Download Proton-motive force

PROTON-MOTIVE
FORCE
Falling Water → electrical Energy
Protons “Falling” → ATP
Energy Balance
• Overall energy balance
• At cellular conditions, assume ∆G’ =54.3 kJ/mole for ATP
hydrolysis to ADP + Pi
• We know that the oxidation of glucose (e.g. in a calorimeter) yields
∆G’ = -2937 kJ/mole
• Assuming that 32 moles of ATP are produced per mole of glucose,
this gives an efficiency of:
effic 
32 moles ATP x 54.3 kJ / mole ATP
2937 kJ
effic  ca 60 %
( without O2 effic ~ 4%)
• NADH + (1/2) O2 + H+ ⇄ H2O + NAD+ ΔGo’ = -220 kJ/mole
• ADP + Pi + H+ ⇄ ATP + H2O
ΔGo’ = -30.5 kJ/mole
• Remainder “lost” as heat which we warm-blooded species
use to keep warm
How is NADH Oxidation Coupled to ADP
Phosphorylation? First Attempts
• Extend ideas from metabolism: transfer the high free
energy electrons in NADH to a compound with high
phosphoryl transfer potential
• Researchers spent a couple of decades looking for an
intermediate
• Couldn’t find one
Couldn’t find an Intermediate – so new
hypothesis
• In 1961 Peter Mitchell proposed a chemiosmotic
mechanism in which entropy maximization tends to drive
protons from the inter-membrane space to the matrix
• Might add: he was ridiculed for his proposal, but
eventually proved correct and was awarded the 1978
Nobel Prize (Chemistry)
The Mitochondrian
Proton Motive Force
• Proton motive force is the
sum of a chemical gradient
and a charge gradient
• pH is 1.4 units lower in
inter-membrane as
compared to matrix (ie
inter-membrane is 25 x
higher)
• Charge gradient is 0.14
volts
• Totaling these two gives
PMF free energy of 20.8
kJ/mole e-
ATP Synthase
• Fo subunit “stick” proton channel
• 10-14 c subunits “rotor”
• Single a subunit on outside “stator”
• F1 subunit “ball”
• 3  units and 3  units in alternate ring
to make up 33 hexamer “stator”
• ,  stalk rotate with c ring “rotor shaft”
• b2 and  anchor the F1 unit to the
membrane
ATP Synthase
• EC 3.6.3.14
ATP Synthase
• The 8.5 nm “dots” are the
F1 subunit of ATP Synthase
• Thinnest human hair
~20m or 20,000 nm
• ~2500 F1 subunits would
span a human hair
ATP Synthase
Side view of F1
Top view of F1
 subunit is pink
ATP Synthase – F1 unit
• ADP3- + HPO42- + H+ → ATP4- + H2O
• Only  sites are catalytic
accept or release
open=
= loose, binds ADP and Pi
= tight, makes ATP
ATP Synthase – F1 unit
• The rotation of 
1
2
subunit drives the
inter-conversion
of the  subunits
• Subunit-sequence
1 - L→T →O →L →T…
2 - O→L →T →O →L…
3 - T→O →L →T →O…
•  unit moves counterclock wise
3
There is a Defined Order to Sequence
• Note that there is a proper order to this sequence (O →L →T)
• Open – molecules of ATP leave and ADP and Pi enter  unit
• Loose – ADP and Pi are bound to  unit enzyme
• Tight – the  unit interacts with b enzyme to squeeze ADP and
Pi together to make ATP
• Open
F1 Animation
File_ATPsyn.htm
Rotational Proof of Molecular Motor
• Immobilize the 33 on glass slide
• Link  subunit to fluorescently labeled actin filament
• Add ATP – hydrolysis of ATP caused filament to rotate
Forming ATP Requires Free Energy
• Remember that ATP hydrolysis to ADP and Pi is
spontaneous:
• ATP + H2O → ADP + Pi
ΔGo’ = -30.5 kJ/mole
+ H2O →
+
Forming ATP Requires Free Energy
• Reasons for spontaneity:
• Phosphate groups are all negative, hence they electrostatically
repelled
• ADP and Pi have more resonance states than ATP, hence they are
more stable.
• For all of these reasons, free energy input is required to
force Pi and ADP back together to make ATP
• ADP3- + HPO42- + H+ → ATP4- + H2O
• This energy is provided by conformationally changing
enzymes driven by  –subunit rotor which is in turn
