Photo-CIDNP MAS NMR spectra obtained from 13C-labeled BBY, thylakoids and plants
In Fig. 2, Spectra a-c, shown in red, are obtained under continuous illumination of selectively 4-ALA 13C-isotope labelled BBY (a), thylakoid membranes (b) and from entire plants (c) of the duckweed Spirodella oligorrhiza. The same Figure shows in black Spectra a’ to c’ obtained from the same samples in the dark. The dark spectra show only weak and broad positive signals in the aliphatic region between 0 and 50 ppm and, due to the C-α of the amino acids of the protein backbone, between 60 and 80 ppm.
The 15 signals that are light-induced by the solid-state photo-CIDNP effect can be straightforwardly recognized. Figure 3 shows an expansion of the spectral region for the light-induced signals in Fig. 2. The data are presented with the tentative assignment (see below) to the labelled carbons in the Chl a donor and Phe a acceptor. For convenience, the isotope label patterns of the Chl a donor (green) and Phe a acceptor (purple, numbering in Italics) obtained by biosynthetic labeling with 4-ALA are indicated with numbering at the red dots on top of Fig. 3. The data show that:
(i) The solid-state photo-CIDNP effect allows to observe the two cofactors forming the SCRP directly from entire plants without any further isolation (Fig. 4). This implies that 13C photo-CIDNP MAS NMR is able to study selectively microscopic structures in the Ångström range within macroscopic units up to the dimension of centimeters. Thus, we propose that such “in-plant” photo-CIDNP MAS NMR could be considered as a new application in a growing field of in-cell solid-state NMR13,14,15.
(ii) The comparison of the 13C photo-CIDNP MAS NMR spectra of BBY particles (spectrum a), thylakoid membrane (spectrum b) and intact leaves (spectrum c) does not reveal any significant difference, neither in the chemical shift nor in the intensity. Minor changes in the relative intensity of the signals might occur at 51.0 and 147.2 ppm, although they are in the limits of the noise. Hence, both, the chemical shift values and the overall intensity pattern are highly conserved among spectra obtained from these three levels of isolation. The consistency observed with the highly sensitive NMR spectroscopy provides clear experimental evidence that the electronic states of the cofactors forming the SCRP are not affected by the preparation procedure. Thus, the data obtained from a D1D2 RC preparation7,10 indeed reflect the genuine natural state occurring in intact thylakoid membranes and plants.
(iii) The below discussed assignment of the light-induced signals corroborates the concept of a SCRP formed by a single donor Chl a and a single acceptor Phe a. While 13C photo-CIDNP MAS NMR intensities are correlated to local electron-spin densities16, in case of 13C labelling, the polarizations are equilibrated by spin-diffusion. On the other hand, the spread of intensity allows to observe nuclei which are otherwise difficult to detect17. Spin-diffusion allows for example for the detection of the nearby aliphatic signals for the two C-17 carbons from the donor and acceptor. While in unlabelled samples, all signals are conveniently assigned to a single Chl a cofactor, i.e. the donor7, upon 4-ALA 13C-labelling also acceptor signals are observed. Effects of selective 13C isotope labelling on the spin-dynamics have also been observed in heliobacterial RCs18 but are not yet theoretically permeated. The donor signals are in general stronger than the acceptor signals, suggesting an additional contribution of the DR mechanism19.
No signals from PS1
It is remarkable that the light-induced spectra of BBY on the one hand, and of thylakoids and plants on the other hand are very similar, although thylakoid and plant samples contain the full photosynthetic machinery, including both PS2 and PS1, while BBY contains PS2 only. The absence of photochemically active PS1, which shows an entirely emissive light-induced 13C photo-CIDNP MAS NMR, can be due to several reasons: (i) The position of the quinones: while the quinones on PS2 are easily accessible and instantaneously reduced upon addition of sodium dithionite, the quinones in PS1 are not expected to be readily reduced upon direct freezing and measurement. To successfully reduce PS1, incubation at room temperature after addition of the reductant and exposure to light at room temperature and during freezing are necessary. (ii) The low pH of the sample environment (~pH 4.5): in vivo, the active site of PS1 is situated at the alkaline (stroma) side of the thylakoid membrane (pH 8), while PS2 functions at the acidic lumen side (pH 4) of the membrane. Acidic conditions strongly decrease PS1 stability and activity, while both donor and acceptors side of PS2 are known to remain intact under strong acidic conditions.
