BIOENERGETICS:
OXIDATIVE
PHOSPORYLATION
Student Edition
10/23/14 Version
Dr. Brad Chazotte
213 Maddox Hall
chazotte@campbell.edu
Web Site:
http://www.campbell.edu/faculty/chazotte
Original material only ©2000-14 B. Chazotte
Pharm. 304
Biochemistry
Fall 2014
Goals
•Learn the basic tenets of the chemiosmotic hypothesis and how mitochondrial
structure is consistent with the hypothesis.
•Consider how thermodynamics describes electrochemical gradient & oxidative
phosphorylation.
•Learn the quantitative description of the proton electrochemical gradient and its
components. Be able to calculate the phosphorylation potential.
•Be able to calculate the free energy of hydrolysis.
•Learn the basic components involved in pumping proton across the mitochondrial
inner membrane. Understand the concept and meaning of the P:O ratio.
•Learn the structure of the F1Fo-ATPase and how Boyer’s binding exchange
involves this structure in the synthesis of ATP.
•Understand how fluorescent dyes can report on the (mitochondrial) membrane
potential in living cells and how changes in the potential due to substrates and
inhibitors can be monitored.
•Understand how gradients involve the use or storage of energy.
CHEMIOSMOTIC HYPOTHESIS
Tenets
1.
Energy transducing membranes are vesicular, sealed, and impermeable to protons
except for the pathways involved in redox mediated or protein-catalyzed H+
translocation.
2.
Energy is stored in a pH gradient or membrane potential which are energetically
equivalent, with the electrochemical potential.
3.
The DmH+ is formed by vectorially alternating H+ and e- carriers in the electron
transport chain.
4.
An H+ flux is coupled to the ATP synthase/ATPase catalyzed by the large multisubunit
F0F1 ATPase. Each reaction is anisotropic with respect to this flux. The synthesis
reaction is coupled to an H+ flux driven by the DmH+ from the DmH+–positive side of
the membrane.
5.
Uncouplers of energy transduction were predicted to be lipid-soluble weak acids or
bases that can catalyze the equilibration of H+ of OH- across the membrane.
Cramer & Knaff 1990
THERMODYNAMICS
Brief Description
Chemical Work and Chemical Potential
The chemical potential mi, of compound I, is the free energy per mole
G
 ni  T, ,p, ,n  n
j
i
 mi
 is the partial derivative with respect to ni when temperature, pressure, and
other n are held constant.
m is important for transport problems because it is the change in free energy
of a system per mole of component moved in or out of the system.
Cramer & Knaff 1990
Proton Electrochemical Gradient (DmH+) #1
The electrochemical difference of protons across the mitochondrial inner
membrane provides the high energy state to drive ATP synthesis, ion and
substrate transport, transhydrogenation, and other energy requiring reactions.
One can write for hydrogen transport across, e.g., the mitochondrial inner
membrane, where F= Faraday constant and DY = membrane potential in mV
DmH+= F DY – 2.303 RT log (Hi+/Ho+)
DmH+= F DY – 2.303 RT DpH
DmH+ is composed of two components:
a membrane potential (charge difference; electrical potential): DY
a pH gradient (concentration difference; chemical potential): D pH
In mitochondria DY is the bulk of the contribution to DmH+
Components of Mitochondrial Proton Gradient
Topic:OxPhos Alberts et al Fig 14-19
Proton Electrochemical Gradient (DmH+) #2
DmH+ = F DY
- 2.303 RT D pH
F =Faraday constant = 96,487 coulombs/mole = 96.5 kJ mol-1 V-1 = 23.06 kcal mol-1 V-1
R = gas constant = 8.3143 J deg-1 mol-1 = 1.9872 cal deg-1 mol-1 = 0.082056 liter atm
deg-1 mol-1
T = absolute temperature
Represents the free energy change in kJ/mole when 1 mole of H+ moves into
the mitochondrion.
DY is the bulk of the contribution to DmH+
Expressing the proton electrochemical gradient in millivolts is called the
phosphorylation potential (D p).
D p= DmH+ / F = DY - 2.303 (RT/F) D pH
Determination of DpH
• Calculate from equilibrium distribution of weak
bases.
