Diller, A.
Citation
Diller, A. (2007, September 18). Photo-CIDNP MAS NMR Studies on photosynthetic reaction centers. Retrieved from https://hdl.handle.net/1887/12365
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Chapter 6
Current view and Outlook
In this thesis, new insights into the photochemical machinery of plant reaction centers and on the mechanism of the solid-state photo-CIDNP effect have been presented. These new insights also provide perspec- tives on future studies on photosynthetic RCs as well as applications of the solid-state photo-CIDNP effect in NMR spectroscopy.
6.1 The donor of photosystem II
The central aim of the Ph.D. thesis is to resolve the origin of the uniquely high redox potential of PS II. A ’hinge model’ for the donor of PS II has been inferred from the pattern of electron spin density distribution, accomplished by photo-CIDNP studies on uniformly la- beled 15N and unlabeled 13C of plant PS I and PS II. In this model, the shift of the electron spin density as a source of the high redox potential is based on a tilt of the axial histidine towards pyrrole ring IV causing π-π overlap of both aromatic systems. The model shows how the matrix involvement can cause a dramatic shift on the active cofactor. This is well in line with propositions that a local electro- static field in a protein can be caused by hydrogen bonding, chemical modifications or transient mesoscopic changes. Hence, the model en- courages to perform experiments on histidine mutants or on selectively
15N- or on 13C isotope labeled histidine, or inversely labeled samples to explore the matrix involvement. The chemical shift assignments presented in this work are based on 1D NMR data, and are tenta- tive. A comprehensive map of the ground-state electronic structure of the photochemically active cofactors can be achieved by performing
13C-13C dipolar correlation photo-CIDNP MAS NMR. For bacterial RCs of Rb. sphaeroides, the complete ground state electronic struc- ture has been revealed by obtaining 2D homonuclear 13C-13C dipolar correlation spectra using radio frequency driven recoupling and pro-
ton driven spin diffusion pulse sequences on site-directed 13C labeled samples (Prakash et al., 2007). Using a similar strategy, the tentative assignments for the photosynthetic active cofactors presented in this thesis can be validated and the proposed matrix involvement may be verified.
Another interesting experimental approach is to study the Chl-His model with vibrational spectroscopy. As this study would concen- trate on the out-off-plane modes, information about the ruffling of the macrocycle can be obtained (Shelnutt et al., 1998; Spiro and Was- botten, 2005). A better understanding of the model can be provided as well by studying the axial Mg-His stretching modes, which have been observed in bacterial RCs (Czarnecki et al., 1997). The applica- tion of Resonance Raman Spectroscopy to elucidate the protonation state of the axial histidine is of particular interest. For instance, in plant heme peroxidases, Resonance Raman spectroscopy studies have already provided crucial insights on the control of the redox properties of the cofactor by variation of hydrogen bonding of the axial histidine (Smulevich et al., 2005).
The ’hinge model’ also stimulates theoretical studies for further investigation. Preliminary results of quantum-chemical computations of this model already showed that depending on the tilt angle up to 40% of spin density can be localized on the axial histidine ligand.
Slight tilts, which do not cause a significant energy increase, lead to spin densities around 18%, which would be well in agreement with the proposed model (Gunnar Jeschke, personal communication). System- atic studies of the energetics and spin density distribution at different tilt angles could provide a complete theoretical understanding of the
’hinge model’ and its effect on the redox properties. This studies are already in progress in the group of Prof. Gunnar Jeschke at the Uni- versity of Konstanz.
The relevance of the ’hinge-model’ lies not only in the understand- ing of the uniqueness of the redox potential but may also trigger ideas in order to increase the redox force in artificial photosynthetic devices.
