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Transcript
84 6 / * 5 t T H E H NAD+ Nicotinamide P O O C H + N H O –O F O U N D AT I O N S O F M I C R O B I O L O G Y NADH + H+ NH2 H Ribose OH O –O P N O O CH2 N Ribose OH OH N NH2 + H+ H NH2 OH O O C H O CH2 HH 2H N N Adenine MINIQUIZ R NAD+/ a cell requires relatively large amounts of a primary electron donor (the substance that was oxidized to yield NADH) and a terminal electron acceptor (such as O2), it needs only a tiny amount of NAD+ and NADH because they are constantly being recycled (Figure 3.11). NADP+ is a related redox coenzyme in which a phosphate group is added to NAD+. NADP+/NADPH typically participate in redox reactions distinct from those that use NAD+/NADH, most commonly in anabolic (biosynthetic) reactions in which oxidations and reductions occur (Sections 3.14–3.16). NADH E 0 ′ = –0.32 V In NADP+, this OH has a phosphate attached. t In the reaction H2 + 12 O2 S H2O, what is the electron donor and what is the electron acceptor? t Why is nitrate (NO3- ) a better electron acceptor than fumarate? t Is NADH a better electron donor than H2? Is NAD+ a better acceptor than 2 H+? How do you determine this? Figure 3.10 The oxidation–reduction coenzyme nicotinamide adenine dinucleotide (NAD+). NAD+ undergoes oxidation–reduction as shown and is freely diffusible. “R” is the adenine dinucleotide portion of NAD+. 3.7 Energy-Rich Compounds the NAD+/NADH couple is - 0.32 V, which places it fairly high on the electron tower; that is, NADH is a good electron donor while NAD+ is a rather weak electron acceptor (Figure 3.9). Coenzymes such as NAD+/NADH increase the diversity of redox reactions possible in a cell by allowing chemically dissimilar electron donors and acceptors to interact, with the coenzyme acting as the intermediary. For example, electrons removed from an electron donor can reduce NAD+ to NADH, and the latter can be converted back to NAD+ by donating electrons to the electron acceptor. Figure 3.11 shows an example of such electron shuttling by NAD+/NADH. In this reaction, NAD+ and NADH facilitate the overall redox reaction but are not consumed in a net fashion as are the original donor and terminal acceptor. In other words, although Energy released from redox reactions must be conserved by the cell if it is to be used to drive energy-requiring cell functions. In living organisms, chemical energy released in redox reactions is conserved primarily in phosphorylated compounds. The free energy released upon removal (hydrolysis) of the phosphate in these energy-rich compounds is significantly greater than that of the average covalent bond in the cell, and it is this released energy that is used by the cell. Phosphate can be bonded to organic compounds by either ester or anhydride bonds, as illustrated in Figure 3.12. However, not all phosphate bonds are energy-rich. As seen in the figure, the ∆G0 ′ of hydrolysis of the phosphate ester bond in glucose 6-phosphate is only - 13.8 kJ/mol. By contrast, the ∆G0 ′ of hydrolysis of the phosphate anhydride bond in phosphoenolpyruvate is -51.6 kJ/mol, NAD+ reduction NAD+ Active binding site site 1. Enzyme I reacts with e– donor and oxidized form of coenzyme, NAD+. Enzyme–substrate complex Enzyme I NAD+ + 4. NAD+ is released. + Substrate (e– donor) NADH + Product Product Active site NADH binding site Enzyme II NADH oxidation Figure 3.11 3. Enzyme II reacts with e– acceptor and reduced form of coenzyme, NADH. Enzyme–substrate complex NAD+/NADH cycling. A schematic example of redox reactions in which two different enzymes are linked by their requirement for either NAD+ or NADH. Substrate (e– acceptor) 2. NADH and reaction product are formed. $ ) " 1 5 & 3 t M I C R O B I A L M E TA B O L I S M 85 NH2 – CH2 C COO– O –O P O– O P O P O P O O O Ester bond OHCH HCOH HCOH O– CH2 O P O– O OH OH Phosphoenolpyruvate N O O CH2 O Glucose 6-phosphate Adenosine triphosphate (ATP) Compound Thioester bond Anhydride bond O CH3 C~S HCOH N O– O –O Anhydride bond – CHO N N Ester bond O O (CH2)2 H N C (CH2)2 H N C H CH3 C C CH2 O R OH CH3 Coenzyme A Acetyl H3C UNIT 1 Anhydride bonds O O– C O P O– O Acetyl phosphate Acetyl-CoA ∆G0′ > 30kJ Phosphoenolpyruvate 1,3-Bisphosphoglycerate Acetyl phosphate ATP ADP Acetyl-CoA ∆G0′ < 30kJ AMP Glucose 6-phosphate G0′ kJ/mol –51.6 –52.0 –44.8 –31.8 –31.8 –35.7 –14.2 –13.8 Figure 3.12 Phosphate bonds in compounds that conserve energy in bacterial metabolism. Notice, by referring to the table, the range in free energy of hydrolysis of the phosphate bonds highlighted in the