Download Electric field effects on the protein and electron transfer dynamics of

Peter Hildebrandt – TU Berlin
Electric field effects on the protein and electron
transfer dynamics of immobilized heme proteins –
vibrational spectroscopic approaches
Acknowledgements
People involved:
Anja Kranich, TU Berlin
Khoa H. Ly
Murat Sezer
Ingo Zebger
Inez Weidinger
Jiu-Ju Feng
Diego Millo
Gal Schkolnik
Tillmann Utesch
Maria Andrea Mroginski
Nattawadee Wisitruangsakul, Bangkok
Lisa Lorenz, Frankfurt
Daniel H. Murgida, Buenos Aires
Hainer Wackerbarth, Göttingen
Financial support:
Deutsche Forschungsgemeinschaft
Sfb498, Cluster of Excellence „Unicat“
Collaborations
Laszlo Zimanyi, Szeged
Andreas Knorr, Berlin
Content
I.
Why studying proteins at interfaces?
II. Vibrational spectroscopies
III. How to probe electric field effects?
IV. What do we learn from SERR and SEIRA spectroscopy of
biomimetic systems?
V. Interfacial processes of cytochrome c
1. Dynamics of protein orientation and electron transfer
2. Dynamics of redox-linked protein structural changes
3. Electric field dependence of electron transfer mechanism and dynamics
4. Electric field dependence of electron tunneling
5. Electric field dependence of protein dynamics
6. Electric field dependence of the interfacial electron transfer process
VI. Cytochrome c in apoptosis
Why studying proteins at
interfaces?
Why studying proteins at interfaces?
Most biological processes occur at interfaces:
electron transfer sequence in the respiratory chain
High electric fields (up to 109 V/m)
at the reaction sites due to the
¾ transmembrane potential
¾ surface potential
¾ dipole potential
Possible effects on the
9 Protein/cofactor structure and interactions
9 Alignment and induction of dipoles
9 Alteration of pKa´s
9 Activation energies
9 Energy levels
Why studying proteins at interfaces?
Electron transfer occurs within an electrostatic complex
cytochrome c
e-
cytochrome c
oxidase
Vibrational spectroscopies
Vibrational Spectroscopies
Raman and IR spectroscopies probe molecular structures…
……..but are inherently insensitive!
Requirements for appropriate methodology:
¾ High sensitivity
¾ High selectivity
¾ Adaptation to specific environments
¾ Adequate time resolution
¾ Molecular structure information
Resonance Raman:
probes exclusively the vibrational bands of the cofactor due to selective
enhancement of upon resonant excitation
IR difference:
probes selectively the vibrational bands of those parts of the macromolecule
that undergo reaction-induced changes
Vibrational Spectroscopies
Sensitivity and selectivity of RR and IR difference spectroscopy is not
sufficient for probing proteins (sub-)monolayers
¾ Surface enhanced Raman spectroscopy: SERR
¾ Surface enhanced infrared absorption spectroscopy: SEIRA
Dipole-dipole coupling of the radiation field with the surface plasmons:
¾ SEIRA: enhancement factor of ca. 102
¾ SER: enhancement factor of ca. 105
Requirements:
Nano-scale roughness
Match of plasmon resonance and incident wavelength
Restricted to Au and Ag
Advantages:
Vibrational spectroscopy of monolayers in proximity to
metal surfaces
Vibrational Spectroscopies
Surface enhanced vibrational spectroscopy
SERRS (= SER + RR):
selectively probes the chromophores solely of the adsorbed
molecules
SEIRA (difference mode):
Selectively probes the structural/orientational changes adsorbed
molecules/molecular building blocks
Electrodes as SER/SEIRA-active materials:
probing potential-dependent processes
Combination with the potential-jump technique:
™
time-resolved SERR
™
time-resolved SEIRA (rapid scan, step scan)
Vibrational Spectroscopies
Limitations of SERRS/SEIRA ......