6.2 Photo-CIDNP as a tool for functional
screening of RCs and spectral editing
Photosynthesis is carried out by many different organisms, ranging from plants to bacteria, which provide a wide range of different pho- tosynthetic RCs. The RCs show a large range of electronic properties at both, the donor and acceptor sides. The donor side can be formed of various cofactors, e.g. BChl a, Chl a or Chl a’, which can be arranged as monomers, asymmetric dimers and symmetric dimers. Also the ac- ceptor side can vary, e.g., two parallel branches have been proposed on the acceptor side of PS I (Li et al., 2006). Studies performed in this work add further to this variation, and contribute to converging evi- dence that the protein matrix is involved on the donor side (Chapter 4). For understanding the whole picture of light reactions of the pri- mary process of photosynthesis, it is therefore important to analyze a wide range of RCs. Traditional methods for spectroscopic or structural characterization of RCs depend, however, on the feasibility of growth, isolation, labeling or crystallization of RCs. Therefore, only few RCs, such as the RCs of the purple bacterium of Rb. sphaeroides have been studied extensively. The occurrence of photo-CIDNP appears to be a property of all photosynthetic RCs (Roy, 2007) and appears to be linked to the high efficiency of the initial light-induced electron trans- fer (Daviso et al., 2007). In this thesis, it has been shown that high- quality photo-CIDNP data can be obtained from membrane-bound RCs. Hence, even if a RC isolation is not feasible, photo-CIDNP MAS NMR still can be applied for the analysis of the electronic structure.
In this thesis it also has been demonstrated that from uniformly 15N labeled RCs, an in-depth analysis of essential details of the photochem- ical machinery of a complex RC is possible, providing information that is otherwise inaccessible. In fact, photo-CIDNP is the only technique known until now, which has the capability to provide a picture of the electronic ground state of RCs (Schulten et al., 2002; Prakash et al., 2007). Since uniformly 15N labeling is rather easy to achieve, photo- CIDNP MAS NMR on uniformly 15N labeled and reduced membranes and cells may provide an option for fast screening of functional proper- ties of RCs from various photosynthetic organisms. The experiments on chromatophores presented in this thesis revealed an inversion of
donor signals from emissive (negative) signals to absorptive (positive) signals upon isotope labeling while the acceptor signals remain unaf- fected. It is possible that a parameter change on the spin-chemical machinery may be responsible for this phenomenon. Further studies on entire cells and with chromatophores having different label patterns and label concentration as well as having different chemical treatment may elucidate the origin of this remarkable effect. It may be useful to combine microscopy, optical methods and magnetic resonance tech- niques in order to understand the origin of the effect on a molecular level. The properties distinguishing between donor and acceptor sig- nals might help to develop a understanding of spin-chemical switches, which can be used to analyze spin properties of active cofactors found in binding pockets or play a role in e.g. spintronics applications. In solid-state NMR, molecular mechanisms controlling the sign-change between e.g. donor and acceptor signals may open new possibilities for spectral editing.
6.3 Orientational effects in Photo-CIDNP
MAS NMR
For the explanation of the solid-state photo-CIDNP effect a combi- nation of several mechanisms: the TSM, the DD and the DR, has been put forward (Jeschke and Matysik, 2003; Daviso et al., 2007).
Nuclear polarization of both TSM and DD mechanisms derives from anisotropic interactions, namely the anisotropy of the hf coupling, the difference between the two electron g tensors, and in case of the TSM also the anisotropic dipole-dipole coupling between the two electron spins. This leads to an orientational dependence of the photo-CIDNP effect (Polenova and McDermott, 1999). In contrast to the two co- herent mechanisms, the TSM and the DD, in the DR mechanism the contribution to the net nuclear polarization with steady state illumi- nation is generated by stochastic relaxation processes which appear to be isotropic and involve moderate time scales≈100 μs. Photo-CIDNP MAS NMR on macroscopically ordered photosystems would enable to visualize the anisotropic effect and to correlate the observed electronic structure of the cofactors with their protein environment. Cofactors
B
0 ZrZD
m
r
Figure 6.1: Principle of MAOSS. Circular glass plates with uniformly aligned membranes are assembled to form a stack and mounted inside a rotor for MAOSS rotating with the frequency,ωr. The membrane normal ZDis parallel to the rotor axis Zrwhich is tilted with respect to B0by the magic angleθm=54.7◦.
with different orientation in respect to the membrane normal, such as the donor and acceptor may result in distinct changes of the sign of photo-CIDNP signals and therefore providing a possibility for spec- tral editing. Due to the fact that the DR contribution can open up a relaxation path (discussed in Chapter 2), which may cause quenching or enhancement of signals and the fact that DR looses orientational properties, photo-CIDNP signals derived from RCs involving not only the TSM and the DD, but also the DR mechanism are expected to be different.