compounds. The “R” group of acetyl-CoA is a 3′-phospho ADP group. almost four times that of glucose 6-phosphate. Although theoretically either compound could be hydrolyzed in energy metabolism, cells typically use a small group of compounds whose ∆G0 ′ of hydrolysis is greater than - 30 kJ/mol as energy “currencies” in the cell. Thus, phosphoenolpyruvate is energy-rich whereas glucose 6-phosphate is not. Adenosine Triphosphate The most important energy-rich phosphate compound in cells is adenosine triphosphate (ATP). ATP consists of the ribonucleoside adenosine to which three phosphate molecules are bonded in series. ATP is the prime energy currency in all cells, being generated during exergonic reactions and consumed in endergonic reactions. From the structure of ATP (Figure 3.12), it can be seen that only two of the phosphate bonds (ATP S ADP + Pi and ADP S AMP + Pi) are phosphoanhydrides and thus have free energies of hydrolysis greater than - 30 kJ. By contrast, AMP is not energy-rich because its free energy of hydrolysis is only about half that of ADP or ATP (Figure 3.12). Although the energy released in ATP hydrolysis is -32 kJ, a caveat must be introduced here to define more precisely the energy requirements for the synthesis of ATP. In an actively growing Escherichia coli cell, the ratio of ATP to ADP is maintained at about 7:1, and this affects the energy requirements for ATP synthesis. In an actively growing cell, the actual energy expenditure (that is, the ∆G, Section 3.4) for the synthesis of 1 mole of ATP is on the order of - 55 to - 60 kJ. Nevertheless, for the purposes of learning and applying the basic principles of bioenergetics, we will assume that reactions conform to “standard conditions” (∆G0 ′), and thus we will assume that the energy required for synthesis or hydrolysis of ATP is 32 kJ/mol. Coenzyme A Cells can use the free energy available in the hydrolysis of energyrich compounds other than phosphorylated compounds. These include, in particular, derivatives of coenzyme A (for example, acetyl-CoA; see structure in Figure 3.12). Coenzyme A derivatives contain thioester bonds. Upon hydrolysis, these yield sufficient free energy to drive the synthesis of an energy-rich phosphate bond. For example, in the reaction Acetyl-S-CoA + H2O + ADP + Pi S acetate+ HS-CoA + ATP + H+ the energy released in the hydrolysis of coenzyme A is conserved in the synthesis of ATP. Coenzyme A derivatives (acetyl-CoA is just one of many) are especially important to the energetics of anaerobic microorganisms, in particular those whose energy metabolism depends on fermentation (see Table 3.4). We will return to the importance of coenzyme A derivatives in bacterial bioenergetics many times in Chapter 13. Energy Storage ATP is a dynamic molecule in the cell; it is continuously being broken down to drive anabolic reactions and resynthesized at the expense of catabolic reactions. For longer-term energy storage, microorganisms produce insoluble polymers that can be catabolized later for the production of ATP. Examples of energy storage polymers in prokaryotes include glycogen, poly-β-hydroxybutyrate and other polyhydroxyalkanoates, and elemental sulfur, stored from the oxidation of H2S by sulfur chemolithotrophs. These polymers are deposited within the cell as granules that can be seen with the light or electron 86 6 / * 5 t T H E F O U N D AT I O N S O F M I C R O B I O L O G Y microscope ( Section 2.14). In eukaryotic microorganisms, starch (polyglucose) and simple fats are the major reserve materials. In the absence of an external energy source, a cell can break down these polymers to make new cell material or to supply the very low amount of energy, called maintenance energy, needed to maintain cell integrity when it is in a nongrowing state. MINIQUIZ t How much energy is released per mole of ATP converted to ADP + Pi under standard conditions? Per mole of AMP converted to adenosine and Pi? t During periods of nutrient abundance, how can cells prepare for periods of nutrient starvation? III t Fermentation and Respiration F ermentation and respiration are two major strategies for energy conservation in chemoorganotrophs. Fermentation is a form of anaerobic catabolism in which an organic compound is both an electron donor and an electron acceptor. By contrast, respiration is the form of aerobic or anaerobic catabolism in which an electron donor is oxidized with O2 or an O2 substitute as the terminal electron acceptor. One can look at fermentation and respiration as alternative metabolic options. When O2 is available, respiration will take place because, as we will see, much more ATP is produced in respiration than in fermentation. But if conditions will not support respiration, fermentation can supply enough energy for an organism to thrive. We begin by examining a major metabolic pathway for microbial fermentations, the glycolytic pathway. 