• Use Henderson-Hasselbalch equation
[HA]in = [HA]out
@ equilibrium
K a = ([H+]in [A-]in)/[HA]in = ([H+]out [A-]out)/[HA]out
DpH = log([A-]in / [A-]out)
Note: mitochondrial DpH is typically less than 1 pH unit
Calculation using Phosphorylation Potential Dp
Problem: Calculate the pH gradient at 37 ºC required across the mitochondrial
inner membrane to equal a membrane potential of -150 mV.
The relevant equation for the phosphorylation potential in mitochondria, which
is in mV units already, is:
D p = DmH+ / F = DY - 2.303 (RT/F) DpH
0 = -150 mV - 2.303 (8. 3143 J deg-1 mol-1*310.15 ºK/96.5 kJ mol-1 V-1) DpH
0 = -150 mV - 2.303 (8. 3143 J deg-1 mol-1*310.15 ºK/96,500 J mol-1 V-1) DpH
0.150 V = - 2.303 (2.672*10-2 V-1) DpH
0.150 V = - 0.0616 V-1 DpH
DpH = -2.44
Proton Motive Force in Oxidative
Phosphorylation
Horton et al 2012 Figure 14.9
Uses of the Proton Gradient
Berg, Tymoczko, & Stryer 2012 Fig 18.44
PROTON PUMPING & OXIDATIVE
PHOSPHORYLAYION
Schematic of Electron Transport Enzyme Complexes
H+ ions transported across a membrane
per unit area to generate DY = 100 mV
DY = Q/C where C is the specific membrane capacitance. Q is the charge per unit area.
For biological membranes C ~ 1 mfarad/cm2.
Thus, if DY = 0.1V and C= 10-6 coulombs/cm2,
Q = 1.0 10-7 coulombs/cm2.
Charge on one proton
= 1.6 10-19 coulombs
# protons translocated per unit area
= 6 x 1011/cm2.
# protons translocated per sq micron
= 6 x103
For a 300 Å diameter vesicle the translocation of 20 protons would generate a 100 mV
potential.
For a typical rat liver mitochondrion estimate:
6 x 1011 protons /cm2  520.6 cm2/mg protein  8.7 x 109 mitochondria/mg protein =
35,903 protons/mitochondrion
Cramer and Knaff 1990
“THE” CHEMIOSMOTIC EXPERIMENT
Berg, Tymoczko, & Stryer 2012 Fig 18.23
Factors Controlling the Partition of Dp
Components D Y and DpH
Nicholls & Ferguson Bioenergetics 2 1992
Proton Pumping in Electron
Transport
Lehninger 2000 Fig 19-15
OXPHOS OVERVIEW
Lehninger 2000 Fig 19-16
Topic:Electron Transport
Q Cycle Schematic
Lehninger 2000 Fig 19-11
Voet, Voet, & Pratt 2013 Fig 18.15
Control of Oxidative
Phosphorylation
P: O Ratios Revisited in the
Chemiosmotic World
P:O, ATP:O, ATP/2e-, 2H+/e- !
The ratio of electrons transported to hydrogen ions pumped is an
important number in oxidative phosphorylation.
It is generally agreed now that FOUR protons are consumed to produced 1 ATP.
One of those protons is used in transporting ATP, ADP and Pi.
P:O Ratios in Electron Transport
ATP synthesis is tightly coupled to the proton gradient.
Possible to express the amount of ATP synthesized in terms of the
substrate molecules oxidized.
Experiments had shown approximately 2.5, 1.5 , and 0.5 ATP
synthesized with oxidations of NADH (via complex I), FADH2 (via
complex II) and TMPD (via Complex IV, artificial 2e- donor).
P:O ratio relates amount of ATP synthesized to amount oxygen reduced.
Years of controversy over ratios. Integer or non-integer. Likely noninteger. Chemiosmotic hypothesis unlike other theories does not need
whole numbers.
