structures of photosynthetic pigment-protein complexes and probing
TRANSCRIPT
Osaka City University
Physics of Biological Materials Lab.
Hideki Hashimoto,1 Ritsuko Fujii,1 Satoru Suzuki,1Katsunori Nakagawa,2 Mamoru Nango,2
Aleksander W. Roszak,3 Alastair T. Gardiner,3 Richard J. Cogdell,3Shin-ichi Adachi,4,6 and Shin-ya Koshihara 5,6
1 Department of Physics, Osaka City University, Japan2 Department of Materials Engineering, Nagoya Institute of Technology, Japan
3 Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK4 Institute of Materials Structure Science, KEK, Japan
5 Department of Materials Science, Tokyo Institute of Technology, Japan6 Non-equilibrium Dynamics Project, ERATO/JST, Japan
E-mail; [email protected]
Structures of photosynthetic pigmentStructures of photosynthetic pigment--protein protein complexes and probing electrostatic field inside complexes and probing electrostatic field inside
them by Stark spectroscopythem by Stark spectroscopy
Osaka City University
Physics of Biological Materials Lab.
Contents
• A comparative look at the structural requirements for photosynthetic light-harvesting
• Structural study of the RC of purple photosynthetic bacteria
• Stark spectroscopy on the native and reconstituted pigment-protein complexes of purple photosynthetic bacteria
Osaka City University
Physics of Biological Materials Lab.
Osaka City University
Physics of Biological Materials Lab.
•The structure of all photosynthetic reaction centres is strongly conserved.
•This is because the structural constraints on electron transfer are very strict.
•A comparison of the structures of photosynthetic light-harvesting complexes shows that they form a very heterogeneous group. Why is this?
Osaka City University
Physics of Biological Materials Lab.
LHCII from spinach
Osaka City University
Physics of Biological Materials Lab.
Peridinin-Chlorophyll a complex
Osaka City University
Physics of Biological Materials Lab.
LH2 complexes
Osaka City University
Physics of Biological Materials Lab.
The physics of energy transfer is rather tolerant. This means that there are many structural solutions to the problem of building an efficient light-harvesting complex.
Osaka City University
Physics of Biological Materials Lab.
Absorption spectrum of the chromatophores of Rb. sphaeroides 2.4.1
400 600 800 10000
1
Wavelength / nm
Abs
orba
nce
Car
Qx
B
QyB
800
B85
0LH2
B87
5 (L
H1)
Osaka City University
Physics of Biological Materials Lab.
ATP synthase
ATPADP + Pi
H+
H+
H+
H+
LH2 LH1
RC
Light
Cyt c
Q
Q
Q
Q
e-
e-e-
B800
B850 B880
Cytochrome b/c1
Cytoplasm
Periplasm
QB
Q
QAQB
System of pigments in RC
Photosynthetic system of purple bacteria in the intracytoplasmic membrane
Fe
Osaka City University
Physics of Biological Materials Lab.
Osaka City University
Physics of Biological Materials Lab.
Contents
• A comparative look at the structural requirements for photosynthetic light-harvesting
• Structural study of the RC of purple photosynthetic bacteria
• Stark spectroscopy on the native and reconstituted pigment-protein complexes of purple photosynthetic bacteria
Osaka City University
Physics of Biological Materials Lab.
Shown above is the structure of RC from Rhodobacter sphaeroides, Roszak, Hashimoto, Cogdell, and Frank, et al. (2004) Structure, 12, 765.
Structure of the photosynthetic RC
H-subunit
L-subunitM-subunit
P
BABB
HA
HB
QAQB
Car
Fe
Osaka City University
Physics of Biological Materials Lab.
Electron transfer in the RC
P*P P+•
hν LM
3ps
200ps
150µs
Fe2+QB
QA
Bchla ‘Primary donor’ P
Accessory Bchla
Pheophytin
Spheroidene
Ubiquinone-10
Osaka City University
Physics of Biological Materials Lab.
Our high-resolution (1.95 Å) X-ray crystal structure analysis of the wild-type RC from Rba. sphaeroides
strain 2.4.1
Osaka City University
Physics of Biological Materials Lab.
