Cellular Biophysics
SS 20007
Manfred Radmacher
Ch. 05 Photosynthesis Part 2
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Bacterial reaction centre
The trimeric Photosystem I reaction center
from
Synechococcus elongatus at 2.5 Å
resolution. View is
from the stromal side looking down into the
3-fold
symmetry axis. The chlorophylls are
depicted in yellow
and the stromal proteins PsaC, PsaD and
PsaE are
depicted in magenta, light-blue and cyan,
respectively,
From: P. Jordan, Ph.D. thesis, Freie
Universität, Berlin.
From: John H. Golbeck "Photosynthetic Reaction
Centers: So little time, so much to do" http://
www.biophysics.org/education/topics.htm
Cellular Biophysics
Ch. 05 Photosynthesis Part II
2
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Bacterial reaction centre
Figure 6 Depiction of the Rhodobacter sphaeroides
reaction center. The a-helices are depicted in
red/yellow; b-sheets in cyan; the bacteriochlorophyll
a and bacteriopheophytin a molecules in green; and
the non-heme iron in blue. The menbrane thickness
can be estimated by the length of the membranespanning
a-helices. From the Jena Image Library
for Biological Molecules; http://www.imbjena.
de/IMAGE.html
From: John H. Golbeck "Photosynthetic Reaction
Centers: So little time, so much to do" http://
www.biophysics.org/education/topics.htm
Cellular Biophysics
Ch. 05 Photosynthesis Part II
3
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Bacterial reaction centre
Figure 7. Cofactors of the bacterial reaction
center. The edge-to-edge distances between the
cofactors are depicted along with the electron
transfer rates. The distances are only
approximate and refer to the closest approach of
the aromatic rings (sufficiently accurate for the
purpose of this discussion). Note the pseudo C2
axis of symmetry that spans the
bacteriochlorophyll a dimer and the non-heme
iron.
From: John H. Golbeck "Photosynthetic Reaction
Centers: So little time, so much to do" http://
www.biophysics.org/education/topics.htm
Cellular Biophysics
Ch. 05 Photosynthesis Part II
4
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Bacterial reaction centre
BacterioChlorophyll-Dimer adsorbs light
P865 -> P865*
Electron Charge transfer to BChla (6Å, 2.8 ps)
P865* + BChla -> P865+ + BChla*Electron Charge transfer to BPhea (5.5Å, 2.3 ps)
BChla*- + BPhea -> BChla + BPhea*Electron Charge transfer to Qa (10Å, 200 ps)
BPhea*- + Qa -> BPhea+ Qa*Electron Charge transfer to Qb (15Å, 100 µs)
Qa*- + Qb -> Qa + Qb*-
Cellular Biophysics
Ch. 05 Photosynthesis Part II
5
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Bacterial reaction cycle
Figure 5. Cyclic operation of the bacterial reaction
center. Note that QA is only capable of a single
electron reduction to the semiquinone radical
state, whereas QB is capable of double reduction
followed by double protonation to the fullyreduced
hydroquinone state. The reduced
hydroquinone is loosely bound to its site and is
displaced by an oxidized quinone for another
round of light-induced turnover.
From: John H. Golbeck "Photosynthetic Reaction
Centers: So little time, so much to do" http://
www.biophysics.org/education/topics.htm
Cellular Biophysics
Ch. 05 Photosynthesis Part II
6
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Redoxpotentials in electron transfer chain
Figure 12. The components of the Photosystem II
reaction center depicted with redox potential on the
y-axis and rate of electron transfer on the x-axis.
Note the similarity in the identity of the cofactors
and the electron transfer rates with the bacterial
reaction center.
Absorption of photon has a
quantum yield of 1 at a 100%
conversion rate,
however, energy is lost in
electron transfer chain
otherwise recombination would
occur
Cellular Biophysics
Ch. 05 Photosynthesis Part II
From: John H. Golbeck "Photosynthetic Reaction
Centers: So little time, so much to do" http://
www.biophysics.org/education/topics.htm
7
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Why do we need two photons?
