Download Succinate

CHAPTER 17 - ELECTRON TRANSPORT AND OXIDATIVE
PHOSPHORYLATION
Introduction
- The complete aerobic catabolism of glucose can be represented as:
C6H12O6 + 6 O2 6 6 CO2 + 6 H2O
- The reactions of glycolysis and the citric acid cycle have resulted in the degradation of glucose
to CO2, but the electrons have been transferred to the redox coenzymes NAD+ and FAD:
Glucose
2 Glyceraldehyde 3-phosphate
2 1,3-bisphosphoglyerate
2
2
NAD+
NADH
Pyruvate
NAD+
2
2
NADH
Acetyl CoA
Citrate
Oxaloacetate
2 NADH
Isocitrate
2 NAD+
2 NAD+
2 NADH
Alpha ketoglutarate
Malate
Succinate
2 NADH
Succinyl CoA
Fumarate
2 FADH2
Succinate
2 FAD
2 NAD+
C6H12O6 + 10 NAD+ + 2 FAD 6 10 NADH + 2 FADH2 + 6 CO2
- We now need to study how these reduced electron carriers transport their electrons through
the mitochondrial electron transport system (electron transport) to the ultimate electron acceptor, O2,
thereby generating sufficient energy to synthesize ATP (oxidative phosphorylation).
- Since the transfer of electrons occurs with a positive DeltaE, the resulting free energy change
will be negative (DeltaG - nFDeltaE). This free energy is stored in a proton gradient which is then used
to drive a membrane-bound ATP-ase in reverse (chemiosmotic principle):
H+
Out
NADH
O2
Electron transport system
+
NAD
H2O
FADH2 FAD
In
ADP + Pi
H+
ATP
H+
- The overall DeltaE0' for electron transport can be determined from the standard reduction
potentials for NAD+ and O2:
- (NAD+ + H+ + 2 e- 6 NADH, DeltaE0' = -0.315 Volts)
½ O2 + 2 H+ + 2 e- 6 H2 O, DeltaE0' = 0.815 Volts
NADH + ½ O2 + H+ 6 H2O + NADH, DeltaE0' = -(-0.315 V) + 0.815 V = 1.130 V
DeltaG0' = -nF DeltaE0' = -(2)(96,485 J/(V mole))(1.130 V) = -218,000 J/ mole = - 218kJ/mole
- Electron transport and oxidative phosphorylation take place in the mitochondria. See text,
pp, 493-4 for description (Inner and outer membranes, matrix, cristae).
Mitochondrial Electron Transport
- Note that since electron transport takes place in the mitochondria, the NADH produced
during glycolysis must be transported into the matrix. Since the inner membrane is impermeable to
NADH only the reducing power of NADH can enter, via either the malate (Figure 15-27) or glycerol
phosphate (Figure 17-4) shuttles. In the latter case cytosolic NADH is exchanged for FADH2 which,
as we will see, results in the formation of fewer ATP than is the case for NADH. Note from Table 13-
3, p. 373, that the standard reduction potential for FAD is less negative than
for NAD+ (-0.219 vs. -0.315 V). This means that NADH has a more positive oxidation potential and
hence is a better reducing agent than FADH2, thus yields more energy when it coughs up its electrons,
resulting in greater ATP production.
Types of electron transport components:
- Cytochromes contain heme groups (like hemoglobin and myoglobin), whose Fe atoms
reversibly cycle between the ferrous (+2) and ferric (+3) states. See p. 505
- Fe-S proteins also contain Fe which cycles between the +2 and +3 states. In these proteins
Fe is coordinated to either organic (cysteine ) or elemental sulfur. See Figure 17-9 for a picture of an
iron-sulfur cluster, even though this figure depicts ferredoxin, a bacterial protein.
