7 CH 9meta2

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Chapter 9
Metabolism: Energy Release and Conservation
An Overview of Metabolism
metabolism total of all chemical reactions occurring in cell catabolism breakdown of larger, more complex molecules into smaller, simpler ones energy is released and some is trapped and made available for work anabolism synthesis of complex molecules from simpler ones with the input of energy
Think about biodegradation..
Central Metabolism
Glucose catabolism through 1) Main glycolysis 2) Hexose monophosphate pathway 3) Tricarboxylic acid cycle Generating 1) Carbon skeleton 2) ATP and 3) NADPH required for biosynthesis.
4
Metabolic intermediates used as carbon skeletons for biosynthesis.

Carbon skeleton from Precursor for
Glucose-6-phosphate Fructose-6-phosphate Ribose-5-phosphate Erythrose-4-phosphate Triose-phosphate 3-phosphoglycerate phosphoenolpyruvate pyruvate acetyl-CoA 2-ketoglutarate succinyl-CoA oxaloacetate.
EMP EMP HMP HMP EMP EMP EMP EMP Pyruvate TCA TCA TCA
Polysaccharides Murein Nucleic aicds Amino acids Lipids Amino acids Amino acids Amino acids Fatty acids Amino acids Amino acids Amino acids 5
Sources of energy
electrons released during oxidation of chemical energy sources must be accepted by an electron acceptor
microorganisms vary in terms of the acceptors they use
Figure 9.1
6
Electron acceptors for chemotrophic processes

Figure 9.2
exogenous electron acceptors
Chemoorganotrophic metabolism
Fermentation
energy source oxidized and degraded using endogenous electron acceptor which means without the participation of the exogenous electron acceptors often occurs under anaerobic conditions but can occus under aerobic conditions limited energy made available
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aerobic respiration
energy source degraded using oxygen as exogenous electron acceptor yields large amount of energy, primarily by electron transport activity
The way to conserve energy

For chemotroph, organisms that use chemicals as electron donor in energy metabolism, two mechanisms for energy conservation are known, fermentation and respiration. The final result in each case is the same, the synthesis of ATP (catabolism). In terms of redox reactions, fermentation and respirations differ as follows: 1) In fermentation, the redox process occurs in the absence of usable terminal electron acceptors 2) In respiration, oxygen or other electron acceptor is present as terminal electron acceptor. The ways to synthesize ATP 1) In fermentation, ATP is produced by a process called substrate-level phosphorylation (SLP). ATP is synthesized during the steps in the catabolisms of an organic compound. 2) In respiration, oxidative phosphorylation in which ATP is produced at the expense of the proton motive force. 3) photophosphorylation
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Madigan MT, Martinko JM and Parker J (2003) Brock biology of microorganisms
The way to conserve energy

For chemotroph, organisms that use chemicals as electron donor in energy metabolism, two mechanisms for energy conservation are known, fermentation and respiration. The final result in each case is the same, the synthesis of ATP (catabolism).
In terms of redox reactions, fermentation and respirations differ as follows: 1) In fermentation, the redox process occurs in the absence of usable terminal electron acceptors, while 2) In respiration, oxygen or other electron acceptor is present as terminal electron acceptor.
The ways to synthesize ATP 1) In fermentation, ATP is produced by a process called substrate-level phosphorylation (SLP). ATP is synthesized during the steps in the catabolisms of an organic compound. 2) In respiration, oxidative phosphorylation in which ATP is produced at the expense of the proton motive force. 3) photophosphorylation
Fig. 5.13 Energy conservation in fermentation and respiration (a) Substrate level phosphorylation (b) Oxidative phosphorylation
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Fermentation
12
Fermentation
In a typical fermentation, most of the carbon is excreted as a partially reduced end product of energy metabolism and only a small amount is used in biosynthesis. Many fermentations involve an anoxic environment, so decomposition of organic materials occur anaerobically. There are two problems an organism faces if it is to catabolize organic compounds in energyyielding metabolism: 1) Conserve some of the energy released as ATP.: In fermentation, ATP syntesis generally occurs by SLP, a mechanism by which high energy phosphate bonds from organic intermediates of fermentation are transferred to ADP.
2) Disposing of electrons removed from the electron donor.: The second problem is solved by production and excretion by the organism of fermentation products generated from the original substrate. 13
Glycolysis and TCA cycle as Fermentation
TCA cycle
Glycolysis
Madigan MT, Martinko JM and Parker J14 (2003) Brock biology of microorganisms
Aerobic respiration (see PMF and oxidative phosphorylation)
15
Aerobic respiration (see PMF and oxidative phosphorylation)
NADH is oxidized by a series of catalytic redox carriers that are integral proteins of the inner mitochondrial membrane. The free energy change in several of these steps is very exergonic. Coupled to these oxidation reduction steps is a transport process in which protons (H+) from the mitochondrial matrix are translocated to the cytosol. The redistribution of protons leads to formation of a proton gradient across the mitochondrial membrane. The size of the gradient is proportional to the free energy change of the electron transfer reactions. The result of these reactions is that the redox energy of NADH is converted to the energy of the proton gradient. In the presence of ADP, protons flow down their thermodynamic gradient from outside the mitochondrion back into the mitochondrial matrix. This process is facilitated by a proton carrier in the inner mitochondrial membrane known as ATP synthase. As its name 16 implies, this carrier is coupled to ATP synthesis. Electron flow through the mitochondrial electron transport assembly is carried out through several enzyme complexes.
Go' of
osmotic pressure
Nutrient concentrated over 100 times into the cell.
Osmotic pressure across the semipermeable cytoplasmic membrane.

