Lecture 4

1. Lecture 4 Coupling

2. How Fuel made into ATP WORK ATP ADP H+ ATP ADP Fuels H+ NAD NADH H+ H+ CO2 H2O H/e H/e O2 H+ H+

3. Terms & Principles • Oxidative Phosphorylation • Use of the proton gradient to make ATP • Electron Transport • Use of NADH to make proton gradient • COUPLING • Interdependence of Oxidative Phosphorylation and Electron Transport • One generally cannot occur without the other • RATE OF FUEL OXIDATON IS COUPLED TO RATE OF ATP CONSUMPTION • Rate of ATP generation is very finely linked to the rate of ATP usage • Can’t burn fuels without doing work!

4. Important Rules I • ATP is made when H+ come through the F0F1ATPase • Note that ATP is made from ADP and P • BUT H+ will ONLY come in through the F0F1ATPase IF ADP is available and is being made into ATP • The movement of protons and the synthesis of ATP at the F0F1ATPase are all part of the same ‘reaction’ and can’t occur independently • The proton gradient is made as electrons/hydrogens move down the electron transport chain from NADH to oxygen • Note that the H+ that are pumped out come from the matrix and not necessarily the Hs on NADH • Indeed, the one hydride on NADH causes the pumping of 10 H+ as it goes down the chain • BUT electrons/hydrogens can ONLY move down the electron transport chain IF protons are simultaneously pumped out • The movement of H/e- and the proton movement from the matrix to the cytoplasm are part of the same ‘reaction’ and can’t occur independently

5. Important Rules II • The bigger the pressure of protons (ie, the bigger the size of the proton gradient), the harder it is to pump protons out of the matrix. • So the bigger the gradient, the slower the movement of H/e down the chain from NADH to oxygen • The carriers in the process are in limited supply • NAD – our initial carrier of Hs and electrons – rips Hs/electrons out of fuels and necessary for continued fuel oxidation • ADP – our carrier of phosphate – made into ATP • Electron and Hydrogen carriers in the ‘electron transport chain’ – will alternately collect and pass-on the H/e to each other • CoA – a carrier involved in fatty acid oxidation and the full oxidation of glucose

6. No Work • Imagine a cell is not doing any work • Obviously this never actually happens, but it’s a good example of one extreme! • If we do no work, we don’t make ADP • Remember ATP is quite stable and it will only ‘break down’ if an enzyme tells it to • If ATP is not used then no ADP is regenerated • All the adenine nucleotides in the form of ATP • ADP not available for new ATP synthesis • H+ cannot pass through F0F1ATPase • Proton gradient stays high • It’s not being dissipated • H+ cannot be pumped out since the H+ gradient is so big • H/e- won’t be able to move down the chain • Because the back pressure is so large • If H/e- cannot move down the chain, NADH cannot pass its H/e to the ETC complexes • So no regeneration of NAD – all NAD stays as NADH • And no oxygen consumption • Since there are limited amount of NAD, if no NAD is regenerated, then no carriers will be available to pick up H from fuels  so there will be no fuel oxidation

7. Doing Work • Now ATP will start to be used • ADP gets made • Now proton gradient can be dissipated • Proton pressure relieved • Pumping starts again • Hs move down the chain to oxygen • Oxygen consumption increases • NADH now re-oxidised to NAD • Availability of NAD allows fuel oxidation to occur again • Rate of fuel oxidation is matched to the rate at which the ATP is used • Rate of oxygen consumption a good indicator of the rate of fuel oxidation and energy expenditure

8. Uncoupling • Imagine if the protons didn’t have to go through the F0 channel to get back into the matrix • If there was a leak/hole in the membrane • Or if something carried the H+ across… • A short circuit! • No driving force for ATP synthesis • No back-pressure to stop H+ pumping • No restriction on H/e movement down the transport chain to oxygen • Instant regeneration of NAD from NADH • Massive fuel oxidation rate • Massive oxygen consumption • But no ATP synthesis  • So ATP levels and cell death… • Unless it can be controlled!!!!!!

9. Dinitrophenol (DNP) • Hydrophobic - can move freely across membrane • H on OH group can come on/off easily • Weak acid • When H comes off, -ve charge can be delocalized • Through nitro group • Dissipates the H+ gradient • Allows uncoupling of fuel oxidation from oxidative phosphorylation • Energy in fuels all lost as heat • Non-specific - affects every tissue

10. DNP mechanism • In matrix, DNP loses H+ • Because the pH in there is relatively high as many protons have be pumped out • Outside mitochondria, DNP picks up H+ • Because the pH in the cytoplasm is relatively low • The DNP can shuttle across the membrane quite rapidly • After H+ comes off from DNP in mitochondria, the negatively charged DNP leaves mitochondria and goes back out to pick up another H+ • First documented as affecting munitions workers • Massive weight loss and heat production • Later used as weight loss agent

11. Natural Uncoupler - Thermogenin • A.k.a. UCP-1 (Uncoupling protein-1) • Found only in brown adipose tissue • Its function is to generate heat • Especially in small mammals and hibernating animals • High in neonates, lost as we grow up • But perhaps more of it than we think! • Under hormonal control – noradrenalin binds 3-receptors on brown adipocytes to switch on thermogenin