BENG0004 Ox Phos and ETC PDF Lecture Notes

BENG0004 Biochemistry and Molecular Biology Oxidative phosphorylation and the electron transport chain Dr. James Allen Lecture 20 Aims Introduction to chemiosmotic theory What is it Why is it important Oxidative Phosphoryl...

BENG0004 Biochemistry and Molecular Biology Oxidative phosphorylation and the electron transport chain Dr. James Allen Lecture 20 Aims Introduction to chemiosmotic theory What is it Why is it important Oxidative Phosphorylation The electron transport chain Complex I Fe-S centres Coenzyme Q Complex II Complex III Complex IV ATP synthase What is Oxidative Phosphorylation? Oxidative Relating to the process of being oxidised Phosphorylation Process of adding a phosphoryl The addition of phosphoryl groups (ADP +Pi →ATP) using an oxidation reaction Important in mammalian mitochondria to generate a useable source of energy for cellular reactions. Early Life – problem with energy, why is oxidative phosphorylation important CO2 abundant, O2 scarce Useful hydrocarbons scarce There are only 6 known ways biology can turn CO2 into hydrocarbons Some examples: Calvin cycle Used by all plants Requires additional energy from light Early Life – problem with energy Wood–Ljungdahl pathway (aka reductive Acetyl-CoA pathway) Shown here taking CO2 and fixing it to generate pyruvate Doesn’t require an input of additional energy (e.g. sunlight in the Calvin cycle) Early Life – problem with energy Wood–Ljungdahl pathway (aka reductive Acetyl-CoA pathway) Wood-ljungdahl uses 1 ATP and provides enough energy to generate 1.5 ATP. Problem? Early Life – problem with energy Wood–Ljungdahl pathway (aka reductive Acetyl-CoA pathway) Only way that doesn’t require an input of energy (e.g. sunlight in the Calvin cycle) Wood-ljungdahl uses 1 ATP and provides enough energy to generate 1.5 ATP. Problem? Need to store this energy for half an ATP until there is enough to make a full one  Chemiosmotic Theory In 1961 the British biochemist Peter Mitchell published a hypothesis paper describing his ideas on the chemiosmotic theory. Peter was awarded the Nobel prize in 1978 for discovering how all cells store the energy from catabolic reactions and then use it to make ATP. His work showed that the energy was stored as a membrane potential (a proton gradient). To make this membrane potential protons have to be actively pumped across the membrane and this requires energy. The energy comes from catabolism. https://www.nature.com/scitable/topicpage/why -are-cells-powered-by-proton-gradients- 14373960/ Energy in mammals - revision Eukaryotic cells primarily convert carbon to useable energy in mitochondria Pyruvate from glycolysis is transported to the mitochondrial matrix Acetyl-CoA then enters TCA cycle TCA cycle What is the output of the TCA cycle? High energy reduced compounds CO2 Single ATP Now the cells have a reduced compound to provide energy for processes. What next? TCA cycle – Energy storage problem NADH and FADH are not the most convenient energy sources for cellular reactions Two issues with NADH and FADH2 production: The transfer of electrons from NADH to O2: NADH, FADH2 and ATP are not ΔG°′= -52.5 kcal/mol for each pair of electrons interchangeable transferred Would need specific enzymes for each energy Same problem as reductive Acetyl-CoA source for each reaction pathway Cellular reactions often need far less hydrolysis of ATP to ADP plus phosphate (Pi): ΔG°′= -7.3 kcal/mol This amount is much more useful to cellular reactions TCA cycle – Energy storage problem Better to convert high energy reduced compounds: To something more widely accepted as an energy donor To something with a more appropriate energy potential But how to match the free energies to avoid wastage? How do you easily convert with minimal energy wastage What are the best ways to store/use energy? Mitochondria – a chemical battery A bit of bonus calculations How many ATP molecules does an average human body need to generate per second? A bit of bonus calculations There is only about 0.1 mole of ATP in the Human body. The majority of ATP is not usually synthesised de novo, (from scratch) but is generated from ADP. Thus, at any given time, the total amount of ATP + ADP remains fairly constant. The energy used by human cells requires the hydrolysis of 100 to 150 moles of ATP daily which is around 50 to 75 kg. Typically, a human will use up their body weight of ATP over the course of the day. This means that each ATP molecule is recycled 1000 to 1500 times during a single day (100 / 0.1 = 1000). ATP cannot be stored; hence its consumption closely follows its synthesis. You roughly