3.8 Glycolysis A nearly universal pathway for the catabolism of glucose is glycolysis, which breaks down glucose into pyruvate. Glycolysis is also called the Embden–Meyerhof–Parnas pathway for its major discoverers. Whether glucose is fermented or respired, it travels through this pathway. In fermentation, ATP is synthesized by substrate-level phosphorylation. In this process, ATP is synthesized directly from energy-rich intermediates during steps in the catabolism of the fermentable substrate (Figure 3.13a). This is in contrast to oxidative phosphorylation, which occurs in respiration; ATP is synthesized here at the expense of the proton motive force (Figure 3.13b). The fermentable substrate in a fermentation is both the electron donor and electron acceptor; not all compounds can be fermented, but sugars, especially hexoses such as glucose, are excellent fermentable substrates. The fermentation of glucose through the glycolytic pathway can be divided into three stages, each requiring several independent enzymatic reactions. Stage I comprises “preparatory” reactions; these are not redox reactions and do not release energy but instead form a key intermediate of the pathway. In Stage II, redox reactions occur, energy is conserved, and two molecules of pyruvate are formed. In Stage III, redox balance is achieved and fermentation products are formed (Figure 3.14). Stage I: Preparatory Reactions In Stage I, glucose is phosphorylated by ATP, yielding glucose 6-phosphate. The latter is then isomerized to fructose 6-phosphate, and a second phosphorylation leads to the production of fructose 1,6-bisphosphate. The enzyme aldolase then splits fructose 1,6-bisphosphate into two 3-carbon molecules, glyceraldehyde 3-phosphate and its isomer, dihydroxyacetone phosphate, which is converted into glyceraldehyde 3-phosphate. To this point, all of the reactions, including the consumption of ATP, have proceeded without any redox changes. Stage II: Production of NADH, ATP, and Pyruvate The first redox reaction of glycolysis occurs in Stage II during the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglyceric acid. In this reaction (which occurs twice, once for each of the two molecules of glyceraldehyde 3-phosphate produced from glucose), the enzyme glyceraldehyde-3-phosphate dehydrogenase reduces its coenzyme NAD+ to NADH. Simultaneously, each glyceraldehyde 3-phosphate molecule is phosphorylated by the addition of a molecule of inorganic phosphate. This reaction, in which inorganic phosphate is converted to organic form, sets the stage for energy conservation. ATP formation is possible because 1,3-bisphosphoglyceric acid is an energy-rich compound (Figure 3.12). ATP is then synthesized when (1) each molecule Intermediates Pi A B Energy-rich intermediates B~P ADP ATP C~P D (a) Substrate-level phosphorylation + ++ + + + + + + + + + + + + + + + + + + – – – – – – – – – – – – – – – – –– + + –– – + – – + + – – + – – + –– – – – – – – – – – – – – – – – Energized + + ++ + membrane ++ + + + + + + + + + + + + + + + + ADP + Pi Dissipation of proton motive force coupled ATP to ATP synthesis + + + + + + + + + + + – – – – – – – – – – + Less energized – + – membrane – + + – – – – – – – – – + + + + + + + + + + + (b) Oxidative phosphorylation Figure 3.13 Energy conservation in fermentation and respiration. (a) In fermentation, substrate-level phosphorylation produces ATP. (b) In respiration, the cytoplasmic membrane, energized by the proton motive force, dissipates energy to synthesize ATP from ADP + Pi by oxidative phosphorylation. 