the mitochondria?

• inner mitochondrial membrane (IMM)

• outer membrane

• intermembrane space (IMS)

• matrix

describe the structure of the IMM

• highly folded (cristae)

• high surface area for enzymes

• less permeable

• very selective transporters (bind specific substances)

describe the structure of the outer membrane

• highly permeable - lots of large channels that let most substances through

describe the composition of the IMS

• solution is very similar to cytosol solution (due to permeable outer membrane)

• high H⁺ concentration (low pH)

describe the composition of the matrix

• low H⁺ concentration (high pH)

• very different to IMS solution due to very selective IMS

why is the pH difference between the IMS and matrix important?

• forms a proton gradient

• proton gradient is needed for the ETC and ATP synthetase to function

what reactions (in the matrix) are the three main sources of reduced coenzymes for oxidative phosphorylation?

• fatty acid oxidation (matrix)

• amino acid oxidation (matrix)

• citric acid cycle (matrix)

reduced coenzymes from these reactions can directly take part in oxidative phosphorylation

what reaction (in the cytosol) is the main source of reduced coenzymes for oxidative phosphorylation?

• glycolysis

• there is no transporter for NADH in the IMM so this NADH molecule cannot directly take place in oxidative phosphorylation

what are shuttles?

biochemical systems that move electrons from NADH made in the cytosol across the inner mitochondrial membrane

use carrier molecules to transfer electrons across the IMM so they can participate in oxidative phosphorylation

how do shuttles work?

• use NADH to reduce a compound that can be transported into the matrix

• that compound is then used to produce NADH in the matrix by reducing NAD

what is an example of a shuttle?

malate-aspartate shuttle

outline the pathway of the malate aspartate shuttle

• oxaloacetate in the cytosol is reduced by malate dehydrogenase (using NADH) to make malate

• malate is transported in to the matrix

• another malate dehydrogenase uses malate to reduce NAD to NADH in the matrix (malate → oxaloacetate)

• oxaloacetate transported back out into the cytosol

malate aspartate shuttle diagram

what is the electron transport composed of?

3 protein complexes (all enzymes) embedded in IMM

Complex I, III and IV

what does complex I do?

catalyse transfer of electrons from NADH to coenzyme Q

• NADH --> NAD (oxidised)

• Coenzyme Q --> Coenzyme QH₂ (reduced)

what does complex III do?

Catalyses transfer of e- from Coenzyme QH₂ to cytochrome C

• Coenzyme QH₂  --> Coenzyme Q (oxidised)

• Cytochrome C --> reduced cytochrome C (reduced)

what does complex IV do?

Catalyses transfer of e- from cytochrome C to oxygen

• Reduced cytochrome C ---> cytochrome C (oxidised)

• Oxygen ---> water (reduced)

are the ETC reactions (catalysed by the complexes) endothermic or exothermic?

exothermic

what is the energy from the exothermic ETC redox reactions used for?

used to pump protons from the matrix into the IMM

energy is required to do this because the protons are being transported against their concentration gradient

what is a half-reaction?

individual oxidation or reduction part of a redox reaction

two complementary half reactions occur simultaneously and represent the specific transfer of electrons between substances

what do the oxidation and reduction half equations show respectively?

oxidation = loss of electrons / loss of hydrogen

reduction = gain of electrons / gain of hydrogen

what is a redox couple?

a pair of chemical species consisting of the oxidised and reduced forms of the same substance

(An electron donor and its corresponding oxidized form; for example, NADH and NAD+)

what is reduction potential (E₀)?

measure of a chemical species' tendency to acquire electrons and be reduced (aka likelihood of a compound to be reduced)

what does a positive reduction potential mean?

• reduced form of compound is preferred

• 'the oxidised compound has a higher potential to get reduced'

• 'reduction of oxidised compound is relatively spontaneous process'

what are the thermodynamics (ΔG, ΔH) of a half-reaction with a positive E₀?

• positive ΔG

• negative ΔH (exothermic process)

• reduction of oxidised compound is relatively spontaneous

what does a negative reduction potential mean?

• prefers oxidised form of compound

• vice versa of positive reduction potential

what does the magnitude of the difference in reduction potential (ΔE₀) mean?

• small difference in reduction potential = not much energy produced by reaction

• large difference in reduction potential = lots of energy produced (very exothermic)

how are half equations and reduction potentials linked to oxidative phosphorylation?

• reduction of oxygen to water (complex IV)

• O₂ + NADH → H₂O + NAD

• oxygen and water are a redox couple, NADH and NAD are a redox couple

what is the ΔE₀ for the reaction for the reduction of oxygen?

• large difference in reduction potentials

• very exothermic, produces lots of energy

• if NAD/FAD involved there is a transfer of TWO electrons

what is the equation for ΔG° (standard gibbs free energy) using E₀?

• n = number of electrons transferred

• F = Faraday's constant (will be given but is 96.5 kJ mol-1 V-1)

• E = difference in reduction potential

what is ΔG° for the reduction of oxygen?

-220kJ mol^-1 (lots of energy)

what are the key electron carriers (coenzymes) in the ETC?

