Making energy! ATP The Point is to Make ATP! 2005-2006

Chapter 9 Warm-Up Define: Glycolysis Respiration Chemiosmosis Phosphorylation Fermentation Electrochemical gradient FAD  FADH2 NAD+ NADH Explain “glycolysis”. Where does it occur? How does it “work”?

Chapter 9 Warm-up What is the chemical equation for cellular respiration? Remember OILRIG In the conversion of glucose and oxygen to CO2 and H2O, which molecule is reduced? Which is oxidized? What happens to the energy that is released in this redox reaction? NAD+ is called a(n) ________________. Its reduced form is _________.

Chapter 9 Warm-Up Why is glycolysis considered an ancient metabolic process? Where in the cell does glycolysis occur? What are the reactants and products of glycolysis? Which has more energy available: ADP or ATP? NAD+ or NADH? FAD+ or FADH2?

Chapter 9 Warm-Up Where does the Citric Acid Cycle occur in the cell? What are the main products of the CAC? How is the proton gradient generated? What is the purpose? Describe how ATP synthase works. In cellular respiration, how many ATP are generated through Substrate-level phosphorylation? Oxidative phosphorylation?

Chapter 9 Warm-Up In fermentation, how is NAD+ recycled?

What you need to know: The summary equation of cellular respiration. The difference between fermentation and cellular respiration. The role of glycolysis in oxidizing glucose to two molecules of pyruvate. The process that brings pyruvate from the cytosol into the mitochondria and introduces it into the citric acid cycle. How the process of chemiosmosis utilizes the electrons from NADH and FADH2 to produce ATP.

Chapter 9. Cellular Respiration and Fermentation

What’s the point? ATP The Point is to Make ATP!

Harvesting stored energy Energy is stored in organic molecules heterotrophs eat food (organic molecules) digest organic molecules serve as raw materials for building & fuels for energy controlled release of energy series of step-by-step enzyme-controlled reactions “burning” fuels carbohydrates, lipids, proteins, nucleic acids We eat to take in the fuels to make ATP which will then be used to help us build biomolecules and grow and move and… live! heterotrophs = “fed by others” vs. autotrophs = “self-feeders” 2005-2006

Harvesting energy stored in glucose Glucose is the model catabolism of glucose to produce ATP glucose + oxygen  carbon + water + energy dioxide C6H12O6 6O2 6CO2 6H2O ATP  + + heat respiration Movement of hydrogen atoms from glucose to water combustion = making heat energy by burning fuels in one step respiration = making ATP (& less heat) by burning fuels in many small steps ATP fuel (carbohydrates) 2005-2006 CO2 + H2O + heat CO2 + H2O + ATP (+ heat)

How do we harvest energy from fuels? Digest large molecules into smaller ones break bonds & move electrons from one molecule to another as electrons move they carry energy with them that energy is stored in another bond, released as heat, or harvested to make ATP They are called oxidation reactions because it reflects the fact that in biological systems oxygen, which attracts electrons strongly, is the most common electron acceptor. Oxidation & reduction reactions always occur together therefore they are referred to as “redox reactions”. As electrons move from one atom to another they move farther away from the nucleus of the atom and therefore are at a higher potential energy state. The reduced form of a molecule has a higher level of energy than the oxidized form of a molecule. The ability to store energy in molecules by transferring electrons to them is called reducing power, and is a basic property of living systems. loses e- gains e- oxidized reduced + – + + e- oxidation reduction e- 2005-2006

How do we move electrons in biology? Moving electrons in living systems, electrons do not move alone electrons move as part of H atom loses e- gains e- oxidized reduced + – + + H Energy is transferred from one molecule to another via redox reactions. C6H12O6 has been oxidized fully == each of the carbons (C) has been cleaved off and all of the hyrogens (H) have been stripped off & transferred to oxygen (O) — the most electronegative atom in livng systems. This converts O2 into H2O as it is reduced. The reduced form of a molecule has a higher energy state than the oxidized form. The ability of organisms to store energy in molecules by transferring electrons to them is referred to as reducing power. The reduced form of a molecule in a biological system is the molecule which has gained a H atom, hence NAD+  NADH once reduced. soon we will meet the electron carriers NAD & FADH = when they are reduced they now have energy stored in them that can be used to do work. oxidation reduction H C6H12O6 6O2 6CO2 6H2O ATP  + oxidation reduction H 2005-2006