This pressure can be expressed as free energy as in equation 5.7.
The energy needed to transport a mole of the solute under the given conditions.
Go' = 2.303 RTlog[S]i/[S]o (equation 5.7)
R : gas constant (8.314 J/K.mole) T : temperature in oK (25oC =298oK) 17 [S]o, [S]i : solute concentration of outside and inside, respectively
Biological energy transduction
Free energy generated from the catabolism is conserved in the forms of ATP and the protonmotive force, which provide energy needed in the 18 anabolism.
The structure Adenosine triphosphate (ATP)
19
High energy bond metabolic intermediates

Intermediate Phosphoenolpyruvate carbamoyl phosphate 1,3-diphosphglycerate acetyl phosphate creatine phosphate arginine phosphate acetyl-CoA Go' (KJ/mole) -61.9 -51.5 -49.4 -47.3 -43.1 -38.1 -34.5
Free energy of ATP hydrolysis

ATP + H2O ----------- AMP + PPi (Go' = -32.2 KJ/mole) ATP + H2O ----------- ADP + Pi (Go' = -30.5 KJ/mole) ADP + H2O ----------- AMP + Pi (Go' = -30.5 KJ/mole) PPi + H2O ------------ 2Pi (Go' = -28.8 KJ/mole)
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ATP as currency in biological metabolism

Catabolism generating energy Adenosine triphosphate (ATP) and protonmotive force Anabolism consuming energy ATP as currency; Energy needed for its synthesis smaller than released from catabolisms bigger than most of the energy consuming anabolisms ATP is a general intermediate for nucleic acids synthesis.
21
Relationship of adenylate energy charge (EC) and the relative concentration of ATP, ADP and AMP
ATP hydrolysis to AMP + PPi or ADP + Pi

ATP synthesis from ADP + Pi adenylate kinase ATP + AMP 2 ADP (Go' = 0KJ)
22
Regulation of catabolism and anabolism byAdenylate Energy Charge (EC)

EC = ([ATP]+1/2[ADP]) / ([ATP]+[ADP]+[AMP])
Adenylate energy charge controls the overall growth of microbes regulating catabolism synthesizing ATP, and anabolism consuming it.
23
Phosphorylation Potential (Gp)

ATP hydrolysis (synthesis) Standard conditions (Go') 30.5 KJ/mole Physiological conditions : Phosphorylation Potential (Gp) 51.8 KJ/mole. Gp = Go' + 2.303 log([ADP][P ]/[ATP]) (equation 5.9)
Metal ions such as Mg2+ which bind the nucleotide increase Gp value. Inter-conversion of ATP and protonmotive force (p)
Substrate-level phosphorylation (SLP) : ATP Electron transport phosphorylation : p
p ATP
24
Protonmotive force (p)

proton gradient : inside alkaline and outside acidic membrane potential : inside negative and outside positive
p = + ZpH
: membrane potential (mV) pH : pHgradient Z : 2.303 RT/F, and R : gas constant T : temperature in oK (25oC = 298oK) F : Faraday constant Actively growing cells maintain p of 0.15 - 0.45 V
25
Protonmotive Force.
Protonmotive force (p) is a biological form of energy facilitating ATP synthesis, transport and motility. p consists of inside alkaline and outside acidic proton gradient, and inside negative and outside positive membrane potential. Electron transport phosphorylation (ETP) during the respiration and photosynthesis builds p (lower part). p is consumed to synthesize In a fermentative cell the membrane-bound ATPase 26 hydrolyzes ATP to export H+ to develop p (upper part).
Acidophilicity and alkalophilicity

Neutrophile Acidophile : the H+ gradient of 103 106. They maintain low or inside positive membrane potential to compensate the large potential rendered by the H+ gradient (Figure 5.14). The inside positive membrane potential gives an additional benefit to the organisms
Alkalophile : Alkalophile maintain the neutral internal pH at the external pH over 10. Their growth is Na+-dependent. Na+ plays an important role to maintain the internal neutral pH in alkalophiles. H+ exported by ETP is exchanged with Na+ with the action of Na+/H+ antiporter (Figure 5.14). A Na+/H+ antiporter mutant (nhaC-) of an alkalophilic Bacillus firmus cannot grow at pH 10. In some bacteria Na+ is exported instead of H+ through the Na+dependent ETP including Na+-dependent NADHquinone reductase complex
27
Protonmotive force in acidophiles and alkalophiles.