Energy of photon is ~ 1.6 eV
useful energy at the end of electron transfer chain: ~ 1 eV
energy needed for dissociation of water: ~ 1.3 eV
=> two photons
(sulfur bacteria dissociate H2S, one photon does the job)
Cellular Biophysics
Ch. 05 Photosynthesis Part II
8
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
In plants there are two Photosystems
Figure 14-47. Changes in redox
potential during photosynthesis. The
redox potential for each molecule is
indicated by its position along the
vertical axis. In photosystem II, the
excited reaction center chlorophyll has a
redox potential high enough to withdraw
electrons from water, by means of a
specially organized cluster of four
manganese atoms. Photosystem II
closely resembles the reaction center in
purple bacteria, and it passes electrons
from its excited chlorophyll to an
electron-transport chain that leads to
photosystem I. Photosystem I then
passes electrons from its excited
chlorophyll through a series of tightly
bound iron-sulfur centers. The net
electron flow through the two
photosystems in series is from water to
NADP+, and it produces NADPH as
well as ATP. The ATP is synthesized by
an ATP synthase that harnesses the
electrochemical proton gradient
produced by the three sites of H+
activity that are highlighted in Figure
14-46. This Z scheme for ATP
production is called noncyclic
photophosphorylation, to distinguish it
from a cyclic scheme that utilizes only
photosystem I (see the text).
From: Alberts et al, "Molecular Biology of the Cell"
Cellular Biophysics
Ch. 05 Photosynthesis Part II
9
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Photosynthetic units in different organisms
Green Bacteria
Cyanobacteria
Dinoflagellates
Green Plants
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
10
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
To enhance absorption cross section light harvesting complexes
are used
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
11
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Simulation of excitation processes in reaction centre
Cellular Biophysics
Ch. 05 Photosynthesis Part II
12
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Organization of pigments in LHC II and LHC I
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
13
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Light harvesting complex II (LHC II) of purple bacteria
The energy levels in LHCs are fine tuned by overlap of electronic levels between molecules in a periodic arrangement.
In these rings excitons (excited, delocalized electron-hole pairs)
Different orientations of B800 and B850
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
14
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Light harvesting complex I (LHC I) of purple bacteria
Exciton in LHI rings of BChl
ring -> reaction centre:
Förster transfer (distance too large for electron exchange)
PaPb -> BA:
electron exchange (overlap of wavefunctions)
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
15
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Excitation transfer between LHC II and LHC I
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
16
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Energy Levels in PSUs
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
17
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Protection mechanisms by carotenoid interactions
3O
2
+ 3BChl* -> 1O2 + 1BChl
Triplett Oxygen (ground state of oxygen)
quenches fluorescence and
produces highly reactive singuelett oxygen
-> Carotenoides quench this process
From:
X. Hu, A. Damjankovic, T. Ritz and K. Schulten, "Architecture and mechanism of the light-harvesting apparatus of purple bacteria", PNAS
(1998), 95(5935-5941
Cellular Biophysics
Ch. 05 Photosynthesis Part II
18
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
The reaction centre shows some analogy with a photodiode
From:
http://www.imo.physik.uni-muenchen.de/
Cellular Biophysics
Ch. 05 Photosynthesis Part II
19
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
How to measure ps-timescale chemical reactions?
From:
http://www.imo.physik.uni-muenchen.de/
Cellular Biophysics
Ch. 05 Photosynthesis Part II
20
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
How to measure ps-timescale chemical reactions?
From:
http://www.imo.physik.uni-muenchen.de/
Cellular Biophysics
Ch. 05 Photosynthesis Part II
21
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Summary of the primary processes of photo-synthesis
From:
http://www.imo.physik.uni-muenchen.de/
Cellular Biophysics
Ch. 05 Photosynthesis Part II
22
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Mitochondria
Muscle Cell Mitochondria (TEM x190,920)
Mitochondrion from a heart muscle cell showing numerous cristae
Cellular Biophysics
Ch. 05 Photosynthesis Part II
23
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Chloroplasts and Mitochondria share many similarities
Figure 14-1. Harnessing energy for life. (A) The essential
requirements for chemiosmosis are a membrane—in which
are embedded a pump protein and an ATP synthase, plus a
source of high-energy electrons (e-). The protons (H+) shown
are freely available from water molecules. The pump
harnesses the energy of electron transfer (details not shown
here) to pump protons, creating a proton gradient across the
membrane. (B) This proton gradient serves as an energy store
that can be used to drive ATP synthesis by the ATP synthase
enzyme. The red arrow shows the direction of proton
movement at each stage.
From: Alberts et al, "Molecular Biology of the Cell"
Cellular Biophysics
Ch. 05 Photosynthesis Part II
24
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Chloroplasts and Mitochondria share many similarities
Figure 14-3. Electron transport processes. (A) The mitochondrion converts energy from chemical fuels. (B) The
chloroplast converts energy from sunlight. Inputs are light green, products are blue, and the path of electron flow is
indicated by red arrows. Each of the protein complexes (orange) is embedded in a membrane. Note that the electronmotive force generated by the two chloroplast photosystems enables the chloroplast to drive electron transfer from
H2O to carbohydrate, and that this is opposite to the energetically favorable direction of electron transfer in a
mitochondrion. Thus, whereas carbohydrate molecules and O2 are inputs for the mitochondrion, they are products of
the chloroplast.
