Diller, A.
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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 4
15 N-photo-CIDNP MAS NMR
analysis of the electron donor of
photosystem II
In natural photosynthesis, the two photosystems that operate in se- ries to drive electron transport from water to carbon dioxide are quite similar in structure and function, but operate at widely different po- tentials. In both systems the photochemistry is initiated in the same way, by photo-oxidation of a Chl a. Considering that the midpoint potential in PS II is ∼0.7 eV higher than that in PS I, the electronic structures of the photo-oxidized Chl donors are expected to be very different. Using RCs from 15N-labeled spinach, these electronic struc- tures are compared by photo-CIDNP MAS NMR measurements. The results show that the electron spin distribution in PS I, apart from its known delocalization over 2 Chl molecules, reveals no marked dis- turbance, whereas the pattern of electron spin density distribution in PS II shows a strong asymmetry of electron spin density towards the pyrrole ring IV in the oxidized radical state. A model for the donor of PS II is presented explaining the inversion of electron spin density based on a tilt of the axial histidine towards pyrrole ring IV causing π-π overlap of both aromatic systems.
The contents of this chapter have been published in Proceedings of the National Academy of Sciences USA (2007) 104: 12767-12771.
4.1 Introduction
In photosynthesis, the two photosystems that operate in series to drive electron transport from water to carbon dioxide are similar in struc- ture and function, but they operate at widely different potentials. In both photosystems the photochemistry starts by photo-oxidation of a Chl a. The oxidized electron donor of PS II is the strongest oxidiz- ing agent known in living nature, having a redox potential of +1.2V, which is required for water oxidation (van Gorkom and Schelvis, 1993).
The electronically excited donor of PS I, probably the most reducing compound in living nature, initiates the dark reaction (Webber and Lubitz, 2001). The question arises what factors tune those electronic properties. The spatial structure of PS II (Figure 4.1) shows two in- ner Chls, PD1and PD2, two accessory Chls, ChlD1 and ChlD2, two Phe a cofactors and two quinones in an arrangement similar to that in bacterial RCs of purple bacteria (Chapter 1, Figure 1.2). The protein binding pocket and the Chl-Chl interactions for the donor molecule might play a role for the different redox potentials, since the energy and oscillator strength of the electronic transitions and the redox po- tential of a molecule depend on the energies of its HOMO and LUMO molecular orbitals, which are in turn determined by local environment and aggregation state of the molecule (Hanson, 1991; Watanabe and Kobayashi, 1991).
Photo-CIDNP MAS NMR is an optical solid-state NMR method using the non-Boltzmann spin order in the correlated electron pair and allows for a strong increase of sensitivity and selectivity of NMR signals allowing to study the electronic structure of photosynthetic co- factors in great detail (Jeschke and Matysik, 2003; Daviso et al., 2007).
The effect of solid-state photo-CIDNP, discovered in 1994 (Zysmilich and McDermott, 1994), is based on the contribution of different mech- anisms known as TSM (Jeschke, 1997), DD (Polenova and McDermott, 1999) and DR (Goldstein and Boxer, 1987; McDermott et al., 1998).
Recently, the contribution of these three mechanisms has been ana- lyzed by field-dependent measurements on unlabeled RCs of the purple bacterium Rb. sphaeroides (Prakash et al., 2005, 2006). The chem- ical shift refers to the electronic ground-state after the photocycle, the photo-CIDNP signal intensity is linked to the intermediate radi-
Q
AFe Q
BPhe
D1Phe
Chl
D1Chl
DP
D1P
D2Y
ZY
DMn
4Chl
ZD1D2
2
Cyt
Chl
b5
ZD 59
2
Figure 4.1: Schematic structure of the PS II core complex of Thermosynechococ- cus elongatus obtained with the program PYMOL (DeLano Scientific, South San Francisco, CA). The arrangement of cofactors of the electron transfer chain lo- cated in subunits D1 and D2 are Chls PD1, ChlD1, ChlZD1 and Chls PD2, ChlD2, ChlZD2, respectively. In addition the position of the electron acceptors QA and QB, and the Fe, as well as the location of YD and the Mn4 cluster, are depicted (Ferreira et al., 2004).
cal state. Analytical expressions imply that for short lifetimes of the radical pair the nuclear polarization created by the TSM mechanism is proportional to the square of the pseudo-secular hyperfine coupling and thus to the square of the hyperfine anisotropy (Jeschke, 1998).