Acidophiles maintain the internal pH neutral at the external acidic pH with a large pH. They have low or inside positive membrane potential to have an adequate p compensating the large pH. The internal positive membrane potential renders an additional benefit to the bacteria preventing H+ leak due to the large concentration gradient.
Alkalophiles grow optimally at alkaline pH with the neutral internal pH. They have a high Na+/H+ antiporter activity. H+ exported by ETP is exchanged with Na+ to prevent alkalization of the cytoplasm. They maintain sodiummotive force instead of protonmotive force. Alkalophiles and halophile have Na+dependent ETP in addition 28 to the Na+/H+ antiporter. This is referred to as primary sodium pump.
Inhibitor of electron transport phosphorylation (ETP)

Three mechanisms: 1) uncoupler, 2) direct inhibition to ETS system, 3) inhibition to synthesize ATP
NADH (by rotenone, amytal) Coenzyme Q (by HQNO) Cytochrome b (by antimycinA) Cytochrome o or d (by cyanide and azide) O2
Useful way to determine electron transport system (ETS) in microorganisms involved in the microbial fuel cell
29
anaerobic respiration energy source oxidized and degraded using molecules other than oxygen as exogenous electron acceptors can yield large amount of energy (depending on reduction potential of energy source and electron acceptor), primarily by electron transport activity
Chemolithotrophic metabolism
anaerobic respiration Inorganic electron donor Inorganic electron acceptor
30
Overview of aerobic catabolism
three-stage process
large molecules (polymers) small molecules (monomers) initial oxidation and degradation to pyruvate oxidation and degradation of pyruvate by the tricarboxylic acid cycle (TCA cycle)
31
Many different energy sources are funneled into common degradative pathways
ATP made primarily by oxidative phosphorylation

Figure 9.3
32
Two functions of organic energy sources
oxidized to release energy supply carbon and building blocks for anabolism
amphibolic pathways
function both as catabolic and anabolic pathways
Figure 9.4
33
The Breakdown of Glucose to Pyruvate
Three common routes

Glycolysis (Embden-Meyerhof pathway) Pentose phosphate pathway Entner-Doudoroff pathway
34
The Glycolytic Pathway
also called Embden-Meyerhof pathway occurs in cytoplasmic matrix of both procaryotes and eucaryotes
35
addition of phosphates primes the pump
oxidation step generates NADH high-energy molecules used to synthesize ATP by substrate-level phosphorylation
Figure 9.5
36
Summary of glycolysis
glucose + 2ADP + 2Pi + 2NAD+
2 pyruvate + 2ATP + 2NADH + 2H+
37
Phosphorylation of glucose
Group translocation Glucose (active transport)
hexokinase glucose + ATP ADP glucose-6-phosphate +
From glycogen
phosphorylase [glucose]n + Pi glucose-1-phosphate (glycogen) [glucose]n-1 +
(glycogen)
phosphoglucomutase glucose-1-phosphate
glucose-6-
38
EMP pathway
Glucose is phosphorylated to glucose-6-phosphate by PEP:glucose phosphotransferase (1) during the group translocation or by hexokinase (2) after transported via active transport system. 3, glucose-6-phosphate isomerase; 4, phosphofructokinase; 5, fructose diphosphate aldolase; 6, triose-phosphate isomerase; 7, glyceraldehyde-3-phosphate dehydrogenase; 8, 3-phosphoglycerate kinase; 9, phosphoglycerate mutase; 10, enolase; 11, pyruvate kinase. Irreversible reactions; 1, 2, 4, 11
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Enzymes unable to catalyze reverse reaction in EMP pathway

Phosphofructokinase - key enzyme Pyruvate kinase Enzymes unable to catalyze the reverse reaction Due to thermodynamic reason. Subject to regulations
40
Methylglyoxal bypass, a modified EMP pathway.
Under phosphate-limited conditions Escherichia coli oxidize dihydroxyacetone phosphate to pyruvate. 1, methylglyoxal synthase; 2, glyoxalase; 3, lactate oxidase. 41
Modified EMP pathway in Pyrococcus furiosus

