pH-Sensing Machinery of Excitation Energy Transfer in Diatom PSI

Letter Cite This: J. Phys. Chem. Lett. 2019, 10, 3531−3535

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pH-Sensing Machinery of Excitation Energy Transfer in Diatom PSI− FCPI Complexes Ryo Nagao,*,¶ Makio Yokono,† Yoshifumi Ueno,⊥ Jian-Ren Shen,¶ and Seiji Akimoto*,⊥ ¶

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Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan † Innovation Center, Nippon Flour Mills Co., Ltd., Atsugi 243-0041, Japan ⊥ Graduate School of Science, Kobe University, Kobe 657-8501, Japan ABSTRACT: Excitation energy-transfer processes in photosynthetic light-harvesting complexes are strongly affected by the surrounding environments of pigments. Here we report on the effects of pH changes on excitation energy dynamics in both diatom photosystem I-fucoxanthin chlorophyll a/c-binding protein (PSI−FCPI) and PSI core complexes by means of fluorescence spectroscopies. The steady-state fluorescence spectra of the PSI−FCPI showed similar features among three samples at pH 5.0, 6.5, and 8.0. However, fluorescence decay-associated spectra of the pH 5.0- and 8.0-adapted PSI−FCPI within 100 ps exhibit peak shifts to longer and shorter wavelengths, respectively, than the peaks in the pH 6.5 spectra. Because such spectral changes hardly occur in the PSI complexes, the peak shifts at pH 5.0 and 8.0 in the PSI−FCPI can be ascribed to alterations of pigment−pigment and/or pigment−protein interactions around/within FCPI caused by the pH changes. These findings provide novel physical insights into the pH-sensing light-harvesting strategy in diatom PSI−FCPI.

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deprotonation of the nearby residues, thereby causing structural changes of the pigment environment. In general, the pH values in the lumenal and stromal sides in thylakoid membranes are changed to approximately 5 and 8, respectively, during illumination.10,11 The contribution of the pH changes to excitation energy dynamics in LHCs have been extensively studied in the green-lineage oxyphototrophs;12−16 however, little is known about the effects of pH changes on the energytransfer mechanisms in the red-lineage oxyphototrophs. Among the red lineages, diatoms are one of the successful phytoplanktons in marine and freshwater environments17 and have unique LHCs, fucoxanthin Chl a/c-binding proteins (FCPs). The pigment and polypeptide compositions of FCPs are remarkably different from those of the Chl a/b-binding LHCs in the green lineages.18 Recently, the crystal structure of a FCP dimer from a marine pennate diatom Phaeodactylum tricornutum was determined.19 The structure revealed the binding of seven Chl a, two Chl c, seven fucoxanthin, and one diadinoxanthin in each FCP monomer and showed close and complicated interactions of Chl molecules with fucoxanthin and diadinoxanthin. The structure also revealed the presence of several acidic residues in the lumenal surface, which may change their protonation states in response to light-induced pH changes, resulting in possible structural changes of the protein and alteration of excitation energy-transfer events.19 However, the fluorescence properties of FCPs were less affected under acidic pH conditions.20 Because the structural

xygenic photosynthetic organisms have developed lightharvesting complexes (LHCs) to capture solar energy, which is subsequently transferred to the reaction center (RC) of the two photosystems (PSI and PSII) to initiate chargeseparation reactions.1 While the protein subunits and pigments of the two photosystems are relatively well conserved among different photosynthetic organisms, LHCs have a wide variety of pigment compositions including different types of chlorophylls (Chls) and carotenoids (Cars) as well as different protein sequences and structures. On the basis of the main pigments that they contain, LHCs are mainly separated into two branches during the evolutionary process, namely, green and red lineages.2 Despite the complicated pigment−protein networks in the green and red lineages, excitation energy transfer in LHCs and the two photosystems is all regulated by the energy levels of the pigments, e.g., Chls with energy levels higher or lower than that of the RC Chls in PSI and PSII will transfer excitation energy to the RC Chls by downhill or uphill processes, respectively.1,3−5 By contrast, close interactions of Chls with Cars often give rise to excitation energy quenching by energy transfer and/or charge transfer among the pigments.6−9 Thus, the energy levels of the individual Chls and Cars will determine the fate of the energy that they absorbed, e.g., either excitation energy transfer or quenching in the photosystems and LHCs. The energy levels of pigments in the photosynthetic apparatus depend on the environment surrounding the pigments, e.g., distances of Chl−Chl and Chl−Car interactions and orientation of pigments in the protein environment. The pigment environment is affected by exogenous factors, one of which is pH that may induce either protonation or © 2019 American Chemical Society

