Download 3.7 Energy-Rich Compounds

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