Coupling oxidation & reduction Redox reactions in respiration release energy as breakdown molecules break C-C bonds strip off electrons from C-H bonds by removing H atoms C6H12O6  CO2 = fuel has been oxidized electrons attracted to more electronegative atoms in biology, the most electronegative atom? O2  H2O = oxygen has been reduced release energy to synthesize ATP  O2 O2 is 2 oxygen atoms both looking for electrons LIGHT FIRE ==> oxidation RELEASING ENERGY But too fast for a biological system C6H12O6 6O2 6CO2 6H2O ATP  + oxidation reduction 2005-2006

Oxidation & reduction Oxidation Reduction  removing H loss of electrons releases energy exergonic Reduction adding H gain of electrons stores energy endergonic C6H12O6 6O2 6CO2 6H2O ATP  + oxidation reduction

Overview of cellular respiration 4 metabolic stages Anaerobic respiration 1. Glycolysis respiration without O2 in cytosol Aerobic respiration respiration using O2 in mitochondria 2. Pyruvate oxidation 3. Kreb’s cycle 4. Electron transport chain C6H12O6 6O2 6CO2 6H2O ATP  + 2005-2006 (+ heat)

What’s the point? ATP The Point is to Make ATP!

Glycolysis “sugar splitting” Believed to be ancient (early prokaryotes- no O2 available) Occurs in cytosol Partially oxidizes glucose (6C) to 2 pyruvates (3C) Net gain: 2 ATP and 2 NADH Also makes 2H2O No O2 required

glucose      pyruvate Glycolysis Breaking down glucose “glyco – lysis” (splitting sugar) most ancient form of energy capture starting point for all cellular respiration inefficient generate only 2 ATP for every 1 glucose in cytosol why does that make evolutionary sense? glucose      pyruvate 6C 2x 3C Why does it make sense that this happens in the cytosol? Who evolved first?

Evolutionary perspective Life on Earth first evolved without free oxygen (O2) in atmosphere energy had to be captured from organic molecules in absence of O2 Organisms that evolved glycolysis are ancestors of all modern life all organisms still utilize glycolysis You mean, I’m related to them?! The enzymes of glycolysis are very similar among all organisms. The genes that code for them are highly conserved. They are a good measure for evolutionary studies. Compare eukaryotes, bacteria & archaea using glycolysis enzymes. Bacteria = 3.5 billion years ago glycolysis in cytosol = doesn’t require a membrane-bound organelle O2 = 2.7 billion years ago photosynthetic bacteria / proto-blue-green algae Eukaryotes = 1.5 billion years ago membrane-bound organelles! Processes that all life/organisms share: Protein synthesis Glycolysis DNA replication 2005-2006

Glycolysis Stage 1: Energy Investment Stage Cell uses ATP to phosphorylate compounds of glucose Stage 2: Energy Payoff Stage Two 3-C compounds oxidized For each glucose molecule: 2 Net ATP produced by substrate-level phosphorylation 2 molecules of NAD+  NADH

Overview glucose C-C-C-C-C-C fructose-6P P-C-C-C-C-C-C-P DHAP P-C-C-C activation energy 10 reactions convert 6C glucose to two 3C pyruvate produce 2 ATP & 2 NADH 2 ATP 2 ADP fructose-6P P-C-C-C-C-C-C-P DHAP P-C-C-C PGAL C-C-C-P 1st ATP used is like a match to light a fire… initiation energy / activation energy. Destabilizes glucose enough to split it in two 2 NAD+ 2 NADH 4 ADP 4 ATP Substrate level phosphorylation pyruvate C-C-C 2005-2006

Glycolysis summary endergonic invest some ATP exergonic harvest a little more ATP & a little NADH Glucose is a stable molecule it needs an activation energy to break it apart. phosphorylate it = Pi comes from ATP. make NADH & put it in the bank for later.