A way to perform photo-CIDNP experiments on oriented RCs is the MAOSS method, in which the sample is aligned on circular glass plates to form a stack and mounted inside a 7-mm sapphire MAS rotor (Figure 6.1) (Glaubitz and Watts, 1998). Here the first photo-CIDNP MAS NMR experiments on oriented RCs of photo- synthetic Rb. sphaeroides WT, its carotenoid-less mutant R-26 and on plant PS II are presented to illuminate the effect of orientation on the photo-CIDNP signal. The RCs were incorporated into L-α- phosphatidylcholine (egg, chicken; obtained from Avanti Polar-Lipids, Inc., Alabama, USA) bilayers, keeping a lipid/protein weight ratio of usually 1:1. The protein-lipid mixture has been prepared by reconsti- tuting the purified protein in a detergent solutions in buffer, followed
by detergent removal. Drops of about 22 μL containing around 0.38 mg protein were spread onto round glass plates with a diameter of 5.4 mm and 0.1 mm thickness (Marienfeld GmbH, Lauda-K¨onigshofen, Germany) and dried. The glass plates were mounted into the MAS rotor and rehydrated to facilitate hydration-induced self-assembly of lipids, as described (Glaubitz and Watts, 1998; Watts, 2005; Lopez et al., 2007).
13C Photo-CIDNP MAOSS NMR on WT RCs of a randomly oriented and oriented quinone-depleted sample of Rb. sphaeroides un- der continuous illumination using white light, are shown in Figure 6.2 A and B. The light-induced photo-CIDNP signals emerging from the aromatic region have been assigned as depicted in Table 6.1 by 2D photo-CIDNP NMR (Schulten et al., 2002; Prakash et al., 2007). The three signals at 134.0, 132.8, 119.4 ppm have been attributed to axial histidines (Alia et al., 2001). The signals of the oriented sample, Fig- ure 6.2B are in contrast to the spectrum of randomly oriented RCs, as both enhanced absorptive and emissive signals are observed. In addi- tion several signals are more pronounced in the oriented spectrum.
In the randomly oriented sample the two strongest emissive peaks are assigned to M19 and M14 of the donor BChls, respectively, while as the strongest emissive peaks in the oriented sample arise from M2 and M11 of the donor BChl. The methine carbons φ-5, φ-10 and φ20 appear as very strong emissive signals upon orientation, as well. In the oriented spectrum additionally peaks appear at 106.2 and 101.8 ppm, which can be attributed to methine carbons of the BPhe acceptor.
The most downfield shift at 189.4 ppm, attributed to the carbonyl of the BPhe acceptor, C-φ31 is only visible in the oriented Figure 6.2B, but it has been observed also in randomly oriented samples at higher spinning frequencies (Prakash et al., 2005). In addition, the signal at 134.0 ppm assigned to φ1, and the signal at 132.8 ppm attributed to φ2, C-13 of BChl a or BPhe a, are more pronounced upon orientation. The weak positive features only found in the oriented spectrum indicated by triangles, at around 155, 152 and 118, appear to be caused by strong dispersive emissive signals assigned to L9/M9, L11 and φ12, respectively. Also the signal at 160.1 ppm, assigned to M14 appears to be slightly dispersive. In addition weak positive features appear at around 142, 105 ppm upon orientation, which may
as well result form dispersion of emissive signals.
In order to further investigate whether the positive signals are caused by dispersive emissive signals, it is worth considering simi- lar effects, such as the crystallite effect observed in NMR, when the magnetization of the crystallite interferes with the echo (Olejniczak et al., 1984). Further experiments would be needed to clarify these aspects.
Simulations for randomly oriented and oriented RCs are in prin- ciple possible (Figure 6.2 C and D), but it appears to be difficult to reproduce the orientational effect in WT RCs in the simulations. In fact, the simulations suggest that the effect of MAOSS on the photo- CIDNP spectra of WT RCs is mainly manifested by changes in the relative peak intensity. In the simulations only the integral polariza- tion over all orientations was considered. Phase distortions are beyond the scope of the current simulation approach. The simulations do not consider the above mentioned crystalline effect, further validation of the experimental orientational effects taking the crystalline effect into account will be required.