Received: May 8, 2019 Accepted: June 7, 2019 Published: June 7, 2019 3531

DOI: 10.1021/acs.jpclett.9b01314 J. Phys. Chem. Lett. 2019, 10, 3531−3535

Letter

The Journal of Physical Chemistry Letters and biochemical studies of FCPs were performed with isolated FCP complexes, there is no evidence for the effects of protonation/deprotonation of photosystem−FCP supercomplexes on the excitation energy dynamics. We have so far investigated excitation energy-transfer mechanisms in PSI−FCPI complexes by comparing timeresolved fluorescence (TRF) properties between the PSI− FCPI membranes and PSI core-like complexes isolated from a marine centric diatom, Chaetoceros gracilis.21−23 These studies clearly showed that FCPI functionally serves as the excitation energy donor for the PSI cores.22,23 In this study, we examined the effects of the pH changes on the excitation energy dynamics in the PSI−FCPI by fluorescence spectroscopies. It was found that while the 77 K steady-state fluorescence spectra of the PSI−FCPI adapted to pH 5.0 and 8.0 were similar to those at pH 6.5, fluorescence decay-associated (FDA) spectra showed significant differences in the early time ranges after excitation at the three different pH values. On the basis of these results, we discuss the physiological significance of the spectral perturbations induced by the pH changes in the diatom PSI−FCPI. The PSI−FCPI membranes and PSI core-like complexes were isolated as described previously.21 The PSI core-like complexes have almost no FCPI subunits but with slight contamination of Chl c. For adaptation of the PSI preparations to different pHs, the PSI−FCPI membranes were suspended in a buffer of 50 mM Mes-NaOH (pH 5.0), 50 mM Mes-NaOH (pH 6.5), or 50 mM Hepes-NaOH (pH 8.0) containing 0.2 M sucrose, 5 mM EDTA, and 10 mM NaCl, whereas the PSI complexes were suspended in each of the three pH buffers without EDTA but with addition of 0.03% (w/v) n-dodecyl-βD-maltoside. The Chl concentrations of the PSI−FCPI membranes and PSI complexes were adjusted to 10 and 50 μg mL−1, respectively, and were incubated for 30 min in the dark before being frozen in liquid nitrogen. Steady-state fluorescence spectra at 77 K were measured by a spectrofluorometer (FP-6600; JASCO, Japan). The excitation wavelength was set to 459 nm. The TRF spectra were recorded by a time-correlated single-photon counting system with a wavelength interval of 1 nm/channel and a time interval of 2.44 ps/channel.24 The excitation source was a picosecond pulse diode laser (PiL047X; Advanced Laser Diode Systems, Germany) operated at 459 nm with a repetition rate of 3 MHz. The FDA spectra were constructed by global analysis according to the previous method.25 Figure 1 shows steady-state fluorescence spectra of the PSI− FCPI membranes and PSI complexes adapted to pH 5.0 (black lines), 6.5 (red lines), and 8.0 (green lines) measured at 77 K. The spectra of the PSI−FCPI exhibit a peak at 710 nm with similar shapes under the three pH conditions. Because the isolated FCPI complexes displayed a fluorescence maximum at around 680 nm,26 the present result indicates that FCPIs are not dissociated from the PSI−FCPI membranes employed here. By contrast, the PSI spectra also showed a main peak at 708 nm with similar shapes, although an extra peak appears at around 640 nm. The 640 nm peak originates from free Chl c that slightly contaminates the isolated PSI due to the process of FCPI removal.21,22 Figure 2 shows the FDA spectra of the PSI−FCPI membranes and PSI complexes adapted to pH 5.0 (black lines), 6.5 (red lines), and 8.0 (green lines) at 77 K. Four lifetime components were necessary to fit the fluorescence decay and rise profiles. The fluorescence decay and rise

Figure 1. Steady-state fluorescence spectra of the PSI−FCPI membranes (left panel) and PSI complexes (right panel) at 77 K. The spectra were recorded upon excitation at 459 nm. Black, red, and green lines indicate samples adapted to pH 5.0, 6.5, and 8.0, respectively. The spectra are normalized by the peak intensity at 708− 710 nm.

