Lecture 03.pdf

BIOL371: Microbiology Lecture 3 – Microbial metabolism 1 Topics of today 1. Fundamentals of metabolism 2. Catabolism: chemoorganotrophs 3. Catabolism: electron transport and metabolism Materials covered:  Chapter 3.1-3.11  Figures 3.1-3.6, 3.8-3.12, 3.14-3.22 2 1. Fundamentals of metabolis...

BIOL371: Microbiology Lecture 3 – Microbial metabolism 1 Topics of today 1. Fundamentals of metabolism 2. Catabolism: chemoorganotrophs 3. Catabolism: electron transport and metabolism Materials covered:  Chapter 3.1-3.11  Figures 3.1-3.6, 3.8-3.12, 3.14-3.22 2 1. Fundamentals of metabolism 1. 2. 3. 4. Defining the Requirements for Life Electron Transfer Reactions Calculating Changes in Free Energy Catalysis and Enzymes 3 What is metabolism  Life requires: 1) liquid water, 2) source of energy to do work, 3) source of electrons to perform biochemical reactions, and 4) nutrients to build macromolecules  Metabolism – the sum total of all chemical reactions needed for life  Catabolism – reactions used to break complex molecules to obtain energy  Anabolism – reactions used to synthesize cellular materials  Relies of electron donors directing electrons to electron acceptors  Energy is neither created nor destroyed  Cells conserve energy (for growth) by converting energy from their surroundings to a form that do the work  By generating energy rich compounds (e.g., adenosine triphosphate (ATP)) that can be released to drive cellular processes 4 Types of metabolism defined by source of energy Metabolic classes of microorganisms  Autotrophs obtain carbon from CO2  Heterotrophs obtain carbon from organic matters  Chemoorganotrophs (heterotrophic)  obtain energy and reducing power from organic matter  Chemolithotrophs (autotrophic) obtain energy and reducing power from inorganics 5 Energy conservation and electron flow  Catabolism uses exergonic (release of free energy) reaction to drive the synthesis of ATP  Anabolism uses endergonic reactions, which consume ATP, to drive the biosynthesis of cellular material  Reducing power, in the form of a reduced electron donor, is required to carry out both catabolic and anabolic reactions Energy conservation Electron flow 6 Principles of energy conservation - bioenergetics  Free energy (G) – the energy released during a chemical reaction that is available to do work, measured in kilojoules (kJ)  ΔG0’ – change in free energy during a reaction at standard conditions (pH 7, 25oC, 1 atm, 1M reactant and products)  Exergonic: reactions with –ΔG0’ , release of free energy  Endergonic: reactions with +ΔG0’ , require energy 7 Free energy  Catabolic pathways: exergonic processes that generate free energy  Free energy produced is conserved by synthesizing energy-rich molecules, e.g., ATP  ATP requires ΔG0’ = 31.8 kJ/mole  Aerobic respiration of 1 mole of glucose could produce 91 moles of ATP under standard conditions, though 38 moles actually produced; some lost as heat  Anabolic pathways: endergonic processes in cellular synthesis require energy  Energy comes from ATP  Catabolic and anabolic reactions are fundamentally linked 8 Reduction-oxidation (redox) reactions  Transfer of electrons is required for both catabolic and anabolic reactions  Electron transfer reactions are called redox reactions as it comprises two half reactions  Electrons are transferred from an electron donor in one half reaction to an electron acceptor in the other half reaction  The loss of electrons is called oxidation, hence the electron donor (commonly called energy source) is oxidized in the redox reaction  The gain of electron is called reduction, hence the electron acceptor is reduced In this example of redox reaction, the two half reactions are:  Oxidation of glucose (electron donor) to CO2  Reduction of O2 (electron acceptor) to H2O 9 Reduction potential  Catabolism depends on the directed flow of electron, from electron donor to electron acceptor  Reduction potential (E0’): affinity of substance for electrons  Redox Reactions and Reduction Potentials  Electrons cannot exist in solution  Must be transferred form one atom (or molecule) to another directly in redox reactions  Redox reactions occur in pairs, therefore are called half reactions or redox couple  By convention, in expressing the redox reaction, the reactant that is oxidized is written on the left, and the reactant that is reduced is written on the right with a forward slash in between (e.g., CO2/glucose and ½ O2/H2O) 10 The redox tower – examples of redox couples in nature  In this redox tower, redox couples are arranged from the strongest electron donors at the top to the strongest electron acceptors at the bottom.  