Photo-CIDNP in PS2 preparations containing the OEC
To allow for the solid-state photo-CIDNP effect, a SCRP on PD1 and Phe a with sufficiently long lifetime, i.e., some 10 s of ns, is required. One might assume that such long lifetime cannot sustain in the presence of the OEC20. Our results demonstrate the occurrence of the same SCRP in the preparations of larger PS2 complexes (with the OEC present) with sufficiently long lifetime as in the D1D2 preparation (where the OEC is lost). There are, indeed, arguments for the interruption of the OEC activity in the present set of experiments: (i) The electron transfer from the OEC requires an intact hydrogen bonding network around TyrZ, the intermediary electron carrier between the OEC and PD1 Chl a21. The rate of re-reduction of PD1+ decreases at low pH22. This effect has been suggested to be linked to a distortion or breakage of the hydrogen bond between TyrZ and the nearby D1-His190, which has an estimated pKa of 4.5-5.323,24,25 (for review see Styring et al.26). (ii) Below a pH of 5.5, the nanosecond kinetics of electron transfer quickly slows down to the microsecond. The pH dependence provides a natural mechanism for physiological regulation of electron transfer from the OEC, allowing for dissipation of excess excitation energy by the RC at high light conditions27. In all experiments shown here, after reduction with Na2S2O3 in the rotor, a pH below 5.0 has been reached in preparations of the larger PS2 complexes, i.e., in plants, BBY and core preparations. Furthermore, acidification of the lumen space down to a pH of 5.0 or slightly below occurs in vivo under strong light conditions28. Hence, the pH allows for lifetimes of the SCRP sufficiently long to induce the solid-state photo-CIDNP effect. (iii) The temperature of the experiment (~235 K) blocks the re-reduction from the OEC. The reaction cycle of the OEC29 is strongly inhibited at 230 K30,31,32. If we assume that the OEC remains blocked in its S2 state, even at pH 6–7.5, the re-reduction rate would slow down to 250 ns33. (iv) Also in core preparations, QB is lost while QA remains bound to the protein pocket34. In the preparations of larger PS2 complexes, both quinones are, at least initially, present. It is possible that QB is lost upon reduction prior to the measurement. In this case, after some photocycles, QB will be saturated and the light-induced electron transfer becomes cyclic. At high light intensities, also under natural conditions, photo-reduction of quinones has been shown to occur leading to double reduction of QA which finally can result in the release of QA as QAH2 (up to 63% in 80 min)35. In core preparations, double reduction of QA can be significantly promoted by the addition of a strong reductant and subsequent illumination36,37. In BBY preparations, illumination under reductive conditions has been demonstrated to cause 100% double reduction of QA38. Hence, the experimental conditions allow for observation of the SCRP of PS2 despite the OEC being present.
13C photo-CIDNP MAS NMR on isolated PS2 RCs from spinach and duckweed
As a next step, we will compare PS2 data from spinach and duckweed. The impossibility to obtain D1D2 RCs from duckweed forces us to compare Core particles to D1D2 RC preparations from spinach. In Fig. 5, shown in red, the 13C photo-CIDNP MAS NMR Spectra a and b are obtained under continuous illumination of a D1D2 preparation from unlabelled spinach (a) and of core particles from unlabelled duckweed (b). Spectra a’ and b’, depicted in grey, show the corresponding dark spectra. As expected, the dark spectra show signals in the aliphatic region between 0 and 50 ppm as well as a broad signal between 60 and 80 ppm. The dark signals are due to the C-α of the amino acids of the protein backbone and the (glycine) buffer used for sample preparation. In the photo-CIDNP MAS NMR spectra in Fig. 5, absorptive (positive) and emissive (negative) light-induced signals occur in the region between 80 and 170 ppm. Although the isolated RC shows stronger light-induced features than the Core complex, chemical shifts and intensity patterns between both light-induced spectra are very similar, proving that the PS2 from both organisms function in a similar fashion. The data provide strong evidence that both the electronic ground-state structure as well as the radical-pair structure of the photochemical machinery of PS2 remain unchanged when comparing the two different plant species and upon isolation of D1D2 from the Core complex. This is in line with an optical study using femtosecond transient absorption spectroscopy, which revealed a conservation of the efficient electron transfer rate constants upon isolation of the D1D2 complex from PS2 Core39. Also the mechanism of electron transfer, with ChlD1 acting as the primary electron donor and PheD1 as the primary acceptor, was found to be the same in both systems39. Hence, our data demonstrate that the photochemical machinery of PS2 is robust against various states of isolation. The highest level of isolation, the D1D2 RC, is not disturbed, and essentially functioning in the same way as in full plants. Furthermore, we have shown that this machinery is very similar in PS2 of spinach and duckweed.