The hyperfine anisotropy in turn is roughly proportional to the spin densityρpin the 2pz orbital on the aromatic atom under consideration.
In related work, numerical computations have performed considering both the TSM and DD mechanisms of13C photo-CIDNP for eight car- bon atoms of both the donor and acceptor in bacterial RCs. At a field
of 4.7 T we found that the polarization Δp0 generated by a single pho- tocycle is given by Δp0 = (-0.216±0.086)ρ2p (Chapter 2, this thesis).
It is thus expected that also for15N in plant RCs the amplitude of sig- nals of the same cofactor is roughly proportional to the square of the spin density in the pz orbital of each aromatic atom. Until now, plant PSs have been investigated only by13C photo-CIDNP MAS NMR and without isotope enrichment (Matysik et al., 2000a; Alia et al., 2004;
Diller et al., 2005). As the origin of the high redox power in PS II, a local electrostatic field causing the asymmetric electron distribution has been proposed, however, the origin of this field remained unclear (Matysik et al., 2000a). The possibilities of local matrix involvement (Diller et al., 2005) and of general charge effects of the D1D2 pro- tein on all inner Chls (Ishikita et al., 2005) have been discussed. The
13C signal patterns of unlabeled RCs are complex and difficult to in- terpret. On the other hand, the interpretation of 15N photo-CIDNP MAS NMR of isotope labeled RCs is more straightforward, as each
15N signal can be assigned easily to one of the four pyrrole subunits.
Until now, studies of plant RCs were hampered by the difficulty to label plants. Here we present the15N photo-CIDNP MAS NMR data of uniformly 15N labeled plant RCs of spinach (Spinacia oleracea).
4.2 Material and Methods
4.2.1 Sample preparation
Spinach plants were cultured on half-strength Gamborg’s B5 basal media (Gamborg et al., 1968). (15NH4)2SO4 and K15NO3 have been used as the source of isotope labeled nitrogen. The preparation of PS I (PS I-110) and PS II (D1D2-cytb559), is described in Matysik et al.
2000a and Alia et al. 2004, respectively.
4.2.2 MAS NMR measurements
The NMR experiments on PS I were performed with a DMX-400 NMR spectro-meter; on PS II with a DMX-200 NMR spectrometer (Bruker Biospin GmbH, Karlsruhe, Germany). The illumination setup is de- scribed elsewhere (Matysik et al., 2000b; Daviso et al., 2007). The
light and dark spectra have been collected with a Hahn echo pulse se- quence and two pulse phase modulation proton decoupling (Bennett et al., 1995). All NMR spectra were obtained at a temperature of 240 K and with a spinning frequency of 8 kHz. Each spectrum was measured within two days. Chemical shifts are given relative to liquid
15NH3, using the response of solid 15NH4NO3 at δ = 23.5 ppm as an external reference.
4.3 Results and Discussion
4.3.1
15N photo-CIDNP MAS NMR on PS I
Spectrum A in Figure 4.2 has been obtained from PS I in the dark.
Only absorptive (positive) signals occur, a broad response between 120-130 ppm from the protein backbone as well as some weak features below 100 ppm which can be attributed to the15N in arginine and ly- sine residues (Prakash et al., 2004). Under illumination, enhanced ab- sorptive (positive) and emissive (negative) signals occur (Figure 4.2B).
