Download Activation parameters for ET

Photosynthetic bacterium
(Rhodobacter sphaeroides)
University of
Szeged,
Hungary
Reaction center
protein
Szeged
Department
of Medical
Physics
Proton channel
Proton transport in
bioenergetic proteins
PÉTER MARÓTI
Mitochondrion: proton pump coupled to
electron transport
Theory of
biological
oxidation (Albert
Szent-Györgyi,
Nobel Prize,
1937)
Chemiosmotic
theory (Peter
Mitchell, Nobel
Prize, 1978)
The proton gradient across
the membrane is used for
biological work (e.g. ATP
synthesis).
Respiratory cytochrome oxidase
catalyses „soft” reduction of oxygen in cell respiration and
pumps hydrogen ions simultaneously out of the mitochondrion
4 e   8 H  (in)  O2  2 H 2O  4 H  (out)
The proton uptake is
- either vectorial (via the ordered D channel) or scalar (via
the provisional K channel) and
-. Coupled tightly to electron transport.
+
H -ATPase
The cross membrane proton gradient
covers the energy cost of
1) the rotatory movement of the protein,
2) the conformational changes (like
respiratory movement) and
3) the synthesis of ATP from ADP and
inorganic phosphate.
membrane
Boyer, Walker and Skou,
Nobel Prize, 1997.
Vectorial transport of protons through the membrane:
membrane
The rotating subunits of the rotor
constitute a Brown-rachet, whose
rectified rotation takes the protons to the
stator and across the membrane.
H+/K+ ATPase proton pump
causes the exchange of a proton against a potassium ion through the membrane:
H  (in)  K  (out)  ATP  H 2 O  H  (out)  K  (in)  ADP  Pi
.
This pump is present in
the colon, the kidney,
but especially the
stomach where it is
particularly active:
controls the secretion of
protons into the gastric
fluid which becomes
acid. It generates a
gradient of pH of more
than 6 pH units:
whereas the blood pH
is 7.3 that of the gastric
fluid is about 1
Human carbonic anhydrase
catalyzes the rapid interconversion of carbon dioxide
to bicarbonate and protons to maintain acid-base
balance in blood and other tissues, and to help the
transport of carbon dioxide out of the tissues:
The proton transfer occurs over a
distance of 8-10 Å and is
associated with the regeneration of
the active site Zn2+ -OH– complex:
The active site of carbonic
anhydrase II. The reactive
water/hydroxyl is bound to a
zinc(II) ion (black), which is
liganded by three histidines. The
fourth histidine, His64, is at the
entrance of the active site cleft
and is observed in two distinct
configurations – the “out” position
is essentially in the bulk phase
and the „in” position is connected
to the Zn2+ ion by four bridging
water molecules (red).
CO2  H 2O  HCO3  H 
Zn 2  - OH2  His 64  Zn 2  - OH-  His 64 H 
M2 proton channel of the flu virus
For cultivation, the virus needs the transmembrane pH gradient of the host cell. The
M2 proton channel leads protons to the interior of the virus.
H+
Structure of M2TM′.
Structure and
mechanism of
proton transport
through the
transmembrane
tetrameric M2
protein bundle of
the influenza A
virus
Rudresh Acharya et al. PNAS 2010;107:15075-15080
©2010 by National Academy of Sciences
It is important to understand the operation of the proton channel to
- design drogs against viruses, as amantadine (Symmetrel) and rimantadine
(Flumadine),
- to figure out how the drog resistance is established.
Light driven proton pump
Upon light excitation, proton electrochemical gradient coupled to cyclic electron transfer
is established in membrane of photosynthetic bacteria.
Periplasmic side
light
membrane
Reaction center (RC)
Cytoplasmic side
Reaction center of bacterial photosynthesis
Arrangement of the cofactors
Reduction cycle of the quinones
Q  2 e-  2 H   QH2
.
Bohr-protons
Chemical protons
The reduction of QB is the first half of the
proton translocation through the membrane
The proton is moving from the aqueous phase into the membrane.
This is an energy consuming process.
cytoplasm
periplasm
light
The free energy is delivered by the absorbed light.
Half of a protonpump: the path of H+ ion to QB
H+
Fuel injection:
His(H128),
His(H126) and
Asp(H124)
Bifurcation:
at Asp(L213),
where the
paths of the
first and
second proton
uptake are
separated H+(1)
secondary
quinone
Proton gate:
Blocking: with
divalent cations
Chain of
protonatable
amino acids with
structural water
molecules
H+(2)
Modification: with
mutants
Basic properties of the proton channel
