MBC - Cell Integrity Flashcards
substrate-level phosphorylation
glycolysis pathway, TCA cycle -> ATP or ATP equivalents such as GTP can be generated directly by the action of a variety of kinases
oxidative phosphorylation
bulk of cellular ATP is generated within the mitochondria
Mitochondria
have an outer membrane (which limits the size of the organelle) and an inner membrane (folds that project inward called cristae), the reactions of oxidative phosphorylation take place in the inner membrane, the Krebs Cycle reactions occur in the matrix, folds within the cristae increase the surface area upon which oxidative phosphorylation can take place
NADH and FADH2
co-enzymes, re-oxidised by molecular oxygen in mitochondria in the overall reactions 1) NADH + H+ + ½ O2 → NAD+ + H20 (ΔG = -223 kJ/mol), FADH2 + H+ + ½ O2 → FAD + H20 (ΔG = -170 kJ/mol), ΔG for ATP hydrolysis is -31 kJ/mol so the energy released from the re-oxidation of NADH and FADH2 is enough to generate several phosphoanhydride bonds, much of this energy is recovered by the components of the electron transport chain and used to synthesise ATP
Electron transport chain - components
Membrane proteins:
- Complex I (a.k.a NADH-Q oxidoreductase or NADH dehydrogenase)
- Complex II (a.k.a. Succinate-Q reductase or succinate dehydrogenase)
- Complex III (a.k.a. Q-cytochrome C oxidoreductase)
- Complex IV (a.k.a. cytochrome c oxidase)
Mobile Carriers:
- Co-enzyme Q (a.k.a. ubiquinone)
- Cytochrome C
Electron transport chain - process
Complexes I, III and IV accept electrons and in doing so a proton (H+) from the aqueous solution, as electrons pass through each of these complexe, a proton is passed or ‘pumped’ to the intermembrane space, Succinate dehydrogenase (Complex II above) is an enzyme of the TCA cycle and sits in the inner mitochondrial membrane that uses FAD as a cofactor and can communicate directly with Coenzyme Q which is also in the membrane, as electrons are passed from FADH2 to Coenzyme Q it also picks up a pair of protons, thereby regenerating FAD and forming QH2, since we have effectively bypassed complex I fewer protons are pumped to the inter membrane space, when FADH2 is reoxidised compared with NADH, protons flowing back into the matrix via ATP synthase are used to generate ATP so fewer ATP molecules are generated from the reoxidation of FADH2 compared to NADH, electrons donated by other FADH2 molecules such as those generated in the glycerol phosphate shuttle and the β-oxidation pathway all bypass complex I in this fashion
Redox reactions govern oxidative phosphorylation
electron transfer reactions involving a reduced substrate (which donates electrons and therefore becomes oxidised) and an oxidised substrate (or oxidant) which accepts electrons and becomes reduced in the process, a substrate that can exist in both oxidised and reduced forms is known as a redox couple (NAD+/ NADH, FAD / FADH2, Fe3+/Fe2+, ½ O2/H2O), the ability of a redox couple to accept or donate electrons is known as the reduction potential or redox potential, standard redox potentials (E0) can be measured experimentally using the hydrogen electrode as a reference, a negative E0 implies that the redox couple has a tendency to donate electrons and therefore has more reducing power than hydrogen (ie NAD+/NADH, E0 = -0.32 V), a positive E0 implies that the redox couple has a tendency to accept electrons and therefore has more oxidising power than hydrogen (ie Fe3+/ Fe2+, E0 = +0.77 V; ½ O2 +2 H+/ H2O, E0 = +0.82 V), the transfer of electrons from one complex to another is energetically favourable and as they progress along the chain the electrons lose energy
ATP synthase
multimeric enzyme consisting of a membrane bound part (F0) and a F1 part which projects into the matrix space, rotation of the enzyme drives transitions states with altering affinities for ATP and ADP, as a consequence conformational energy flows from the catalytic subunit into the bound ADP and Pi to promote the formation of ATP (chemical energy), direction of proton flow dictates ATP Synthesis v ATP Hydrolysis (depending on the direction of the flow of protons through the ATP synthase the complex can either generate ATP or consume it)
The oxygen electrode
device that measures the oxygen concentration in a solution housed within a small chamber, base of the chamber is formed by a teflon membrane permeable to oxygen and a compartment containing two electrodes, a Platinum (Pt) cathode and a silver (Ag) anode underneath, a small voltage of around 0.6 volts is applied between the anode (+) and cathode (-), oxygen diffuses through the teflon membrane and is reduced to water at the platinum cathode (O2 + 4H+ + 4e- → 2 H2O), the circuit is completed at the silver anode, which is slowly oxidised to AgCl by the KCl electrolyte (4 Ag0 + 4Cl- → AgCl + 4e-), the resulting current is therefore proportional to the oxygen concentration in the sample chamber, we can use the oxygen electrode to dissect various components of the electron transport chain, the first step is to prepare a suspension of mitochondria from a tissue and place them into the chamber of the oxygen electrode, if the oxygen consumption of the suspension is then monitored for a set time period, the effects of various substrates and inhibitors on the electron transport chain can be determined
ATP consumption
A sedentary male of around 70 kg (154 lbs) requires about 8,400 kJ for a day’s worth of activity which equates to 83 kg of ATP, each of these ATP molecules has a lifespan of between 1-5 mins, humans possess only about 250 g of ATP at any given moment with each ATP molecule recycled approximately 300 times per day, any interruption to the process of oxidative phosphorylation /ATP synthesis, means that a cell rapidly becomes depleted of ATP and is likely to die, the most common cause of a failure of oxidative phosphorylation is simply a lack of oxygen (ie hypoxia (diminished), anoxia (total)), depending on the cell type and their metabolic requirements, death will be within a few minutes (neurons) or a few hours (muscle), uptake of oxygen by mitochondria is controlled by the components of ATP production: Inorganic phosphate (Pi) and ADP, this is known as respiratory control and allows the body to adapt oxygen consumption to actual energy requirements
Metabolic poisons
molecules that interfere with either the flow of electrons along the ETC or the flow of protons through ATP synthase
- cyanide (CN-) and azide (N3-) bind with high affinity to the ferric (Fe3+) form of the haem group in the cytochrome oxidase complex blocking the final step of the ETC
- malonate closely resembles succinate and acts as a competitive inhibitor of succinate dehydrogenase and slows down the flow of electrons from succinate to ubiquinone by inhibiting the oxidation of succinate to fumarate
- Rotenone is a isoflavone found in the roots and seeds of some plants, it inhibits the transfer of electrons from complex I to ubiquinone
- Oligomycin is an antibiotic produced by Streptomyces that inhibits oxidative phosphorylation by binding to the ‘stalk’ of ATP synthase and blocking the flow of protons through the enzyme
- Dinitrophenol (DNP) is a proton ionophore which can shuttle protons across the inner mitochondrial membranes
Dinitrophenol
Dinitrophenol can induce weight loss by transporting protons across the mitochondrial membrane uncoupling oxidative phosphorylation from ATP production and markedly increasing the metabolic rate and body temperature, use of it in slimming drug was abandoned in 1937, still used in pesticides and food dye