87 $ ) " 1 5 & 3 t M I C R O B I A L M E TA B O L I S M GLYCOLYSIS HOCH2 H HO O H OH H ATP P OCH2 O H H H OH OH 1 A H OH H OH Glucose H O P OCH2 H OH H H 2 B OH OH OH C O O P OCH2 H2COH OH P OCH2 D ATP H H 3 H C HO H2CO P HO H2COH 2 NAD+ 5 OH 4 HC O H E HC OH H2CO P Stage II 2 O– 2 O C Pyruvate O C 10 O C 2 O– O C 9 P O C CH3 O C P O CH2 G 2 lactate 11 2 Pyruvate Stage III 7 OH C H HO CH2 H 2 P 2 O– 8 P O C CH2 I 2 ATP 2 O– 6 O C O P OH C H P OCH2 F 2 ATP + 2 NADH NADH consumption NADH production 12 13 2 ethanol + 2 CO2 GLYCOLYTIC INTERMEDIATES AND ENZYMES Intermediates F 1,3-Bisphosphoglycerate Enzymes 7 Phosphoglycerokinase A Glucose 6-P G 3-P-Glycerate 1 Hexokinase 8 Phosphoglyceromutase H 2-P-Glycerate 2 Isomerase 9 Enolase Phosphoenolpyruvate 3 Phosphofructokinase 10 Pyruvate kinase B Fructose 6-P C Fructose 1,6-P D Dihydroxyacetone-P 4 Aldolase 11 Lactate dehydrogenase E Glyceraldehyde-3-P 5 Triosephosphate isomerase 12 Pyruvate decarboxylase 6 Glyceraldehyde-3-P dehydrogenase 13 Alcohol dehydrogenase Energetics Yeast Lactic acid bacteria I Glucose 2 ethanol + 2 CO2 –239 kJ Glucose 2 lactate –196 kJ Figure 3.14 Embden–Meyerhof–Parnas pathway (glycolysis). (Top) The sequence of reactions in the catabolism of glucose to pyruvate and then on to fermentation products. Pyruvate is the end product of glycolysis, and fermentation products are made from it. (Bottom) Intermediates, enzymes, and contrasting fermentation balances of yeast and lactic acid bacteria. of 1,3-bisphosphoglyceric acid is converted to 3-phosphoglyceric acid, and (2) each molecule of phosphoenolpyruvate is converted to pyruvate (Figure 3.14). During Stages I and II of glycolysis, two ATP molecules are consumed and four ATP molecules are synthesized (Figure 3.14). Thus, the net energy yield in glycolysis is two molecules of ATP per molecule of glucose fermented. Stage III: Redox Balance and the Production of Fermentation Products During the formation of two 1,3-bisphosphoglyceric acid molecules, two NAD + are reduced to NADH (Figure 3.14). However, recall that NAD + is only an electron shuttle, not a net (terminal) acceptor of electrons. Thus, the NADH produced in glycolysis must be oxidized back to NAD+ in order for another round of glycolysis to occur, and this is accomplished when pyruvate is reduced by NADH to fermentation products (Figure 3.14). For example, in fermentation by yeast, pyruvate is reduced to ethanol (ethyl alcohol) with the subsequent production of carbon dioxide (CO2). By contrast, lactic acid bacteria reduce pyruvate to lactate. Many other possibilities for pyruvate reduction exist depending on the organism (see next section), but the end result is the same: NADH is reoxidized to NAD+ , and this allows earlier reactions of the pathway that require NAD+ to continue. Catabolism of Other Sugars and Polysaccharides Many microorganisms can ferment disaccharides. For example, lactose (milk sugar) and sucrose (table sugar) are common UNIT 1 Stage I 88 6 / * 5 t T H E F O U N D AT I O N S O F M I C R O B I O L O G Y disaccharides widely used by fermentative anaerobes. With either substrate, the first step in its fermentation is to break the disaccharide into its components. For lactose, this is glucose and galactose as a result of activity of the enzyme β-galactosidase, and for sucrose, this is glucose and fructose resulting from invertase activity. Fructose and galactose are then converted to glucose by isomerase enzymes and fermented by the glycolytic pathway. Polysaccharides are important structural components of microbial cell walls, capsules, slime layers, and storage products, and many polysaccharides can be fermented. Cellulose and starch are two of the most abundant natural polysaccharides. Although both these polysaccharides are polymers of glucose, the glucose units in the polymer are bonded differently. This makes cellulose more insoluble than starch and less rapidly digested. Cellulose is attacked by the enzyme cellulase and starch by the enzyme amylase. The activities of these enzymes release glucose from the polymer; the glucose can then be fermented. Many other sugars can also be fermented. But since glucose is the starting substrate of the glycolytic pathway, these sugars have to be converted to glucose first before they enter the pathway. MINIQUIZ t Which reactions in glycolysis involve oxidations and reductions? t What is the role of NAD + /NADH in glycolysis? t Why are fermentation products made during glycolysis? a second option for catabolizing glucose—respiration—by contrasting the metabolic patterns of the common baker’s yeast, an organism that can either ferment or respire, depending on its environmental conditions. Fermentative Diversity Fermentations are classified by either the substrate fermented or the products formed, and with rare exception, all generate ATP by substrate-level phosphorylation. Table 3.4 lists some of the major fermentations of glucose on the basis of the products formed, including the production of alcohol or lactic acid, as we have just detailed. Other categories include propionic acid, mixed acid (acetic acid, formic acid, lactic acid), butyric acid, or butanol. All of the organisms listed in Table 3.4 use the glycolytic pathway to