Direct contact of proteins with metal surfaces may cause denaturation
Vibrational Spectroscopies
Limitations of SERRS/SEIRA and how to overcome them
Direct contact of proteins with metal surfaces may cause denaturation
Biocomaptible coating of metal
surfaces by (e.g.) self-assembled
monolayers of lipid analogues
(functionalised mercaptanes)
Vibrational Spectroscopies
Limitations of SERRS ......
Wavelength dependence of the enhancement effect
polydisperse nanostructures
Ag
Preferred spectral range for
studying cofactor-protein
complexes for combining RR
and SER
Au
But: Au displays a much
broader electrochemical
potential range and is
chemically more stable
Vibrational Spectroscopies
Limitations of SERRS and how to overcome them
Layered hybrid devices
Electrochemical roughening of
Ag electrode
Coating by a dielectric layer
(SAM, SiO2)
Deposition of a metal film or
semiconductor film (Au, Pt, TiO2)
0.3 V
-0.3 V
75 nm
Functionalisation of the outer
metal layer for protein binding
C-O
Metal film: ca. 20 nm
dielectric layer: 2 – 30 nm
Nanostructured Ag
Ag
Vibrational Spectroscopies
Limitations of SERRS and how to overcome them
Distance-dependence of the enhancement
experimental
theoretical
CO
Ag
25
20
Ag-spacer-Au
Cyt
413 nm
g0
15
10
Ag
5
0
0.6
0.8
1.0
r/R
1.2
1.4
How to probe electric field
effects?
How to probe electric field effects?
Approach: Mimicking the protein-protein complex by model systems
electron transfer
e-
eS
S
S
S
S
S
S
S
electrode
SAM –
membrane
model
electrostatic binding
cytochrome c
How to probe electric field effects?
Quantification of the local electric field strength
ω-carboxyl alkanethiol
eCyt-c
Parameters controlling the local electric field
strength
™ chain length (distance) - dc
™ charge density at head groups σc
™ electrode potential - E
™ ionic strength - κ
How to probe electric field effects?
Determining the interfacial electric field strength by using the field-induced
frequency shift of the CN stretching (vibrational Stark effect - VSE)
Probing the VSE
by SERS (Ag) and
SEIRA (Au) as a
function of E, dc,
σc, and κ
r
r ⎞
1 r
⎛ r r
~
Δ ν = ν ( E F ) − ν ( 0 ) = ⎜ Δμ ⋅ E F + E F ⋅ Δ α ⋅ E F ⎟
2
⎝
⎠
How to probe electric field effects?
Mapping the electric field in the SAM/protein interface & on the protein surface
Cyt-c
S
S
S
S
S
S
S
S
¾ Introducing cysteine residues at
different positions of the Cyt-c surface
¾ chemical modification by
mercaptobenzonitrile
SAM
¾ Measuring the VSE
solution
How to probe electric field effects?
K39C
r
F = −1.5 ⋅ 108 V / m
Δν = ν SEIRA − ν IR
r r
r r
= Δμ ⋅ F ( x) = Δμ ⋅ F ( x) ⋅ cos(α )
experiment
⎛ Δν ( K 39C ) ⎞
⎜⎜
⎟⎟ = 0.4
⎝ Δν ( K 8C ) ⎠ exp
MD
simulations
⎛ Δν ( K 39C ) ⎞
⎜⎜
⎟⎟ = 0.24
⎝ Δν ( K 8C ) ⎠ calc
r
F = −6.2 ⋅108V / m
K8C
How to probe electric field effects?
Determining the electric field from the VSE and by theoretical calculations
™
Electrostatic calculations
™
Molecular dynamics simulations and QM/MM hybrid methods
SAM
solution
metaldependent
Electric field strength increases with
¾ decreasing distance from the electrode
¾ with increasing potential difference with respect to the potential of zero-charge Epzc
What do we learn from SERR
and SEIRA spectroscopy of
biomimetic systems?
