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Part III => METABOLISM and ENERGY
§3.6 Oxidative Phosphorylation
§3.6a Electron Transport
§3.6b ATP Synthesis
Section 3.6a:
Electron Transport
Synopsis 3.6a
- During processes such as glycolysis and Krebs cycle, oxidation of macronutrients results in
the release of electrons that are ultimately captured in the form of reduced NADH/FADH2
- In order to recover the free energy of these electrons stored in NADH and FADH2, they are
funneled into a series of redox protein complexes collectively referred to as the “electron
transport chain (ETC)”—located within the inner mitochondrial membrane (IMM)
- ETC in turn couples the free energy of incoming electrons with the transfer of protons (H+)
across the IMM culminating with the reduction of O2 to H2O via a series of redox reactions—
the electrochemical energy stored in the resulting proton gradient across the
IMM is then utilized to synthesize ATP directly from ADP and Pi
- Simply put, the energy released from the oxidation of NADH and FADH2 (ultimately
from the nutrients) is coupled to phosphorylation of ADP directly with Pi (HPO42-) to
generate ATP in a process referred to as “Oxidative Phosphorylation”
- Oxidative phosphorylation (OP) is in stark contrast to “substrate-level phosphorylation”—
whereby the transfer of a phosphoryl group from a “high-energy” compound (eg
phosphoenol pyruvate) to ADP is used to synthesize ATP (see §3.1 and §3.2)
- Be aware that ETC and OP are often used synonymously, or sometimes considered as two
distinct processes—but, in reality, oxidative phosphorylation is ultimately an overall
consequence of ETC!
Mitochondrion: The Cell’s Power Plant
OMM harbors large non-selective channels such
as voltage-dependent anion channels /porins,
which enable facilitated diffusion of most
metabolites into the intermembrane space!
IMM is much more restrictive with respect to
the non-selective diffusion of metabolites but
harbors metabolite-specific transporters!
Both the Krebs cycle and oxidative
phosphorylation occur within the
mitochondrial matrix!
IMM  Inner (Mitochondrial) Membrane
OMM  Outer (Mitochondrial) Membrane
Oxidative Metabolism
Glycerol-3-phosphate Shuttle
IMM
- NADH and FADH2 produced during
glycolysis and Krebs cycle enter the ETC
- While glycolysis occurs in the cytosol,
Krebs cycle and ETC take place within
the mitochondrial matrix
- Glycolytic NADH cannot diffuse through
IMM but transfers its electrons to
mitochondrial FAD producing NAD+ (to
be reused in glycolysis) and FADH2
(which ultimately supplies electrons to
ETC) via the “glycerol-3-phosphate
shuttle”
Coupling of ETC to ATP Synthesis
NADH and FADH2 essentially act like miniature “batteries” in that the flow of
electrons from them “powers up” various transmembrane vehicles (eg complexes
I, III and IV) that in turn “pump” protons across the IMM against their
concentration gradient in a manner akin to active transporters (see §1.5)
Krebs
cycle
V
ETC Components: A Closer Look
ETC is primarily comprised of protein complexes I-IV,
coenzyme Q (CoQ), and cytochrome c (CytC)—all of which
are highly dynamic and move freely within the IMM
2
NADH
Krebs
cycle
2
NAD+
FADH2
FAD
The eflux of protons (H+) is associated with complexes I, III
and IV (that essentially act as proton pumps)—the free
energy captured in the resulting electrochemical proton
gradient is ultimately harnessed to synthesize ATP
ETC Components: Standard Reduction Potentials (ε°)
- ETC components are arranged such
that their ε° increases progressively
from electron injectors NADH and
FADH2 to the terminal electron
acceptor O2
H2
- ETC thus essentially acts like an
“electron gradient”—with the
electrons having the tendency to
flow “downhill” (in the direction of
increasing ε°) in a manner akin to
the rolling of a stone down a hill (or
the flow of electrons across an
electrochemical cell—see §3.1)
- The free energy resulting from the
dissipation of such electron
gradient is usurped to pump
protons across the IMM—thereby
setting up a proton gradient, which
is ultimately harnessed to
synthesize ATP