Substrate-level Phosphorylation In the last step of glycolysis, where did the P come from to make ATP? P is transferred from PEP to ADP kinase enzyme ADP  ATP I get it! The P came directly from the substrate! 2005-2006

Energy accounting of glycolysis 2 ATP 2 ADP glucose      pyruvate 6C 2x 3C 4 ADP 4 ATP All that work! And that’s all I get? And that’s how life subsisted for a billion years. Until a certain bacteria ”learned” how to metabolize O2; which was previously a poison. But now pyruvate is not the end of the process Pyruvate still has a lot of energy in it that has not been captured. It still has 3 carbons bonded together! There is still energy stored in those bonds. It can still be oxidized further. Net gain = 2 ATP some energy investment (2 ATP) small energy return (4 ATP) 1 6C sugar  2 3C sugars

Heck of a way to make a living! Is that all there is? Not a lot of energy… for 1 billon years+ this is how life on Earth survived only harvest 3.5% of energy stored in glucose slow growth, slow reproduction Heck of a way to make a living! So why does glycolysis still take place?

We can’t stop there…. Glycolysis NADH glucose + 2ADP + 2Pi + 2 NAD+  2 pyruvate + 2ATP + 2NADH Going to run out of NAD+ How is NADH recycled to NAD+? without regenerating NAD+, energy production would stop another molecule must accept H from NADH NADH 2005-2006

How is NADH recycled to NAD+? Another molecule must accept H from NADH anaerobic respiration ethanol fermentation lactic acid fermentation aerobic respiration NADH

Anaerobic ethanol fermentation Bacteria, yeast 1C 3C 2C pyruvate  ethanol + CO2 NADH NAD+ beer, wine, bread at ~12% ethanol, kills yeast Animals, some fungi Count the carbons!! Lactic acid is not a dead end like ethanol. Once you have O2 again, lactate is converted back to pyruvate by the liver and fed to the Kreb’s cycle. pyruvate  lactic acid 3C NADH NAD+ cheese, yogurt, anaerobic exercise (no O2) 2005-2006

Pyruvate is a branching point fermentation Kreb’s cycle mitochondria

Glycolysis (summary)

What’s the point? ATP The Point is to Make ATP!

Mitochondrion Structure

Cellular Respiration

Glycolysis is only the start

pyruvate    acetyl CoA + CO2 Oxidation of pyruvate Pyruvate enters mitochondria 3 step oxidation process releases 1 CO2 (count the carbons!) reduces NAD  NADH (stores energy) produces acetyl CoA Acetyl CoA enters Krebs cycle where does CO2 go? NAD NADH 3C 2C 1C pyruvate    acetyl CoA + CO2 [ 2x ] Waiting to exhale?

Pyruvate oxidized to Acetyl CoA reduction oxidation Yield = 2C sugar + CO2 + NADH

Krebs cycle

reduction of electron carriers and Count the carbons and electron carriers! pyruvate 3C 2C acetyl CoA citrate 4C 6C NADH x2 4C 6C This happens twice for each glucose molecule CO2 reduction of electron carriers and oxidation of sugars NADH 5C 4C CO2 FADH2 4C 4C NADH ATP

Whassup? So we fully oxidized glucose C6H12O6  CO2 & ended up with 4 ATP! What’s the Point? 2005-2006

What’s so important about NADH? NADH & FADH2 Krebs cycle produces large quantities of electron carriers NADH FADH2 stored energy! go to ETC What’s so important about NADH? 2005-2006

Citric Acid Cycle (Krebs) Occurs in mitochondrial matrix Acetyl CoA→ Citrate→ Net gain: 2 ATP, 6 NADH, 2 FADH2 (electron carrier) ATP produced by substrate-level phosphorylation CO2

So why the Krebs cycle? If the yield is only 2 ATP, then why? value of NADH & FADH2 electron carriers reduced molecules store energy! to be used in the Electron Transport Chain

What’s the point? ATP The Point is to Make ATP!

Cellular respiration

There’s got to be a better way! ATP accounting so far… Glycolysis  2 ATP Kreb’s cycle  2 ATP Life takes a lot of energy to run, need to extract more energy than 4 ATP! There’s got to be a better way! What’s the Point?