What do we learn from SERR and SEIRA spectroscopy?
electrode
SAM
cytochrome c
S
S
S
S
S
S
S
S
Electron transfer
SERR:
Excitation in resonance
with the Soret transition
of the heme:
Probing oxidation
marker bands
What do we learn from SERR and SEIRA spectroscopy?
electrode
SAM
Stationary SERR spectra
cytochrome c
S
S
S
S
S
S
S
S
Electron transfer
equilibria
Met
Met
Fe2+
Fe3+
His
His
What do we learn from SERR and SEIRA spectroscopy?
electrode
SAM
cytochrome c
S
S
S
S
S
S
S
S
Potential jump
technique
Electron transfer dynamics
Met
Met
Fe2+
Fe3+
His
His
Time-resolved
SERR spectra
one-step
relaxation process
What do we learn from SERR and SEIRA spectroscopy?
electrode
SAM
cytochrome c
S
S
S
S
S
S
S
S
Redox-linked protein
structural changes
SEIRA:
Difference spectra „oxidised“
minus „reduced“
Monitoring redox-sensitive
amide bands
Time-resolved SEIRA:
(step & rapid scan)
What do we learn from SERR and SEIRA spectroscopy?
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Protein re-orientation
Differential enhancement of
totally symmetric (A1g) and nontotally symmetric modes (B1g)
depending on the heme
orientation
What do we learn from SERR and SEIRA spectroscopy?
electrode
SAM
cytochrome c
1361
B1Red
S
S
S
S
S
S
S
S
1491
Met
1591
2+
His
1371
Met
Electric-field
induced structural
changes
B1Ox
1500
1581
3+
His
1369
3+
coordination and
spin state changes
of the heme
(SERR)
Met80
B2Ox
HS
1577
1487
His
1374
His
3+
His18
B2Ox
LS
1504
1586
His
and
protein structural
changes (SEIRA)
His26
1300 1350 1400 1450 1500 1550 1600 1650
His33
-1
ν / cm
What do we learn from SERR and SEIRA spectroscopy?
Stationary potential-dependent SERR and SEIRA spectroscopy:
¾ Heme structure
¾ Conformational and redox equilibria
¾ Orientational distribution
Time-resolved potential-jump SERR and SEIRA spectroscopy:
¾ electron transfer
¾ protein and cofactor dynamics
¾ re-orientation
Interfacial processes of
cytochrome c
1. Dynamics of protein orientation and electron transfer
Re-orientation
Protein re-orientation:
intensity ratio of modes of
different symmetry
Heme reduction:
oxidation marker bands
heme reduction
S
S
S
S
S
S
S
S
Cyt c at C-15 SAM:
Electron tunnelling is rate limiting
1. Dynamics of protein orientation and electron transfer
Protein re-orientation:
intensity ratio of modes of
different symmetry
Re-orientation
Heme reduction:
oxidation marker bands
heme reduction
S
S
S
S
S
S
S
S
Cyt c at C-5 SAM:
Protein dynamics is rate limiting
2. Dynamics of redox-linked protein structural changes
β-turn III
aa 67-70
Protein structural changes monitored
by SEIRA spectroscopy
Lys 86,87
Lys 72,73
heme
Amide I band changes of the β-turn III
segment 67-70 occur simultaneously
with electron transfer
3. Electric-field dependence of electron transfer mechanism and dynamics
Re-orientation slows down with distance and increasing field strength:
Electron tunneling
k ET = A ⋅ exp(− dβ )
3. Electric-field dependence of electron transfer mechanism and dynamics
Change of ET mechanism with increasing the electric field strength
Low electric field:
High electric field:
Electron tunnelling rate-limiting
Gated electron transfer
4. Electric-field dependence of electron tunnelling
Electron tunneling regime at C-15 SAMs: TR-SERR data
Marcus-DOS
⎛ λ + eη
erfc ⎜
⎜ 4λ k T
k ET (η )
B
⎝
=
k ET (0)
⎛
λ
erfc ⎜
⎜ 4λ k T
B
⎝
⎞
⎟
⎟
⎠
⎞
⎟
⎟
⎠
Reorganisation energy
λ = 0.22 eV
4. Electric-field dependence of electron tunnelling
Electron tunneling regime at C-15 SAMs on different electrodes
SERR
SERR
SEIRA
Metal-specific overpotential
dependencies
4. Electric-field dependence of electron tunnelling
Electric-field dependent
retardation of electron tunnelling