catabolize glucose, the major difference in the fermentations being in what happens to pyruvate (Figure 3.14). The mechanism for the reduction of pyruvate by each organism is what leads to the different fermentation products (Table 3.4). In addition to the two ATP produced in glycolysis, some of the fermentations listed in Table 3.4 allow for additional ATP to be formed. This occurs when the fermentation product is a fatty acid because the fatty acid is formed from a coenzyme-A precursor. Recall that CoA derivatives of fatty acids, such as acetyl-CoA, are energy-rich (Section 3.7 and Figure 3.12). Thus, when Clostridium butyricum forms butyric acid, the final reaction is Butyryl-CoA + ADP + Pi S butyric acid + ATP + CoA 3.9 Fermentative Diversity and the Respiratory Option Besides using the glycolytic pathway to ferment glucose to ethanol plus CO2, as in yeast, or to lactic acid, as in lactic acid bacteria (Figure 3.14), many other fermentative bacteria use the glycolytic pathway as a mechanism for conserving energy and generating fermentation products. We conclude our focus on fermentations by briefly considering fermentative diversity and then introduce This can significantly increase the yield of ATP from the fermentation of glucose, although the yield falls far shy of what we will see is possible in glucose respiration. Some fermentations are classified on the basis of the substrate fermented rather than the fermentation products generated, and these fermentations typically occur through pathways other than glycolysis. For instance, some endospore-forming anaerobic bacteria (genus Clostridium) ferment amino acids, the breakdown products of proteins, and others ferment purines and pyrimidines, the products Table 3.4 Common bacterial fermentations and some of the organisms carrying them out Type Reaction Alcoholic Hexosea S 2 ethanol + 2 CO2 Organisms − Yeast, Zymomonas + Homolactic Hexose S 2 lactate + 2 H Heterolactic Hexose S lactate− + ethanol + CO2 + H+ - Streptococcus, some Lactobacillus - Leuconostoc, some Lactobacillus - Propionic acid 3 Lactate S 2 propionate + acetate + CO2 + H2O Propionibacterium, Clostridium propionicum Mixed acidb,c Hexose S ethanol + 2,3-butanediol + succinate2- + lactate- + acetate− + formate- + H2 + CO2 Enteric bacteria including Escherichia, Salmonella, Shigella, Klebsiella, Enterobacter Butyric acidc Hexose S butyrate- + 2 H2 + 2 CO2 + H+ Clostridium butyricum Butanolc 2 Hexose S butanol + acetone + 5 CO2 + 4 H2 − - Clostridium acetobutylicum − + Caproate/Butyrate 6 Ethanol + 3 acetate S 3 butyrate + caproate + 2 H2 + 4 H2O + H Clostridium kluyveri Acetogenic Fructose S 3 acetate- + 3 H+ Clostridium aceticum a Glucose is the starting substrate for glycolysis. However, many other C6 sugars (hexoses) can be fermented following their conversion to glucose. b Not all organisms produce all products. In particular, butanediol production is limited to only certain enteric bacteria. The reaction is not balanced. c Other products include some acetate and a small amount of ethanol (butanol fermentation only). $ ) " 1 5 & 3 t M I C R O B I A L M E TA B O L I S M 89 Barton Spear MINIQUIZ Figure 3.15 Common food and beverage products resulting from the alcoholic fermentation of Saccharomyces cerevisiae. of nucleic acid breakdown. Some fermentative anaerobes even ferment aromatic compounds. In many cases, these fermentations are carried out by a single group of anaerobic bacteria; in a few cases, only a single bacterium is known to ferment a particular substance. These bacteria are metabolic specialists, having evolved the capacity to ferment a substrate not catabolized by other bacteria. Although they may seem to be metabolic oddballs, these and other fermentative bacteria are of great ecological importance in degrading the remains of dead plants, animals, and other microorganisms in anoxic environments in nature. We investigate the principles behind some of these unusual fermentations in Chapter 13. Saccharomyces cerevisiae : Fermentation or Respiration? During glycolysis, glucose is consumed, ATP is made, and fermentation products are generated. For the organism the crucial product is ATP; fermentation products are merely waste products. However, fermentation products are not waste products to humans. Instead, they are the foundation of the baking and fermented beverage industries (Figure 3.15) and are key ingredients in many fermented foods. In the baking and alcohol industries, the metabolic capacities of the key player, the baker’s and brewer’s yeast Saccharomyces cerevisiae, are