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BIOL 3702 Lecture Outline Chapter 9: Metabolism: Energy, Release, and Conservation Fueling Processes ◆ Chemoorganotrophs differ in the types of electron acceptors they use in their various energy-yielding processes ◆ The process involving exogenous (external) acceptors in the electron transport chain is termed respiration ✳ Final electron acceptor is oxygen, the process is termed aerobic respiration ✳ Final electron acceptor is not oxygen, the process is termed anaerobic respiration ◆ The electron transport system uses the donated electron to make ATP via oxidative phosphorylation ◆ When the electron transport system is not used and endogenous (internal) acceptors are employed, the process is termed fermentation and ATP is formed via substratelevel phosphorylation Aerobic Respiration ◆ Aerobic catabolism can be divided into three different stages: ✳ Stage I - larger molecules are broken down into their constituent parts with little energy released ✳ Stage II - either an aerobic or anaerobic process whereby even simpler molecules are produced as is ATP as well as NADH and/or FADH2 ✳ Stage III - complete oxidation of molecules under aerobic conditions to form CO2 as well ATP, NADH, and FADH2 (the latter two molecules generate even more ATP through the electron transport system ◆ Some of the pathways in metabolism are amphibolic, i.e., they function both catabolically and anabolically Breakdown of Glucose ◆ Microbes use a number of pathways to catabolize glucose to pyruvate, among which include the following: ✳ Glycolysis ✳ Pentose phosphate pathway ✳ Entner-Doudoroff Pathway Page 1 of 9 BIOL 3702 Lecture Outline Chapter 9 ◆ Glycolysis (Embden-Meyerhof or Glycolytic Pathway) ✳ Most common pathway found in all major groups of microbes ✳ Functions in the absence or presence of oxygen ✳ Located in the cytoplasmic matrix of both procaryotes and eucaryotes ✳ Divided into two parts ● ● Six carbon stage - glucose (6 carbon [C6] molecule) is phosphorylated twice and converted to fructose 1,6-bisphosphate [C6] – Input of 2 ATP molecules – No energy produced Three carbon stage - catabolism of fructose 1,6-bisphosphate [C6] to two molecules of pyruvate [C3] and generate per pyruvate – One molecule of NADH – Two molecules of ATP by substrate-level phosphorylation ✳ Substrate-level phosphorylation is the synthesis of ATP by coupling ADP phosphorylation with an exergonic reaction ✳ Overview of process ● ● Six-carbon stage – ATP + Glucose [C6] = glucose-6-phosphate [C6] – Glucose-6-phosphate [C6] + ATP = fructose-1,6-bisphosphate [C6] Three-carbon stage – Fructose-1,6-bisphosphate [C6] cleaved into glyceraldehyde-3-phosphate [C3] – Glyceraldehyde-3-phosphate [C3] converted to pyruvate [C3] + 2 ATP + NADH (this process occurs twice - balance the carbon atoms!!! 2 x C3 = C6) ✳ Summary of glycolysis ● ● ● 2 pyruvate molecules 2 ATP molecules - sum total: 2 used to begin glycolysis, 4 produced by substrate-level phosphorylation 2 NADH molecules ◆ Pentose Phosphate Pathway (Hexose monophosphate pathway) ✳ May be used simultaneously with glycolysis and Enter-Doudoroff pathway ✳ Operates under either aerobic or anaerobic conditions ✳ Important in both catabolism and biosynthesis Page 2 of 9 BIOL 3702 Lecture Outline Chapter 9 ✳ Summary of pentose phosphate pathway ● ● NADPH formed serves as a source of electrons for reduction of molecules during biosynthesis Four- and five-carbon sugars are synthesized that will be used for a variety of purposes ● Intermediates are used to produce ATP ● Serves as a catabolic pathway for five-carbon sugars ● More important as an anabolic pathway than as a catabolic pathway to produce energy ◆ Entner-Doudoroff Pathway ✳ Begins the same way as the pentose phosphate pathway to produce glyceraldehyde-3-phosphate ✳ Glyceraldehyde-3-phosphate catabolized to pyruvate via the 3-carbon stage of glycolysis ✳ Total yield per glucose molecule ● One ATP molecule ● One NADH molecule ● One NADPH molecule Tricarboxylic Acid Cycle ◆ Tricarboxylic Acid Cycle (Krebs Cycle, TCA Cycle, Citric Acid Cycle) is widely distributed among microbes ◆ Comprised of five basic steps: ✳ Oxidation of pyruvate [C3] to acetyl-coenzyme A (AcCoA) [C2] with the release of CO2 and the formation of NADH ✳ AcCoA [C2] condenses with oxaloacetate (OAA) [C4] to form citric acid (citrate) [C6], the first compound of the 6-carbon stage ✳ Through a series of reactions in the six-carbon stage,citric acid [C6] loses a carbon as CO2 to form α-ketoglutarate [C5] while generating one NADH molecule ✳ In the five-carbon stage, α-ketoglutarate [C5] loses a carbon as CO2 to form succinyl coenzyme A (succinyl-CoA) [C4] while generating one NADH molecule ✳ Through a series of reactions in the four-carbon stage, succinyl-CoA [C4] is converted to OAA [C4] while generating one NADH molecule, one FADH2 molecule, and