Electron densities of the surfactant molecules surrounding the RC
Osaka City University
Physics of Biological Materials Lab.
‘Solvent’ molecules surrounding the RC
Osaka City University
Physics of Biological Materials Lab.
Contents• A comparative look at the structural requirements for
photosynthetic light-harvesting
• Structural study of the RC of purple photosynthetic bacteria
• Stark spectroscopy on the native and reconstituted pigment-protein complexes of purple photosynthetic bacteria
Osaka City University
Physics of Biological Materials Lab.
Research Background
Osaka City University
Physics of Biological Materials Lab.
Research Background
Great success of X-ray crystallographyon pigment-protein complexes of purple photosynthetic bacteria
Ultra-fast laser spectroscopy to understand each step of energy and electron transfer
Osaka City University
Physics of Biological Materials Lab.
Research Background
It is important to understand the exact mechanisms of electrostatic interaction between pigments and their
surrounding proteins in order to clarify the true functional mechanisms of the pigments in photosynthesis
Osaka City University
Physics of Biological Materials Lab.
Characteristics of electroabsorption (Stark effect) spectroscopy
• Pigments in pigment-protein complexes are under the influence of internal electric field produced by the electrostatic potential of apoprotein
• Electroabsorption spectroscopy is one of the most promising methods to probe the pigment-protein interaction modulated by the external electric field
Osaka City University
Physics of Biological Materials Lab.
Optical absorption spectra under the influence of external or internal electric field
Intensity change
Abs
orba
nce
Photon energy
∆A
Spectral shift
Abs
orba
nce
Photon energy
∆A
Broadening & Shrinkage
Abs
orba
nce
Photon energy
∆A
Osaka City University
Physics of Biological Materials Lab.
Liptay equation
),()2
)()()0,((
)0,(),()(
2int22
2
int
ζχννν
νννν
ννν
REdhAdH
hdAdFDA
AEAA
++=
−=∆
,22
2
MB
MAD += ,
22 α∆
+µ∆
=MAF 2µ∆=H
intintintint
intintintint
22
21)0()(
)(15/))1cos3)(1cos3(5(),(
∆αEE∆µE
BEEAEMM
−−ν=ν
++=−χ−ζ+=ζχ
mm hFh
FR
Stark effect
Osaka City University
Physics of Biological Materials Lab.
Experimental setup for electroabsorption spectroscopy
Monochromater
Photo-diode
Dual-phase Lock-in amp
DC
fSample cell
2f
PC
Pre-amplifier
Bipolar poweramplifier
AC
Osaka City University
Physics of Biological Materials Lab.
Contents of this presentation
1. Local electrostatic field induced by carotenoid in the reaction centre of purple photosynthetic bacteria [ J. Phys. Chem. B 109 (2005) 992-998]
2. Stark spectroscopy on the LH2 complex from Rhodobacter sphaeroides strain G1C; frequency and temperature dependence [J. Phys. Chem. B 108 (2004) 10334-10339]
Osaka City University
Physics of Biological Materials Lab.
Contents of this presentation
Local electrostatic field induced by carotenoid in the reaction centre of purple photosynthetic bacteria
[ J. Phys. Chem. B 109 (2005) 992-998]
Osaka City University
Physics of Biological Materials Lab.
ObjectiveEvaluation of electrostatic effect of Car on Bchls (especially P) in RC
Electroabsorption (EA) spectra of two reaction centres were recorded
One is prepared from Rb. sphaeroidesstrain R26.1 (R26.1-RC), which lacks carotenoid
R26.1-RC
Osaka City University
Physics of Biological Materials Lab.
ObjectiveEvaluation of electrostatic effect of Car on Bchls (especially P) in RC
The other one is a reconstituted RC (R26.1 -RC+ Car) which was prepared by re-incorporating synthetic Car (3,4-dihydrospheroidene) into R26.1-RC
Electroabsorption (EA) spectra of two reaction centres were recorded
R26.1-RC+Car
Osaka City University
Physics of Biological Materials Lab.