on center stage. However, S. cerevisiae can carry out two modes of glucose catabolism, fermentation, as we have discussed, and respiration, which we will consider next. As a rule, cells carry out that form of metabolism that most benefits them energetically. The energy available from a molecule of glucose is much greater if it is respired to CO2 than if it is fermented. This is because unlike CO2, organic fermentation products such as ethanol still contain a significant amount of free energy. Thus, when O2 is available, yeast respire glucose rather t Which fermentation products are produced by Lactobacillus and which by Clostridium species? Which would you find in fermented milk products, such as yogurt? t Which yeast fermentation product is the desired agent in bread and what is its function in bread-making? 3.10 Respiration: Electron Carriers Fermentation is an anaerobic process and releases only a small amount of energy. By contrast, if pyruvate is fully oxidized to CO2 rather than reduced to some fermentation product, a far higher yield of ATP is possible. Oxidation using O2 as the terminal electron acceptor is called aerobic respiration; oxidation using other acceptors under anoxic conditions is called anaerobic respiration (Section 3.13). Our discussion of respiration covers both carbon transformations and redox reactions and focuses on two issues: (1) how electrons are transferred from the primary electron donor to the terminal electron acceptor and how this process is coupled to energy conservation, and (2) the pathway by which organic carbon is oxidized into CO2. We begin with a consideration of electron transport, the series of reactions that lead to the proton motive force. NADH Dehydrogenases and Flavoproteins Electron transport occurs in the membrane, and several types of oxidation–reduction enzymes participate in electron transport. These include NADH dehydrogenases, flavoproteins, iron–sulfur proteins, and cytochromes. Also participating are nonprotein electron carriers called quinones. The carriers are arranged in the membrane in order of increasingly more positive reduction potential, with NADH dehydrogenase first and the cytochromes last (Figure 3.9). NADH dehydrogenases are proteins bound to the inside surface of the cytoplasmic membrane and have an active site that binds NADH. The 2 e- + 2 H+ from NADH are transferred from the dehydrogenase to a flavoprotein, the next carrier in the chain. This forms NAD+ that is released from the dehydrogenase and can react with another enzyme (Figure 3.11). UNIT 1 than ferment it, and the major product is CO2 (from activities of the citric acid cycle, see Figure 3.22). Only when conditions are anoxic do yeasts switch to fermentation. This fact has practical significance. Since the brewer and baker need the products of yeast fermentation rather than yeast cells themselves, care must be taken to ensure that the yeast is forced into a fermentative lifestyle. For example, when grapes are squeezed to make wine, the yeast at first respire, making the juice anoxic. Following this, the vessel is sealed against the introduction of air and fermentation begins. Yeast also serves as the leavening agent in bread, although here it is not the alcohol that is important, but CO2, the other product of the alcohol fermentation (Table 3.4). The CO2 raises the dough, and the alcohol produced along with it is volatilized during the baking process. We discuss fermented foods in more detail in Chapter 31. F O U N D AT I O N S O F M I C R O B I O L O G Y Isoalloxazine ring Redox site Fe3+) (Fe2+ O P H3C N H3C N H H H H C C C C H OH OH OH Heme NH N O 2H H CH2 Ribitol Oxidized (FMN) O H3C N H3C N N R H COO– COO– CH2 CH2 CH2 CH2 CH3 H3C N NH O N Fe H2C N N CH3 C Reduced (FMNH2) CH3 E0′ of FMN/FMNH2 (or FAD/FADH2) = –0.22 V C H2C Cytochrome (b) (a) Figure 3.16 Flavin mononucleotide (FMN), a hydrogen atom carrier. The site of oxidation–reduction (dashed red circle) is the same in FMN and the related coenzyme flavin adenine dinucleotide (FAD, not shown). FAD contains an adenosine group bonded through the phosphate group on FMN. Flavoproteins contain a derivative of the vitamin riboflavin (Figure 3.16). The flavin portion, which is bound to a protein, is a prosthetic group (Section 3.5) that is reduced as it accepts 2 e- + 2 H+ and oxidized when 2 e- are passed on to the next carrier in the chain. Note that flavoproteins accept 2 e- + 2 H+ but donate only electrons. We will consider what happens to the 2 H+ later. Two flavins are commonly found in cells, flavin