different for Au and Ag
) + e[a
(
⎛ λ + e E − E0
erfc ⎜
⎜
k ET (E )
⎝
=
k ET (0)
⎛ λ + e a0
erfc ⎜
⎜
⎝
[
]
+ a1 (E − E pzc ) ⎞
⎟
⎟
4λ k B T
⎠
+ a1 E 0 − E pzc ⎞
⎟
⎟
4λ k B T
⎠
(
0
)]
5. Electric-field dependence of protein dynamics
Low electric field:
High electric field:
Electron tunnelling rate-limiting
Gated electron transfer
5. Electric-field dependence of protein dynamics
Molecular dynamics simulation and electron coupling analysis
Low affinity binding site – strong electronic coupling – fast ET
High affinity binding site – weak electronic coupling – slow ET
5. Electric-field dependence of protein dynamics
General description: orientational distribution and rate distribution
Preferred orientation
Optimum ET
5. Electric-field dependence of protein dynamics
Electric field effect on protein dynamics:
viscosity and H/D dependence
H2O
viscosity
1.2 cp
D2O
viscositycorrected
KIE of 2.0
5. Electric-field dependence of protein dynamics
Gating regime:
Increasing electric field slows down protein reorientation,
rearrangement of hydrogen bond network,
and electron tunnelling
S
S
S
S
S
S
S
S
e-
5. Electric-field dependence of protein dynamics
Gating via protein re-orientation: a general phenomenon
8
Observed for different
6
proteins but with different
4
ln k
0
app
limiting rates even at the
same electric field strength
2
-
1
-
2
Ag-S-(CH2)-CO2 Cyt c
Au-S-(CH2)-CO2 Cyt c
0
3
Au-S-(CH2)-CH3 Azurin
4
Au-S-(CH2)-Py Cyt c
+
-2
Au-S-(CH2)-NH3
5
Cyt b562
6
Au-S-(CH2)-CH3 / -OH Cyt c6
7
-4
Au-S-(CH2)-CH3 / -OH CuA
0
2
4
6
8
10
12
14
16
Number of methylenes
18
20
22
6. The interfacial redox process of cytochrome c
6. The interfacial redox process of cytochrome c
oxidation of cytochrome c
6. The interfacial redox process of cytochrome c
oxidation at low electric fields
Fast protein
structural
change
Electron tunnelling is
rate-limiting
Fast re-orientation
6. The interfacial redox process of cytochrome c
oxidation at high electric fields
Retardation of
electron
tunneling &
coupled protein
structural
changes
Protein dynamics is
rate-limiting
slow re-orientation
6. The interfacial redox process of cytochrome c
processes at very high electric fields
Major protein structural change including the
protein and the heme pocket
Switch from the redox to the apoptotic function
Cytochrome c
in apoptosis
Cytochrome c in apoptosis
Observations:
™ Cyt-c catalyses peroxidation of cardiolipin
™ Increased permeability of the inner
mitochondrial membrane
™ Cyt-c release to the cytosol
™ Involvement of Cyt-c in caspase-dependent
apoptotic pathway
What makes Cyt-c act as a peroxidase?
Cytochrome c in apoptosis
Cyt-c at very high electric fields
(electrode, lipid versicles):
Conformational changes involving
the dissociation of the Met ligand
from the heme – state B2
¾ very low redox potential
(cannot accept electrons from
complex III)
¾ strongly increased peroxidase
activity
Met
3+
His
3+
His
Met80
His
3+
His
His18
His26
His33
Cytochrome c in apoptosis
Transition from the
native B1 state to the
state B2 corresponds to
a switch from the redox
to the peroxidase
function
This switch is electric
field dependent
S
S
S
S
S
S
S
S
Conclusions
1.
Proteins at biomimetic interfaces: providing novel insight into
molecular processes under natural reaction conditions, such as
electric field effects
2.
SERR/SEIRA allow disentangling interfacial redox processes in
terms of
• protein (re-)orientation
• electron tunneling
• redox-linked structural changes
3.
Coated electrodes allow probing the electric field dependent switch
from the redox to the apoptotic function
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