powered by H+ returning to the matrix
How Does H+ Flow Cause  to Rotate?
• Take a closer look at the a and c subunits of Fo
Opening at top
Opening at bottom
How does the c-ring turn?
Animation
matrix
Intermembrane space
How Does H+ Flow Cause  to Rotate?
H+ poor matrix side
H+ rich cytoplasmic side
How Does H+ Flow Cause  to Rotate?
• A closer look at the rotating c units
+
+
+
+
+
+
The protein residues in the c-ring-a complex
Aspartate, asp
C-ring
H
H+
H+
a-unit
Serine, ser
Asparagine, asn
Inlet H+ channel
Arginine, arg
Outlet H+ channel
Rotational Summary
Photomicrograph of C-ring
Rates of Energy Production
• A resting human being requires 85 kg of ATP each day
• Molar mass ATP is 507.2
• Gives 167.6 moles of ATP/day
• With ratio of 3 moles of electrons per mole of ATP
generated
• Gives 502.8 moles of electrons and with 6.02 x 1023
electrons per mole yields 3 x 1026 protons/day
• Or 3.3 x 1021 protons/sec
Getting ATP out into the Cytoplasm
• ATP is made in the mitochondria matrix
• It needs to get out into the cytoplasm in order to provide
•
•
•
•
•
power
Remember that the inner membrane is itself only
permeable to H2O, CO2 and O2
How do we get these highly charged ATP and ADP
molecules in and out of matrix
Need a transporter protein called ATP-ADP translocase
ATP/ADP flows are coupled: ATP flows out only if ADP
flows in and vice versa (antiporter)
Cost approximately 1 H+ in proton motive force to make
this happen
Getting ATP out into the Cytoplasm
Approximate ATP Yield
NADH
Only NADH
FADH2
Total
NADH = FADH2
Complex III & IV
Per e- carrier
Complex I
4
4
Complex II
------
0
Complex III
------
1
1
2
2
2
4
7
3
10
e- carriers
10
2
Protons pumped
70
6
ATP Synthase @ 3H+/ATP
23
2
Complex IV
Total
0
Per 1 molecule Glucose
25
ATP Glycolysis & TCA
4
ATP Total
29
Yield of ATP from Glucose Oxidation Pathway
ATP Yield
per Glucose
MalateGlycerol-3Phosphate
Aspartate
Shuttle
Shuttle
Glycolysis: glucose to pyruvate (cytosol)
Phosphorylation of glucose
Phosphorylation of fructose-6-phosphate
Dephosphorylation of 2 molecules of 1,3-BPG
Dephosphorylation of 2 molecules of PEP
Oxidation of 2 molecules of glyceraldehyde-3phosphate yields 2 NADH
(see below)
Pyruvate conversion to acetyl-CoA (mitochondria)
2 NADH (see below for ATP yield)
Citric acid cycle (mitochondria)
2 molecules of GTP from 2 molecules
of succinyl-CoA
Oxidation of 2 molecules each of isocitrate,
-ketoglutarate, and malate yields 6
NADH (see below for ATP yield)
Oxidation of 2 molecules of succinate yields 2
-1
-1
+2
+2
+2
+2
+3
+5
+5
+5
+3
+3
+15
30
+15
32
[FADH2]
Oxidative phosphorylation (mitochondria)
2 NADH from glycolysis yield 1.5 ATP each if NADH
is oxidized by glycerol-phosphate shuttle; 2.5 ATP by
Oxidative decarboxylation of 2 pyruvate to 2 acetyl-CoA:
2 NADH produce 2.5 ATP each
2 [FADH2] from each citric acid cycle produce 1.5 ATP
6 NADH from citric acid cycle produce 2.5 ATP each
Net Yield
-1
-1
+2
+2
malate-aspartate shuttle
each
P/O Values
• P/O ratio is the number of ATP molecules formed/pair of e• The table above assumes P/O = 2.5 for mitochondrial
NADH
• How do we get this?
• Assume a 10 c-subunits in the ATP Synthase rotor
• So 10 H+ are required for one complete rotation which makes 3 ATP
or about 3H+/ATP
• Add 1 H+ to power ATP-ADP translocase gives a total of 1 ATP/4 H+
• In ETC about 10 H+ pumped for every 2 e- of NADH/FADH2
→ NAD+/FAD:
1 ATPcyto 10 H 
ATPcyto
10

 2.5


4H
pair e
4
pair e 
Control
• Controlled by need of cell for ATP
• Electrons flow through the electron transport chain only if
protons are constantly being “used” to convert ADP to
ATP