Figure 2. FDA spectra of the PSI−FCPI membranes (left panel) and PSI complexes (right panel) at 77 K. Black, red, and green lines indicate samples adapted to pH 5.0, 6.5, and 8.0, respectively. The spectra are normalized by the total fluorescence intensity of each sample.

components are represented as positive and negative peaks, respectively. A pair of positive and negative amplitudes reflects energy transfer from a pigment with positive amplitudes to one with negative amplitudes.27,28 It should be noted that when negative or positive peaks are superimposed by higher magnitudes of positive or negative peaks, respectively, a FDA spectrum does not necessarily exhibit a set of positive and negative peaks.29 The pH 6.5 FDA spectra of the PSI−FCPI and PSI are virtually identical to those reported recently.22 In the PSI complexes, the first FDA spectra (15−30 ps) exhibit similar peak positions of positive and negative bands at 3532

DOI: 10.1021/acs.jpclett.9b01314 J. Phys. Chem. Lett. 2019, 10, 3531−3535

Letter

The Journal of Physical Chemistry Letters

717 nm decay component is faster than the lifetime of the S1 state of Chl a at ∼5 ns, suggesting nonphotochemical quenching around/within FCPI. However, it should be noted that the nonphotochemical quenching component is unlikely to be a major process in the excitation energy transfer in PSI−FCPI because of a very low amplitude of such ns components in the absolute FDA spectrum of the PSI−FCPI observed previously.22 To highlight the alteration of the FDA spectral properties within 100 ps, the first and second FDA spectra were summed under individual conditions (Figure 3). In PSI−FCPI, both pH

692 and 711−713 nm, respectively, reflecting the excitation energy transfer in the PSI cores from Chls having a relatively high energy to low-energy Chls. However, the relative magnitude of the 711−713 nm bands to the 692 nm band increases in the order of pH 5.0 > pH 6.5 > pH 8.0, i.e., there seem to be apparent effects of the pH changes on the energy supply and acceptance in the PSI cores. The second FDA spectra (65−80 ps) showed a positive peak but with different band shapes, and the third FDA spectra (280−380 ps) also showed a single positive peak. Because the time constants from tens to hundreds of ps appear to represent the event of energy trapping at the RC Chls in PSI30−33 and may also include an energy quenching process by the interactions of Chls with Cars,6−9 the presence of only positive peaks without distinct negative peaks in the second and third FDA spectra is attributable to either photochemical quenching by the trapping events or nonphotochemical quenching by Chl−Car interactions. It should be noted that the contribution of free Chl c to the excitation energy-transfer events in the PSI cores is negligibly low because of its much longer lifetime.22 In the PSI−FCPI membranes, the first FDA spectra (20−35 ps) exhibit different peak positions of both positive and negative bands with different relative magnitudes under the three pH conditions. The pH 5.0 FDA spectrum shows a set of 688 nm positive and 709 nm negative bands; the pH 6.5 FDA spectrum exhibits a set of 684 nm positive and 701 nm negative bands; and the pH 8.0 FDA spectrum exhibits a set of 680 nm positive and 698 nm negative bands. The peak shifts are larger upon an increase in the pH values. In addition, the peak positions of the band sets are shifted to shorter wavelengths than those of the positive and negative bands observed in the first FDA spectra of PSI, suggesting larger effects of pH changes on FCPI. The second FDA spectra (65−100 ps) show different spectral shapes in response to the pH changes. The pH 5.0, 6.5, and 8.0 FDA spectra exhibit broad positive bands at 701, 696, and 695 nm, respectively. The positive bands are shifted to shorter wavelengths than those in the second FDA spectra of PSI, and the tendency of the peak shifts is consistent with that of the first FDA spectra of the PSI−FCPI. It is interesting to note that the pH 8.0 FDA spectrum shows a small negative band at 722 nm in addition to the large 695 nm positive band. The 722 nm rise component is a prominent feature for the pH 8.0 FDA spectrum because there is no apparent negative band in the other second FDA spectra. By contrast, the pH 5.0 FDA spectrum shows a distinct positive shoulder at 710−730 nm. These features are different from those observed with the PSI core-like complexes, suggesting alteration of excitation energytransfer pathways by the protonation and deprotonation of FCPI. The third FDA spectra (210−410 ps) shows similar spectral features with a positive band at 708−709 nm irrespective of the pH changes. Because, as mentioned in the FDA spectra of PSI, the time constants of the fluorescence decays from tens to hundreds of ps appear to represent the energy trapping to the RC Chls and the energy quenching by Chl−Car interactions,6−9,30−33 the result suggests that most of the energy is related to either photochemical or nonphotochemical quenching. The forth FDA spectra (1.3−1.9 ns) also show similar spectral features with a peak at 716−717 nm irrespective of the pH changes. The time constant of a few ns hardly contributes to the trapping phase in the RC Chls because uphill energy transfer is suppressed at 77 K. The time constant of the 716−