Direction of
Electron Flow
ETC Components: Points of ATP Synthesis
ε° / V
Parallel Pathways
for injecting
electrons into ETC
FADH2
FAD
Complex II
∆ε°’ = +0.085 V
∆G = -16.4 kJ.mol-1
(-0.040 V)
- NADH and FADH2 do not directly
donate electrons to O2 but rather
via a series of membrane-bound
protein complexes called I-IV
located within the IMM
- While NADH donates electrons to
Complex I, Complex II serves as an
electron acceptor for FADH2—both
cofactors ultimately donate their
electrons to coenzyme Q (CoQ)
- Electron transport from NADH and
FADH2 to O2 via complexes I-IV is an
highly exergonic process
- Free energy (∆G) of electron
transport is coupled to the
generation of a proton gradient
across the IMM
- Recall that ∆G is given by (see §3.1):
∆G = -zF∆ε
Complex I (NADH Dehydrogenase): Structure
Peripheral arm
 Electron transport
Transmembrane arm  Proton pump
Mitochondrial Matrix
IMM
Intermembrane Space
Complex I (NADH Dehydrogenase): Function
- The peripheral arm of Complex I harbors various cofactors
such as flavin mononucleotide (FMN) and nine iron-sulfur
Clusters—two conforming to [2Fe-2S] geometry (N1a
and N1b), while the rest to [4Fe-4S]—see §3.1
NADH
- By virtue of such cofactors, the peripheral arm of
Complex I facilitates the transfer of electrons from
NADH to coenzyme Q (CoQ)—also known as ubiquinone—in a
series of redox steps (from FMN via iron-sulfur clusters N1-N7)
culminating with the overall reaction:
NADH + CoQ + H+  NAD+ + CoQH2
∆ε° = +0.360V => ∆G° = -70 kJ/mol
- The reaction generates sufficient free energy to pump four
protons across the IMM—how?!
- The transfer of electrons from NADH to CoQ via the peripheral
arm of Complex I induces conformational changes within its
transmembrane arm
- Such structural transition is coupled to an alteration of hydrogen
bonding network that allows protons to “hop” across the
transmembrane arm of Complex I within the IMM from the
mitochondrial matrix to the intermembrane space, thereby
setting up a proton gradient—that will be ultimately coupled to
ATP synthesis (next section)
CoQ
Complex II (Succinate Dehydrogenase): Structure
Hydrophilic domain
(electron transport)
FAD(H2)
[2Fe-2S]
[4Fe-4S]
[3Fe-4S]
Mitochondrial
Matrix
CoQ
Heme b
Transmembrane domain
(facilitates electron transport
by directly binding to CoQ)
Intermembrane Space
IMM
Complex II (Succinate Dehydrogenase): Function
- The hydrophilic domain of Complex II harbors various cofactors such
as flavin adenine dinucleotide (FAD) and three iron-sulfur clusters
designated [2Fe-2S], [4Fe-4S], and [3Fe-4S]
H2
- Complex II catalyzes the oxidation of succinate to fumarate (Step 6
of Krebs cycle) by virtue of its succinate dehydrogenase activity coupled
with the reduction of its FAD cofactor to FADH2
- Next, Complex II facilitates the transfer of electrons from FADH2 to
CoQ—in a series of redox steps (from FADH2 via three iron-sulfur
clusters) culminating with the overall reaction:
FADH2 + CoQ  FAD + CoQH2
∆ε° = +0.085V => ∆G° = -16 kJ/mol
- This reaction does not generate sufficient free energy to pump protons across the IMM via the
transmembrane domain of Complex II—this step is however important in that it injects electrons
directly into CoQ so that the energy carried by electrons can be utilized by Complexes III and IV in
generating the proton gradient
- Importantly, the transmembrane domain directly binds to CoQ and thus ensures its proximity to
the iron-sulfur clusters which pass the electrons downhill to CoQ
- Transmembrane domain also binds heme b, which is believed to attune electron transit between
Complex II and CoQ
Complex III (Cytochrome c Reductase): Structure
Homodimeric
Complex
Mitochondrial
Matrix
CoQH2
Cytochrome bH
IMM
Transmembrane
domain
Cytochrome bL
Stigmatellin
[2Fe-2S]
Intermembrane
Space
Cytochrome c
Cytochrome c1
Complex III (Cytochrome c Reductase): Function
Q Cycle
- Complex III harbors four redox centers: cytochrome bH,
cytochrome bL, cytochrome c1, and a single [2Fe-2S] ironsulfur cluster within the so-called Rieske iron-sulfur protein
(ISP)—recall from §3.1 that cytochromes harbor heme
(Fe3+) cofactor!