The ratio of enhanced absorptive and emissive signals depends on the magnetic field (Figure 4.4A and B). Both, the two emissive as well as the enhanced absorptive set of signals can be assigned befittingly to Chl cofactors (Table 5.1), based on assignment obtained on Chls in solution (Boxer et al., 1974).
At the donor site, which has been shown to be very rigid, slow sig- nal recovery is expected (Fischer et al., 1992; Alia et al., 2004). Hence, an experiment with very short cycle delay of 0.4 seconds (Figure 4.4C), showing a strong decay of the emissive compared to the enhanced ab- sorptive signals, suggests that the emissive signals originate from the donor Chl. The emissive signal at 211.5 ppm is broadened by a shoul- der at ∼215 ppm (Table 5.1 and Figure 4.3), indicating involvement of a second donor Chl a species, which can be due to the epimeriza- tion at the C-132 and the different hydrogen bonding at the 131-keto group. Hydrogen bonding of the protein to the carbonyl group of the 3-acetyl and the 131-keto group, which are both part of the conju- gated π-system, has been show to have a significant influence on the electronic structure and redox potential (Witt et al., 2002). The in-
300 250 200 150 100 50 0 350
C
D
B
A
15N chemical shift (ppm)
Figure 4.2: 15N photo-CIDNP MAS NMR spectra of PS I and PSII. Overview spectra of PS I (A: dark, B: light) were obtained at 9.6 Tesla (400 MHz proton frequency). Spectra of PS II (C: dark, D: light) have been measured at 4.7 Tesla (200 MHz proton frequency). PS I-110 and D1D2 sample preparations were studied at 240 K and with a cycle delay of 4 s.
volvement of a second donor Chl a species, might also cause the split of the N-IV signal (250.3 and 254.9 ppm), while no effect is observed on the remote N-I position (186.2 ppm). The N-III appears at an absorptive signal at 193.2 ppm. The four absorptive signals (Figure 4.4B) can be assigned to a single Chl cofactor, probably the primary electron acceptor A0 (Alia et al., 2004). The enhanced absorptive sig- nal at 233.3 ppm indicates a strong local disturbance at a pyrrole ring IV in the electronic ground-state. On the other hand, the intensity patterns demonstrate that PS I in the radical-pair state is assembled by Chl cofactors with nuclear polarization patterns similar to that of
Table 4.1: 15N chemical shifts of the photo-CIDNP signals in compar- ison to chemical shift data reported in literature. All chemical shifts are referenced to liquid ammonia with use of an external standard of solid 15NH4NO3 (δ= 23.5).
Assignment Solution PS I PS II data
cofactor atom σliqa σsolidb σsolidb
Chl a N-I 186.0 186.2(e) 190.9(a)
N-II 206.5 206.1(a) 211.5(e) 211.5(e)
N-III 189.4 193.2(a) 195.3(e) N-IV 247.0 233.3(a) 247.6(e)
250.3(e) 254.9(e) Phe a N-I 125.5
N-II 241.5
N-III 133.9 138.3(e)
N-IV 295.8 295.0(e)
a Chemical shift in ppm. Measured in CDCl3(Boxer et al., 1974).
b Chemical shift in ppm, this work.
a = absorptive (positive), e = emissive (negative).
isolated Chls having their maximum on pyrrole ring II (K¨aß et al., 1995, 1998; K¨aß and Lubitz, 1996) (Figure 4.5A).
The assignment of the emissive signals to the donor implies a dimeric donor having an asymmetric electron spin distribution be- tween two Chl cofactors as it has been found with ENDOR (Rigby et al., 1994; Krabben et al., 2000; K¨aß et al., 2001) as well as with quantum-chemical calculations (Plato et al., 2003). The Chl electron spin density pattern of the PS I donor resembles two undisturbed Chl cofactors, hence the pattern is similar to that of isolated Chls (K¨aß et al., 1995, 1998; K¨aß and Lubitz, 1996). In PS II the electron spin density shows remarkable differences with PS I.