Photo-CIDNP MAOSS NMR on R-26 RCs of a randomly oriented and oriented sample are shown in Figure 6.3 A and B, respec- tively. The enhanced absorptive (positive) signals originating from the donor cofactors show no significant differences. This observation is in line with the theory, hence, in R-26 RCs, the absence of a carotenoid as a triplet quencher causes a long-living donor triplet state and the production of net nuclear polarization by the DR mechanism (Prakash et al., 2006). The DR mechanism, which is based on a stochastic re- laxation process appears to therefore destroy the orientational effect caused by the TSM and the DD mechanism (Chapter 2). The acceptor signals of BPhe a, caused by the coherent mechanisms TSM and DD solely, don’t show a pronounced effect, either. Stronger orientational effects might still occur if the polarization for the dominant orienta- tions would have a different sign than the polarization averaged over all orientations. However, this neither applies to the donor nor the ac- ceptor signals (G. Jeschke, unpublished). Even for a perfect MAOSS alignment the magnetic field B0 vector is distributed over a cone that includes an angle of 35.26◦ with the membrane plane. Hence, with respect to the membrane plane, only the C2 pseudo-symmetry axis of
Table 6.1: 13C chemical shifts of the photo-CIDNP signals
photo-CIDNP photo-CIDNP
carbon WTa,b R-26c carbon WTa,b R-26c
φ131 189.4 - L4 - 136.8
L6 164.0 164.4 A φ3 134.8 -
M19 162.3 162.5 A φ2 134.0 133.7 E
M14 160.1 161.0 A M13, L13 131.0 - L9, M9 158.7 158.8 A φ13 126.4 - M16 150.9 151.3 A L12, M12 - 124.6 A
L11 153.6 153.7 A φ12 119.4 119.7 E
M1 148.2 148.6 A φ15 108.5 106.8 E
M11 145.3 145.6 A φ10 - 101.3 E
M2 143.4 143.8 A φ5 97.4 97.8 E
φ1 138.3 138.8 E φ20 94.9 95.2 E
a Assignment for carbons C-1, C-3, C-6, C-8, C-11, C-13, C-17 and C-19 from 2D assignments (Schulten et al., 2002)
b Assignment for carbons C-4, C-5, C-9, C-10, C-14, C-15, C-16 and C-20 from 2D assignments (Prakash et al., 2007).
c Assignment based on tentative assignments (Prakash et al., 2006).
φ = BPhe,
L = BChl P2located on the active A branch,
M = BChl P1 located on the B branch (Prakash et al., 2007).
the RCs is fixed. The distribution of orientations of the magnetic field B0 vector in the molecular frame of the RC in an ideal MAOSS exper- iment is thus characterized by a fixed angle θ between the magnetic field B0 vector and the C2 axis but a uniform distribution of angles corresponding to the rotation about the C2 axis. Simulations suggest that the orientational effect might be more pronounced by applying an orientation parallel to the rotor axis instead of rectangular as in MAOSS experiments (G. Jeschke, unpublished). The latter can be tested experimentally be aligning the sample on polymer films, which are rolled up and inserted into the MAS rotor (Sizun and Bechinger, 2002).
Photo-CIDNP MAOSS NMR on RCs of PS II shows no indication for any change of positive or negative signals assigned to the Chl a donor (Figure 6.3). There is, however, a distinctive reduction
L-6 M-19 M-14 L-9,M-9 L-11 M-16 M-1 M-1
1
M-2 C-1f C-2,f fC-2/His
C-12/Hisf C-15f C-?f C-10f C-5f C-20f
C-3f11
140 120 100 80
160 180 200
A
B
13C chemical shift (in ppm)
M/L/-13/ C-2//Hisf f
140 120 100 80
160 200 180
C
D
13C chemical shift (ppm)
L-6 M-19 M-14 L-9,M-9 L-11
M-16 M-1 M-1
1
M-2 C-1f C-2,f fC-2/His
C-12/Hisf C-15fC-?f C-10f C-5f C-20f
C-3f11 M/L/-13/ C-2//Hisf f
Figure 6.2: Olefinic and carbonyl region of13C photo-CIDNP MAS NMR spectra of RCs of Rb. sphaeroides WT. Experimental data obtained at 233 K, 4.7 T and MAS frequency of 3.9 kHz with continuous illumination, on the left. MAS data from an isotropic powder sample in a rotor (A) and MAOSS data from a sample oriented on glass discs (B) is shown. Simulated spectra obtained by G. Jeschke, are shown on the right for a randomly oriented sample (C) and an oriented sample (D).