Figure 3. Summed FDA spectra of the PSI−FCPI membranes (left panel) and PSI complexes (right panel) at 77 K. Black, red, and green lines indicate samples adapted to pH 5.0, 6.5, and 8.0, respectively. These spectra were summed using the first and second FDA spectra depicted in Figure 2.

6.5 and 8.0 spectra showed comparable magnitudes between positive peaks at 687−689 nm and negative peaks at 710 nm. However, the pH 5.0 FDA spectrum exhibited a relatively high amplitude of the positive peak at 692 nm with a very small negative magnitude at 718 nm. These results strongly indicate that the significant energy quenching in PSI−FCPI is induced under acidic pH conditions. Such spectral alteration induced by the pH changes hardly appears in the FDA spectra of the PSI core-like sample, suggesting that the pH-induced spectral changes occur around/within the FCPI. To verify the energy quenching under the pH 5.0 conditions in PSI−FCPI, we reconstructed decay curves from the FDA spectra (Figure 4). The decay curves were reconstructed with amplitudes at 688 nm, where the maximum amplitude was observed in the first FDA spectrum at pH 5.0 (Figure 2). The pH 5.0 decay curve clearly exhibits faster fluorescence decay than the curves at the other two pH conditions, confirming the rapid energy quenching in the PSI−FCPI membranes adapted to pH 5.0. The time constants in the pH 5.0 FDA spectra in all of the time regions examined are longer than those in the pH 6.5 and 8.0 FDA spectra (Figure 2). This contrasts with the observations of the significant quenching from the reconstructed decay curve. This can be explained as follows. The first FDA spectra at pH 6.5 and 8.0 have the rise components at 698−701 nm, whereas the pH 5.0 FDA spectrum loses the 700 nm rise components but with the longer-wavelength components at 709 nm (Figure 2). Because fluorescence from lower-energy Chls has relatively long time constants, it is suggested that the rapid energy-transfer pathways from higherenergy Chls to the 700 nm Chls are suppressed at pH 5.0. The 3533

DOI: 10.1021/acs.jpclett.9b01314 J. Phys. Chem. Lett. 2019, 10, 3531−3535

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The Journal of Physical Chemistry Letters