ObjectiveEvaluation of electrostatic effect of Car on Bchls (especially P) in RC
The electrostatic effect of Car on P was evaluated from the difference of these two spectra
Osaka City University
Physics of Biological Materials Lab.
A.W. Roszak, K. McKendrick, A.T. Gardiner, I.A. Mitchell, N.W. Isaacs, R.J. Cogdell, H. Hashimoto, and H.A. Frank, Structure, 12, 2004, 765.
Gatekeeper (Phe M162) and locking (Trp M75) amino acid residues in the site of RC where Car is re-incorporated
Trp M75
Phe M162
Osaka City University
Physics of Biological Materials Lab.
Influence of static-electric field on the excited state of Bchls caused by the presence of Car
Electrostatic field F around Bchls in RC
cpa FFFF ++=
Fa: applied electric fieldFp: pocket fieldFc: static-electric field due to Car
Osaka City University
Physics of Biological Materials Lab.
Absorption change due to applied electric field
Absorption change (∆A) due to applied electric field can be written as,
22
22*
61
61
31
adAd
dAdFDAA F∆µ ⎟⎟
⎠
⎞⎜⎜⎝
⎛++=∆
νν
νν
( ) 021 22 ≈+= YµX
µeg
eg
D [ ] [ ]∆α∆µµ
Xµ∆α TrTrF ≈+= *2 2
eg
eg
cpint* F∆αF∆α∆µ∆µ ⋅+⋅+=
Osaka City University
Physics of Biological Materials Lab.
Influence of Car on Bchl
Change of ∆µ value by static-electric field due to the presence of Car
cc F∆α∆µ ⋅=
This change is expected to be observed by EA spectroscopy
The contribution of ∆µ is detected as the second-order derivative waveform of absorption spectra in EA spectra.
Therefore, the second derivative waveform should be observed in the difference EA spectra between R26.1-RC and
R26.1-RC+Car.
Osaka City University
Physics of Biological Materials Lab.
Electroabsorption spectra of RC’s from Rb. sphaeroides R26.1 at 79 KIn-phase Quadrature-phase
10000 12000 14000–3
0
3–10
–5
0
5
–5
0
5
0
1
Nor
mal
ized
abso
rban
ceN
orm
aliz
ed ∆
Ain
[×10
–6]
(a) Rb. sphaeroides R26.1
(b) Rb. sphaeroides R26.1+ Car
(c) (a)–(b)
P
BH
at 79 K
Wave number [cm–1]
–10
–5
0
5
–5
0
5
0
1
10000 12000 14000–3
0
3N
orm
aliz
edab
sorb
ance
Nor
mal
ized
∆A
quad
[×10
–6]
(d) Rb. sphaeroides R26.1
(e) Rb. sphaeroides R26.1+ Car
P
BH
at 79 K
Wave number [cm–1]
(f) (d)–(e)
Osaka City University
Physics of Biological Materials Lab.
Electroabsorption spectra of RC’s from Rb. sphaeroides R26.1 at 293 KIn-phase Quadrature-phase
10000 12000 14000–3
0
3
–5
0
5
10
–5
0
5
100
1
Nor
mal
ized
abso
rban
ce
(a) Rb. sphaeroides R26.1
(b) Rb. sphaeroides R26.1+ Car
(c) (a)–(b)
Nor
mal
ized
∆A
in [×
10–6
]
Wave number [cm–1]
P
BH
at 293 K
10000 12000 14000–3
0
3–5
0
5–5
0
50
1
Nor
mal
ized
abso
rban
ce
(d) Rb. sphaeroides R26.1
(e) Rb. sphaeroides R26.1+ Car
(f) (d)–(e)N
orm
aliz
ed ∆
Aqu
ad [×
10–6
]
Wave number [cm–1]
P
BH
at 293 K
Osaka City University
Physics of Biological Materials Lab.