one GTP molecule (equivalent to ATP; produced via substrate-level phosphorylation) Page 3 of 9 BIOL 3702 Lecture Outline Chapter 9 ◆ Summary of TCA cycle per molecule of pyruvate ✳ 4 NADH molecules ✳ 1 FADH2 molecule ✳ 1 ATP (as GTP) molecule ✳ 3 CO2 molecules Electron Transport Chain ◆ In eucaryotes, the electron transport chain is located in the mitochondrial inner membrane ✳ Comprised of a series of electron carriers arranged into four complexes connected by coenzyme Q and cytochrome c ✳ Electrons flow from electron carrier to another ✳ Final electron acceptor is to O2 ◆ Energy that is released during these transfers is captured as ATP via the formation of proton and electrical gradients ◆ The ATP is made via the process of oxidative phosphorylation - ATP formation driven by electron transfer between carriers ✳ NADH will drive the synthesis of 3 ATPs ✳ FADH2 will drive the synthesis of 2 ATPs ◆ The same principles apply to the electron transport system in procaryotes, but the structural details differ as do physiological responses ✳ Vary in cytochrome composition ✳ Electrons can enter at several different points in the chain ✳ Chains may be branched ✳ Different branches may operate under different environmental conditions Oxidative Phosphorylation ◆ Several theories have been postulated for this mechanism of ATP synthesis ◆ Most widely accepted theory is the chemiosmotic theory ✳ During electron transport in eucaryotes, protons move outward from the mitochondrial matrix and electrons are transported inward ✳ Results in the formation of a gradient of protons and a membrane potential due to the unequal distribution of charges ✳ This unequal distribution of charges, known as the proton motive force, drives the formation of ATP when protons return to the mitochondrial matrix ✳ ATP synthesis occurs through the action of ATP synthase (F1 particle) attached to Page 4 of 9 BIOL 3702 Lecture Outline Chapter 9 the inner mitochondrial membrane ◆ In procaryotes, a similar process takes place with the F1 particle located in the plasma membrane ◆ Inhibitors of aerobic ATP synthesis fall into one of two categories: ✳ Blockers - prohibit electron transport from one carrier to another ✳ Uncouplers - permit electron transport, but disconnect the link to oxidative phosphorylation (energy is released as heat) Yield of ATP ◆ Eucaryotes theoretically generate 36-38 total ATP molecules per glucose molecule catabolized ◆ Procaryotes may produce less ATP (about 30 molecules per glucose molecule catabolized) due to less efficient electron transport chain, but still produces more energy in this manner than in anaerobic respiration Anaerobic Respiration ◆ Anaerobic respiration is an energy-yielding process that involves the use exogenous electrons acceptors other than O2 ◆ Major electron acceptors in anaerobic respiration include nitrate, sulfate, and CO2, though various metals and organic compounds also function as final electron acceptors ◆ Nitrate as an electron acceptor - - ✳ Dissimilatory nitrate reduction - nitrate (NO3 ) is reduced to nitrite (NO2 ) via the transfer of two electrons ✳ Not very efficient in the production of ATP because only two electrons are donated - ✳ NO2 is also toxic and must be eliminated - ✳ Some bacteria use the process of denitrification to convert NO3 to nitrogen gas (nontoxic) ◆ Some obligate anaerobes use CO2 (or carbonate) or sulfate as final electron acceptors ✳ Methanogens are able to reduce CO2 to methane (CH3) - - ✳ Sulfur bacteria reduce SO4 to sulfide (S2 ) or hydrogen sulfide (H2S) ◆ In summary, anaerobic respiration is less efficient than aerobic respiration in the synthesis of ATP Page 5 of 9 BIOL 3702 Lecture Outline Chapter 9 Fermentations ◆ In the absence of aerobic and anaerobic respiration, NADH is not oxidized by the electron transport chain due to the absence of an external electron acceptor ◆ NADH must still be oxidized back to NAD+ or glycolysis will stop ◆ Many microbes use pyruvate or its derivatives as the electron acceptor ◆ Various types of fermentations exist, but all have two common themes: ✳ NADH is oxidized to NAD+ ✳ Electron acceptor is often pyruvate or a derivative thereof that, when partially oxidized, can lead to ATP production ◆ Some chemoorganoheterotrophs will only produce energy via fermentation even though exogenous electron acceptors are available Carbohydrate Catabolism ◆ Microbes catabolize other types of carbohydrates than glucose ✳ Monosaccharides ✳ Disaccharides ✳ Polysaccharides ◆ Monosaccharide sugars are initially phosphorylated, then fed into glycolysis (sometimes after slight modifications) ◆ Disaccharides are first cleaved into monosaccharides by hydrolysis (addition of water across a covalent bond) or phosphorolysis (phosphate attack on covalent bond), then enter glycolysis ◆ Polysaccharides are similarly cleaved, usually via the catalysis of exogenous