In the simulation dispersive effects are only very minor (see text). Centerbands are indicated with dashed lines. For the assignment see Table 6.1.
of the intensity of the emissive signals at 142.5 and 129.2 ppm in the spectrum of the oriented sample (Figure 6.3B). These signals have been attributed to a histidine (Alia et al., 2001). Such an assignment is in line with the ’hinge-model’ presented in Chapter 4.
Initially, significant orientational effects were expected of photo- CIDNP (Polenova and McDermott, 1999). The experiments suggest that the orientational effect may be diminished by destructive inter- ference of the two coherent mechanisms or erased by the incoherent DR mechanism.
Recently SLF NMR experiments on oriented samples rotating at the magic angle were reported (Lopez et al., 2007). This combination would be very fruitful on photosynthetic RCs, as it investigates the orientation of specific molecular groups in the sample. In this two- dimensional experiment the dipolar couplings are correlated with the isotropic chemical shifts. The information drawn from this experiment considers the size and orientation of hetero-nuclear dipolar couplings,
L-6 M-19 M-14 L-9,M-9 L-1
1
M-16 M-1 M-1
1
M-2 f-1? L-4 ? L-12,M-12 F-12 F-15 F-10 F-5 F-20
140 120 100 80
160 180 200
A
B
13C chemical shift (ppm)
F-2 His His
200 150 100 80
13C chemical shift (ppm) C
D
Figure 6.3: 13C photo-CIDNP MAS NMR spectra of quinone-depleted RCs of Rb. sphaeroides R-26 (A, B) and RCs of PS II (C, D). Spectra of quinone- depleted RCs of Rb. sphaeroides R-26 are obtained at 233 K under illumination with continuous white light at 7.4 T and 8 kHz spinning on randomly oriented samples (C) and 3.9 kHz spinning on oriented samples in (D). The spectra of the D1D2 preparation of PS II of spinach are obtained at 233 K under illumination with continuous white light at 4.7 T and 3.6 kHz spinning frequency. Randomly oriented samples are shown in (A) and oriented samples in (B).
which can be directly related to the orientation of molecular groups in the sample. During the experiment, the sample is spun slowly, but still provides reasonable chemical shift resolution. The dipolar coupling is restored during the t1 evolution period. The 1H-X dipolar couplings, which are so useful in the SLF experiments, may be obtained for var- ious resolved sites simultaneously just by performing one experiment.
This is due to the fact that they can be directly obtained from the FID in the indirect dimension sampled over one rotor period. Preliminary experiments of photo-CIDNP MAS NMR in combination with SLF studies are shown in Figure 6.4. Hence, it is in principle possible to obtain orientational information of the cofactors from photo-CIDNP studies if the system provides sufficient anisotropy of the solid-state photo-CIDNP effect.
Photo-CIDNP proves to be therefore a valuable tool, not only from technical perspective such as signal enhancement, spectral editing and functional screening of photosynthetic RCs. In addition it is valuable
N N
N N
O
C M g
O C
I II
III IV
20
4 5
9 10
14 15 16n
N N
N N
O
COOPhytyl M g
O COOMe
I II
III IV
20
4 5
9 10
14 15 16n
5- C ALA13
acq.
TPPM p/2
1H
13C
Light Continuous illumination
p
LG TPPM
tr tr
A B
C
t1
D
Figure 6.4: 13C photo-CIDNP SLF experiments on randomly oriented 5-13C ALA labeled RCs of Rb. sphaeroides WT. A shows the 5-13C ALA isotope label pattern of BChl a. The pulse program used is shown in B (TPPM = proton decoupling, LG = phase modulated Lee-Goldburg, during which the magnetization evolves for a variable time interval t1,τr= rotor period). C shows a detailed view of the 2D spectrum in the region of the isotope labeled P1-C10 methine. The dipolar evolution curve derived from the intensity modulation during t1 over one full rotor period, of the P1-C10 methine is shown in D is fitted as described in reference Lopez et. al 2007. The dots show the signal intensities obtained by spectral deconvolution with the fit of 8 Gaussian peaks to the 1D slice of each increment of the SLF experiment, the solid line indicates the experimental fit.
for the understanding of the primary process of natural photosynthesis and can be use to develop efficient artificial photosynthetic devices.
The author therefore concludes with Berthold Brecht (1964): ’Wir stehen selbst entt¨auscht und sehn betroffen den Vorhang zu und alle Fragen offen.’
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