Nonlinear optical parameters of P bandP band D [10-18
(m/fLV)2]Tr[∆α] [Å3/fL
2]∆µ∗ [D/fL]
R26.1-RC -1.5 ± 0.4 (2.2 ± 0.3)·102 6.5 ± 0.3
R26.1+Car-RC -1.8 ± 0.4 (2.4 ± 0.8)·102 6.5 ± 0.3
R26.1-RC 3.9 ± 0.4 -(1.7 ± 0.1)·103 10.8 ± 0.4R26.1+Car-RC 3.6 ± 0.4 -(1.7 ± 0.4)·103 9.0 ± 0.4
293 K
79 K
Fext is externally applied electric field.fL is a local field correction factor.exta FF ⋅= Lf
Even if the experimental errors are taken into account, the difference of ∆µ∗ between R26.1-RC and R26.1-
RC+Car at 293 K is estimated to be ~1.0 [D].
Osaka City University
Physics of Biological Materials Lab.
Nonlinear optical parameters of B band
B band D [10-18
(m/fLV)2]Tr[∆α] [Å3/fL
2]∆µ∗ [D/fL]
R26.1-RC 0.9 ± 0.2 -(1.2 ± 0.4)·102 2.8 ± 0.3
R26.1+Car-RC 1.4 ± 0.5 -(1.1 ± 0.2)·102 2.9 ± 0.2
R26.1-RC -7.1 ± 0.5 -(5.3 ± 3.6)·10 2.1 ± 0.1R26.1+Car-RC -7.0 ± 1.7 -(5.8 ± 2.4)·10 1.5 ± 0.2
293 K
79 K
∆µ∗ of B band is different between R26.1-RC and R26.1-RC+Car at 293 K.
Osaka City University
Physics of Biological Materials Lab.
H band D [10-18
(m/fLV)2]Tr[∆α] [Å3/fL
2]∆µ∗ [D/fL]
R26.1-RC 0.4 ± 0.2 -(8.4 ± 7.2)·10 4.4 ± 0.3R26.1+Car-RC 0.4 ± 0.1 -(0.9 ± 2.0)·10 4.3 ± 0.3
R26.1-RC 0.8 ± 0.2 (9.5 ± 2.3)·10 6.2 ± 0.4
R26.1+Car-RC 0.8 ± 0.3 (10.0 ± 4.0)·10 5.7 ± 0.5
293 K
79 K
Nonlinear optical parameters of H band
No significant difference is observed between R26.1-RC and R26.1+Car-RC in the nonlinear optical parameters of H band
Osaka City University
Physics of Biological Materials Lab.
Estimation of static-electric field induced by the presence of carotenoid
]/[ 0.1 Lcc fDF ≈⋅∆=∆ αµ
]cmV[ 101 5×≈cF fL = 2.2b
Reported localized field around P is ~1×106 [V/cm].a
The presence of Car causes ~10 % change of the electrostatic environment around P. According to our results of X-ray crystallography of RC,c carotenoid binding pocket becomes smaller when Car is absent. This may cause some rearrangements of amino acid residues around P. As a consequence, the above ~10 % changeshould be occurred.
[a] T.R. Middendorf et al., BBA, 1143, 1993, 223.[b] M. Loche et. al., PNAS, 84, 1987, 7537.[c] A.W. Roszak et al., Structure, 12, 2004, 765.
Osaka City University
Physics of Biological Materials Lab.
Conclusions on RC
Carotenoid bound to the RC was found to affect on the electrostatic environment around P and B (BB).
The static electric field on P due to the presence of carotenoidwas estimated to be ~ 1×105 [V/cm]. It corresponds to as high as 10% of the local electric field around P.
Carotenoid could be one of the important factors that regulate the function of P (vice versa).
Osaka City University
Physics of Biological Materials Lab.
Contents of this presentation
1. Local electrostatic field induced by carotenoid in the reaction centre of purple photosynthetic bacteria [ J. Phys. Chem. B 109 (2005) 992-998]
2. Stark spectroscopy on the LH2 complex from Rhodobacter sphaeroides strain G1C; frequency and temperature dependence [J. Phys. Chem. B 108 (2004) 10334-10339]
Osaka City University
Physics of Biological Materials Lab.