(Extremophile. 1998 2:201-205)
Oligosaccharides produced by the extracellular -amylase (1) are transported into the cell to be hydrolyzed to glucose by -glucosidase (2). Glucose and fructose-6-phosphate are phosphorylated by ADP-dependent glucokinase (3) and ADP-dependent phosphofructokinase (4). Glyceraldehyde-3-phosphate is reduced to 3-phosphoglycerate by glyceraldehyde-3phosphate:ferredoxin oxidoreductase (5). Pyruvate is reduced as an electron acceptor to alanine. 6, pyruvate kinase; 7, pyruvate:ferredoxin oxidoreductase; 8, acetyl-CoA synthetase; 9, alanine aminotransferase
42
Gluconeogenesis
When microbes grow on substrates other than carbohydrate, they have to convert the substrate to glucose-6-phospate to supply carbon skeletons. They cannot employ EMP pathway since some enzymes do not catalyze the reverse reactions. phosphofructokinase Fructose-6-phosphate + ATP Fructose-1.6-diphosphate + ADP ( Go' = -14.2KJ/mole) pyruvate kinase Phosphoenolpyruvate + ADP pyruvate + ATP ( Go' = -23.8KJ/mole)
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PEP from pyruvate

PEP synthetase Pyruvate + ATP pyruvate + ATP + Pi PEP + AMP + Pi PEP + AMP +PPi pyruvate-phosphate dikinase
PEP from TCA cycle intermediates

PEP carboxykinase oxaloacetate + ATP malate enzyme malate + NAD pyruvate + NADH + CO2 PEP + CO2 + ADP
Fructose-6-phosphate
fructose diphosphatase
fructose-1.6-diphosphate
fructose-1.6-diphosphate + Pi
fructose-6-phosphate + Pi
fructose-6-phosphate + PPi
44
PPi-dependent phosphofructokinase
The Pentose Phosphate Pathway
also called hexose monophosphate pathway can operate at the same time as glycolytic or Entner-Doudoroff pathways can operate aerobically or anaerobically an amphibolic pathway
45
HMP (pentose phosphate) pathway

1, hexokinase 2, glucose-6-phosphate dehydrogenase 3, lactonase 4, 6-phosphogluconate dehydrogenase 5, ribulose-5-phosphate-3epimerase 6, ribose-5-phosphate isomerase 7, transketolase 8, transaldolase. In a glucose mineral medium, Escherichia coli metabolizes glucose through EMP pathway (72%) and HMP pathway (28%).
Glucose is oxidized to ribulose-5-phosphate coupled to the reduction of NADPH (a). Isomerase and epimerase convert ribulose-5-phosphate to ribose-5-phosphate and xylulose-5-phosphate (b). Transaldolase and transketolase rearrange pentose-5-phosphates into glucose-6-phosphate and glyceraldehydes-3-phosphate involving erythrose-4-phosphate (c). Nucleotide synthesis starts with ribose-5-phosphate, and the aromatic amino acids are produced from erythrose-4-phosphate and phosphoenolpyruvate. NADPH supplies 46reducing power during the biosynthetic processes.
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The Pentose Phosphate Pathway
oxidation steps produce NADPH, which is needed for biosynthesis
sugar transformation reactions

produce sugars needed for biosynthesis sugars can also be further degraded
Figure 9.6
47
Key Biochemical Reactions for The Pentose Phosphate Pathway
Figure 9.7
48
Summary of pentose phosphate pathway
glucose-6-P + 12NADP+ + 7H2O 6CO2 + 12NADPH + 12H+ Pi

-NADPH synthesis for source of electrons during biosynthesis -4 or 5-carbons for other purpose erythrose 4-P: aromatic amino acids and pyridoxal(vitamin B6) pentose ribose 5-P: nucleic acids ribulose 1,5-bisP: CO2 acceptor in photosynthesis -Production for ATP entering glycolytic pathway
49
The Entner-Doudoroff Pathway

reactions of pentose phosphate pathway
KDPG
yield per glucose molecule:

1 ATP 1 NADPH 1 NADH
Figure 9.8
50
Key intermediate
reactions of glycolytic pathway
Pseudomonas, Rhizobium, Azotobacter, Agrobacterium
Fermentations
oxidation of NADH produced by glycolysis pyruvate or its derivative used as endogenous electron acceptor ATP formed by substrate-level phosphorylation Oxygen is not needed
Figure 9.9
51
Homolactic fermenters; Glucose to pyruvate to lactic acid
alcoholic fermentation
alcoholic beverages, bread, etc.
O OH O
Heterolactic fermenters; Glucose to pyruvate to mixture acids
food spoilage yogurt, sauerkraut, pickles, etc.
Pyruvate
OH OH O
Lactate
O
Figure 9.10
52
OH
Acetoin
Two types of Formic acid fermentation: Mixed acid and Butandiol Fermentations
53
methyl red test detects pH change in media caused by mixed acid fermentation
Butanediol fermentation
Voges-Proskauer test detects intermediate acetoin Methyl red test and VogesProskauer test important for distinguishing pathogenic members of Enterobacteriaceae
54
Fermentations of amino acids
Stickland reaction
oxidation of one amino acid with use of second amino acid as electron acceptor
Figure 9.11
55
Pyruvate dehydrogenase complex

24 molecules of pyruvate dehydrogenase with thiamine pyrophosphate (TPP), 24 molecules of dihydrolipoate acetyltransferase with dihydrolipoate 12 molecules of dihydrolipoate dehydrogenase with flavin adenine dinucleotide (FAD) NAD coenzyme A Irreversible Activated by pyruvate and AMP Repressed by NADH and ATP Common properties in 2-keto acid dehydrogenase complexes (2-ketoglutarate, 2-ketobutyrate) controlled differently
56
Oxidative decarboxylation of pyruvate by dehydrogenase complex.
Pyruvate dehydrogenase (E1) decaroxylates pyruvate and its prosthetic group thiamine pyrophosphate (TPP) binds the resulting hydroxyethyl group, which binds lipoate (Lip) of dihydrolipoate acetyltransferase (E2) reducing its disulphide. E2 transfers acetyl group to coenzyme A to form acetyl-CoA. Dihydrolipoate dehydrogenase (E3) transfers 57 electrons of reduced lipoate to NAD+.
The Tricarboxylic Acid Cycle

also called citric acid cycle and Krebs cycle completes oxidation and degradation of glucose and other molecules common in aerobic bacteria, free-living protozoa, most algae, and fungi amphibolic
provides carbon skeletons for biosynthesis
58
oxidation steps form NADH and FADH2
energy drives condensation high-energy of acetyl molecule group with oxaloacetate oxidation and decarboxylation steps complete oxidation and degradation also form NADH
Figure 9.12
substrate-level phosphorylation
59
Acetyl-CoA oxidation through TCA cycle.

1. citrate synthase; 2. aconitase; 3. isocitrate dehydrogenase;
4. 2-ketoglutarate dehydrogenase complex;

5. succinate thiokinase(succinyl-CoA synthetase); 6. succinate dehydrogenase; 7. fumarase(fumarate hydratase);
8. malate dehydrogenase.
60 Enzymes of the reactions 1, 4 and 6 cannot catalyze the reverse reactions.
Summary
for each acetyl-CoA molecule oxidized, TCA cycle generates:
2 molecules of CO2 3 molecules of NADH one FADH2 one GTP
61
Electron Transport and Oxidative Phosphorylation
only 4 ATP molecules synthesized directly from oxidation of glucose to CO2 most ATP made when NADH and FADH2 (formed as glucose degraded) are oxidized in electron transport chain (ETC)
62
The Electron Transport Chain (ETC)
series of electron carriers that operate together to transfer electrons from NADH and FADH2 to a terminal electron acceptor electrons flow from carriers with more negative E0 to carriers with more positive E0
63
Electron transport chain
as electrons transferred, energy released some released energy used to make ATP by oxidative phosphorylation
as many as 3 ATP molecules made per NADH using oxygen as acceptor
P/O ratio = 3
P/O ratio for FADH2 is 2

i.e., 2 ATP molecules made
64
NAD/NADH
large difference in E0 of NADH and E0 of O2
large amount of energy released DGo = -nFDEo

1/2O2/H2O
1.14 volts from reduction potential from NAD to O2
65
Figure 9.13
Mitochondrial ETC
Chemiosmotic Hypthothesis Proton gradient
Figure 9.14 electron transfer through electron transport chain accompanied by proton movement across inner mitochondrial membrane through membrane bound protein complex I, III, and IV
66
NADH: 2 electrons transfer Flavopeoteins (FAD or FMN), CoQ: 1 or 2 electrons transfer Cytochromes/Fe-S/Copper:1 electron
67
Procaryotic ETCs
located in plasma membrane some resemble mitochondrial ETC, but many are different
different electron carriers may be branched may be shorter may have lower P/O ratio
68
ETC of E. coli
branched pathway for terminal oxidase
Upper branch Cytochrome d -Working in stationary phase and low aeration -High affinity for oxygen -Does not actively pump protons lower branch Cytochrome o -Working in log phase and high aeration -Moderately high affinity for oxygen -Actively pump protons
Figure 9.15
69
ETC of Paracoccus denitrificans