mononucleotide (FMN, Figure 3.16) and flavin adenine dinucleotide (FAD). In the latter, FMN is bonded to ribose and adenine through a second phosphate. Riboflavin, also called vitamin B2, is a source of the parent flavin molecule in flavoproteins and is a required growth factor for some organisms (Table 3.1). Cytochromes, Other Iron Proteins, and Quinones The cytochromes are proteins that contain heme prosthetic groups (Figure 3.17). Cytochromes undergo oxidation and reduction through loss or gain of a single electron by the iron atom in the heme of the cytochrome: d cytochrome ¬ Fe3+ - eCytochrome ¬ Fe2+ S Several classes of cytochromes are known, differing widely in their reduction potentials (Figure 3.9). Different classes of cytochromes are designated by letters, such as cytochrome a, cytochrome b, cytochrome c, and so on, depending upon the type of heme they contain. The cytochromes of a given class in one organism may differ slightly from those of another, and so there are designations such as cytochromes a1, a2, a3, and so on among cytochromes of the same class. Cytochromes of different classes also differ in their reduction potentials (Figure 3.9). Occasionally, cytochromes form complexes with other cytochromes or with iron–sulfur proteins. An important example is the cytochrome bc1 complex, which contains two different b-type cytochromes and one c-type cytochrome. The cytochrome bc1 complex plays an important role in energy metabolism, as we will see later. Porphyrin ring Richard Feldmann 90 6 / * 5 t T H E Figure 3.17 Cytochrome and its structure. (a) Structure of heme, the iron-containing portion of cytochromes. Cytochromes carry electrons only, and the redox site is the iron atom, which can alternate between the Fe2+ and Fe3+ oxidation states. (b) Space-filling model of cytochrome c; heme (light blue) is covalently linked via disulfide bridges to cysteine residues in the protein (dark blue). Cytochromes are tetrapyrroles, composed of four pyrrole rings. In addition to the cytochromes, in which iron is bound to heme, one or more proteins with nonheme iron typically participate in electron transport chains. These proteins contain prosthetic groups made up of clusters of iron and sulfur atoms, with Fe2S2 and Fe4S4 clusters being the most common (Figure 3.18). Ferredoxin, a common nonheme iron–sulfur protein, has an Fe2S2 configuration. The reduction potentials of iron–sulfur proteins vary over a wide range depending on the number of iron and sulfur atoms present and how the iron centers are embedded in the protein. Thus, different iron–sulfur proteins can function at different locations in the electron transport chain. Like cytochromes, nonheme iron–sulfur proteins carry electrons only. Quinones (Figure 3.19) are hydrophobic molecules that lack a protein component. Because they are small and hydrophobic, quinones are free to move about within the membrane. Like the flavins (Figure 3.16), quinones accept 2 e - + 2 H + but transfer only 2 e - to the next carrier in the chain; quinones typically participate as links between iron–sulfur proteins and the first cytochromes in the electron transport chain. Cys Fe Cys Cys Fe S S Fe S Cys Cys E0′ of iron-sulfur proteins, ~ –0.2 V S Fe S Fe Cys Fe S Cys Cys (a) (b) Figure 3.18 Arrangement of the iron–sulfur centers of nonheme iron–sulfur proteins. (a) Fe2S2 center. (b) Fe4S4 center. The cysteine (Cys) linkages are from the protein portion of the molecule. $ ) " 1 5 & 3 t M I C R O B I A L M E TA B O L I S M O CH3O C C C C CH3 C (CH2 CH O Oxidized E0′ of CoQ (ox/red) ~ 0 V CH3 C CH2)nH 2H OH CH3O C CH3O C C C C CH3 C R OH Reduced Figure 3.19 Structure of oxidized and reduced forms of coenzyme Q, a quinone. The five-carbon unit in the side chain (an isoprenoid) occurs in multiples, typically 6–10. Oxidized quinone requires 2 e - and 2 H + to become fully reduced (dashed red circles). MINIQUIZ t In what major way do quinones differ from other electron carriers in the membrane? t Which electron carriers described in this section accept 2 e- + 2 H+? Which accept electrons only? 3.11 Respiration: The Proton Motive Force The conservation of energy in respiration is linked to an energized state of the membrane (Figure 3.13b), and this energized state is established by electron transport. To understand how electron transport is linked to ATP synthesis, we must first understand how the electron transport system is organized in the cytoplasmic membrane. The electron transport carriers we just discussed (Figures 3.16–3.19) are oriented in the membrane in such a way