Dr Agnieszka Adamczewska L6 – Cellular respiration Summer School 2015 Images from Wikimedia Commons

Major Concepts 1.Identify the three end-product options for glycolysis, and under what conditions these end-products form 2.State in words (not chemical formulae) the overall reaction of the glycolytic pathway, understand parts that are common and different 3.Understand how the overall balance sheet for glycolysis is obtained, and show the methods of reaction “coupling” that the cell uses 4.Say where the enzymes of the glycolytic pathway are to be found in eukaryotic cells and in prokaryotic cells 5.Explain what oxidation reduction reactions are and the special role of the coenzyme NAD + /NADH

Major Concepts 6.Give the names of, and recognise the equations for, the overall reactions of, the two major parts of the cellular respiration sequence - glycolysis and tricarboxylic acid (TCA) cycle. 7.Describe how a H + pumping mechanism is coupled to a proton-driven ATP synthase. 8.State how many ATP molecules are produced per glucose molecule in the glycolytic pathway and in the whole respiratory pathway. 9.Describe where in the respiratory pathway CO 2 is released, and where O 2 is consumed.

Macromolecules store energy Energy is stored in carbon-carbon bonds e.g. glycogen or starch, fats and oils Get energy out of food through catabolic reactions Image from Campbell Biology 8e Australian Version © Pearson Education Inc.

Metabolism: chemical reactions that occur within cells Catabolism – breaking down organic matter to release energy Anabolism – using energy to produce cellular components Catabolism Complex moleculesSimple molecules Anabolism ADPATPPiPi Metabolism (L3)

ATP: energy carrier Made through metabolism of energy rich molecules. - carbohydrates are converted into glucose - lipids are processed by β-oxidation Catabolism Complex moleculesSimple molecules ADPATPPiPi

ATP: Adenosine triphosphate Hydrolysis ADP Adenosine diphosphate

Energy conversions in cells Energy from macromolecules is released by cellular respiration. Initial breakdown of macromolecules produces simple sugars, fatty acids, glycerol, and amino acids. Subsequent gradual oxidation of the fuel molecules by removal of electrons from C-C and C-H bonds releases energy: Energy conversions located in the cytosol 1. Glycolysis converts glucose to pyruvate 2. Fermentation to lactate and alcohol Energy conversions located in mitochondria in the presence of O 2 3.  -oxidation of lipids produces acetyl CoA 4. Citric acid cycle converts pyruvate to acetyl CoA and finally CO 2 5. Electron transport chain (NADH, FADH 2 > O 2 > H 2 O) drives proton pumps, proton gradient is coupled to synthesis of ATP

Energy conversion pathways Cellular respiration CYTOSOL MITOCHONDRION

Pathway described in 1930's - a major biochemical triumph - involves a number of steps one glucose (C6) two pyruvate (2 x C3) Glycolysis

Glucose (6C) 2 ATP 2 ADP 3 steps Fructose 1,6-bisphosphate (unstable) G3P (3C) NAD + NADH 2 ATP 2 ADP Pyruvate (3C) NAD + NADH 2 ATP 2 ADP Pyruvate (3C) 5 steps Mitochondria image from Wikimedia Commons glyceraldehyde 3 phosphate Cytosol

Glycolysis Energy Conversions Net yield 2 NADH 2 ATP

Glycolysis “Splitting glucose” Glucose from hydrolysis of polysaccharides (L2) enters cell via facilitated diffusion - Glucose-Na + symport (L5) Cytosol – 10 enzymes Glucose (6C) + 2 ATP + 2 NAD ADP + 2 Pi  2 Pyruvate (3C) + 4 ATP + 2 NADH Net yield: 2 ATP

NAD+ Nicotinamide Adenine Dinucleotide Electron carrier (coenzyme ) Image from Wikimedia Commons NAD + reduced to NADH by transfer of H + from food

1. Glycolysis: Processing of glucose to pyruvate a) Glucose (6C) is phosphorylated using 2 ATP and split into two molecules of glyceraldehyde 3-phosphate (3C). Total 5 steps, consuming 2 ATP b) Oxidation in another 5 steps to 2 molecules of pyruvate (3C) and production of 4 ATP (net yield =2 ATP/glucose) Pyruvate can be converted in the absence of O 2 by alcoholic fermentation to ethanol (in yeast and bacteria) or by lactate fermentation to lactate (muscle tissue) In the presence of O 2 pyruvate enters mitochondria, is converted (decarboxylated) to a 2C compound acetyl CoA (a substrate for citric acid cycle) CO 2, and NADH;