aerobic
CH2NH2
Think about succinate
Figure 9.16a
70
ETC of P. denitrificans
Anaerobic; highly branched ETC chain Cyt aa3 is not working
Figure 9.16b
example of anaerobic respiration
71 Nar: nitrate reductase, Nir:nitrite reductase, Nor:nitric oxide reductase, Nos:Nitrous oxide reducatse
Oxidative Phosphorylation
chemiosmotic hypothesis by Peter Mitchell
most widely accepted explanation of oxidative phosphorylation postulates that energy released during electron transport is used to establish a proton gradient and charge difference across membrane
called proton motive force (PMF)
In Prokaryotic, PMF uses ATP synthesis, Flagella rotation, and Transport of molecules across membrane
72
Central Role of PMF
73
PMF drives ATP synthesis diffusion of protons back across membrane (down gradient) drives formation of ATP ATP synthase
enzyme that uses proton movement down gradient to catalyze ATP synthesis
74
movement of protons establishes PMF

Figure 9.17
75
ATP synthase uses proton flow down gradient to make ATP
1 a subunits
9-12 c subunits
Figure 9.19a
76
Figure 20.25 A model of the F1 and F0 components of the ATP synthase, a rotating molecular motor. The a, b, and d subunits constitute the stator of the motor, and the c, g, and e subunits form the rotor. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in a and b that synthesize ATP. H+-Asp61 on c rotor Once it forms, it rides the rotor until other proton access channel on a
c subunit: antiparallel transmembrane helics
77
NADH pump 3H+ in 3ATP
Figure 20.27 The binding change mechanism for ATP synthesis by ATP synthase (a3b 3) This model assumes that F1 has three interacting and conformationally distinct active sites on b subunits. The open (O) conformation is inactive and has a low affinity for ligands; the L conformation (with loose affinity for ligands) is also inactive; the tight (T) conformation is active and has a high affinity for ligands. Synthesis of ATP is initiated (step 1) by binding of ADP and Pi to an L site. In the second step, an energy-driven
conformational change converts the L

site to a T conformation and also converts T to O and O to L. In the third step, ATP is synthesized at the T site and released from the O site. Two additional passes through this cycle produce two more ATPs and return the enzyme to its original state.
LT O L
78 3H+ make full one turn of b units 3ATP by 1 NADH
Inhibitors of ATP synthesis

blockers
inhibit flow of electrons through ETC
uncouplers
allow electron flow, but disconnect it from oxidative phosphorylation many allow movement of ions, including protons, across membrane without activating ATP synthase
destroys pH and ion gradients
some may bind ATP synthase and inhibit its activity directly
79
The Yield of ATP in Glycolysis and Aerobic Respiration
aerobic respiration provides much more ATP than fermentation Pasteur effect
decrease in rate of sugar metabolism when microbe shifted from anaerobic to aerobic conditions occurs because aerobic process generates greater ATP per sugar molecule They do not need many sugar to get ATP as they did in anaerobic conditions
80
81
ATP yield
amount of ATP produced during aerobic respiration varies depending on growth conditions and nature of ETC under anaerobic conditions, glycolysis only yields 2 ATP molecules
82
Anaerobic Respiration
uses electron carriers other than O2 generally yields less energy because E0 of electron acceptor is less positive than E0 of O2 More efficient than fermentation ATP synthesis by ETC and oxidative phosphorylation in the absence of O2
More oxidized endogenous organic molecules
83
Fermentation
An example
dissimilatory nitrate reduction
use of nitrate as terminal electron acceptor by nitrate reductase (O2 repressed synthesis of nitrate reductase) denitrification
reduction of nitrate to nitrogen gas in soil, causes loss of soil fertility
NO3
84
NO2
NO
N2O
N2
Neurotransmitter Regulate Blood Pressure Used by Macrophages to kill bacteria and tumor
2NO3 + 10e- + 12H+
N2 + 6H2O
ETC of P. denitrificans
Anaerobic; highly branched ETC chain Cyt aa3 is not working
Figure 9.16b
example of anaerobic respiration
85 Nar: nitrate reductase, Nir:nitrite reductase, Nor:nitric oxide reductase, Nos:Nitrous oxide reducatse
Succession
O2 NO3SO42Mn2+ Fe3+ CO2
O2 users first come. Why? Then what element will be used Then the next? Next?
Oxygen first used because Oxygen inhibits synthesis of nitrate reductase