that, as electrons are transported, protons are separated from electrons. Two electrons plus two protons enter the electron transport chain from NADH (through NADH dehydrogenase) to initiate the process. Carriers in the electron transport chain are arranged in the membrane in order of their increasingly positive reduction potential, with the final carrier in the chain donating the electrons plus protons to a terminal electron acceptor such as O2. During electron transport, H+ are extruded to the outer surface of the membrane. These originate from two sources: (1) NADH and (2) the dissociation of H2O into H+ and OH- in the cytoplasm. The extrusion of H + to the environment results in the accumulation of OH - on the inside of the membrane. However, despite their small size, neither H + nor OH - can diffuse through the membrane because they are charged and highly polar ( Section 2.8). As a result of the separation of H + and OH - , the two sides of the membrane differ in both charge and pH; this forms an electrochemical potential across the membrane. This potential, along with the difference in pH across the membrane, is called the proton motive force (pmf) and causes the membrane to be energized, much like a battery (Figure 3.13b). Some of the potential energy in the pmf is then conserved in the formation of ATP. However, besides driving ATP synthesis, the pmf can also be tapped to do other forms of work for the cell, such as transport reactions, flagellar rotation, and other energy-requiring reactions in the cell. Figure 3.20 shows a bacterial electron transport chain, one of many different carrier sequences known. Nevertheless, three features are characteristic of all electron transport chains regardless of which specific carriers they contain: (1) the carriers are arranged in order of increasingly more positive E0 ′, (2) there is an alternation of electron-only and electron-plus-proton carriers in the chain, and (3) the net result is reduction of a terminal electron acceptor and generation of a proton motive force. Generation of the Proton Motive Force: Complexes I and II The proton motive force develops from the activities of flavins, quinones, the cytochrome bc1 complex, and the terminal cytochrome oxidase. Following the oxidation of NADH + H+ to form FMNH2, 4 H+ are released to the outer surface of the membrane when FMNH2 donates 2 e- to a series of nonheme iron proteins (Fe/S), forming the group of electron transport proteins called Complex I (Figure 3.20). These groups are called complexes because each consists of several proteins that function as a unit. For example, Complex I in Escherichia coli contains 14 separate proteins. Complex I is also called NADH: quinone oxidoreductase because the overall reaction is one in which NADH is oxidized and quinone is reduced. Two H + from the cytoplasm are taken up by coenzyme Q when it is reduced by the Fe/S protein in Complex I (Figure 3.20). Complex II simply bypasses Complex I and feeds electrons from FADH2 directly into the quinone pool. Complex II is also called the succinate dehydrogenase complex because of the specific substrate, succinate (a product of the citric acid cycle, Section 3.12), that it oxidizes. However, because Complex II bypasses Complex I (where electrons enter at a more negative reduction potential), fewer protons are pumped per 2 e - that enter at Complex II than enter at Complex I (Figure 3.20); this reduces the ATP yield by one per two electrons consumed. Complexes III and IV: bc1 and a-Type Cytochromes Reduced coenzyme Q (QH2) passes electrons one at a time to the cytochrome bc1 complex (Complex III, Figure 3.20). Complex III consists of several proteins that contain two different b-type hemes (bL and bH), one c-type heme (c1), and one iron–sulfur center. The bc1 complex is present in the electron transport chain of almost all organisms that can respire and also plays a role in photosynthetic electron flow in phototrophic organisms ( Sections 13.3 and 13.4). The major function of the cytochrome bc1 complex is to move e - from quinones to cytochrome c. Electrons travel from the bc1 complex to cytochrome c, located in the periplasm. Cytochrome c functions as a shuttle to transfer e− to the high-redox-potential cytochromes a and a3 (Complex IV, Figure 3.20). Complex IV functions as the terminal oxidase and reduces O2 to H2O in the final step of the electron transport chain. Complex IV also pumps protons to the outer surface of the membrane, thereby increasing the strength of the proton motive force (Figure 3.20). UNIT 1 CH3O C 91