1) Surface proton antenna: increases the proton flux.
An effective selection
(positive discrimination) for H+
ions is required. To get
imagination, consider the
typical concentrations:
Na+: ~ 100 mM = 10-1 M
H+: ~ 0.1 μM = 10-7 M
(pH 7)
His(H128),
His(H126) and
Asp(H124)
Basic properties of the proton channel
2) Protonated semiquinone is required for the second ET: rhodosemiquinone
at the QB binding site
Proton-activated electron transfer
RQH
RQ–
~pK
(2)
kAB
 f (QB (H  ))  kET
After pioneering work of the San Diego group conducted
by G. Fehér, Mel Okamura and Mark Paddock.
As RQ is a low potential quinone at
QB, mutant (M265IT) of low potential
QA was applied.
Maróti és mtsi (2015) Biochemistry 54(12) 2095-2103.
3) UQB has strong acidic character (pKa< 4.5) in the environment of an
acidic cluster of amino acids.
The protonatable groups are (pH) titrated according to the Henderson-Hasselbalch
expression in solution.
However, the QB/QBH● redox couple is not in solution, but in acidic environment whose
spatial and electrostatic structure is not constant, but changes with pH.
The value of pKa is pretty low (pK ≈ 4.0), and the redox couple does not titrate according
to the simple Henderson-Hasselbalch equation. However, we can keep formally the H-H
expression if a pH-dependent pK is introduced.
Henderson-Hasselbalch equation
The acidic cluster in
the QB pocket
introduces a strongly
pH-dependent
interaction with two
consequences, at
least:
acidic cluster
1) pH-dependent pK
value and
shift
2) pK shift upon
mutations.
pK downshift of the UQ/UQ- redox couple
upon mutations in the QB pocket
Basic properties of the proton channel
4) How buried is QB-? About 15 Å is the depth that is an optimum distance
between successful protection of the semiquinone from spontaneous oxidation
and still efficient (but long distance) H+ supply.
5) Electrostatics: the transport of e– and H+ ions needs opposite electrostatics.
The primary goal is the stabilization of the semiquinone (that requires +
potential), and the transport of protons (that requires – potential) adapt to
these circumstances.
Maróti and Govindjee (2015) Photosynth. Res.
Electrostatical potential at the QB binding site
blue: positive
red: negative
potential
The pK value of the
antibiotics
stigmatelin
bound to the QB site
is sensitive to the
electrostatics.
On the reversed way,
the electrostatic
potential can be
determined by
detection of the
actual pK value of the
stigmatellin.
Gerencsér and et al. (2015)
Biophysical Journal 108,
379–394
Basic properties of the proton channel
6) The proton transport chain can be
blocked at different levels (depths).
Deep inside
L213
Below the surface
M17, L210
On the surface
H126/H128
How to rescue the proton transfer by
NaN3?
pH 8.0, 100 μM Cd2+
How to measure and to block the 2nd ET?
NiCl2
Gerencsér and Maróti (2001) Biochemistry 40, 1850-1860.
Activation free energy of the 2nd electron transfer
WT and ET-limited mutants
A combination of activation
parameters of proton- and
electron transfer can be
observed.
If the electron transfer is the
rate limiting step, then the
very fast proton preequilibrium (acid association)
parameters will combine with
those of the true activation
step to give observed or
„total” activation energetics:
#
#
Gtotal
 GH0'  GET
#
#
 GET
 Gtotal
 GH0'
H
 H
#
total
 H  H
0'
H
#
ET
#
ET
 H
#
total
 H
0'
H
#
#
#
#
T  S total
 T  S H0'  T  S ET
 T  S ET
 T  S total
 T  S H0'
Thermodynamics of Acid Dissociation and Association*
Oxy-acid dissociation (pH=0)* for pKa ≈ 4.5 Oxy-acid association (pH=0) for pKa≈ 4.5
kcal
 0  2.303  R T  pK a
mol
kcal
H H0  2
0
mol
GH0  6
T  S H0  4
kcal
0
mol
kcal
H H0  2
0
mol
kcal
T  S H0  4
0
mol
GH0  6
kcal
0
mol
 2.303  R T  pK a
Oxy-Acid Dissociation Heats vs. pK
Oxy-acid association (pH=7.5, pKa≈ 4.5):
GH0'  2.303  R T  (pK a  pH)  4
H H0'  H H0  2
kcal
 170 meV
mol
kcal
 90 meV
mol
T  S H0'  T  S H0  2.303  RT  pH  6
kcal
 260 meV
mol
There is a strong linear correlation
between pKa and HH0’ (and hence also
T·SH0’, because pKa is linear with GH0’).
*Data from: Christensen, J.J., Hansen, L.D., and Izatt, R.M. (1976) Handbook of Proton Ionization Heats and
Related Thermodynamic Quantities, Wiley-Interscience, John Wiley & Sons, New York
Analyses of Temperature Dependence of the Rate of the 2nd ET
in electron transfer limited RC
The Rate of Electron Transfer derived from Marcus Theory
2 V
2
#
#
 Gtotal