From: Alberts et al, "Molecular Biology of the Cell"
Cellular Biophysics
Ch. 05 Photosynthesis Part II
25
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Chloroplasts and Mitochondria share many similarities
Figure 14-29. Redox potential changes along the
mitochondrial electron-transport chain. The redox potential
(designated E′0) increases as electrons flow down the
respiratory chain to oxygen. The standard free-energy change,
ΔG°, for the transfer of each of the two electrons donated by an
NADH molecule can be obtained from the left-hand ordinate
(ΔG = -n(0.023) ΔE′0, where n is the number of electrons
transferred across a redox potential change of ΔE′0 mV).
Electrons flow through a respiratory enzyme complex by
passing in sequence through the multiple electron carriers in
each complex. As indicated, part of the favorable free-energy
change is harnessed by each enzyme complex to pump H+
across the inner mitochondrial membrane. It is thought that the
NADH dehydrogenase and cytochrome b-c1 complexes each
pump two H+ per electron, whereas the cytochrome oxidase
complex pumps one. It should be noted that NADH is not the
only source of electrons for the respiratory chain. The flavin
FADH2 is also generated by fatty acid oxidation (see Figure
2-77) and by the citric acid cycle (see Figure 2-79). Its two
electrons are passed directly to ubiquinone, bypassing NADH
dehydrogenase; they therefore cause less H+ pumping than the
two electrons transported from NADH.
From: Alberts et al, "Molecular Biology of the Cell"
Cellular Biophysics
Ch. 05 Photosynthesis Part II
26
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Fluorescence Labelling is a very important technique in modern
biology and biophysics
triple stain:
red: actin
green: micro tubules
blue: DNA
from:
http://www.itg.uiuc.edu/technology/atlas/structures/
Cellular Biophysics
Ch. 05 Photosynthesis Part II
27
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Examples of fluorophores
Fluorescent conjugates with polypeptides
A: A) Alexa 488-SP,
B) BODIPY Fl-SP,
C) fluorescein-SP,
D) Oregon Green 488-SP
E) tetramethylrhodamine-SP
from: Vicki J Bennett and Mark A Simmons "Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation", BMC Chemical
Biology Volume 1
Cellular Biophysics
Ch. 05 Photosynthesis Part II
28
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Examples of fluorophores
Fluorescent conjugates with polypeptides
A: A) Alexa 488-SP,
B) BODIPY Fl-SP,
C) fluorescein-SP,
D) Oregon Green 488-SP
E) tetramethylrhodamine-SP
from: Vicki J Bennett and Mark A Simmons "Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation", BMC Chemical
Biology Volume 1
Cellular Biophysics
Ch. 05 Photosynthesis Part II
29
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
GFP opens new labelling strategies
From: http://www.tsienlab.ucsd.edu/Images.htm
From: http://papilio.ab.a.u-tokyo.ac.jp/bioresource/shimada/index-e.html
Cellular Biophysics
Ch. 05 Photosynthesis Part II
30
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Optical Microscopy - STED
From:
http://www.mpibpc.gwdg.de/abteilungen/200/
Introduction to Biophysics
Ch. 2 Cells & Organelles
31
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Optical Microscopy - STED
From:
http://www.mpibpc.gwdg.de/abteilungen/200/
Introduction to Biophysics
Ch. 2 Cells & Organelles
32
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Optical Microscopy - STED
From:
http://www.mpibpc.gwdg.de/abteilungen/200/
Introduction to Biophysics
Ch. 2 Cells & Organelles
33
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
There is much more to fluorescence
The Fluorescent Toolbox for Assessing Protein Location and Function Ben N. G. Giepmans,1,2 Stephen
R. Adams,2 Mark H. Ellisman,1 Roger Y. Tsien2,3* SCIENCE VOL 312 14 APRIL 2006
Introduction to Biophysics
Ch. 2 Cells & Organelles
34
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
There is much more to fluorescence
The Fluorescent Toolbox for Assessing Protein Location and Function Ben N. G. Giepmans,1,2 Stephen
R. Adams,2 Mark H. Ellisman,1 Roger Y. Tsien2,3* SCIENCE VOL 312 14 APRIL 2006
Introduction to Biophysics
Ch. 2 Cells & Organelles
35
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Spectroscopy - inhomogenous line broadening
From: Moerner "Examining Nanoenvironements in solids on the scale of single, isolated impurity
molecule", Sience 1994, Vol, 265
Introduction to Biophysics
Ch. 2 Cells & Organelles
36
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Remember light scattering and Fourier transforms
decay
process
diffusion
Lorentzian
in homogenous line broadening
Gaussian
homogenous line broadening
Introduction to Biophysics
Ch. 2 Cells & Organelles
37
