II. Energetics, Enzymes and Redox 3.3 Energy/Carbon Source Classes of Microorganisms 3.4 Bioenergetics 3.6 Electron Donors and Electron Acceptors 3.7 Energy-Rich Compounds

3.3 Energy Classes of Microorganisms Metabolism The sum total of all of the chemical reactions that occur in a cell Catabolic reactions (catabolism) Energy-releasing metabolic reactions Anabolic reactions (anabolism) Biosynthetic metabolic reactions

Broad Overview of Metabolism Prokaryotes will not make something if they can import it There are only a few key precursor molecules (but lots of ways to make them) Energy sources vary

3.3 Energy/Carbon Source Classes of Microorganisms Microorganisms have a variety of ways to conduct their metabolism Grouped into carbon source classes Heterotrophs rely on reduced carbon Autotrophs fix CO2 or reduce CO2 Grouped into energy source classes Chemotrophs use chemical energy Chemolithotrophs use inorganic compounds Chemoorganotrophs use carbon compounds Phototrophs use light energy

3.3 Energy Classes of Microorganisms Autotrophs may be chemotrophs or phototrophs with respect to energy source Phototrophic autotrophs (photoautotrophs) are familiar Cyanobacteria (Sec.14.3) Photosynthesize using chlorophyll a Fix CO2 to carbohydrate using Calvin Cycle Chemotrophic autotrophs (chemoautotrophs) are less familiar Methanogens (Sec.13.20) = Archaea; sediments, animals Reduce CO2 to CH4 with unusual enzymes for energy Utilize methanol or acetate from CH4 for carbon

3.3 Energy Classes of Microorganisms Heterotrophs may be chemotrophs or phototrophs with respect to energy source Chemotrophic heterotrophs (chemoheterotrophs) are very common Phototrophic heterotrophs (photoheterotrophs) also exist Heliobacter (Sec. 14.8) a gram + rod Carries out photosynthesis using bacteriochlorophyll g Relies on pyruvate, lactate, butyrate, acetate for carbon

3.4 Bioenergetics In any chemical reaction, some energy is lost as heat but some energy (Free energy = G): energy released that is available to do work The change in free energy during a reaction at standard conditions is referred to as ΔG0′ ΔG: free energy that occurs under actual conditions in a cell

3.6 Electron Donors and Electron Acceptors Energy from oxidation–reduction (redox) reactions is used in synthesis of energy-rich compounds (e.g., ATP) Redox reactions occur in pairs (two half reactions; Figure 3.8) Electron donor: the substance oxidized in a redox reaction (loses electrons) Electron acceptor: the substance reduced in a redox reaction (gains electrons)

3.6 Electron Donors and Electron Acceptors Half reaction donating e– Electron donor Electron acceptor Formation of water Net reaction Half reaction accepting e– Figure 3.8 Example of an oxidation–reduction reaction. Energy from oxidation–reduction (redox) reactions is used in synthesis of energy-rich compounds (e.g., ATP) Figure 3.8

3.6 Electron Donors and Electron Acceptors Reduction potential (E0′): measurement of energy transfer in redox reactions or tendency to donate electrons Expressed as volts (V) Half reactions with highly positive reduction potentials occur easily Reduced substance with a more negative E0′ donates electrons to the oxidized substance with a more positive E0′

3.6 Electron Donors and Electron Acceptors The redox tower represents the range of possible reduction potentials (Figure 3.9) The reduced substance at the top of the tower donates electrons The oxidized substance at the bottom of the tower accepts electrons The farther the electrons "drop," the greater the amount of energy released

Figure 3.9 The redox tower. Figure 3.9

3.6 Electron Donors and Electron Acceptors Redox reactions usually involve reactions between intermediates (carriers) Electron carriers are divided into two classes Prosthetic groups (attached to enzymes) Example: heme Coenzymes (diffusible) Examples: NAD+, NADP

3.7 Energy-Rich Compounds Chemical energy released in redox reactions is primarily stored in certain phosphorylated compounds (Figure 3.12) ATP; the prime energy currency Phosphoenolpyruvate Glucose 6-phosphate Chemical energy also stored in coenzyme A, a high energy sulfur compound

Anhydride bonds Ester bond Ester bond Anhydride bond Phosphoenolpyruvate Adenosine triphosphate (ATP) Glucose 6-phosphate Compound G0′kJ/mol Thioester bond Anhydride bond ΔG0′< 30kJ Phosphoenolpyruvate –51.6 1,3-Bisphosphoglycerate –52.0 Acetyl phosphate –44.8 Figure 3.12. Phosphate bonds in compounds that conserve energy in bacterial metabolism. ATP –31.8 ADP –31.8 Acetyl Coenzyme A Acetyl phosphate Acetyl-CoA –35.7 Acetyl-CoA ΔG0′< 30kJ AMP –14.2 Glucose 6-phosphate –13.8 Figure 3.12

3.7 Energy-Rich Compounds Long-term energy storage involves insoluble polymers that can be oxidized to generate ATP Examples in prokaryotes Glycogen Poly-β-hydroxybutyrate and other polyhydroxyalkanoates Elemental sulfur Examples in eukaryotes Starch Lipids (simple fats)

3.7 Energy-Rich Compounds An energy rich compound that also serves as an electron carrier Reduced NAD carries high energy electrons Provides “reducing power” for biosynthesis

Modes of ATP Synthesis Substrate level phosphorylation Transmembrane gradient (eg proton gradient or pmf) Respiration Aerobic Anaerobic Photosynthesis Anoxygenic Oxygenic

Direct transfer of high energy phosphate

Example: pyruvate kinase reaction Part of catabolic pathway called glycolysis or EMP pathway PEP is a product of another reaction Substrate level phosphorylations are common in all organisms

Metabolic pathways that use a gradient to make ATP Respiration Aerobic-use oxygen as final electron acceptor Anaerobic-use something other than oxygen as final acceptor Photosynthesis Anoxygenic-use something other than water as original electron donor Oxygenic-use water as original electron donor

Transmembrane gradient Figure 3.20 Generation of the proton motive force during aerobic respiration. Figure 3.20

MICROBES CAN USE MANY OTHER SUBSTANCES AS DONORS OR ACCEPTORS!!!!!! Transmembrane gradient Fig. 3.20 shows NADH as the electron donor And oxygen as the electron acceptor But…………. MICROBES CAN USE MANY OTHER SUBSTANCES AS DONORS OR ACCEPTORS!!!!!!

