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Transcript
Principles of
BIOCHEMISTRY
Third Edition
HORTON
MORAN
Prentice Hall c2002
OCHS
Chapter 14
RAWN
SCRIMGEOUR
1
Chapter 14 - Electron Transport and
Oxidative Phosphorylation
• The cheetah, whose capacity for aerobic
metabolism makes it one of the fastest animals
Prentice Hall c2002
Chapter 14
2
14.1 Superoxide Anions
• Superoxide anions (.O2-) (a by-product of
oxygen metabolism) can cause cell damage
• Superoxide dismutase catalyzes the very fast
dismutation of 2 superoxide anions
2 .O2- + 2H+
H2O2 + O2
• Catalase decomposes the hydrogen peroxide
2 H2O2
Prentice Hall c2002
2 H2O + O2
Chapter 14
3
Fig 14.1 Coupled electron transport
reactions catalyzed by NADPH oxidase
• Superoxide anions can be formed by an NADPHdependent enzyme in plasma membranes (via a
short electron transport chain shown below)
NADPH + 2 O2
NADP+ + 2 .O2- + H+
2 O2
. -
2 O2
Prentice Hall c2002
Chapter 14
4
14.2 Cytochrome P450
• Cyt P450 is a hydroxylase (monooxygenase)
that catalyzes the incorporation of an oxygen
atom (derived from O2) into an organic molecule
• Involved in many reactions: hydroxylations,
oxidations, epoxidations, dealkylations
• Xenobiotics (foreign compounds such as drugs,
anaesthetics, dyes pesticides) are substrates for
cytochrome P450
Prentice Hall c2002
Chapter 14
5
Eukaryotic cytochrome P450
• Cyt P450 is part of a two enzyme sequence
that includes a flavoprotein dehydrogenase
• Dehydrogenase contains both FMN and FAD
• Overall reaction: hydroxylation of substrate RH
RH + O2 + NADPH + H+
Prentice Hall c2002
ROH + H2O + NADP+
Chapter 14
6
Fig 14.2 Mammalian cytochrome P450
is an integral membrane protein
• Globular heme
domain (blue shere)
• One or two
transmembrane
peptides
Prentice Hall c2002
Chapter 14
7
14.3 Hydrogenase and Fumarate Reductase
Fig 14.3 Sugar fermentation by C. pasteurianum.
The transfer of electrons from reduced ferridoxin
(Fdred) to H+ is catalyzed by hydrogenase
Prentice Hall c2002
Chapter 14
8
Fumarate Reductase
• E. coli can use fumarate as a terminal electron
acceptor when growing anaerobically
• Fumarate reductase reaction is reverse of
succinate dehydrogenase reaction
• Oxidation-reduction enzyme cofactors are
aligned in the sequence below: (QD and QP are
menaquinone molecules)
QD-QP-[3Fe-4S]-[4Fe-4S]-[2Fe-2S]-FAD
Prentice Hall c2002
Chapter 14
9
Fig 14.4 Menaquinone (abbreviated Q)
Prentice Hall c2002
Chapter 14
10
14.4 Oxidative Phosphorylation
in Mitochondria
• Reduced coenzymes NADH and QH2 from:
(1) Aerobic oxidation of pyruvate by the citric
acid cycle
(2) Oxidation of fatty acids and amino acids
• Oxidative phosphorylation is the process by
which NADH and QH2 are oxidized and ATP is
formed
Prentice Hall c2002
Chapter 14
11
Mitochondrial oxidative phosphorylation
(1) Respiratory electron-transport chain (ETC)
Series of enzyme complexes embedded in
the inner mitochondrial membrane, which
oxidize NADH and QH2. Oxidation energy is
used to transport protons creating a proton
gradient
(2) ATP synthase uses the proton gradient
energy to produce ATP
Prentice Hall c2002
Chapter 14
12
Overview of oxidative phosphorylation
Fig 14.5
Prentice Hall c2002
Chapter 14
13
14.5 The Mitochondrion
• Final stages of aerobic oxidation of biomolecules
in eukaryotes occur in the mitochondrion
• Site of citric acid cycle and fatty acid oxidation
which generate reduced coenzymes
• Contains electron transport chain to oxidize
reduced coenzymes
Prentice Hall c2002
Chapter 14
14
Fig 14.6 Structure of the mitochondrion
Prentice Hall c2002
Chapter 14
15
Fig 14.6 (continued)
Prentice Hall c2002
Chapter 14
16
Location of mitochondrial complexes
• Inner mitochondrial membrane:
Electron transport chain
ATP synthase
• Mitochondrial matrix:
Pyruvate dehydrogenase complex
Enzymes of the citric acid cycle
Enzymes catalyzing fatty acid oxidation
Prentice Hall c2002
Chapter 14
17
14.6 The Chemiosmotic Theory
• Proposed by Peter Mitchell in the 1960’s
(Nobel Prize 1978)
• Chemiosmotic theory:
A proton concentration gradient serves
as the energy reservoir for driving ATP
formation
Prentice Hall c2002
Chapter 14
18
Respiration by mitochondria
• Oxidation of substrates is coupled to the
phosphorylation of ADP
• Respiration (consumption of oxygen)
proceeds only when ADP is present
• The amount of O2 consumed depends
upon the amount of ADP added
Prentice Hall c2002
Chapter 14
19
Fig 14.7 Coupled nature of respiration
in mitochondria
(a) O2 consumed only with ADP, excess Pi
(b) (+) Uncoupler DNP (O2 consumed without ADP)
Prentice Hall c2002
Chapter 14
20
Uncouplers
• Uncouplers stimulate the oxidation of
substrates in the absence of ADP
• Uncouplers are lipid-soluble weak acids
• Both acidic and basic forms can cross the inner
mitochondrial membrane
• Uncouplers deplete any proton gradient by
transporting protons across the membrane
Prentice Hall c2002
Chapter 14
21
Fig 14.8 2,4-Dinitrophenol: an uncoupler
• Because the negative charge is delocalized over the
ring, both the acid and base forms of DNP are
hydrophobic enough to dissolve in the membrane
Prentice Hall c2002
Chapter 14
22
Mitchell’s postulates for chemiosmotic theory
1. Intact inner mitochondrial membrane is
required (to maintain a proton gradient)
2. Electron transport through the ETC generates
a proton gradient (pumps H+ from the matrix
to the intermembrane space)
3. The membrane-spanning enzyme, ATP
synthase, catalyzes the phosphorylation of
ADP in a reaction driven by movement of H+
across the inner membrane into the matrix
Prentice Hall c2002
Chapter 14
23
14.7 The Protonmotive Force
• Protonmotive force (Dp) is the energy of the
proton concentration gradient
• Protons that are translocated into the
intermembrane space by electron transport, flow
back into the matrix via ATP synthase
• H+ flow forms a circuit (similar to an electrical
circuit)
Prentice Hall c2002
Chapter 14
24
Fig 14.9 Analogy of electromotive
and protonmotive force
Prentice Hall c2002
Chapter 14
25
Free-energy change from proton movement
1. Chemical contribution
DGchem = nRT ln ([H+]in / [H+]out)
(n = number of protons translocated)
2. Electrical contribution: Dy=membrane potential
DGelect = zFDy
(z = charge (1.0 for H+), F =96,485 JV-1mol-1 )
Prentice Hall c2002
Chapter 14
26
Protonmotive force (Dp)
• From the two previous equations the
protonmotive force (Dp) is:
Dp = Dy - (0.059 V) DpH
• Dy = difference in charge across the
membrane (V) in volts (DY = DYin - DYout)
• DpH = pHin - pHout
Prentice Hall c2002
Chapter 14
27
14.8 Overview of Electron Transport
• Five oligomeric assemblies of proteins
associated with oxidative phosphorylation are
found in the inner mitochondrial membrane
• Complexes I-IV contain multiple cofactors, and
are involved in electron transport
• Complex V is ATP synthase
Prentice Hall c2002
Chapter 14
28
Table 14.1
Prentice Hall c2002
Chapter 14
29
A. Complexes I-IV
• Electrons flow through the ETC components in
the direction of increasing reduction potentials
NADH (strong reducing agent, Eo’ = -0.32 volts)
O2 (terminal oxidizing agent, Eo’ = +0.82 volts)
• Mobile coenzymes: ubiquinone (Q) and
cytochrome c serve as links between ETC
complexes
• Complex IV reduces O2 to water
Prentice Hall c2002
Chapter 14
30
Fig 14.10 Mitochondrial electron transport
Prentice Hall c2002
Chapter 14
31
Table 14.2
Prentice Hall c2002
Chapter 14
32
Table 14.3
Prentice Hall c2002
Chapter 14
33
Inhibitors can block electron transfer
through specific complexes in the ETC
• Complex I: blocked by rotenone