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Spectroscopy - spectral diffusion
From: Moerner "Examining Nanoenvironements in solids on the scale of single, isolated impurity
molecule", Sience 1994, Vol, 265
Introduction to Biophysics
Ch. 2 Cells & Organelles
38
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Spectroscopy - spectral hole burning
From: Moerner "Examining Nanoenvironements in solids on the scale of single, isolated impurity
molecule", Sience 1994, Vol, 265
Introduction to Biophysics
Ch. 2 Cells & Organelles
39
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Spectral Hole burning
From: Frauenfelder & Wolynes, "Biomolecules: where the physics of complexity and simplicity meet", Physics Today,
Febr. 1994, 58-64
Introduction to Biophysics
Ch. 5 Protein Structure
40
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Spectral Hole burning
From: Frauenfelder & Wolynes, "Biomolecules: where the physics of complexity and simplicity meet", Physics Today,
Febr. 1994, 58-64
Introduction to Biophysics
Ch. 5 Protein Structure
41
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Spectroscopy - blinking molecules
From: Thomas Basche & Christoph Bräuchle "Single molecule
spectroscopy in solids: quantum optics and spectral dynamics"
Introduction to Biophysics
Ch. 2 Cells & Organelles
42
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
One reason for blinking is being trapped in the triplet state
Figure 2.
Three-level energy scheme describing single molecule
fluorescence. S0 and S1 are the singlet ground and excited
states; T1 the first excited triplet state. The repeated S1-S0
excitation-emission photo-cycle yields the fluorescent signal.
Occasionally the molecule can drop into the T1 state
(intersystem crossing) where it gets trapped because the T1
lifetime is much longer than the S1 lifetime.
Figure 3.
Time image with 3.5 s of fluorescence blinking. Bright
streaks are due to S0-S1 cycling; dark streaks represent
residence in the T1 state. At first impression one sees noise,
yet detailed analysis reveals stochastic quantum jumps
governed by the triplet lifetime and singlet-triplet crossing
rate of the individual molecule.
From: María García-Parajó http://ot.tnw.utwente.nl/project.php?projectid=23&submenu=16
Introduction to Biophysics
Ch. 2 Cells & Organelles
43
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Fluorescence
Fig. 1. Labeling schemes (left) and physical observables (right). (A) Localization of a macromolecule labeled with a single
ßuorophore F with nanometer accuracy. The point-spread-function (PSF) can be localized within a few tenths of a
nanometer. (B) Colocalization of two macromolecules labeled with two noninteracting ßuorophores, F1 and F2. Their
distance can be measured by subtracting the center positions of the two PSFs. (C) Intramolecular detection of
conformational changes by spFRET. D and A are donor and acceptor; ID and IA are donor and acceptor emission intensities;
t is time. (D) Dynamic colocalization and detection of association or dissociation by intermolecular spFRET. Donor and
acceptor intensities are anticorrelated both in (C) and (D).
From: Shimon Weiss "Fluorescence Spectroscopy of
Single Biomolecules" SCIENCE 1999 p. 1676 VOL 283
Introduction to Biophysics
Ch. 2 Cells & Organelles
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INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Fluorescence
(E) The orientation of a single immobilized dipole can be determined by modulating the excitation polarization. The
ßuorescence emission follows the angle modulation. (F) The orientational freedom of motion of a tethered ßuorophore can
be measured by modulating the excitation polarization and analyzing the emission at orthogonal s and p polarization
detectors. IS and IP are emission intensities of s and p detectors. (G) Ion channel labeled with a ßuorescence indicator I.
Fluctuations in itsintensity II report on local ion concentration changes. (H) Combination of (C)and (G). D and A report on
conformational changes whereas I reports on ion ßux.
From: Shimon Weiss "Fluorescence Spectroscopy of
Single Biomolecules" SCIENCE 1999 p. 1676 VOL 283
Introduction to Biophysics
Ch. 2 Cells & Organelles
45
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Fluorescence
From: Shimon Weiss "Fluorescence Spectroscopy of
Single Biomolecules" SCIENCE 1999 p. 1676 VOL 283
Introduction to Biophysics
Ch. 2 Cells & Organelles
46
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher
Single Molecule Fluorescence combined with other techniques
From: Shimon Weiss "Fluorescence Spectroscopy of
Single Biomolecules" SCIENCE 1999 p. 1676 VOL 283
Introduction to Biophysics
Ch. 2 Cells & Organelles
47
INSTITUT FÜR
BIOPHYSIK
Universität Bremen
Prof. Manfred Radmacher