inhibits growth of strict anaerobic sulfate reducers and methanogens
Mn2+ and Fe3+ will be the next SO42- used by sulfate reducers because it is better electron acceptor than CO2 and better consumer for Hydrogen 86 Methanogens used finally after SO42- is exhausted totally
Catabolism of Carbohydrates and Intracellular Reserves
many different carbohydrates can serve as energy source carbohydrates can be supplied externally or internally (from internal reserves)
87
Carbohydrates
monosaccharides converted to other sugars that enter glycolytic pathway disaccharides and polysaccharides cleaved by hydrolases or phosphorylases
88
Reserve Polymers
used as energy sources in absence of external nutrients
e.g., glycogen and starch
cleaved by phosphorylases (glucose)n + Pi (glucose)n-1 + glucose-1-P glucose-1-P enters glycolytic pathway
e.g., PHB(poly-b-hydroxybutyrate)
PHB acetyl-CoA acetyl-CoA enters TCA cycle
89
Lipid Catabolism
triglycerides
common energy sources hydrolyzed to glycerol and fatty acids by lipases
glycerol degraded via glycolytic pathway fatty acids often oxidized via -oxidation pathway
Figure 9.21
90
-oxidation pathway
enters TCA cycle or used for biosynthesis
to ETC
Figure 9.22
91
Protein and Amino Acid Catabolism

protease
hydrolyzes protein to amino acids
deamination
removal of amino group from amino acid resulting organic acids converted to pyruvate, acetyl-CoA, or TCA cycle intermediate
can be oxidized via TCA cycle can be used for biosynthesis
92
deamination often occurs by transamination
Figure 9.23
transfer of amino group from one amino acid to -keto acid
93
Oxidation of Inorganic Molecules

carried out by chemolithotrophs electrons released from energy source through
Oxidation of Inorganic Molecules transferred to terminal electron acceptor by ETC
ATP synthesized by oxidative phosphorylation
Terminal electron acceptors: O2, SO42-, and NO3-
94
Consequence of Chemolithotroph
Chemolithotroph bacteria are autotrophic and employ Calvin cycle to fix CO2 which requires 3 ATP and 2 NADPH Much less energy is available from oxidation of inorganic molecules - P/O ratios for oxidative phosphorylation in chemolithotrophic = 1 So? They need to use vast amount of inorganic compounds to get proper ATP for their life Then, How about the environmental impact with this bacteria?
95
usually aerobic
96
Nitrifying bacteria
oxidize ammonia to nitrate NH3 NO2 NO3

Ammonia and nitrate is more positive potential than NAD How do they make ATP and NADH?
requires 2 different genera

ATP
Nitrosomonas, Nitrosospira
Nitrobacter and Nitrococcus
NH4+ + 3/2O2 NO2- + H2O + 2H+
97
Electrons to flow up the reduction potential gradient by PMF
PMF is not linked to synthesize ATP, but to pump in reverse electrons
NO2 NO3
Normal ETC
Sulfur-oxidizing bacteria
* *
So, H2S, and S2O32- to SO42-
substrate-level phosphorylation Sulfur-oxidizing bacteria can synthesize ATP by both oxidative phosphorylation and substrate-level phosphorylation
(APS)
(APS)
98
Metabolic flexibility of chemolithotrophs many switch from chemolithotrophic metabolism to chemoorganotrophic metabolism many switch from autotrophic metabolism (via Calvin cycle) to heterotrophic metabolism
99
Autotrophic growth by chemolithotrophs
Calvin cycle requires NADH as electron source for fixing CO2

many energy sources used by chemolithotrophs have E0 more positive than NAD/NADH
use reverse electron flow to generate NADH
100
Photosynthesis
light reactions
energy from light trapped and converted to chemical energy
dark reactions
chemical energy used to reduce CO2 and synthesize cell constituents (discussed in Chapter 10)
101
oxygenic photosynthesis eucaryotes and cyanobacteria anoxygenic photosynthesis all other bacteria
102
The Light Reaction in Eucaryotes and Cyanobacteria
chlorophylls
major light-absorbing pigments
accessory pigments
transfer light energy to chlorophylls e.g., carotenoids and phycobiliproteins
103
different chlorophylls have different absorption peaks
Light absorption peaks at 645 and 430 nm
Light absorption peaks at 665 and 430 nm
Green light is not absorbed

104
To membrane
Carotenoids
absorb different wavelengths of light than chlorophylls at 470 to 630 nm
In algae
In diatoms and brown algae
In red algae and cyanobacteria
Figure 9.27
105
attached to a protein to form phycobiliprotein
Organization of pigments
antennas
highly organized arrays of chlorophylls and accessory pigments captured light transferred from chlorophyll to chlorophyll to special reaction-center chlorophyll
directly involved in photosynthetic electron transport
Photosystems I and II
antenna and its associated reaction-center chlorophyll
106
Green plant photosynthesis