Reduction potential (E0’): tendency to donate or accept electrons  Expressed in volts (V) compared with reference (typically H2  Electrons are negatively charged, so the reduced substance of a redox couple has a tendency to donate when the redox couple has a negative reduction potential; e.g., glucose in CO2/glucose, E0’ = –0.43 V  Oxidized substance will accept electrons when the redox couple has positive reduction potential; e.g., O2 in ½ O2/H2O  The greater the difference in reduction potentials of 11 the two half reactions, the more energy is released Electron carriers; e.g., NAD+/NADH  In cells, the transfer of electrons from one substance to another rarely takes place in a single step  Movement in electrons proceeds through consecutive reactions at different locations in the cell  Soluble electron carriers such as nicotinamide adenine dinucleotide (NAD+/NADH) needed to carry electrons  NAD+/NADH redox couple = –0.32 V  Reduction requires 2 e– and 1 H+  Reduction of NAD+ results in NADH + H+  12 NAD+/NADH cycling  NAD+/NADH are coenzymes  They are diffusible and mediate many different reactions  They and many other molecules are electron shuttles  Typically involved in catabolic reactions  NADP+/NADPH is another electron shuttle typically involved in anabolic reactions 13 Calculating changes in free energy  Calculating free energy based on the redox tower  ΔE0’ is proportional to ΔG0’  ΔG0’= nFΔE0’ where n is the number of electrons transferred and F is Faraday constant (96.5 kJ/V)  ΔG0’ can also be determined if the free energy of formation (Gf0) is known  See table 3.3 for free energy of formation of select compounds  ΔG0’ tells whether the reaction is exergonic (–ΔG0’) or endergonic (+ΔG0’) 14 Catalysis  Free-energy calculations only reveal energy change during a reaction, nothing about the rate  Consider the formation of water from hydrogen and oxygen  Favorable reaction with ΔG0’ of –237kJ  Reaction will not occur spontaneously  Breaking of the bonds of the two reactants requires energy  Energy of activation – minimum energy required to initiate a chemical reaction  Catalyst is required to overcome the energy of activation  Can increase the reaction rate by 108-1020 times the spontaneous rate 15 Catalysis and enzymes  Enzymes are biocatalysts  Typically proteins, some RNAs  Lowers activation energy, increases enzyme rate  In reaction, enzyme binds substrate, forming enzyme-substrate complex, then releases product and enzyme 16 2. Catabolism: chemoorganotrophs 1. 2. 3. 4. 5. Energy-rich compounds Glycolysis, the Citric Acid Cycle, and the Glyoxylate Cycle Principles of Fermentation Principles of Respiration: Electron Carriers Principles of Respiration: Generating a Proton Motive Force 17 Energy-rich compounds of cells  Energy is released from redox reactions, but how is energy conserved to do work  Adenosine triphosphate (ATP) the most important energy-rich phosphorylated compound  Several others have energy-rich phosphate or sulfur (coenzyme A) bonds  Cells typically use compounds with free energy of hydrolysis (∆G0’) <-30 kJ/mol as energy “currency” 18 Glycolysis pathway  Nearly universal pathway for glucose catabolism  Glucose oxidized to pyruvate: 1) preparative stage, 2) oxidative stage, 3) reductive stage  Can participate in multiple forms of catabolism (fermentation, aerobic respiration, anaerobic respiration) 19 Citric acid cycle  Pathway by which pyruvate is oxidized to CO2; for every pyruvate that enters the cycle the following are produced  1 ATP  3 CO2  4 NADH  1 FADH2 20 Other catabolic pathways of chemoorganotrophs  Glycolysis and citric acid cycle can oxidize several C4-C6 compounds; e.g., glucose, citrate, malate, fumarate, succinate  Some C2 (e.g., acetate) compounds are catabolized through glyoxylate cycle  C3 compounds are carboxylated by pyruvate carboxylase or phosphoenolpyruvate carboxylase 21 Essentials of fermentation  Fermentation of glucose involves substrate-level phosphorylation and redox balance via pyruvate reduction + excretion as waste  Fermentation is a form of anaerobic catabolism  Many fermentation products (waste) are useful for humans (e.g., beer and wine, yogurt, cheese, effect of microbiome on health) 22 Maintaining redox balance NAD+ Balanced Redox NADH NADH Intermediate Substrate ADP ATP NAD+ Fermentation product Cell growth  All fermentations must do two things:  Conserve energy – produce energy-rich compounds (e.g., ATP)  Redox balance – donate electrons back to an electron acceptor derived from original organic donor  Oxidize NADH back to NAD+  Maintain balance by dumping electrons onto some intermediates (e.g., pyruvate), generating fermentation end products (e.g., ethanol, lactic acid) 23 Respiration  Respiration: electrons transferred from reduced electron donors (e.g., glucose) to external electron acceptors (e.g., O2)  Reduced electron shuttles produced by glycolysis and citric acid cycle must be reoxidized for redox balance  Reoxidation in respiration is achieved by:  Electron transport using electron carriers  Electron transport occurs in cytoplasmic membrane (mitochondrial membrane in eukaryotes)  Forms electrochemical gradient (usually protons) that conserves energy through ATP synthesis 24 Respiration and the proton motive force: overview  The coenzyme NADH (generated from the citric acid cycle, glycolysis etc) possesses electrons with high reduction potential  Oxidation of NADH would release a large amount of energy  Electrons from NADH are transferred through a series of redox reactions localized at the cytoplasmic membrane.  As electrons flow from a more negative to a more positive reduction potential, protons (H+) are transported from the inside to the outside of the cellular membrane  Oxidative phosphorylation: synthesis of ATP at the expense of proton motive force 25 Electron carriers in electron transport  Electron carriers are arranged in membrane with increasingly more positive reduction potential  NADH dehydrogenases – active site binds NADH  Two e– and one H+ from NADH plus on H+ from cytoplasm are transferred to a flavoprotein  The regenerated NAD+ returns to participate in glycolysis  Flavoproteins contain flavin (Flavin mononucleotide (FMN) or Flavin adenine dinucleotide (FAD)) as a prosthetic group  Accepts 2 e– and 2 H+ from NADH dehydrogenase and donate one e– with the proton released into the cytoplasm 26 Cytochromes and other electron carriers  Cytochromes contain heme prosthetic groups  Many types depending on the type of heme group  Nonheme iron-sulfur proteins  Quinones – small hydrophobic redox molecules that lack a protein component Nonheme iron-sulfur protein Cytochrome Ubiquinone 27 Generation of proton motive force during aerobic respiration in Paracoccus denitrificans Electrons are flowing from a more negative to a more positive reduction potential Note: this is one wellstudied example, there are many electron transport schemes 28 Complex I: NADH dehydrogenase  Primary electron donor can enter the chain at either Complex I or Complex II  Complex I contains 14 different proteins functioning as a unit  Also called NADH dehydrogenase or NADH:quinone oxidoreductase  NADH oxidized to NAD+, ubiquinone (Q) reduced to ubiquinol (QH2) and diffuses to Complex III  Four H+ released outside – contribute to proton motive force 29 Complex II: succinate dehydrogenase  Complex II – also called succinate dehydrogenase complex  Alternate entry point for primary electron donor  2 e– from FADH2 and 2 H+ from cytoplasm transferred to ubiquinone (Q) to ubiquinol (QH2)  Less energy conserved due to lack of H+ translocation 30 Complexes III and IV: cytochromes  Complex III: cytochrome bc1 complex  Transfer e– from QH2 to cytochrome c  Pumps 2 H+ from QH2 to outside cytoplasmic membrane  Cytochrome c shuttles to Complex IV  Complex IV: cytochrome a and a3  Terminal oxidase, reduces O2 to H2O  Needs 4 e– and 4 H+ from cytoplasm  Pumps 1 H+ per electron to outside 31 Electron transport and proton release  For every 2 e– from NADH to O2, 10 H+ are transferred outside cytoplasmic membrane  4 at Complex I  4 at Complex III  2 at Complex IV  2 consumed in cytoplasm (H2O)  For every 2 e– from FADH2 to O2, 6 H+ are transferred outside membrane  4 at Complex III  2 at Complex IV  2 consumed in cytoplasm (H2O) 32 Generation of ATP from proton motive force  ATP synthase (ATPase)  Uses energy from proton motive force to form ATP  Comprises two components  F1: multiprotein complex extending to the cytoplasm that catalyzes ATP synthesis  F0: membrane-integrated proton-translocating multiprotein complex  H+ passing through F0 is coupled to the rotation of F1  The free energy captured in the rotation is coupled to ATP synthesis  ~3.3 H+ required to produce one ATP  F1/F0 complex catalyzes the reversible reaction between ATP and ADP 33 Energetics of fermentation and aerobic respiration  Respiration conserves more energy than fermentation due to oxidative phosphorylation  Lactic acid buildup in muscle cells after hard workout is the result of anaerobic catabolism Lactic acid fermentation in muscle cells ET: electron transport SLP: substrate-level phosphorylation Ox. Phos.: oxidative phosphorylation 34 Metabolic diversity and its relationship to oxygen  Fermentation uses internal electron acceptor  Respiration require external electron acceptor and generate ATP by oxidative phosphorylation  Aerobic respiration uses oxygen as electron acceptor  Anaerobic respiration uses electron acceptors other than oxygen; e.g., nitrate  Chemolithotrophs use inorganic compounds as electron donors 35