 Gtotal





k
exp  
 k max exp  

 4k BT
 k BT 
 k BT 
is plotted in Eyring representation
#
#
H total
S total
k h
ln


 ln 
kBT
RT
R
where κ is the transmission
coefficient (adiabaticity
parameter) in the transition state
theory (TST):
k max  h

k BT
The experimentally obtained
„total” parameters are listed
below for a couple of electron
transfer mutants:
Activation parameters for ET-limited RCs
#
H total
Strain
kcal/mol
(meV)
WT(R-26)
4.5
(200)
M17DN
L210DN
H173EQ*
*Note that
H173EQ may
be partially PTlimited
12.6
(550)
11.9
(520)
9.5
(410)
#
TS total
kcal/mol
(meV)
-4.2
(-180)
#
Gtotal
kcal/mol
(meV)
8.6
(380)
2.8
(102)
2.0
(90)
-1.4
(-60)
9.7
(420)
9.7
(420)
10.9
(470)
Representation of the observed (total)
activation parameters for ET-limited RCs
Activation parameters for ET-limited RCs
measured
proton pre-equilibrium
temperature dependence
of the rate of 2nd ET
Strain



H total
T  S total
Gtotal
data from oxy-acids
pKa
H H
T  S H
GH
electron transfer
measured-H+ pre-equilibrium

H ET


T  SET
GET
WT
200
-180
380
4.5
-90
-260
170
290
80
210
M17
DN
550
120
430
3.8
-90
-310
220
640
430
210
L210
DN
520
90
430
3.8
-90
-310
220
610
400
210
H173 410
EQ
- 60
470
3.5
-90
-330
240
500
270
230
The mutations do not influence the rate (and free energy) of ET very much,
rather they lower the pKa value of QB-/QBH via electrostatics.
The energy is measured in meV and pH 7.5
This is kept
roughly
constant
The approximate operational pKa value of the UQB/UQBH● redox
couple at different solutional pH
(2)
From pH-dependence of kAB
Maróti és mtsi (2015) Biochim Biophys Acta Bioenergetics 1847, 223–230.
WT activation parameters
1) The transmission coefficient (adiabaticity parameter) κ is:
From the exchange coupling
between QA- and QB- (EPR
data):
kmax ≈ 3.5∙109 s-1

k max  h
k BT
  6 10 4
The rate limiting step is non-adiabatic electron transfer with small κ value.
2) The resulting value of free energy change of actvation of the ET: ΔGETact ≈ 200 meV
gives very good correspondence with expectations for an ET reaction with
0'
GET
 250 meV and   1.2 eV, i.e. from :
act
GET

G

0'
ET

4

2
Note that this relationship is quite sensitive – at least, in so far as we have a narrow
range of ΔGET0’ to fit.
3) The entropy change of the electron transfer, +80 meV is
- positive (the entropy increases) and
- small (relative to the enthalpy change of 290 meV)
Electron transfer limited RC: pH-dependence of the activation parameters
of the electron transfer
Activation energies
Proton pre-equilibrium (see oxy-acids)
GH0'  58 meV  (pKa  pH)
T  SH0'  T  SH0  58 meV  pH
ΔG
ΔH
pH
pKa
T·ΔS
?
Total (measured)
pH dependence of entropy change of activation
Proton pre-equilibrium
Due to entropy mixing, the slope of the pH-dependence should be negative.
Observed (total) activation entropy change
The slope of the pH-dependence of the observed (total) entropy change is
positive (or in some cases slightly negative)
Our data indicate that the magnitude of ΔGET is at least as large as ΔGH, so it is not
inconceivable that the contribution from ΔSET overwhelms that for ΔSH.
ΔSET gets more positive with increasing pH because the increasing negative charge of
the protein binds more ions and these must be rearranged (released or more bound,
depending on solvent effect).
The observed difference between slopes of entropy pH-dependence of proton
equilibrium and of total entropy change is probably due to the pH-dependence of the ET
activation parameters: the higher and higher negative surface potential upon pH
increase introduces extra effect that can change the slope of the pH-dependence of the
entropy change.
Proton transfer limited RCs
The observed slope and interseption in Eyring plot cannot be related to firm (proton
transfer) theory.
Activation parameters of protonation mutants
The point of
WT (8.6;4.5)
is out of this
range
Proton-limited RCs: pH dependence of the
activation enthalpy
Azide
is a common chemical that can rescue
the proton transfer to QB partially by
- its electrostatic influence and/or
- its ability to carry H+ ions.
azide
azide
Kinetic isotope effect in proton-limited RCs
Maróti és mtsi (2015) Biochim Biophys Acta Bioenergetics 1847, 223–230.
Effects of azides and deuterization on the activation parameters
Take home messages
(2)
• The temperature-dependence of kAB was analyzed in ET-limited and
PT-limited cases.
• The activation parameters of ET-limited RCs can be decomposed
into those of proton-preequilibrium (taken from oxy-acid dissociation
data) and of electron transfer.
• The obtained activation parameters for the ET step are in amazingly
good agreement with what we expect for the reaction (free energy
and reorganisation energy).
• A pKa downshift of the QB semiquinone might be deducible for ETlimited cases (M17DN and L210DN).
• The pH dependent local potential might influence the pH
dependence of the activation parameters.
• The activation parameters of PT-limited RCs display changes due to
chemical rescue by small acids/buffers and deuterization of the
solution. The quantitative analysis of the PT-limited cases needs
additional investigations.
Thanks to
Eiji and Kat Takahashi
Colin Wraight (1945-2014)
Gábor Sipka
Mariann Kis
Emese Asztalos
SZEGED
I highly
appreciate your
kind attention.