reconstructed decay curve also highlights the rapid energy quenching under the acidic pH conditions (Figure 4). It is widely accepted that when oxyphototrophs are exposed to excess light, the thylakoid lumen gives rise to acidic pH.10,11 In the green-lineage oxyphototrophs, the acidic pH facilitated excitation energy quenching;12−16 therefore, it is suggested that the low-energy Chls in the region of 710−730 nm are involved in the quenching events. By contrast, there is a set of large positive and small negative bands in the second FDA spectrum under the pH 8.0 conditions (Figure 2). The relatively high fluorescence component at 695 nm in addition to a remarkable positive shoulder at 675−685 nm is mainly related to either the excitation energy trapping to PSI or quenching at 65 ps. However, a part of the energy is likely migrated to the 722 nm Chl component that originates from FCPI because of the absence of apparent peaks at 722 nm in the PSI complexes, and the energy may be subsequently utilized for either trapping to PSI or quenching. The alterations of excitation energy dynamics induced by the basic pH have not been observed in the green-lineage oxyphototrophs. These observations imply that the structural changes of FCPI caused by deprotonation promote energy supply to PSI by forming the high-energy Chls. In conclusion, this study demonstrated pH-induced spectral changes in the diatom PSI−FCPI complex by means of TRF spectroscopy. By comparing spectral properties of the PSI− FCPI membranes with the PSI core-like preparations, we found that the excitation energy dynamics in FCPI are modified in response to the pH changes. This seems to be caused by structural changes of Chl molecules and/or their surrounding proteins in FCPI due to protonation or deprotonation events. The acidic or basic pH-induced structural changes around/within Chl molecules may enhance excitation energy quenching or transfer, respectively, thereby leading to an attractive notion that the roles of some Chl molecules in FCPI in the excitation energy-transfer events are regulated by the pH changes. The pH-sensing machinery of the excitation energy transfer in FCPI would function to maintain the balance of photosynthetic performance in response to the pH changes under different light conditions in thylakoid membranes in diatoms.

Figure 4. Decay curves reconstructed from the FDA spectra of the PSI−FCPI membranes at 688 nm (Figure 2). Black, red, and green lines indicate samples adapted to pH 5.0, 6.5, and 8.0, respectively.

longer time constants in the pH 5.0 FDA spectra may therefore be due to the distinct suppression of the 700 nm Chl pathways. The above results indicate that the pH effects on the excitation energy dynamics are clearly visible in the FDA spectra (Figures 2 and 3) but not in the steady-state spectra (Figure 1). This is likely due to the fast spectral changes only in the early tens of ps, that is, the fluorescence alterations within 100 ps were observed by the FDA spectra. The pHinduced spectral perturbations are larger in PSI−FCPI than those in the PSI core-like complexes. In particular, the apparent peak shifts are characteristic of the PSI−FCPI spectra, whereas the alterations of relative peak intensity mainly occur in the PSI spectra with much smaller peak shifts. These results strongly indicate that the excitation energy transfer around/within FCPI in the early time range after excitation is affected by the pH changes. In the first and second FDA spectra of PSI−FCPI, the main peaks observed under the pH 6.5 conditions are shifted to longer and shorter wavelengths under the pH 5.0 and 8.0 conditions, respectively (Figure 2). The spectral features are clearly verified in the summed FDA spectra (Figure 3). This is due to structural changes in the surrounding environments of Chls, i.e., Chls and/or amino acid residues close to Chl molecules are either protonated or deprotonated under the acidic or basic pH conditions, respectively. Thus, the protonation states of FCPI may contribute to the excitation energy-transfer events, resulting in the apparent peak shifts upon pH changes. Under the pH 5.0 conditions, the broad positive band appears in the second FDA spectrum, which contains a shoulder at 710−730 nm and is absent in the second FDA spectra under the pH 6.5 and 8.0 conditions but appeared in the later FDA spectra (Figure 2). This suggests that the structural perturbations caused by protonation accelerated excitation relaxation of the low-energy Chls to a decay time constant of 100 ps, which may contribute to either energy trapping to the RC Chls in PSI or excitation energy quenching by Chl−Car interactions.6−9 This is emphasized by the summed FDA spectra that showed a weak 718 nm negative peak relative to the 692 nm positive peak (Figure 3). The

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel/Fax: +81-86-2518630 (R.N.). *E-mail: [email protected]. Tel/Fax: +81-78-8035705 (S.A.). ORCID

Ryo Nagao: 0000-0001-8212-3001 Jian-Ren Shen: 0000-0003-4471-8797 Seiji Akimoto: 0000-0002-8951-8978 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

This work was supported by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science KAKENHI JP17K07442 and JP19H04726 (to R.N.), JP17H06433 (to J.-R.S.), and JP16H06553 (to S.A.). 3534

DOI: 10.1021/acs.jpclett.9b01314 J. Phys. Chem. Lett. 2019, 10, 3531−3535

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The Journal of Physical Chemistry Letters

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