- Flavin (FMN)-based dehydrogenase
- Coenzyme Q (CoQ)
H
O
OH
H3 CO
+
CH3
H3 CO
H
(H2C CH C CH2 )10 H
H3 CO
CH3
H3 CO
O
(H2C CH C CH2 )10H
OH
Reduced
Oxidized
Note that CoQ is the only non-protein component of the electron transport chain. A two-electron
transfer is depicted above. The long isoprenoid tail confers lipid solubility to CoQ. The reduced form is
capable of losing a single H atom (H+ + e-) to form a semiquinone radical:
OH
O
H3 CO
H3 CO
CH3
H3 CO
H2C
(
CH C CH2
OH
-H -e
)10 H
CH3
H3 CO
H2C
(
CH C CH2
OH
Note also that since components of the electron transport chain are for the most part either one
)10H
(cytochromes) or two (NADH, FADH2) electron carriers, the ability of CoQ to function as either a oneelectron or two-electron carrier allows it to act as a “go-between” when passing electrons from the twoelectron carriers and one-electron carriers (see, for example, Figure 17-13).
- The sequence of electron transport components is governed by the necessity that each transfer
occur spontaneously, i.e., with a positive DeltaE (and - DeltaG). We have already seen that when
determining the direction of spontaneous electron transfer between two components whose half cell
standard reduction potentials are available (see Table 13-3, p. 373), the electron donor is the
component whose standard reduction potential is the more negative, or the less positive, of the two.
Thus, for example, ubiquinol will donate electrons spontaneously (under standard conditions) to cyt b
which will in turn donate spontaneously to cyt c1, and so on.
- These various components are arranged into 4 large, immobile complexes, labeled complex I
through complex IV (see Figure 17-8). Note that various respiratory inhibitors block the flow of
electrons at various points (see Figure 17-7). These inhibitors include rotenone, a plant toxin, amytal, a
barbiturate antimycin A and cyanide. Note also that CoQ and cytochrome c are mobile carriers which
shuttle electrons from complexes I and II to complex III and from complex III to complex IV,
respectively. Cytochrome c is the only water soluble component of the electron chain. Cytochrome c,
present in all organisms with mitochondrial electron transport chains, evolved more than 1.5 billion years
ago before the divergence of plants and animals. Its function has been preserved throughout this period.
Cytochrome c of any eukaryotic species interacts in vivo with complex IV of any other species tested
thus far, and the reduction potential of all cytochrome c’s is about +0.25 V. Evidently, nature stumbled
on a very efficient structure/function relationship early in the evolutionary development of cytochrome c
and stuck with it.
ATP Synthesis
- By use of Table 13-3, the potential drops across complexes I, III and IV correspond to
sufficiently large and negative free energy changes to make ATP (see Figure 17-7). NADH is a strong
enough reducing agent to pass electrons into the electron transport chain from complexes I to III to IV.
Since FADH2 is not a powerful enough reducing agent to pass electrons through complex I and must
therefore pass electrons in at complex III, bypassing complex I. From an energetic viewpoint, this is why
NADH is “worth” 3 ATP, whereas FADH2 is only worth 2.
- Recall that substrate-level phosphorylations involved high-energy intermediates, such as 1,3bisphosphoglycerate and succinyl CoA. Despite attempts to isolate similar high-energy intermediates
involved in oxidative phosphorylation, none were ever found. Peter Mitchell proposed the
chemiosmotic theory in 1961, and this was eventually accepted. According to this theory, the free
energy of electron transport is conserved by pumping protons (H+) from the mitochondrial matrix to the
intermembrane space (cytosolic side of inner membrane) to create an electrochemical proton gradient
across the inner mitochondrial membrane. Protons then flow back down this gradient through a
membrane-bound ATP-ase (ATP synthase), the free energy released driving the ATP-ase in reverse,
i.e., in the direction of ATP synthesis (see Figure 17-18).
- Although somewhat esoteric, this theory seems more plausible when it is noted that some of the
electron-transport components give off protons when oxidized:
NADH 6 NAD+ + 2e- + H+
CoQH2 6 CoQ + 2 e- + 2 H+
- A favorable membrane potential, (DeltaØ = 0.168V, negative on the inside) also favors proton
flow back into the matrix. This potential is partly due to the fact that the inner leaflet of the membrane
contains acidic phospholipids such as phosphatidyl serine and phosphatidyl ethanolamine, whose head
groups have a formal negative charge, and partly due to the fact that the inner mitochondrial membrane is
impermeable to small ions such as Cl -, which thus cannot equalize the charge imbalance due to proton
pumping. The pH difference across the inner mitochondrial membrane has been measured at 0.75 pH
units. This corresponds to the following free energy change:
DeltaG = DeltaG0' + RT ln Q + ZF DeltaØ = 2.3 RT log Q + DeltaØ
= 2.3RT(log[H+]in - log[H+]out) + DeltaØ = 2.3RT(pHout - pHin) + DeltaØ
= 2.3(8.3145 J/mole)(310 K)(-0.75) + (1)(96485 J/(mole V)(-0.168V)
= -4.45 kJ - 16.21 kJ = -20.7 kJ per mole of protons.