To compare the spectra of the D1D2 preparation of spinach and that of the Core preparation of duckweed in more details, Fig. 6 provides an enlarged view. Spectrum a, originating from the D1D2 preparation of unlabeled spinach, is reproducing well data from the literature7,8. Previously, 23 light-induced signals were observed, most of them were tentatively assigned to the aromatic ring carbons of the Chl a donor. Absence of signal doubling provided a hint for a monomeric donor. The emissive signals between 90 and 130 ppm were identified as the four methine carbons. It has been proposed that the broad emissive response between 140 and 145 ppm and the emissive signal at 129.2 ppm originate from the axial histidine of the Chl a donor10. Spectrum b originates from the Core preparation from unlabelled duckweed. Despite the overall great similarity of chemical shifts and other spectral features, the emissive signal between 129 and 130 ppm is apparently missing. Also, the broad emissive signals at 142.5 and 139.8 ppm might be extinguished. All these emissive features are assigned to the axial histidine8. MAS rotation might lead to orientation effects in membrane-based samples, changing the relative intensity contribution between π-systems having different orientations10. Therefore, the collective absence of these emissivive features in the membrane sample suggests a common origin and backs the assignment to the axial histidine.
Assignment of 13C photo-CIDNP MAS NMR signals of 5-ALA, 4-ALA and 3-ALA labeled samples
As shown above, the possibility to introduce 13C isotope labels allows to observe the effect in larger systems. Furthermore, isotope labelling enables for a more detailed characterization of the SCRP and the identification of possible abnormalities. To this end, we will now aim for signal assignment by using selectively 5-ALA, 4-ALA and 3-ALA labeled samples. Since it allows for efficient label introduction, we prepared thylakoid samples from duckweed with various 13C isotope label patterns to study the structure of the SCRP of PS2. In Fig. 6, Spectra c to e have been obtained from samples were the Chl a and Phe a molecules were specifically 13C labeled according to the 5-ALA (c), 4-ALA (d) and 3-ALA (e) labeling patterns (for labeling patterns, see Supplementary Fig. S1). By using specifically labeled samples, it is possible to selectively highlight eight carbons in each active Chl a or Phe a cofactor and assign them by using literature values obtained from Chl a aggregates40 and plant Phe a reconstituted in bacterial RCs41. In Table 1, we aim to reconstruct the spectra of the unlabeled PS2 core and D1D2 samples (Fig. 6, Spectra a and b) by using the data obtained from selectively 13C-labeled thylakoid membranes (Fig. 6, Spectra c to e). Again, the consistency of many marker lines in labeled and unlabeled samples clearly demonstrates the conservation of the electronic structure of the PS2 Chl a donor upon isolation of core particles from thylakoid membranes. The detailed discussion on the assignment can be found in Supplementary Information. We can conclude from the assignments:
(1) While the aromatic system appears largely undisturbed, the absence of the donor C-131 carbonyl carbon and the occurrence of the two weak, unassigned so far, absorptive signals at 151.8 and 148.3 ppm in the 3-ALA pattern teases for further studies.
(2) The emissive signals at 142.5 and 139.8 ppm, assigned to the axial histidine10,11, do indeed not originate from the Chl a and Phe a cofactors. The emissive signal at 129.2 ppm, however, might be caused by the C-12 carbon of the acceptor, although we cannot rule out that that acceptor signal is overlaying the histidine signal. Thus, the matrix is involved into the formation and evolution of the SCRP.
(i) Chl a protonated at position 131, positively charged, [Chl-OH]+;
(ii) Chl a protonated at position 131, neutral, [Chl-OH];
(iii) a Schiff-base formation at the C-131 of the Chl a donor, [Chl-NH2]+.
Possible chemical modifications of the Chl a donor
The question remains about the missing donor C-131 signal and the possible signals at 151.8 and 148.3 ppm. One might assume that the C-131 signal has been significantly shifted by a chemical modification at the C-131 position. This idea is attractive because it might explain the unchanged optical properties although the electro-chemical properties of these cofactors, esp. the extremely high redox potential, are highly unusual. Possible chemical modifications are protonation and Schiff-base formation at this carbonyl side (see above). To explore the possibility of such chemical modifications, theoretical calculations have been applied. The results of the calculations are listed in Table 2. It can be seen that the calculated shifts are in reasonable agreement with the experimental values for Chl a, with a root-mean-square deviation of 5.3 ppm and a maximum absolute deviation of 10.8 ppm. Changes in these shifts upon chemical modification are expected to be of higher accuracy due to the possibility of error cancellation. This is supported by the observation that these changes are much more consistent with the corresponding changes calculated with the BP86 functional (given in Supplementary Table S1) than the actual Chl a chemical shifts.
In all three calculated structures with a chemical modification, the ppm value of carbon C-131 lowered. The lowest value, around 160 ppm, is found for the Schiff-base. Therefore, from a spectroscopic view, we would not rule out an assignment of the two possible signals around 150 ppm. However, in the structure43, there is no amino acid around the donor Chl a able to form a Schiff base. The two calculated structures with protonated C-131 positions are not very likely from the chemical shift values. Hence, a chemical modification as explanation of the absence of the signals of C-131 from the donor is unlikely.