120 ppm 165
210 255
300
N-I N-III N-II N-IV
PS I Chl a PS II Chl a
N-III N-II N-IV
Chl a
N-III N-II
N-IV N-I
PS II Phe a
N-IV N-III
Phe a
N-IV N-II N-III N-I
Figure 4.3: Assignment scheme of 15N chemical shifts of the photo-CIDNP signals based on Table 5.1. Emissive signals are shown in light gray, absorptive signals in black.
4.3.2
15N photo-CIDNP MAS NMR on PS II
In the dark, due to a low concentration, little signal is detected from the PS II sample (Figure 4.2C). Upon illumination, several emissive signals appear (Figure 4.2D and 4.4D). The pattern demonstrates clearly that the radical pair is formed by a Chl a and a Phe a having well separated signals (Table 5.1, and Figure 4.3). The strongest sig- nal observed in PS II originates from the pyrrole nitrogen N-IV (247.6 ppm). Two other signals of the Chl donor cofactor are detected at 211.5 (N-II) and 195.3 ppm (N-III). No unequivocal signal is observed at the frequency expected for the N-I. In addition, there is no sign, that the N-I signal might be shifted. As the amplitude of signals of the same cofactor is roughly proportional to the square of the spin density in the pz orbital of each aromatic atom, as discussed above, the three light-induced Chl signals reveal a strong asymmetry of electron spin density in the donor of PS II. The derived electron spin density pat- tern, shown in Figure 4.5, depicts a strong asymmetry of electron spin
300 250 150
295.0
A
15N chemical shift (ppm)
200
254.9 250.3 247.
6 243.8 211.5 195.3 138.3233.3 206.1 190.9
C
B
186.2
193.2
D
Figure 4.4: 15N photo-CIDNP MAS NMR spectra of PS I and II. Detailed spectra of PS I (A, B, C) and PS II (D) obtained at 9.6 Tesla (A) and 4.7 Tesla (B, C, D). Cycle delay were 4 (A, B, D) and 0.4 (C) seconds.
density in the form of a shift towards the pyrrole ring IV. This is in-line with the strong asymmetry of electron spin density detected previously by 13C photo-CIDNP MAS NMR, demonstrating maximum electron spin density at the neighboring C-15 methine carbon (Matysik et al.,
131 2
20 N
12 N
13 16 14
17 N 18
19 3
8
O 4
5
Mg
O
O O
R
I II
IV III
V
1
6
9
11
15
10 7
N
A B
I II
IV III
V
Figure 4.5: Electron spin density patterns. Based on the 15N photo-CIDNP intensities, electron spin density pattern of the donor cofactors of PS I (A) and PS II (B) are shown.
2000a). Hence there is a good agreement between photo-CIDNP data obtained from both types of nuclei. Thus, in the donor of PS II the electron spin density pattern is inverted compared to the donor and acceptor cofactors in PS I as well as to isolated Chl cofactors (Fig- ure 4.5B). On the other hand, there is no indication for a significant disturbance of the electronic ground-state. Therefore, the change in the electronic structure appears to be restricted to the photo-oxidised state. Two emissive signals appear at 295.0 and 138.3 ppm, which can be conveniently assigned to N-IV and N-III of the primary electron acceptor, a Phe cofactor. The donor signals are remarkably narrow, FWHH of ∼40 Hz), while the acceptor signals are slightly broader (∼70 Hz), indicating a general feature of photosynthetic RCs having a rigid donor site without structural heterogeneities and more struc- tural flexibility at the acceptor site (Fischer et al., 1992; Alia et al., 2004).