2. Fermentation Anaerobic conversion of pyruvate to alcohol or lactic acid

Alcohol fermentation REDUCTION By yeast and many bacteria. Used by humans for thousands of years in brewing, winemaking, and baking (CO 2 bubbles from baker’s yeast)

Lactic acid fermentation By certain fungi and bacteria Used in dairy industry to make cheese and yogurt A product of muscle exercise, may enhance muscle performance (pain from K + ions) REDUCTION

Fermentation No oxygen Glycolysis then fermentation Fungi, bacteria, animals  lactic acid Yeast, plants  ethanol + CO 2 Glycolysis 2ATP2ADP + 2P i 2NAD + 2NADH + 2H + Glycolysis 2ATP2ADP + 2P i 2NAD + 2NADH + 2H +

Pyruvate A key juncture in catabolism Food Chloroplast

Site of cellular respiration (energy production) Double membrane: permeable outer membrane and impermeable, folded inner membrane (cristae) containing enzymes of respiration Images from Wikimedia Commons Mitochondria (L4)

Mitochondria: number, shape, and subcellular location are highly variable

Glucose + oxygen  carbon dioxide + water + energy ATP NADH Glycolysis NADH FADH 2 Krebs cycle Oxidative phosphorylation Mitochondria image from Wikimedia Commons Fuel in, energy out

Intermediate reaction Pyruvate to Acetyl CoA CoA NAD + NADH + H + CO 2 Mitochondria image from Wikimedia Commons

Other fuel molecules Other carbohydrates (apart from glucose) Fats and proteins can also be broken down and enter pathways  -oxidation Image from Campbell Biology 8e Australian Version © Pearson Education Inc.

3.  -Oxidation of lipids  -oxidation degrades long-chain fatty acids by 2C atoms at a time Last reaction splits off acetyl CoA (energy in C-C bond) and enters Citric acid cycle NADH, FADH2, energy (electron) carriers h

NAD + and FAD reduced to NADH and FADH 2 ATP is formed 8 steps, each catalysed by different enzyme Krebs cycle FADH 2 NADH CO 2 evolved AcetylCoA (2C) Oxaloacetate (4C) Citrate (6C) NADH CO 2 5C NADH CO 2 4C 6C TCA cycle Citric acid cycle 4. Krebs cycle accept e- from intermediates ATP

2× NADH Glycolysis 6× NADH 2× FADH 2 Krebs cycle Mitochondria image from Wikimedia Commons Chemical bonds to electrons glucose CO 2

Chemical bonds to electrons 1 glucose: Glycolysis  2 pyruvate + 2 ATP + 2 NADH 2 pyruvate  2 acetyl CoA + 2 NADH + 2 CO 2 TCA cycle: 2 acetyl CoA  6 NADH + 2 FADH CO ATP 1 glucose  4 ATP + 8 NADH + 2 FADH CO 2

5. Electron transport chain (ETC) (Proton-motive force i.e. the power in movement of protons) NADH and FADH 2 Inner membrane of mitochondria Four protein complexes of acceptors Oxygen needed Mitochondria image from Wikimedia Commons

Intermembrane space Mitochondrial matrix Inner mitochondrial membrane NADH dehydrogenase bc 1 complex Cytochrome oxidase complex H+H+ C H+H+ H+H+ NAD + Q e–e– H+H+ e–e– 5. Electron transport chain FAD FADH 2 NADH Start with NADH (or FADH 2 ) as primary electron donor H2OH2O 2H + + 1/2O 2 Finish with O 2 as terminal electron acceptor H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+

Chain of redox reactions Inner mitochondrial membrane Electrons move to higher redox potentials, towards oxygen with highest electron affinity Energy released is used to pump H + from matrix to inter- membrane space 2e NADH + H + High energy Low energy Cyt a (Fe 3+ ) ½ O 2 + 2H + H2OH2O Cyt a (Fe 2+ ) Cyt c (Fe 3+ ) Cyt c (Fe 2+ ) Cyt b (Fe 3+ ) 2H + + NAD + Cyt b (Fe 2+ ) Energy released Electrons “falling” from NADH to oxygen