- Owing to such redox centers, Complex III mediates the
transfer of electrons from CoQH2 (QH2) to cytochrome c
(CytC) via a series of redox steps—collectively referred to
as the Q Cycle—culminating with the overall reaction:
CoQH2 + 2CytC(Fe3+)  CoQ + 2CytC(Fe2+) + 2H+
∆ε° = +0.190V => ∆G° = -37 kJ/mol
CytC
- The Q cycle is critical in that while CoQH2 is a 2e- carrier,
CytC is a 1e- carrier—thus one molecule of CoQH2 reduces
TWO molecules of CytC!
- In order to facilitate the above reaction, CytC transiently
binds to Complex III on its intermembrane face lying
between IMM and OMM
- The free energy released by the above reaction is used to
pump four protons across the IMM via Complex III—how?!
- Unlike Complex I, proton pumping by Complex III is
primarily mediated by virtue of the ability of CoQ to act as
a proton carrier across the IMM
CytC
IMM
Complex IV (Cytochrome c Oxidase): Structure
Cytochrome a
Cytochrome a3
Mitochondrial
Matrix
IMM
CytC
e-
e-
Intermembrane
Space
CuA
CuB
Homodimeric
Complex
CytC
Complex IV (Cytochrome c Oxidase): Function
- Complex IV contains four redox centers: cytochrome a,
cytochrome a3, a single Cu+ ion (CuB), and a pair of Cu+
ions (CuA)—recall from §3.1 that cytochromes
harbor heme (Fe3+) cofactor!
Intermembrane Space
- Owing to such redox centers, Complex IV catalyzes
the transfer of electrons from cytochrome c (CytC)
to the terminal electron acceptor O2 via a series of
redox steps—culminating with the overall reaction:
2CytC(Fe2+) + 0.5O2 + 2H+  2CytC(Fe3+) + H2O
∆ε° = +0.580V => ∆G° = -112 kJ/mol
- The free energy released by the above reaction is
used to reinforce proton gradient across the IMM
in two ways by Complex IV:
(1) Two protons are pumped across the IMM,
thereby increasing proton concentration
in the intermembrane space
(2) Depletion of two matrix protons by supplying
them for reduction of each half-molecule of O2
- Like Complex I, proton pumping by Complex IV is mediated by
conformational changes that facilitate protons to “hop” along
the transmembrane domain to the intermembrane space
CytC
e-
e-
Redox
Centers
e-
Matrix
Exercise 3.6a
- Describe the route followed by electrons from NADH/FADH2 to O2
- Write the net equation for electron transfer from NADH to O2
- For each of the electron-transport complexes, write the overall
redox reaction
- Position the four electron-transport complexes on a graph showing
their relative reduction potentials, and indicate the path of
electron flow
- List the types of cofactors in Complexes I, II, III, and IV
- Describe the different mechanisms for translocating protons during
electron transport
Section 3.6b:
ATP Synthesis
SPINDLE
Synopsis 3.6b
- Free energy of electrons in the form of NADH/FADH2 released via ETC is
coupled to the generation of an electrochemical proton gradient across the
IMM—the electrochemical potential of such a proton gradient is
subsequently harnessed to drive ATP synthesis directly from ADP and Pi
(strictly, HPO42-) via “oxidative phosphorylation”
- Coupling the free energy stored in the proton gradient to ATP synthesis is
carried out by an enzyme called “ATP synthase”—which can be essentially
viewed as Complex V located downstream of Complexes I-IV in the ETC
- Embedded within the IMM and protruding into the mitochondrial matrix,
ATP synthase is comprised of two components:
(1) Fo component (IMM-embedded)
(2) F1 component (Matrix)
- Fo (capitalized F subscripted with small letter o not zero!) component
includes a c-ring whose rotation is driven by the dissipation of the proton
gradient and drives conformational changes in the F1 component—which in
turn catalyzes ATP synthesis by the so-called “binding change” mechanism
Coupling of ETC to ATP Synthesis
Complex V
(ATP Synthase)
Fo
F1
- Coupling the free energy stored in the proton gradient to ATP synthesis is carried out by
an enzyme called “ATP synthase”—which can be essentially viewed as Complex V located
downstream of Complexes I-IV in the ETC
- ATP synthase can also act as an ATPase—ie it can catalyze exergonic hydrolysis of ATP to
pump protons against their electrochemical gradient!