consume your own body weight in ATP everyday, obviously influenced by your level of activity. A marathon runner can synthesise dozens of kilos of ATP per hour. If we assume you weigh 50 kilos, that makes 50kg of ATP. The molecular weight of ATP is 507.18 g/mol, 50,000g/507.18g/mol = 98.6 mol. 1 mol is roughly 6 x 1023 molecules so that makes 5.9 x 1025 molecules per day. Per second that would make 5.9 x 1025/24 x 3600= 6.8 x 1020 molecules. Since you have about 1013 cells in your body that makes about 68 million ATPs per cell per second, roughly 2 million glucose molecules are needed per second for this. The electron transport chain How to set up a membrane potential? Proton motive force (PMF) The movement of ions across the sets up two forces: Diffusion, caused by a concentration gradient – particles diffuse from higher concentration to lower Electrostatic force, caused by electrical potential gradient - cations - - - - - - - - - - (e.g. H+) diffuse from the positive (P) + + + + + +++ + side of a gradient to the negative (N) + + + + + ++ + + side. These two gradients taken together can be expressed as an electrochemical gradient. PMF is the potential energy stored as this electrochemical gradient The electron transport chain The NADH and FADH2 generated in the catabolic reactions of the cell are reoxidised at the electron transport chain. This is a series of large protein complexes with electron donors and acceptors, Embedded in the inner mitochondrial membrane Electrons from NADH and FADH2 are passed between them down a potential energy hill and at three places protons are pumped across the membrane. The electron transport chain There are 4 enzyme complexes in the electron transport chain. For each NADH oxidised, protons are pumped out via the first, third and fourth complexes. This generates a PMF. At the final step oxygen is reduced by the cytochrome oxidase (complex IV) forming water. This is the sole oxygen requiring step in respiration. ATP Synthase also called the F1F0 ATPase uses this PMF to generate ATP. The electron transport chain Complex I Complex I - NADH:ubiquinone oxidoreductase We have high energy source of NADH from TCA cycle (and fatty acid catabolism) Need to convert to PMF Complex I is large 46 subunit protein How do proteins transfer electrons over long distances? Annu. Rev. Biochem. 2013.82:551-575 https://www.annualreviews.org/doi/pdf/10.1146/annurev-biochem- 070511-103700 Complex I NADH binds to Flavin Mono- nucleotide First electron acceptor Forming reduced flavin`: FMNH2 The electron accepting ring of FMN is identical to FAD Passes electrons one at a time to ‘Fe-S clusters’ Complex I – Fe-S clusters Many different Fe-S clusters used as electron carriers in ETC All share same overall goal, to shuttle electrons between different environments Different cluster orientation and different local environment (residues etc) contribute to different redox potential Allows for subtle changes in reactivity with free electrons Allows for subtle and successive changes in protein architecture Complex I - Ubiquinone Complex I contains pocket for Coenzyme Q binding Q pool inside the membrane is used to transfer electrons and their energy to further complexes in the E.T.C. Hydrophobic Diffuses rapidly in membrane Keeps electrons in membrane but allows them to freely diffuse to further complexes Q = “coenzyme Q” = “ubiquinone” Complex I - Ubiquinone Q = “coenzyme Q” = “ubiquinone” Reduces to ubiquinol via a semiquinone intermediate Accepts single electron at a time from Fe-S clusters Therefore reduced in a two step process QH2 = “reduced coenzyme Q” = “ubiquinol” Electron pumping Two electrons from NADH to ubiquinone causes 4 protons to be pumped out of the matrix In addition to the two protons take by ubiquinone to form ubiquinol All add to PMF The electron transport chain Complex II Complex II – succinate dehydrogenase Same enzyme that is present in the TCA cycle FADH2 doesn’t leave the complex, in fact transfers its electrons directly to Fe-S These in turn generate more QH2 for the Q pool Complex II does not pump ions Consequently FADH2 has less contribution to ATP formation The electron transport chain Complex III Complex III – Q - Cytochrome c oxidoreductase A Cytochrome bc1 protein Homodimeric, 11 distinct polypeptide chains Effectively transports 2 H+ to IMS Contains 2Fe-2S cluster Rieske center Unusual histidine coordination Alters the chemistry, stabilising the reduced form This allows it to better accept QH2 electrons Contains 3 cytochromes: Cytochromes bL and bH (low/high affinity) Cytochrome c Berg, Jeremy, M., Tymoczko, J.L., Gatto, G.J., and