Intermembrane space Mitochondrial matrix Inner mitochondrial membrane NADH dehydrogenase bc 1 complex Cytochrome oxidase complex H+H+ C H+H+ H+H+ NAD + Q e–e– H+H+ e–e– Generating proton gradient FAD FADH 2 NADH Start with NADH (or FADH 2 ) as primary electron donor H2OH2O 2H + + 1/2O 2 Finish with O 2 as terminal electron acceptor H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+

Chemiosmosis couples the ETC to ATP synthesis! FADFADH 2 NAD + NADH + H + H 2 O H + + OH - ½ O 2 + 2H + H2OH2O ADP + P i ATP

complexity of 1 o, 2 o, 3 o and 4 o structure embedded in inner membrane of mitochondria highly conserved in nature ATP synthase Matrix Intermembrane space Inner membrane Image from

ATP synthase Matrix Inner membrane Image from Rotor H + binding H + ions flow down gradient and enter half channel in stator H + ions enter binding sites in rotor, changing shape of subunits so rotor spins Each H + ion makes one complete turn before being released to matrix Spinning of rotor causes internal rod to spin Turning rod activates catalytic sites in knob Catalytic knob Intermembrane space H+H+ H+H+ H+H+ H+H+ ADP + P i ATP

Glucose + oxygen  carbon dioxide + water + energy ATP NADH Glycolysis NADH FADH 2 Krebs cycle Oxidative phosphorylation Mitochondria image from Wikimedia Commons Fuel in, energy out 2x ~34x 2

Aerobic respiration = lots of energy C 6 H 12 O O 2  6 CO H 2 O + ~38 ATP Where are these ATPs from? Glycolysis = 2 ATP TCA = 2 ATP Oxidative phosphorylation (ETC + chemiosmosis) = ~34 ATP

Infoldings of the inner mitochondrial membrane (cristae) greatly increase the number of electron transport chain proteins and ATP synthase proteins Cristae increases surface area

Lack of oxygen? Need oxygen to accept final electrons If no oxygen, complex IV keeps electrons  All protein complexes keep electrons  No pumping of H + into intermembrane space  No H + gradient  No energy for ATP synthase No ATP made in ETC - ATP from glycolysis and TCA not enough Most cells cannot survive long without oxygen

Prokaryotic cell – no mitochondria Still need ATP! Image from Wikimedia Commons

Aerobic Bacteria do it too! Cell membrane Electron carrier ATP synthase complex ATP ADP + Pi H+ NADH NAD + O2O2 H2OH2O H+ Enzymes embedded in bacterial cell membrane

Anaerobic respiration Prokaryotes living in anaerobic environment - no oxygen e.g. waterlogged soils, intestines Nitrate (NO 3 - ) or sulfate (SO 4 2- ) are the terminal electron acceptors Products include CO 2, inorganic substance, ATP e.g. C 6 H 12 O KNO 3 (potassium nitrate)  6 CO H 2 O + 12 KNO 3 (potassium nitrite) + ATP

Summary Cytoplasm and mitochondria are sites of cellular respiration Glucose + oxygen  carbon dioxide + water + energy Aerobic respiration has four stages For one glucose molecule: Glycolysis = 2 ATP, TCA = 2 ATP, Oxidative phosphorylation (ETC, chemiosmosis) = ~34 ATP Chemiosmosis - electrons in ETC used to pump protons into inter-membrane space, this establishes a proton gradient across inner membrane, protons accumulate, lowers pH Redox reactions integral to ETC Fermentation and anaerobic respiration in absence of O 2

Aerobic respiration StageWhere Main starting materials Main end products GlycolysisCytoplasmGlucose Pyruvate, ATP, NADH forming Acetyl CoA Matrix of mitochondria Pyruvate Acetyl CoA, CO 2, NADH Citric acid cycle Matrix of mitochondria Acetyl CoA, H 2 O CO 2, NADH, FADH 2, ATP Oxidative phosphorylation Inner membrane mitochondria O 2, NADH, FADH 2 ATP, H 2 O

Read: Knox B, et al. (2010) Biology: an Australian perspective. - Chapter 6 Harvesting energy