- Simply put, oxidative phosphorylation involves coupling the free energy released by the
oxidation of nutrients to directly synthesize ATP from ADP and Pi (strictly, HPO42-)
ATP Synthase: Mushroom-Like Structure
Embedded within the IMM and
protruding into the mitochondrial
matrix, ATP synthase is comprised
of two components:
(1) IMM-embedded Fo component
(2) Matrix F1 component
Fo component (a1b2c12)
- 1 a subunit
- 2 b subunits
- 12 c subunits (but may vary)
F1 component (α3β3γδε)
- 3 α subunits (catalytic)
- 3 β subunits (catalytic)
- 1 γ subunit
- 1 δ subunit
- 1 ε subunit
The b subunits together with the δ
subunit form a peripheral stalk that
tethers F1 to the a subunit of Fo—a
key feature that underscores the
rotation of c12 subunits (called cring) of Fo relative to F1
Matrix
IMM
c12
Intermembrane
space
- ATP synthase can be envisioned as a:
(1) Rotor (c12)—the C-ring
(2) Spindle (γε)—spinning inside the ….
(3) Stator (ab2δα3β3)
—so what sets the rotor spinning?
ATP Synthase: Rotary Engine
- Protons enter via a hydrophilic channel located
between the a-subunit and the c-ring at the
Stator
intermembrane space:
(1) Proton binding to one of the 12 c-subunits causes
a conformational change that makes the c-ring
rotate counter-clockwise (as viewed from the
matrix) by one c-subunit
(2) Binding of protons to successive c-subunits
makes the c-ring spin smoothly and continuously IMM
Spindle
Matrix
- Such rotary action converts electrochemical energy
stored in the proton gradient across IMM—a form of
potential energy or the proton motive force (pmf)—
into mechanical energy of the spinning rotor
- Spinning of the rotor is coupled to ATP synthesis
occurring at the interface of the α3β3 catalytic
subunits of F1 component (by virtue of yet another
conformational change within α3β3)—the
mechanical energy is ultimately converted back to
chemical energy!
c-ring
Rotor
Intermembrane
space
Hydrophilic channel
ATP Synthase: Basis of Rotation
- The conformational change that spins the rotor is
driven by a mutual attraction between a cationic
arginine on the a-subunit and an anionic
aspartate on the c-subunit—essentially a “salt
bridge” or an “ion pair”
- As the proton concentration rises in the
intermembrane space (thanks to ETC), such
union of opposites is disrupted by the entry
of a proton—since it competes with the arginine
for binding to the aspartate—via the hydrophilic
channel
- Upon binding to aspartate, the proton induces a
structural change within the c-subunit that drives
it in a counter-clockwise manner (as viewed from
the matrix side) so as to allow the next c-subunit
to engage in an arginine-aspartate “bridge” with
the stationary a-subunit
Stator
Spindle
IMM
Matrix
c-ring
- The above process is repeated to drive the rotary
action of the c-ring in a continuous manner until
the proton gradient is fully discharged
- How many protons are required to drive one full
turn of the rotor (c-ring)?!
Rotor
Intermembrane
space
Hydrophilic channel
ATP Synthase: In Action
- One full turn of the rotor (c-ring)
requires 12 protons—one for each
c-subunit!
- Each full turn produces 3 ATP
molecules—one by each of the
three αβ catalytic protomers of
α3β3 subunits of F1 component
- Simply put, 3/12 (or 0.25) ATP
molecules are produced for the
discharge of every proton during
oxidative phosphorylation
- How is the spinning of the rotor
coupled to ATP synthesis occurring
at the interface of the α3β3 catalytic
subunits of F1 component?