Contents of this presentation
Stark spectroscopy on the LH2 complex from Rhodobacter sphaeroides strain G1C; frequency and temperature
dependence [J. Phys. Chem. B 108 (2004) 10334-10339]
Osaka City University
Physics of Biological Materials Lab.
How can we evaluate electrostatic interaction between pigments and apoproteins?
• Most previous reports on the use of Stark spectroscopy to study photosynthetic pigment-protein complexes have only used in-phase signals
• More detailed information on dynamic electrostatic interactions with the environment can be obtained from out-of-phase (quadrature-phase) signal
Osaka City University
Physics of Biological Materials Lab.
How can we evaluate electrostatic interaction between pigments and apoproteins?
By studying the frequency and temperature dependence of the quadrature-phase signals the nature of the pigment-protein interaction
can be investigated!
Osaka City University
Physics of Biological Materials Lab.
Absorption spectra of the LH2 complex from Rb. sphaeroides G1C
300 500 700 9000
10
1
Wavelength [nm]
in PVA matrix
in buffer solution(a)
(b)
Nor
mal
ized
abs
orba
nce
Nor
mal
ized
abs
orba
nce
Osaka City University
Physics of Biological Materials Lab.
Electro-absorption spectra of the LH2 complex from Rb. sphaeroides G1C
10000 12000 14000
–2
0
2
–1
0
0
1
Nor
mal
ized
Abs
orba
nce
∆A
quad
[×
106 ]
Wavenumber [cm–1]
(a)
(b)
(c)
∆A
in [×
105 ]
← in phase
← quadrature phase
Osaka City University
Physics of Biological Materials Lab.
Theoretical model to generate the phase-retarded signals
( ) ( )2
2
2
2*21::
21*
ννν ∂∂⋅
+∂∂∆
+∂∂⋅
=∆ΑA
hA
hA
hE∆µEαEE∆µ
pocketmol E∆α∆µ∆µ :* +=
( ) ( ) xC0ExE pocketpocket ⋅+=
∆µ∗ is dipole-moment change upon photoexcitation∆α is polarizability change upon photoexcitation
Osaka City University
Physics of Biological Materials Lab.
Dependence of the phase-retarded signals on the modulation frequency of the applied
electric field
mq
dtd
dtd Exxx ⋅
+−−= 202
2
ωγ )sin()( tft extL ωEE ⋅=
( )( ) )sin()( 2/122222
0
pextL ωtf
mqt φ
ωγωω+
+−
⋅=
Ex
20
2tanωω
γωφ−
=p
Osaka City University
Physics of Biological Materials Lab.
Absorption change modulated with 2ω frequency
( )
( )2
2cos61
22cos
3x
2ext
2L
2
2
2
2*
2ext
2L0
2
tEfAh
Ah
tEfAhCA
ωνννµ
νννα
φωννναω
⎟⎟⎠
⎞⎜⎜⎝
⎛
∂∂∆
+∂∂∆
+
−∂∂
⋅∆=∆
Osaka City University
Physics of Biological Materials Lab.
In-phase signals detected by a dual-phase lock-in amplifier
( )
( )26
1
2cos2x
61
2ext
2L
22
22*
in
2ext
2L
22
22*0
in
Efh
AhAF
Efh
AAh
αCh
A
⋅⋅⎟⎟⎠
⎞⎜⎜⎝
⎛∂
∂∆+
∂∂
⋅=
⋅⋅⎥⎦
⎤⎢⎣
⎡∂
∂∆+
∂∂
⋅⎟⎠⎞
⎜⎝⎛ ∆⋅⋅
+∆
=∆
νννµ
ννν
νννµ
νννφα
( ) ( )( ) ⎟
⎟⎠
⎞⎜⎜⎝
⎛
+−
−+⋅∆=⋅+⋅∆=
222220
220
0n 21cosx21ωγωω
ωωαφα
mqCCFi
Osaka City University
Physics of Biological Materials Lab.
Quadrature-phase signals detected by a dual-phase lock-in amplifier
( )
261
2sin∆2x
61
2ext
2L
quad
2ext
2L
0quad
EfhAF
EfhAαCA
⋅⋅
∂∂
⋅=
⋅⋅
∂∂
⋅⋅⋅=∆
ννν
νννφ
( ) 222220
0quad 2sinx2ωγωω
γωαφα+−
∆=∆⋅⋅=mqCCF
Osaka City University
Physics of Biological Materials Lab.