• Complex III: blocked by antimycin A
• Complex IV: blocked by cyanide
Prentice Hall c2002
Chapter 14
34
B. Cofactors in Electron Transport
• Electrons enter the ETC two at a time via NADH
• Flavin coenzymes are then reduced
(Complex I) FMN
FMNH2
(Complex II) FAD
FADH2
• FMNH2 and FADH2 donate one electron at a time
• All subsequent steps proceed by one e- transfers
Prentice Hall c2002
Chapter 14
35
Mobile electron carriers
1. Ubiquinone (Q)
Q is a lipid soluble molecule that diffuses within
the lipid bilayer, accepting electrons from I and
II and passing them to III
2. Cytochrome c
Associated with the outer face of the
membrane, transports electrons from III to IV
Prentice Hall c2002
Chapter 14
36
14.9 Complex I
• NADH-ubiquinone oxidoreductase (NADH
dehydrogenase)
• Transfers electrons from NADH to Q
• NADH transfers a two electrons as a hydride ion
(H:-) to FMN
• Electrons are passed through Complex I to Q via
FMN and iron-sulfur proteins
Prentice Hall c2002
Chapter 14
37
Fi. 14.11 Electron transfer and proton
flow in Complex I
• Reduction of Q to QH2 requires 2 e• About 4 H+ translocated per 2 e- transferred
Prentice Hall c2002
Chapter 14
38
14.10 Complex II
• Succinate-ubiquinone oxidoreductase
(or succinate dehydrogenase complex)
• Accepts electrons from succinate and catalyzes
the reduction of Q to QH2
• FAD of II is reduced in a 2-electron transfer of a
hydride ion from succinate
Prentice Hall c2002
Chapter 14
39
Fig 14.12 Electron transfer in Complex II
• Complex II does not contribute to proton
gradient, but supplies electrons from succinate
Prentice Hall c2002
Chapter 14
40
14.11 Complex III
• Ubiquinol-cytochrome c oxidoreductase
• Transfers electrons to cytochrome c
• Oxidation of one QH2 is accompanied by the
translocation of 4 H+ across the inner
mitochondrial membrane
• Two H+ are from the matrix, two from QH2
Prentice Hall c2002
Chapter 14
41
Fig 14.13 Electron transfer and proton
flow in Complex III
• Four H+ are translocated, two from the matrix and
two from QH2
Prentice Hall c2002
Chapter 14
42
Proposed Q cycle.
• Proposal for electron movement between
between QH2 and cytochrome c in Complex III
• One e- transferred to cytochrome c via the
Fe-S protein
• Second e- transferred to cytochrome b then Q
• Three forms of Q are involved: QH2, Q and the
semiquinone anion .QPrentice Hall c2002
Chapter 14
43
Fig 14.14 Q cycle: Step 1
Prentice Hall c2002
Chapter 14
44
Q cycle: Step 2
Prentice Hall c2002
Chapter 14
45
Q cycle: Step 3
Prentice Hall c2002
Chapter 14
46
14.12 Complex IV
• Cytochrome c oxidase
• Catalyzes a four-electron reduction of
molecular oxygen (O2) to water (H2O)
• Source of electrons is cytochrome c
(links Complexes III and IV)
• Translocates H+ into the intermembrane space
Prentice Hall c2002
Chapter 14
47
Fig 14.15 Electron transfer and proton
flow in Complex IV
• Iron atoms (hemes of cyt a) and copper atoms are
both reduced and oxidized as electrons flow
Prentice Hall c2002
Chapter 14
48
Complex IV contributes to the proton gradient
Net effect is transfer of four H+ for each pair of eO2 + 4 e- + 4H+
2 H 2O
1. Proton translocation of 2 H+ for each pair of
electrons transferred (each O atom reduced)
2. Formation of H2O removes 2H+ from the
matrix (contributes to Dp even though no
proton translocation occurs)
Prentice Hall c2002
Chapter 14
49
Fig 14.16 Proposed mechanism for reduction
of molecular oxygen by cytochrome oxidase
Prentice Hall c2002
Chapter 14
50
14.13 Complex V: ATP Synthase
• F0F1 ATP Synthase uses the proton gradient
energy for the synthesis of ATP
• An F-type ATPase which generates ATP
• Composed of a “knob-and-stalk” structure
• F1 (knob) contains the catalytic subunits
• F0 (stalk) has a proton channel which spans the
membrane.