• ubiquinone

• flavin mononucleotide (FMN)

• Iron-Sulfur (FeS) Protein

• Cytochrome

describe ubiquinone

• accepts H atoms and electrons

• can also be referred to as a hydrogen carrier

describe FMN

• accepts H atoms and electrons

• Can also be referred to as a hydrogen carrier

• Similar structure to FAD -- can be reduced to FMNH2 (found in complex 1)

describe FeS protein

• Can only accept SINGLE electron

• Contain iron ion complex with cysteine residues and sulfur atoms

describe cytochrome

• Can only accept SINGLE electron

• Protein with a haem group

how many oxygen atoms does ONE NADH reduce?

½ O₂

how many hydrogen ions are pumped out of the matrix per NADH?

10 H+

what is ATP synthase?

enzyme

c-subunits embedded in IMM, gamma stalk extending into matrix

what does ATP synthase do?

• produce ATP

• use energy flow of protons across a membrane to chemically bond ADP and inorganic phosphate

what are the main structural components of ATP synthase?

• c-subunits

• a-subunits

• beta subunits

• gamma stalk

ATP synthase diagram

how does the rotary motor of ATP synthase work?

• High concentration of protons are built up in the IMS (by pumping), creating a strong electrochemical gradient

• Protons are forced to flow back to matrix through ATP synthase

• This causes the c-subunit of ATP synthase to turn

• As the c-subunits turn they interact with the alpha and beta subunits below (interacting through the gamma stalk)

• As gamma stalk turns it causes the beta subunits to change shape

• Bind ADP and phosphate --> convert it to ATP --> release ATP

describe the structure of c-subunits

• Aspartate residue around the middle of each c-subunit

• Aspartate residues have a COOH group on the end of their R group/side chain

• These residues are protonated and have no charge - therefore easily embeds in IMM (with fatty acid chains of phospholipid bilayer/hydrophobic environment)

describe alpha subunits

• Contains proton channels (hydrophilic) that extend partway into the membrane

• One half proton channel faces an environment with a HIGH concentration of protons (IMS)

• Other half proton channel faces an environment with a LOW concentration of protons (matrix)

how do C and A subunits work together?

• when c-subunits are facing/in contact with proton channels of A-subunits they are easily able to release/accept a proton

• when proton channel is facing IMS (high [H⁺]) aspartate residues on c-subunit are more likely to accept a proton

  • COO → COOH

• when proton channel is facing matrix (low [H⁺]) aspartate residues on c-subunit are more likely to release a proton into the matrix

  • COOH → COO

what are the three different posisble conformations of beta subunits ?

loose

tight

open

• each conformation holds ADP, P_i and ATP in different ways

• each beta subunit cycles through the different conformations as the gamma stalk turns

what can the loose conformation do?

can hold ADP and P_i

allows them to be separate

what can the tight conformation do?

converts substrates to ATP

(cannot release ATP)

what can the open conformation do?

release ATP

bind ADP and Pi

describe a cycle of ATP synthase

• Aspartate residue on c-subunit has COOH group

• C-subunit is facing proton channels of A subunit

• H atom is released because matrix has low concentration of protons

• COOH group on aspartate becomes COO- group

• C-subunit (aka the aspartate residue with a COO- group) it is more likely to want to stay in contact with the A subunit and the proton channels (because they are both slightly hydrophilic/charged)

• Concentration of protons in IMS is high so proton is likely to be accepted by c-subunit

• COO- group becomes COOH group

• C-subunit is no longer charged

• C-subunit turns in order to be in contact with fatty acid chains/hydrophobic environment

• Proton gradient drives the rotation of the c-subunits which causes the gamma stalk to turn

• As gamma stalk turns it interacts with beta subunits

• Beta subunits switch between different conformations forming ATP

how many electrons are required to reduce one oxygen atom?

2 e^-

(both NADH and reduced flavoproteins provide two electrons e.g. one NADH reduces one oxygen atom)

how many protons does one NADH molecule pump out of the matrix?

10 H⁺

how many protons does one FADH₂ molecule pump out of the matrix?

6 H⁺

what is the number of protons needed for a full rotation of ATP synthase determined by?

the number of c-subunits

if there are more c-subunits, more protons required for a full rotation (one proton per subunit for a full rotation)

how many ATPs does a full rotation of ATP synthase produce?

3 ATPs

how many protons are required to import ADP/P_i and export 3 ATP

3 H⁺

what is a P:O ratio?

the number of ATP molecules produced per oxygen atom reduced in cellular respiration

how is the P:O ratio calculated? (using NADH and FADH₂ values)

number of protons pumped (for NADH/FADH₂) ➗ number of protons required per ATP

• number of protons required per ATP = H⁺ re-entering for a full rotation + H⁺ for transport of ADP, P, ATP

how much ATP can be made for every NADH oxidised? (if there are 8 c-subunits)?

what is driving the formation of ATP?

the transfer of electrons from NADH to oxygen along the ETC drives the proton gradient which drives the formation of ATP

example of coupling (in oxidative phosphorylation)?

NADH is needed to make ATP

ATP synthesis is needed for the ETC to function