Temperature dependence of in-phase and quadrature-phase EA spectra
10000 12000 14000–1
0
–1
0
–1
0
–1
0
10000 12000 14000–0.2
0
0.2–0.2
0
0.2–0.2
0
0.2–0.2
0
0.2
79K
200K
270K
293K
Wavenumber [cm–1]
∆A
in
(a)N
orm
aliz
ed
Wavenumber [cm–1]
79K
200K
270K
293K
Nor
mal
ized
(b)
∆A
quad
Osaka City University
Physics of Biological Materials Lab.
Temperature dependence of Fquad / Fin value
⎟⎠⎞
⎜⎝⎛−∝∝
−++−⋅⋅
=
RTH
CqmF
F
r
exp1
])[( 220
222220
τ
ωωωγωω
ωγ
21
in
quad
H is the activation energy that characterizes the relaxation factor γ
Osaka City University
Physics of Biological Materials Lab.
Temperature dependence of Fquad / Fin value
100 200 300
0
0.2
0.4
0.6
Temperature [K]
Fqu
ad /
Fin
H ~ 12 kJ / mol
Quite a number of molecular motions can be plausible candidates that generate the relaxation factor, γ.
For example, local mode relaxation, crankshaft and kink motion, rotation of methyl group, side chain motion, local intermolecular rearrangements etc.
Osaka City University
Physics of Biological Materials Lab.
Temperature dependence of Fquad / Fin value
Inspection of X-ray crystal structure of the LH2 complex from Rps. acidophila strain 10050, suggests that a plausible candidate for the physical origin of γ could be either a local twisting motion of the apoprotein main chain, or rotational motion of apoprotein side chains
100 200 300
0
0.2
0.4
0.6
Temperature [K]
Fqu
ad /
Fin
H ~ 12 kJ / mol
Osaka City University
Physics of Biological Materials Lab.
Frequency dependence of Fquad value
( )( ) 2222222
0
quad44
22ff
fm
qCfFγππω
πγα+−
∆=
fπω 2=
Osaka City University
Physics of Biological Materials Lab.
Dependence of the phase-retarded signals on the modulation frequency
0 500 10000
1
2
Fqu
ad( f
) / F
quad
(500
)
Frequency [ f ]
ω0 ~ 11 kHz
secm sec
μsec
n sec
p sec
f sec
Rotation of an aromatic sidechain of an amino acid residueinside a protein
Stretching vibration
Deformation vibration
Osaka City University
Physics of Biological Materials Lab.
The X-ray crystal structure of the LH2 complex from Rps. acidophila strain 10050 clearly shows that the Mg2+ ions at the centre of the B850 bacteriochlorin rings are liganded to histidine side chains.
These histidine residues are prime candidates for the strong electrostatic interaction suggested by the presence of the phase-retarded signals.
However, the resonance frequency of 11 kHz looks too low for a normal mode reflecting the Mg-His interaction (~1 THz).
LH2 subunit of Rps. acidophila 10050
Osaka City University
Physics of Biological Materials Lab.LH2 subunit of Rps. acidophila 10050
The relevant normal mode could be an overall, coupled mode reflecting the total environment of pigment-protein interaction
Osaka City University
Physics of Biological Materials Lab.
Conclusions on LH2Electroabsorption (Stark effect) signals due to electrostatic interactions between the B850 pigments and surrounding apoproteins were detected as a phase-retarded signal using dual-phase lock-in detectionThe Arrhenius type activation energy (H = 12 kJ/mol) was determined from the temperature dependence of the phase-retarded signals. The resonance frequency (ω0 = 11 kHz) was determined from their frequency dependence.The calculated thermodynamic and kinetic parameters can be accounted for by assuming a strong electrostatic interaction of the B850 pigments with the dynamic environment provided by the apoproteins.
Osaka City University
Physics of Biological Materials Lab.
Thank you for your attention !