- Enter the “binding change”
mechanism
ATP Synthase: Binding Change Mechanism
In the so-called “binding change” mechanism, each of the three αβ catalytic protomers
of the α3β3 subunits of F1 component is envisioned to adopt three distinct conformations
designated O, L and T that are in equilibrium exchange with each other:
O  catalytically-inactive / low affinity for ligands (ATP, ADP, Pi)
L  catalytically-inactive / moderate affinity for ligands
T  catalytically-active / high affinity for ligands
(1) In the absence of the spinning action of the rotor (c12γε), the αβ protomer largely equilibrates
between the O and L states—it cannot synthesize ATP from ADP and Pi—with the latter being able to
accommodate ADP/Pi with moderate affinity
(2) Upon the spinning action of the rotor (c12γε), the free energy released shifts the conformational
equilibrium of the αβ protomers from the L state to the catalytically-active T conformation, enabling it
to “stick” together ADP and Pi to generate ATP
(3) Upon the synthesis of ATP, the T state undergoes conformational change to O state (with low affinity
for ligands), thereby releasing ATP and enabling the αβ protomer to return to its initial state in order to
undergo another catalytic cycle
ATP Synthesis Via OP: Oxidation of Carbs
Free energy of electron transport from
NADH/FADH2 drives ATP synthesis
NADH Oxidation
10 H+/NADH pumped across the IMM
10 H+/NADH * 0.25 ATP/H+ => 2.5 ATP/NADH
FADH2 Oxidation
6 H+/FADH2 pumped across the IMM
6 H+/FADH2 * 0.25 ATP/H+ => 1.5 ATP/FADH2
Aerobic Conditions
Glycolysis
Acetyl-CoA Synthesis
Krebs Cycle
Total
=> 7 ATP/glucose
=> 5 ATP/glucose
=> 20 ATP/glucose
=> 32 ATP/glucose
Anaerobic Conditions
Glycolysis
Acetyl-CoA Synthesis
Krebs Cycle
Total
=> 2 ATP/glucose
=> 0 ATP/glucose
=> 0 ATP/glucose
=> 2 ATP/glucose
ATP Synthesis Via OP: Oxidation of Fats
Palmitic Acid (16:0)
Palmitoyl-CoA
6
7 FADH2
ETC
10.5 ATP
7 NADH
ETC
17.5 ATP
β-Oxidation
- Palmitic acid is a saturated fatty acid harboring
16 carbon atoms (16:0)
- It is the most commonly occurring fatty acid in
living organisms
8 Acetyl-CoA
- So how much energy does β-oxidation of a single
chain of palmitic acid (16 C atoms) generate?
- Complete degradation of palmitic acid would
require 7 rounds of β-oxidation producing 7
FADH2, 7 NADH and 8 acetyl-CoA—the final
round produces 2 acetyl-CoA!
- Further oxidation of each acetyl-CoA via the
Krebs cycle produces 3 NADH, 1 FADH2 and 1
GTP (enzymatically converted to ATP) per
molecule (and there are 8 acetyl-CoA!)—see §3.5
- Oxidation of each NADH and FADH2 via the ETC
respectively produces 2.5 and 1.5 molecules of
ATP—as noted in §3.4
Krebs
cycle
Fat Is hypercaloric!
24 NADH
ETC
60 ATP
8 FADH2
ETC
12 ATP
8 GTP
8 ATP
Total Energy = 108 ATP
ATP Comparison: Carbs vs Fats
Palmitate
Glucose
6
but:
thus:
Mr = 256 g.mol-1
NA = 6x1023 mol-1
m = 256 g.mol-1 / 6x1023 mol-1
m = 43x10-23 g
but:
thus:
Complete Oxidation via the Krebs cycle
Glucose => 32 ATP
=> 32 ATP / 30x10-23 g
Plamitate => 108 ATP
=> 108 ATP / 43x10-23 g
Mr = 180 g.mol-1
NA = 6x1023 mol-1
m = 180 g.mol-1 / 6x1023 mol-1
m = 30x10-23 g
=> 1.07x1023 ATP/g
=> 2.51x1023 ATP/g
- Fats generate more than two-fold greater ATP per unit mass compared to
carbs—ie they serve as more energy-efficient fuels
- The energy-efficient nature of fats in particular is not lost on heart (virtually
devoid of glycogen reserves)—an organ that primarily uses fats to meet its
energy needs (see §3.1)
Exercise 3.6b
- Explain why an intact and impermeable IMM is essential for ATP
synthesis
- Describe the overall structure of the F1 and F0 components of ATP
synthase. Which parts move? Which are stationary? Which are mostly
stationary but undergo conformational changes?
- Summarize the steps of the binding change mechanism
- Describe how protons move from the intermembrane space into the
matrix. How is proton translocation linked to ATP synthesis?