That sounds more like it! There is a better way! Electron Transport Chain series of molecules built into inner mitochondrial membrane mostly transport proteins transport of electrons down ETC linked to ATP synthesis yields ~34 ATP from 1 glucose! only in presence of O2 (aerobic) That sounds more like it!

Mitochondria Double membrane * form fits function! outer membrane inner membrane highly folded cristae* fluid-filled space between membranes = intermembrane space matrix central fluid-filled space * form fits function!

Electron Transport Chain

Remember the NADH? Kreb’s cycle Glycolysis 8 NADH 2 FADH2 2 NADH PGAL 2005-2006

Electron Transport Chain NADH passes electrons to ETC H cleaved off NADH & FADH2 electrons stripped from H atoms  H+ (H ions) electrons passed from one electron carrier to next in mitochondrial membrane (ETC) transport proteins in membrane pump H+ across inner membrane to intermembrane space Oxidation refers to the loss of electrons to any electron acceptor, not just to oxygen. Uses exergonic flow of electrons through ETC to pump H+ across membrane.

electrons flow downhill to O2 But what “pulls” the electrons down the ETC? Pumping H+ across membrane … what is energy to fuel that? Can’t be ATP! that would cost you what you want to make! Its like cutting off your leg to buy a new pair of shoes. :-( Flow of electrons powers pumping of H+ O2 is 2 oxygen atoms both looking for electrons electrons flow downhill to O2

Electrons flow downhill Electrons move in steps from carrier to carrier downhill to O2 each carrier more electronegative controlled oxidation controlled release of energy Electrons move from molecule to molecule until they combine with O & H ions to form H2O It’s like pumping water behind a dam -- if released, it can do work 2005-2006

Why the build up H+? ATP synthase ADP + Pi  ATP enzyme in inner membrane of mitochondria ADP + Pi  ATP only channel permeable to H+ H+ flow down concentration gradient = provides energy for ATP synthesis molecular power generator! flow like water over water wheel flowing H+ cause change in shape of ATP synthase enzyme powers bonding of Pi to ADP “proton-motive” force 2005-2006

ATP synthesis Oxidative phosphorylation Chemiosmosis couples ETC to ATP synthesis build up of H+ gradient just so H+ could flow through ATP synthase enzyme to build ATP Oxidative phosphorylation So that’s the point! 2005-2006

Summary of cellular respiration C6H12O6 6O2 6CO2 6H2O ~36 ATP  + Where did the glucose come from? Where did the O2 come from? Where did the CO2 come from? Where did the H2O come from? Where did the ATP come from? What else is produced that is not listed in this equation? Why do we breathe? Where did the glucose come from? from food eaten Where did the O2 come from? breathed in Where did the CO2 come from? oxidized carbons cleaved off of the sugars Where did the H2O come from? from O2 after it accepts electrons in ETC Where did the ATP come from? mostly from ETC What else is produced that is not listed in this equation? NAD, FAD, heat!

Taking it beyond… What is the final electron acceptor in electron transport chain? O2 So what happens if O2 unavailable? ETC backs up ATP production ceases cells run out of energy and you die! What if you have a chemical that punches holes in the inner mitochondrial membrane?

What’s the point? ATP The Point is to Make ATP!

Any Questions??

Glycolysis Fermentation Respiration O2 present Without O2 Without O2 Fermentation Respiration Keep glycolysis going by regenerating NAD+ Occurs in cytosol No oxygen needed Creates ethanol [+ CO2] or lactate 2 ATP (from glycolysis) Release E from breakdown of food with O2 Occurs in mitochondria O2 required (final electron acceptor) Produces CO2, H2O and up to 32 ATP

Various sources of fuel Carbohydrates, fats and proteins can ALL be used as fuel for cellular respiration Monomers enter glycolysis or citric acid cycle at different points

aerobic cellular respiration ENERGY aerobic cellular respiration (with O2) anaerobic (without O2) glycolysis (cytosol) mitochondria Fermentation (cytosol) pyruvate oxidation ethanol + CO2 (yeast, some bacteria) citric acid cycle substrate-level phosphorylation lactic acid (animals) ETC oxidative phosphorylation chemiosmosis