4.3.3 Matrix involvement
In Spectrum 4.4C, a signal arises at 243.8 ppm. It is possible that this signal arises from a second Chl cofactor having much lower electron spin density. On the other hand, there is no further indication in this spectrum or in the 13C photo-CIDNP MAS NMR data for involve- ment of a second donor Chl cofactor (Matysik et al., 2000a; Diller et al., 2005). In addition, the signal is clearly broader (FWHH of 90-100 Hz) than the other signals assigned to the Chl donor. This indicates that another, structurally more flexible unit close to the Chl donor cofactor also carries electron spin density. Hence, an involve- ment of the protein matrix has to be considered. An assignment to a protonated Schiff base nitrogen (Creemers et al., 1999), as discussed as a chemical modification of the donor Chl (Diller et al., 2005), is not convincing. The chemical shift value is also difficult to reconcile to any aromatic amino acid other than histidine. In fact, a nitrogen N-π of a Type-1 histidine (i.e., carrying a lone pair at the π-position) res- onates at∼250 ppm (Alia et al., 2001). With13C photo-CIDNP MAS NMR, three emissive signals at 142.5, 139.8 and 129.2 ppm have been detected (Diller et al., 2005) which also match to a Type-1 histidine (Alia et al., 2001). As in the15N data, these three signals have roughly a double FWHH as the Chl signal. The nitrogen N-τ in Type-1 his- tidines can be either bound to the Mg of the Chl or protonated (Alia et al., 2001). On basis of the observed chemical shifts, it is not possi- ble to distinguish whether the histidine carrying electron spin density is the axial one. Analysis of the pocket of one of the four central Chl cofactors considering the X-ray structure (Loll et al., 2005) does not provide any possibility for a non-axial histidine among one of the four central Chl cofactors. Thus, we propose that the electron spin density is distributed over both, the donor Chl and its axial histidine.
Hence, the reduction of spin density, observed by EPR and originally interpreted in terms of a weakly coupled dimer having 82% of the spin density on one Chl cofactor (Rigby et al., 1994), could be explained by the model presented here implying that the rest of the spin density are localized on the axial histidine. Since the two accessory Chls, ChlD1 and ChlD2, are not coordinated to histidines (Zouni et al., 2001), the donor must be an inner Chl, either PD1 or PD2. This conclusion is
His198
P
D1Figure 4.6: Model of the electron donor in PS II. Due to the tilt of the axial histidine towards pyrrole ring IV (arrow), the electron spin density pattern is inverted and partially shifted on the axial histidine.
in-line with results of previous pulse EPR studies (Zech et al., 1997;
Lubitz et al., 2002; Kammel et al., 2003).
4.3.4 The hinge model of the donor of PS II
Implying that the signals arise from a Type-1 histidine having a de- protonated π-position, the donor would be a negatively charged [Chl His]−complex in the ground-state, and a neutral radical in the photo- oxidized state. Hence, stabilization of the HOMO would lead to an increased gap to the continuum and, hence, to an increased redox po- tential. Here, we propose a hinge-type model for the donor complex unifying those aspects (Figure 4.6). Slight bending of the axial histi- dine towards pyrrole ring IV and the methine bridge C-15 would lead toπ-π overlap of both conjugated systems and stabilize the negatively charged electronic ground state of the complex. Such lowering of the ground-state would increase the redox potential. Preliminary density functional computations indicate that such a small tilt indeed causes
both, a shift of spin density into pyrrole ring IV and some distribution of spin density into the aromatic ring of the histidine (G. Jeschke, per- sonal communication). A more detailed analysis is currently on the way. The energies of both HOMO and LUMO have to shift equally in the considered theories, to maintain the same energy difference and color. Systematic tests are tedious as both the orientation of the histi- dine ring with respect to the coordinating nitrogens and the tilt angle need to be varied and each structure has to be optimized with respect to deformations of the Chl macrocycle. Nevertheless, the hinge model of the electron donor in PS II can at least in principle explain the observed inversion of the pattern of electron spin density distribution and the spin density on a histidine.
It is well known that a metalloporphyrin macrocycle can conserve its shape during evolution if it is of functional relevance (Shelnutt et al., 1998). It appears that such functional conservation principle can be extended to functional cofactor-matrix units.
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