- Since the estimated DeltaG required for ATP synthesis (from ADP) is +40 - +50 kJ/mole
under physiological conditions, at least two moles of protons must be pumped back inside to provide
enough free energy to synthesize 1 mole of ATP.
- ATP synthase is a multi-subunit transmembrane protein consisting of two functional units, Fo (o
for oligomycin, which blocks proton flow through the channel) and F1. Fo is the water-insoluble
membrane channel through which protons flow. F1 is a water-soluble peripheral protein which forms
“knobs” which protrude into the matrix and which contain the ATP-ase activity (see Figure 17-19).
Recall that no high-energy intermediates such as PEP are known to be involved in oxidative
phosphorylation. Instead, available evidence supports the so-called binding charge mechanism,
according to which flow of protons through Fo down a concentration gradient provides free energy
which is transmitted to F1 in the form of conformational changes which result in the synthesis of ATP.
- The existence of uncoupling agents, such as 2,4-dinitrophenol, which uncouple electron
transport from oxidative phosphorylation, lends support to the chemiosmotic theory (see Figure 17-22).
Control of Oxidative Phosphorylation
- The cytochrome reductase (system IV) reaction is the only component of the electron
transport chain that is noticeably exergonic. Electron exchange between components “upstream” of
system IV, or from NADH to cyt c, are close to equilibrium (DeltaG0' -0):
1/2 NADH + cytochrome c (Fe+3 ) + ADP + P i
+
Keq =
[NAD ]
[NADH]
1/2 NAD+ + cytochrome c (Fe+2 ) + ATP
1/2 [cyt c(Fe+2)]
[ATP]
[cyt c)Fe )] [ADP][Pi}
+3
Since cytochrome reductase receives electrons from cyt c (Fe+2), the above equilibrium expression can
be rearranged to express the availability of substrate (i.e., electrons from cyt c (Fe+2)) from points
“upstream”:
[cyt c(Fe +2)]
+3 = Keq
[cyt c)Fe )]
[NADH]
+
[NAD ]
1/2
[ADP][P i}
[ATP]
The higher this ratio, the greater the flow of electrons through system IV, and vice versa. In times of
energy need (low [ATP], high [ADP]), electron flow can thus be seen to increase through system IV,
thereby increasing ATP production. Similarly, high [NADH], or reducing power, also increases electron
flow.
- Since glycolysis and the citric acid cycle supply the reducing power (NADH and FADH2) to
feed electrons through electron transport, an adequate supply of electrons for electron transport is
provided by regulation of the control points of glycolysis and the citric acid cycle. We know that high
energy has an inhibitory effect on these pathways (high [ATP] inhibits glycolysis and also the citric acid
cycle to a certain extent (ATP inhibits á-ketoglutarate dehydrogenase); high [NADH] inhibits the citric
acid cycle (ATP is an allosteric inhibitor, NADH acts by product inhibition). Thus, ATP and NADH
ultimately inhibit the flow of electrons through electron transport, which subsequently reduces the rate of
oxidative phosphorylatio (ATP production). See Figure 17-23 for a summary of this coordinated
control. Note that from the above discussion NADH is seen both to increase the flow of electrons
through system IV, and to inhibit the citric acid cycle. This is not as inconsistent as it seems. NADH
provides a direct source of electrons for electron transport, hence will increase the rate of electron
transport. At the same time, by inhibiting the citric acid cycle, it inhibits further production of NADH.
Problems: 2, 3, 4 (see Figure 17-7 for the effects of amytal on electron transport), 7, 9