Ps I and II involved in noncyclic electron flow ATP + NADPH made (noncyclic photophosphorylation) Ps I involved in cyclic electron flow ATP made (cyclic photophosphorylation)
107
Antennas: 300 chlorophyll molecules
excited
Pheophytin (Pheo) Plastocyanin (PC) Plastoquinone (PQ)
Chlorophyll a
NADP+
Chlorophyll a
Photosystem I absorbs longer light >680
Antennas Photosystem II Photosystem II absorbs shorter light <680
2 electrons:4 photons
electron flow PMF ATP
Stroma
Thylakoid interior
OEC: 108 oxygen-evolving complex
Dark reaction: Economy

CO2 + 3ATP + 2NADPH + 2H+ + H2O
(CH2O) + 3ADP + 3Pi + 2NADP+

2e- produces 1 NADPH + ATP in the noncyclic system with 4 photons (4 quanta light energy) participation To fix one CO2, we need 3 ATP and 2 NADPH. Therefore, 8 quanta will produce 2ATP and 2NADPH. How about one more ATP? Then independent cyclic photophosphorylation operated? This needs another 2 to 4 quanta to produce 1ATP. Therefore, 10 to 12 quanta of light energy is needed to fix one CO2
109
The Light Reaction in Green and Purple Bacteria
Green sulfur bacteria Bchl a and b absorb at 775 and 790 nm Green nonsulfur bacteria But In vivo absorb at 830-890 and Purple sulfur bacteria 1,020 to 1,040 nm (infrared region) Purple nonsulfur bacteria
110
Purple nonsulfur bacteria

Rhodobacter sphaeroides
electron source for generation of NADH by reverse electron flow
111
Reverse electron flow

more positive E0
more negative E0
Figure 9.32
ATP or PMF used to drive upward (reverse) flow of electrons

112
Green sulfur bacteria

Chlorobium limicola
e-
113

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Think about biodegradation..

Metabolic intermediates used as carbon skeletons for biosynthesis.

Electron acceptors for chemotrophic processes

exogenous electron acceptors

The way to conserve energy

Madigan MT, Martinko JM and Parker J (2003) Brock biology of microorganisms

The way to conserve energy

Madigan MT, Martinko JM and Parker J (2003) Brock biology of microorganisms

Glycolysis and TCA cycle as Fermentation

Aerobic respiration (see PMF and oxidative phosphorylation)

Madigan MT, Martinko JM and Parker J (2003) Brock biology of microorganisms

Aerobic respiration (see PMF and oxidative phosphorylation)

Nutrient concentrated over 100 times into the cell.

Osmotic pressure across the semipermeable cytoplasmic membrane.

Biological energy transduction

The structure Adenosine triphosphate (ATP)

High energy bond metabolic intermediates

Free energy of ATP hydrolysis

ATP as currency in biological metabolism

ATP hydrolysis to AMP + PPi or ADP + Pi

Regulation of catabolism and anabolism byAdenylate Energy Charge (EC)

Phosphorylation Potential (Gp)

Substrate-level phosphorylation (SLP) : ATP Electron transport phosphorylation : p

Protonmotive force (p)

Acidophilicity and alkalophilicity

Protonmotive force in acidophiles and alkalophiles.

Inhibitor of electron transport phosphorylation (ETP)

Overview of aerobic catabolism

ATP made primarily by oxidative phosphorylation

Two functions of organic energy sources

The Breakdown of Glucose to Pyruvate

Three common routes

The Glycolytic Pathway

addition of phosphates primes the pump

Enzymes unable to catalyze reverse reaction in EMP pathway

Methylglyoxal bypass, a modified EMP pathway.

Modified EMP pathway in Pyrococcus furiosus

PEP from pyruvate

PEP from TCA cycle intermediates

The Pentose Phosphate Pathway

HMP (pentose phosphate) pathway

oxidation steps produce NADPH, which is needed for biosynthesis

sugar transformation reactions

Key Biochemical Reactions for The Pentose Phosphate Pathway

Summary of pentose phosphate pathway

glucose-6-P + 12NADP+ + 7H2O 6CO2 + 12NADPH + 12H+ Pi

The Entner-Doudoroff Pathway

yield per glucose molecule:

reactions of glycolytic pathway

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Homolactic fermenters; Glucose to pyruvate to lactic acid

Heterolactic fermenters; Glucose to pyruvate to mixture acids

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Fermentations of amino acids

Pyruvate dehydrogenase complex

Oxidative decarboxylation of pyruvate by dehydrogenase complex.

The Tricarboxylic Acid Cycle

oxidation steps form NADH and FADH2

Acetyl-CoA oxidation through TCA cycle.

4. 2-ketoglutarate dehydrogenase complex;

Electron Transport and Oxidative Phosphorylation