ATP Synthase aka F1F0 Synthase or ATPase Controlled entry of protons drives ATP synthesis 3-4 protons/ATP Can work in reverse

Anaerobic respiration Nitrate (NO3–), ferric iron (Fe3+), sulfate (SO42–), carbonate (CO32–), fumarate, DMSO are examples of acceptors Less energy released compared to aerobic respiration “Primitive” form of respiration?

Figure 13.39 Figure 13.39 Major forms of anaerobic respiration. Figure 13.39

Nitrate Reduction is a Good Example of Anaerobic Respiration (Ch. 13 Nitrate Reduction is a Good Example of Anaerobic Respiration (Ch.13.16 and 13.17) Nitrate Reduction = use of nitrate as electron acceptor in anaerobic respiration (nitrate to nitrite) A dissimilative process Conversion of nitrate to more reduced substances such as N2O or N2 = denitrification

Figure 13.40 (Escherichia coli) stutzeri) Nitrate reduction Nitrate reductase Nitrite reductase Denitrification (Pseudomonas stutzeri) Nitric oxide reductase Figure 13.40 Steps in the dissimilative reduction of nitrate. Gases Nitrous oxide reductase Figure 13.40

Figure 13.41 Respiration and nitrate-based anaerobic respiration.

Lithotroph: type of chemotroph that uses inorganic substance (e. g Lithotroph: type of chemotroph that uses inorganic substance (e.g. a mineral) as a source of energy: in other words as an electron donor. Anaerobic respiration: process in which electrons are transferred to a final acceptor that is not oxygen-can be organic or inorganic

Photosynthesis Uses light energy to remove an electron from an electron donor and boost it to a high energy level Photons captured by pigment molecules Chlorophylls, bacteriochlorophylls, accessory pigments such as carotenoids Absorb different wavelengths

Photosynthesis-Anoxygenic Anoxygenic photosynthesis uses an electron donor other than water H2S is a common donor Green sulfur bacteria, purple bacteria, Heliobacter, some Archaea Use a transmembrane gradient to generate ATP via cyclic photophosphorylation

Photosynthesis-Oxygenic Oxygenic photosynthesis uses water as electron donor Cyanobacteria 2 Photosystems linked by electron carriers Use a transmembrane gradient to generate ATP via either cyclic photophosphorylation or noncyclic photophosphorylation

Makes reducing power in the form of NADPH When reducing power is not needed-cyclic photophosphorylation

III. Fermentation and Respiration Overview 3.8 Glycolysis 3.9 Fermentative Diversity and the Respiratory Option 3.10 Respiration: Electron Carriers 3.11 Respiration: The Proton Motive Force 3.12 Respiration: Citric Acid and Glyoxylate Cycle 3.13 Catabolic Diversity

III. Fermentation and Respiration Overview Two key metabolic pathways Complementary Overlapping Definition depends on context-industrial, medical, biochemical

Fermentation In food science fermentation can refer to the production of foods such as yogurt In chemical engineering it can refer to the production of ethanol as an additive for gasoline In microbiology it refers to the breakdown of carbon compounds (eg glucose) to smaller compounds with a limited harvest of energy through substrate level phosphorylation and no oxygen used

Respiration In medicine or exercise science respiration refers to breathing In microbiology respiration refers to the removal of electrons from a substance and their transfer to a terminal acceptor with a significant harvest of energy through oxidative phosphorylation (redox reactions). Oxygen may be used as the terminal acceptor (aerobic respiration) or not (anaerobic respiration).

Fermentation: substrate-level phosphorylation; ATP is directly synthesized from an energy-rich intermediate Respiration: oxidative phosphorylation; ATP is produced from proton motive force formed by transport of electrons

Fermentation A basic and important process for microorganisms A sugar is the starting material and the end product depends on the species Three Stages (I) Preparation, (II) Energy Harvesting, (III) Reboot Reboot stage is very diverse!

3.8 Glycolysis Stage I and Stage II (Figure 3.14) called Glycolysis (Embden–Meyerhof-Parnas or EMP pathway): a common pathway for catabolism of glucose End product of glycolysis is pyruvate (pyruvic acid) In fermentation pyruvate is processed through Stage III It accepts electrons so is reduced

Figure 3.14 Fermentation with lactic acid produced Figure 3.14 Embden–Meyerhof–Parnas pathway (glycolysis). Figure 3.14

3.9 Fermentative Diversity and the Respiratory Option Fermentations may be classified by products formed (See Sec.13.12) in Stage III Ethanol Lactic acid (homolactic vs heterolactic) Propionic acid “Mixed acids” Butyric acid (extra ATP generated) Butanol

3.9 Fermentative Diversity and the Respiratory Option Fermentations may be classified by substrate fermented (See Sec.13.12) Usually NOT glucose Amino acids Purines and pyrimidines Aromatic compounds

3.9 Fermentative Diversity and the Respiratory Option Fermentation Helps detoxify and eliminate waste products Provides metabolites for other microbes in the environment May help to recover additional ATP Maintains redox balance (page 87 and Fig. 3.14) of NAD and NADH.