Voet, Voet & Pratt 2006 pp577-578
Mitochondria Redox States
(according to Chance and Williams [Adv. Enz. 17 1956])
State 1
State 2
State 3
State 4
State 5
[O2]
>0
>0
>0
>0
0
ADP
low
high
high
low
high
Substrate
low
~low
high
high
high
Respiration
slow
slow
fast
slow
0
Limiting
ADP
substrate
e-trans
ADP
oxygen
Oxidative phosphorylation is occurring during state 3 respiration
Polarographic Determination of P/O Ratio
State 1
State 3
State 4
State 5
COMPONENTS INVOLVED IN
OXIDATIVE
PHOSPHORYLATION
[DIRECTLY & INDIRECTLY]
F1F0 ATP Synthase Polypeptide Structure
Ref:Sarasate Fig 5 Science 283 1999
Voet, Voet, & Pratt 2013 Fig 18.22 (inverted)
OXIDATIVE
PHOSPHORYLATION:
ATP Synthase Binding Model
Topic: OxPos
ATP Synthase Binding Site Model
3-αβ
“subunit
pairs” in
F1. β binds
nucleotide
O - catalytically inactive & very low ligand affinity
L – catalytically inactive & loose ligand binding
2
T – catalytically active & tight ligand binding
1) ADP & Pi bind to site “L”
2) “L” converted to “T” site by energy
driven conformational change
1
3) ATP is synthesized at site “T” and
release as “T” becomes “O” site during
energy driven conformational change
3
1
Lehninger 2000 Fig 19-23 Topic:Ox Phos
Voet, Voet & Pratt 2008 Figure 18.24
ATP Synthase (F1F0) Structure
Cryo EM (F1F0 E. Coli)
Artist Illustration
Negative Stain EM
Voet, Voet & Pratt 2002 Figure 18.26
Proof of
ATP
Binding
Model
Fo
F1
Voet, Voet & Pratt 2013 Fig 18-28
Topic:Ox Phos
OXIDATIVE PHOSPHORYLATION
ADP/ATP Translocator 1
Horton et al 2012 Fig 14.20 Topic: OxPhos
ADP-ATP Translocator:
Conformational Mechanism
OXPHOS TRANSPORTER
RELATIONSHIPS
Lehninger 2000 Fig 19-25
Topic:Electron Transport
e- Transport & Oxidative Phosphorylation
Lehninger 2000 Understand Biochemistry CD
MITOCHONDRIAL OxPhos
Free Energy of Hydrolysis in a Cell
DG°´= –30.5 kJ/mol for ATP. However, that is based on standard
conditions, i.e. 1 molar. pH 7.0, which may not be the conditions
in a living cell. Consider a human erythrocyte
Lehninger 2000 Table 14-5
Free Energy of Hydrolysis in a Cell. II
[ADP]1 [Pi]1
DGp
= DG ´ + 2.303RT log K´eq =
DG ´ + 2.303RT log
[ATP]1
[2.50 x 10-4]1 [1.65 x 10-3]1
= -30,500 J mol –1 + 2.303 x 8.315 J mol –1 K -1 x 298  K * log
[2.25 x 10-3]1
= - 30,500 J mol –1 + (5,707 J mol –1 x -3.737)
= - 30,500 J mol –1 – 21,327 J mol –1
=
and
- 51,827 J mol –1 for hydrolysis
51,827 J mol –1 for ATP synthesis
Lehninger 2000 Box 14-2
Topic:OxPhos
Nernst Equation
DY = -(RT/F) ln(Ain / Aout)
DY = the membrane potential
Ax = probe chemical activity inside or outside
R = gas constant T = absolute temperature
F = Faraday constant
Which at room temperature simplifies to
DY = -59  ln(Cin / Cout)
Cx = the probe concentration inside or outside
Calculation using the Nernst Equation
Given: TMRM concentration = 50 mM inside and 5 nM outside the
mitochondrion at 37  C
DY = - (RT/F) ln(Cin / Cout)
DY = - (8.315 J mol –1  K -1 x 310  K / 96,500 J mol-1 V-1 ) ln(50 µMin
/ 5nMout)
DY = - (8.315 J mol –1  K -1 x 310  K / 96,500 J mol-1 V-1 ) ln(50 µMin
/ 5nMout)
DY = - (8.315 J mol –1  K -1 x 310  K / 96,500 J mol-1 V-1 ) ln(10,000)
DY = - (2,477.87 J mol –1  / 96,500 J mol-1 V-1 ) 9.210
DY = - (0.0267 V ) 9.210 = -0.246 V = - 246 mV
Confocal Image of Human Fibroblast Labeled with TMRM
Graylevel Image
Pseudocolored Image
Cytoplasm
Nucleus
Mitochondrion
6AP16076
Selecting a Region of Interest to Histogram
Human Mononuclear Leukocyte
DY based Pseudocolored Image
Graylevel Image
Mononuclear Leukocyte
ROI
Platelet
Mitochondria
6AP02123
Selected Inhibitors of Mitochondrial
Bioenergetics
•
•
•
•
•
•
•
•
CCCP
Valinomycin
Rotenone
Antimycin a
TTFA
KCN
Oligomycin
2-Deoxyglucose
collapses DpH and DY
collapses DY
inhibits Complex I electron transport.