Stryer, L. (2015). Biochemistry 8th Edition. Cytochromes Cytochromes are redox-active proteins containing a heme cofactor Involved in electron transfer Some cytochromes used to perform really difficult chemistry E.g. P450s Same heme cofactor involved in blood cells Unlike in haemoglobin, iron alternates between 2+ and 3+ oxidation states. This means it is a single electron carrier As opposed to QH2 Complex III – The Q cycle Complex III has complex system of redox reactions 2 Ubiquinol oxidised to form 2 Ubiquinone 1 Ubiquinone is reduced to form 1 Ubiquinol 2 cytochrome c reduced 6 protons in total contribute to gradient Complex III – The Q cycle First QH2 binds at site Q0 1 electron passes to Fe-S and then to cytochrome c 2 reduced cytochrome c generated per 2 Ubiquinol cycle Proton is released to IMS Second electron passes to cyctochrome bL and on to a Ubiquinone to form a semiquinone Second proton released into IMS 1 proton taken from matrix per electron Process is repeated to form full Ubiquinol back in membrane Removing 2 protons from the matrix Cytochrome C Small (104 amino acid) heme containing protein In intermembrane space in mitochondria It shuttles electrons from Complex III to Complex IV. It can bind to a special lipid, cardiolipin, in the inner mitochondrial membrane and is anchored but mobile. Carries the electron from Complex III to Complex IV Cytochrome c is either loosely bound to membrane surface via electrostatic forces or tightly bound via a lipid called cardiolipin. Side view of the heme Cutaway view of the heme in cytochrome c The importance of cardiolipin Cardiolipin carries 4 long acyl chains each with a double bond Constitutes about 20% of the inner mitochondrial membrane. This is the only place it is found in mammalian cells. It is also found in bacterial membranes. Cytochrome c can bind to one of the acyl chains and this means it floats on the surface of the inner mitochondrial membrane. Cardiolipin Cytochrome c Cardiolipin in the inner mitochondrial membrane links Complex III and Complex IV into a supramolecular assembly with cytochrome c molecules Cardiolipin may also act as a proton trap keeping the protons Complex III Complex IV localised near all the electron transfer complexes and the ATP Synthase Also plays a role in apoptosis and response to ROS The electron transport chain Complex IV Complex IV – Cytochrome c oxidase Last of the 3 proton pumping complexes Catalyses transfer of electrons from reduced cytochrome c to molecular oxygen 13 polypeptide chains, 2 heme groups, 3 copper ions 2 interesting Copper ion centres CuA/CuA CuB Transfers a total of 4 electrons to O2, to reduce to 2H2O Complex IV electron transfer CuA/CuA accepts 2 electrons from 2 cytochrome c One goes to CuB and the other to heme a3 Oxygen binds between these Two protons also added to each oxygen atom from the matrix Adding to gradient Further excess energy pumps protons across membrane 2 cycles for each O2 molecule Complex IV Inhibited by cyanide. CN- binds where O2 binds between the Fe in the heme in cytochrome a3 and the Cu. Carbon monoxide, azide (N3) and hydrogen sulphide also inhibit Complex IV at this position. Release of ROS also an issue for mitochondria The electron transport chain - ATP synthase The final stage – ATP Synthase Also called the F1F0 ATPase (and even Gamma subunit Complex V) F1 It is a large enzyme complex of 17 proteins. PMF is used to switch protein conformations and drive ATP synthesis Consists of F0 pore domain and F1 nucleotide binding domain Rotates at about 6,000 rpm Stalk rotates 3 protons traversing the channel in the F0 give enough energy for one molecule of ATP to be F0 made. Example of intricate molecular machine using PMF to generate mechanical force which in turn generates chemical reactive energy to store as ATP Excess of protons ATP synthase F0 10 c subunits arranged in a ring in the membrane These rotate using the force of the proton gradient Protons move between interface of a subunit and c subunit ring Central stalk is attached to middle of c ring Proton binding moves central stalk inside the F1 subunits Stock et al. Science 1999 DOI: 10.1126/science.286.5445.1700 ATP synthase F1 Each binding site consists of one ab subunit 3 ab subunits surround central g stalk g stalk rotation causes switching of each ab subunits between 3 different states ATP bound, ADP + Pi bound, ATP release Oxidative phosphorylation Electrons carry energy derived from catabolism through the ETC Generates a proton motive force by moving protons out of matrix Electrons eventually used to reduce molecular oxygen PMF used to generate ATP, a more useful form of energy for cells