Prentice Hall c2002
Chapter 14
51
ATP Synthase components
• Passage of protons through the Fo (stalk) into
the matrix is coupled to ATP formation
• Estimated passage of 3 H+ / ATP synthesized
• Fo is sensitive to oligomycin, an antibiotic that
binds in the channel and blocks H+ passage,
thereby inhibiting ATP synthesis
Prentice Hall c2002
Chapter 14
52
Structure of ATP synthase
• F1 knobs: inner face of the inner mitochondrial
membrane (subunit composition: a3b3gde)
• Fo subunit composition: a1b2c9-12
(c subunits form cylindrical, membrane-bound
base)
• a3b3 oligomer of F1 is connected to c subunits
by a multisubunit stalk of g and e chains
Prentice Hall c2002
Chapter 14
53
Knob-and-stalk structure
of ATP synthase
Prentice Hall c2002
Chapter 14
54
Prentice Hall c2002
Chapter 14
55
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Prentice Hall c2002
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Prentice Hall c2002
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Mechanism of ATP Synthase
• There are 3 active sites, one in each b subunit
• The c-e-g unit forms a “rotor”
• Rotation of the g subunit inside the a3b3
hexamer causes domain movements in the
b-subunits, opening and closing the active sites
Prentice Hall c2002
Chapter 14
61
ATPase mechanism (continued)
• The a-b-d-a3b3 unit is the “stator” (the Fo
channel is attached to a3b3 by the a-b-d arm)
• Passage of protons through the Fo channel
causes the rotor to spin in one direction and
the stator to spin in the opposite direction
Prentice Hall c2002
Chapter 14
62
Fig 14.17 (b)
Prentice Hall c2002
Chapter 14
63
Fig 14.17 (c)
• Molecular
structure of
rotor and
stator (part)
of ATP
synthase
Prentice Hall c2002
Chapter 14
64
Fig 14.18 Binding-change mechanism
of ATP synthase
1. ADP, Pi bind to an open site
2. Inward passage of protons, conformation change,
ATP synthesis from ADP and Pi
3. ATP released from open site, ADP and Pi form ATP
in the tight site
Prentice Hall c2002
Chapter 14
65
Prentice Hall c2002
Chapter 14
66
Experimental observation of ATP
synthase rotation
• Fluorescent protein
arm (actin) attached
to g subunits
• a3b3 subunits bound
to a glass plate
• Arm seen rotating
when MgATP added
Prentice Hall c2002
Chapter 14
67
Prentice Hall c2002
Chapter 14
68
Prentice Hall c2002
Chapter 14
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14.14 Active Transport of ATP, ADP and Pi
Across the Mitochondrial Membrane
• ATP is synthesized in the mitochondrial matrix
• ATP must be transported to the cytosol, and ADP
and Pi must enter the matrix
• ADP/ATP carrier exchanges mitochondrial ATP4for cytosolic ADP3-
• The exchange causes a net loss of -1 in the
matrix (draws some energy from the H+ gradient)
Prentice Hall c2002
Chapter 14
70
Fig 14.20 Transport of ATP, ADP and Pi
across the inner mitochondrial membrane
• Adenine nucleotide translocase: unidirectional
exchange of ATP for ADP (antiport)
• Symport of Pi and H+ is electroneutral
Prentice Hall c2002
Chapter 14
71
14.15 The P:O Ratio
molecules of ADP phosphorylated
P:O ratio = ----------------------------------------atoms of oxygen reduced
• Translocation of 3H+ required by ATP synthase for
each ATP produced
• 1 H+ needed for transport of Pi, ADP and ATP
• Net: 4 H+ transported for each ATP synthesized
Prentice Hall c2002
Chapter 14
72
Calculation of the P:O ratio
Complex I
III
IV
#H+ translocated/2e4
2
4
Since 4 H+ are required for each ATP synthesized:
For NADH: 10 H+ translocated / O (2e-)
P/O = (10 H+/ 4 H+) = 2.5 ATP/O
For succinate substrate = 6 H+/ O (2e-)
P/O = (6 H+/ 4 H+) = 1.5 ATP/O
Prentice Hall c2002
Chapter 14
73
14.16 Aerobic Oxidation of Cytosolic NADH
• Cytosolic NADH must enter the mitochondria to
fuel oxidative phosphorylation, but NADH and
NAD+ cannot diffuse across the inner
mitochondrial membrane
• Two shuttle systems for reducing equivalents:
(1) Glycerol phosphate shuttle: insect flight
muscles
(2) Malate-aspartate shuttle: predominant in
liver and other mammalian tissues
Prentice Hall c2002
Chapter 14
74
Fig 14.21 Glycerol phosphate shuttle
• Cytosolic NADH transfers 2 e- to FAD, then Q
Prentice Hall c2002
Chapter 14
75
Fig 14.22 (continued)
Prentice Hall c2002
Chapter 14
76
Fig. 14.22 Malate-aspartate shuttle
(continued next slide)
Prentice Hall c2002
Chapter 14
77
14.17 Regulation of Oxidative Phosphorylation
• Overall rate of oxidative phosphorylation
depends upon substrate availability and cellular
energy demand
• Important substrates: NADH, O2, ADP
• In eukaryotes intramitochondrial ratio ATP/ADP
is a secondary control mechanism
• High ratio inhibits oxidative phosphorylation as
ATP binds to a subunit of Complex IV
Prentice Hall c2002
Chapter 14
78
Prentice Hall c2002
Chapter 14
79