inhibits electron transport at Complex III
inhibits Complex II electron transport.
inhibits electron transport at Complex IV
prevents ATP synthesis, increases DY
causes mitochondrial respiratory jump
CCCP Effects
6MA13087
6MA13088
CCCP
Effects of Mitochondrial Inhibitors on IPDs of Human
Mononuclear Leukocytes
HMINH1
2.8 mM, 29 mg/ml, 0.87 mM
BIOENERGETICS OF
CELLULAR TRANSPORT
Topic: Bioeneregtics Transport
Thermodynamics of Ion Gradient
For protons we have written:
mH+ = mo +zF DY + 2.303 RT log (H+)
DY = the membrane potential, R = gas constant, T = absolute
temperature, F = Faraday constant, z = charge (for proton: z = +1)
Likewise for a electrochemical sodium gradient we can write
mNa+ = zF DY + 2.303 RT log (Na+final)
(Na+initial)
Cramer & Knaff 1990 pp18-19
Lehninger 2000 Fig 12-29
Active Transport Processes Driven via the
Mitochondrial Gradient
Topic:OxPhos Alberts et al Fig 7-21
Thermodynamics of mH+–Linked Active Transport
Symport
If all of the free energy available in the mH+ is stored in the electrochemical potential,
then we can write for Dms of substrate accumulation in a symport mechanism. Where S
refers to a solute molecule and n protons to transport one solute molecule
DGtotal = n* mH+ ms = 0
eq 1
DmH+= F DY + 2.303 RT log (Hi+/Ho+)
eq 2
Dms= zF DY + 2.303 RT log (Si+z/So+z)
eq 3
Where “i” is inside and “o” outside & for solute the initial state is outside and the final state is
inside DY = Yi -Yo; then Substituting eqs 2 & 3 into eq 1
0= zF DY + 2.303 RT log (Si+z/So+z) + F DY + 2.303 nRT log (Hi+/Ho+)
Divide by 2.303RT and Rearrange
log (Si+z/So+z) = n D pH – (n+ z) F (DY/z)
Cramer & Knaff 1990, pp 19-20
Thermodynamics of mH+–Linked Active Transport
Antiport
If all of the free energy available in the DmH+is stored on the electrochemical potential then
we can write for Dms of solute accumulation in an antiport mechanism. Where S refers to a
solute molecule and n protons to transport one solute molecule
DmH+= F DY + 2.303 RT log (Hi+/Ho+)
eq2
In antiport initial and finale states are opposite of symport so the terms in the
log expression for solute are inverted:
Dms= zF DY + 2.303 RT log (So+z/Si+z)
eq3
Where i is inside and o outside; then Substituting eqs 2 & 3 into eq 1
0= zF DY + 2.303 RT log (So+z/Si+z) + F DY + 2.303 nRT log (Hi+/Ho+)
Rearrange and Divide by 2.303RT
log (Si+z/So+z) = - n D pH + (n - z) F (DY/z)
Cramer & Knaff 1990, pp20-21
SUMMARY OF TRANSPORT PROCESSES
Lehninger 2000 Fig 12-43
End of Lectures