enzymes produced by microbes, then again fed into the glycolytic pathway ◆ Moreover, internal reserve polymers are processed into monosaccharides prior to being fed into other metabolic pathways ✳ Glycogen and starch are cleaved into one glucose molecule at a time which enters the glycolytic pathway ✳ Poly-β-hydroxybutyrate is converted into acetoacetate which is then converted to acetyl-CoA (enters the TCA cycle) Page 6 of 9 BIOL 3702 Lecture Outline Chapter 9 Lipid Catabolism ◆ Lipids, which are comprised of glycerol and fatty acids, are hydrolyzed into these components ✳ Glycerol is phosphorylated, oxidized to dihydroxyacetone phosphate, then fed into the glycolytic pathway ✳ Fatty acids are oxidized by the β-oxidation pathway into acetyl-CoA, which then feeds into glycolysis; NADH and FADH2 are also produced Protein Catabolism ◆ Some microbes produced proteases that hydrolyze proteins and peptide chains into amino acids which are further catabolized in a two step process: ✳ Deamination - removal of the amino group ✳ Transamination - transfer of the amino group to an α-keto acid acceptor (this step does not always occur) ◆ The resulting product(s) can be used by the TCA cycle or as a carbon source for making other compounds Chemolithotrophy ◆ Chemolithotrophs generate energy using electrons from inorganic nutrients ◆ Commonly used electron donors are hydrogen, reduced nitrogen compounds, reduced sulfur compounds, and ferrous iron (Fe2+) ◆ Final electron acceptor is usually O2 ◆ Three major chemolithotrophic groups: ✳ Hydrogen gas utilizers ✳ Nitrifying bacteria (oxidize ammonia to nitrite, then to nitrate) ✳ Sulfur-oxidizing bacteria ◆ The nitrifying and sulfur-oxidizing bacteria do not generate efficient yields of ATP, but do produce some NADH Photosynthesis ◆ Photosynthesis is the process of trapping light energy and converting it to chemical energy in terms of ATP and NADH (or NADPH) molecules ◆ Photosynthesis if composed of two parts: ✳ Light reactions - trapping light and making chemical energy ✳ Dark reactions - using energy to fix CO2 and make cellular constituents Page 7 of 9 BIOL 3702 Lecture Outline Chapter 9 ◆ Light reactions in eucaryotes/cyanobacteria ✳ Photosynthetic microbes trap light energy using a group of green pigments termed chlorophyll ✳ Accessory photosynthetic pigments include: ✳ ✳ ● Carotenoids (usually yellowish) ● Phycobiliproteins (Cyanobacteria and red algae) of two types: – Red (phycoerythin) – Blue (phycocyanin) in color Accessory proteins function to cover wavelengths of light which are not absorbed by chlorophylls, and transfer the energy to these molecules ● Makes photosynthesis more efficient in capturing light energy ● Helps protect the organism from harmful oxidizing affects of sunlight Chlorophylls and accessory pigments arranged in highly organized arrays that transfer all energy to a reaction-center chlorophyll involved in electron transport ✳ Organized into two photosystems: ● ● Photosystem I - absorbs energy from wavelengths of light ≥680 nm using a special chlorophyll a molecule termed P700 Photosystem II - absorbs energy from wavelengths of light ≤680 nm transferring it to a special chlorophyll molecule termed P680 ✳ The photosystems cooperate to form ATP and NADP+ by two mechanisms ● Cyclic photophosphorylation ● Non-cyclic photophosphorylation ✳ Cyclic photophosphorylation ● ● ● P700, excited by light energy, eventually donates its high-energy electron to ferredoxin Ferredoxin donates the electron through a series of electron carriers to produce ATP - this type of ATP synthesis is termed photophosphorylation Electron returns to P700 Page 8 of 9 BIOL 3702 Lecture Outline Chapter 9 ✳ Non-cyclic photophosphorylation ● ● ● ● P700, excited by light energy, eventually donates its high-energy electron to ferredoxin Ferredoxin donates the electron to reduce NADP+ to NADPH (these electrons are now not available to reduced the oxidized P700) P680, which has also been excited by light energy, donates its electrons to P700 via a series of electron carriers that also produce an ATP molecule Electrons lost from P680 are replace from the oxidation of water to O2 (oxygenic process) ✳ Photophosphorylation takes place in the thylakoid membrane of the chloroplast in eucaryotes and in similar structures in cyanobacteria ✳ ATP production occurs by the previously described chemiosmotic mechanism ◆ Light reaction in green and purple bacteria ✳ Differs significantly from that performed by eucaryotes and cyanobacteria ● Anoxygenic process - do not use water as an electron source or produce O2 ● NADH/NADPH synthesis uses quite different electron donors ● Different photosynthetic pigments (bacteriochlorophylls) are used that are more effective for particular environmental niches ✳ Absence of a photosystem II restricts these microbes to ATP production by cyclic photophos-phorylation Page 9 of 9