Lecture 25
The sources of energy: posphorylation,
oxidation and coupling chemical energy
to work
Peter Mitchell
Why protons?
Why ATP?
Why oxygen?
Most cells use a proton gradient as an energy source across
their plasma membrane. Why do animal cells use a sodium
gradient?
Why protons?
Protons because one single Histidine,
Glutamate or Aspartate residue furnish a
simple and tunable binding site for H+. pKa of
these groups can vary by 2-3 units depending
on the environment. These sites do not bind
metal ions tightly unless work in concert.
It is much more difficult to build a selective
binding site that would discriminate between
Na+, K+, Mg2+, Ca2+, Zn2+, Cu2+, Pb2+, Hg2+,
Fe2+, Fe3+, etc
Protomotive Force has an electrical component and can
couple electrochemical proton gradient to the transport of
other charged substances
  H  RT ln(
 H
[ H ]1
)  F   ... in Joules
[ H ]2
  60  pH    ..... in mV
F
0 mV
-180 mV
pH = 8
PMF = -60 -180 = -240 mV
pH = 7
Measuring the membrane potential…..
0 mV
-180 mV
positive charge
pH = 7
pH = 8
Calibration???
Rhodamine 123
Fluorescent dye Rhodamine 123 (Rh123+) will penetrate
into the vesicles according to electric gradient, increasing
their fluorescence. Increased concentration of Rh123 inside
the vesicle beyond certain point will cause self-quenching.
Measuring the membrane potential…..another way
0 mV
-180 mV
pH = 7
pH = 8
K+
Valinomycin…potassium uniporter
What happens if we add valinomycin?
 
RT
F

ln(
[ K ] out

)
[ K ]in
but val. may affect the potential…
http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm
Measuring the membrane potential…..a better way
val
[KCl] outside mM
fluorescence
0.05
0.1
0.2
time
Null method
Why ATP?
phosphoanhydride bonds
The reaction of ATP hydrolysis is very
favorable
ΔGo = -30.5 kJ/mol = - 7.3 kCal/mol
because:
1. Charge separation of closely packed
phosphate groups provides electrostatic
relief
2. Inorganic Pi, the product of the reaction,
is immediately resonance-stabilized
(electron density spreads equally to all
oxygens)
3. ADP immediately ionizes giving H+ into a
low [H+] environment (pH~7)
4. Both Pi and ADP are more favorably
solvated by water than one ATP molecule.
Mg2+
ATP exists in complex with Mg2+
ATP is not the only:
Phosphoenolpyruvate (PEP)
-61.9 kJ/mol
1,3-Bisphosphoglycerate
-49.3 kJ/mol
Phosphocreatinine
-43.0 kJ/mol
ATP
-30.5 kJ/mol
Pyrophisphate (Pi-Pi) or
Inorganic polyphosphate (polyPi)
-19 kJ/mol
Thioesters (Acetyl CoA)
-31 kJ/mol
In real cells G for ATP hydrolysis is more negative than standard Go
G p  G
o
 RT ln
[ADP][P]
[AT P]
Calculate Gp in erythrocytes if
Gp = -30.5 kJ/mol
[ATP] = 2.3 mM;
[ADP] = 0.25;
[Pi] = 1.65 mM,
In other cells:
Rat myocyte
Rat neuron
E. coli
[ATP]
8
2.6
7.9
[ADP] [AMP]
0.9
0.04
0.73
0.06
1.04
0.82
[Pi] in mM
8.05
2.7
7.9
ATP provides energy to group transfer reactions:
A-P → A + P
G1
B-P → B + P
G2
A-P + B → A + B-P
G = G1-G2
G of hydrolysis
PEP
1,3-BPG
P
E
P
Phosphocreatinie
ATP
Glucose-6-P
Pi
Glycerol-P
Synthesis of any phosphorylated compound can be coupled to ATP
hydrolysis
Transfer reactions:
Phoshoryl transfer; Pyrophosphoryl transfer; Adenylyl transfer
Synthesis of NTPs (dNTPs) from ATP occurs as phosphoryl exchange at
G ~0. The reaction is catalyzed by nucleoside diphosphate kinase which
first phosphorylates its own His, releases ADP and then phosporylates the
incoming NDP or dNDP.
How is the energy of phosphoryl transfer (or removal) imparted to a
conformational change or how the chemical work is done?
Simple binding of ATP to an enzyme (or another effector protein) may
cause massive conformational change by allosteric mechanism
through the stage of binding site rearrangement. Cleavage of the
gamma phosphate would lead to another conformational change. A
complete release of the nucleotide diphosphate returns the protein to
its initial state.
Phosphorylation of certain sites (Tyr, Ser or Thr) promotes recognition by a
counterpart domain (recall SH2 domains)
Why oxygen?
Electron re-distribution from less electronegative to more
electronegative atoms occurs with massive energy release:
Electronegativity of common elements:
H < C < S < N < O …Cl < F
Identify the substance and the
reduction state of the first carbon
(red)
Enthalpies of oxidation (combustion)
hydrogen (MW 2)
H2 + ½O2 → H2O
-286 kJ/mol
methane (MW 16)
CH4 + 3O2 → CO2 + 2H2O
-891 kJ/mol
glucose (MW 180.2)
C6H12O6 + 6O2 → 6CO2 + 6H2O
palmitic acid (MW 256.4)
C16H32O2 + 23O2 – 16CO2 + 16H2O
-2840 kJ/mol
(-680 kCal/mol)
- 9730 kJ/mol
Oxidation-reduction (Red-ox) reactions usually lead to redistribution of electron densities or complete transfer of
electrons resulting in change of ionization state.
Fe2+ + Cu2+ → Fe3+ + Cu+
or in the form of half-reactions: Fe2+ → Fe3+ + e
Cu2+ + e → Cu+
In biological systems oxidation is often coupled to
dehydrogenation.
1. Direct transfer of electrons
2. As a transfer of H atoms or removal of H atoms coupled to
production of H+
3. As a Hydride ion :H–
4. Through direct combination with oxygen
R-CH3 + (½)O2 → R-CH2-OH
Standard Reduction potentials for some half-reactions, Volt
½ O2 + 2H+ + 2e → H2O
+0.816
Fe3+ + e → Fe2+
+0.771
Cytochrome c (Fe3+) + e → Cytochrome c (Fe2+)
+0.254
Fumarate2- +2H+ + 2e → succinate2-
+0.031
2H+ + 2e → H2
0
(standard condition)
Pyruvate + 2H+ + 2e → lactate
-0.185
FAD + 2H+ + 2e → FADH2
-0.219
S + 2H+ + 2e → H2S
-0.243
NAD+ + H+ + 2e → NADH
-0.320
NADP+ + H+ + 2e → NADPH
-0.324
α-ketoglutarate + CO2 + 2H+ + 2e → isocytrate
-0.38
2H+ + 2e → H2
-0.414
(pH 7)
Reduction potentials for mixtures of reductant/oxidant (hlfreaction potentials) are measured using the standard
hydrogen electrode
RT
[electron acceptor]
o
E  E 
ln
nF
[electron donor]
2H+ + 2e → H2
Mg2+ + 2e → Mg (metal)
http://www.chemguide.co.uk/physical/redoxeqia/eomgdiag.gif
Electron transport chain puts reductants and oxidants in specific order
Complex I
II
III
IV
Energy released in forming water is stored as a PMF
Energy is divided into smaller units ~12 protons per water molecule