Cu+ Contributes to the Orange Carotenoid Protein-Related

17 Jun 2019 - Publication History ... (8,9) In cyanobacteria, the orange carotenoid protein (OCP) helps the ... A copper ion can accept and donate a s...
0 downloads 0 Views 1MB Size
Download PDF
Article Cite This: Biochemistry 2019, 58, 3109−3115

pubs.acs.org/biochemistry

Cu+ Contributes to the Orange Carotenoid Protein-Related Phycobilisome Fluorescence Quenching and Photoprotection in Cyanobacteria Wenjing Lou,† Benjamin M. Wolf,† Robert E. Blankenship,†,‡ and Haijun Liu*,†,‡ †

Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130, United States Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States

‡

Downloaded via KEAN UNIV on July 19, 2019 at 07:23:55 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Photosynthesis starts with absorption of light energy by using light-harvesting antenna complexes (LHCs). Overexcitation of LHCs and subsequent photosystems, however, is damaging and can be lethal. The orange carotenoid protein (OCP) protects most cyanobacteria from photodamage by dissipating excessive excitation energy harvested by phycobilisomes (PBS, LHCs) as heat. OCP has two states: the orange, inactive OCP (OCPO) and the red, active OCP (OCPR), with the latter able to bind PBS at a ratio of 2:1 and execute photoprotection. Conversion of OCPO to OCPR is driven by blue light absorption. Previous work indicated that in the presence of Cu2+, photoactivation of OCP can result in it being locked in its red form OCPR. The molecular mechanism of such chemical conversion, however, remains unclear. Here, we demonstrated that Cu+ can convert OCPO to OCPR under anaerobic conditions independent of light illumination. Interestingly, in the presence of Cu2+ and ascorbic acid, a ubiquitous reductant in photosynthetic organisms, the conversion of OCPO to OCPR can also take place spontaneously in the dark, indicative of a locked OCPR−Cu+ complex. Furthermore, our functional and structural studies indicate that OCPR−Cu+ can interact with PBS and trigger PBS fluorescence quenching. We hypothesize that copper ion, a redox-active component, may synergistically play an important role in the regulation of nonphotochemical quenching in cyanobacteria under stress conditions.

C

carotenoid.12 In darkness, OCP is orange (OCPO) with two absorption maxima at 495 and 475 nm and a shoulder at 440 nm (Figure 1A).13 The absorption of blue light converts the inactive, orange form of OCP (OCPO) into its active, red form (OCPR). Compared with OCPO, the absorption spectrum of OCPR has a red shift with absorption maximum peaking at 508−510 nm and loss of the vibrational bands.14,15 It seems that only OCPR can bind to the core of PBS and act as a quencher of excess light energy.16−18 The OCPR is unstable and spontaneously reverts back to OCPO in darkness.14,19 It is known that the fluorescence recovery protein (FRP) can greatly accelerate the conversion of OCPR to OCPO and the detachment of OCPR from PBS, so FRP is a gatekeeper that switches off the OCP-induced fluorescence quenching.16,20−22 Light is the driving force for triggering formation of the active OCP. However, protein stability and functionality in vivo could change dramatically under changing physiochemical conditions. Our previous studies showed that thiocyanate, a kosmotrope in the Hofmeister series, can induce OCP conformational changes that lead to OCP activation.23 It seems that besides light intensity and temperature, there may be other factors that could have a similar effect on the protein

yanobacteria, the endosymbiotic ancestor of the chloroplast in plants and eukaryotic algae, are a group of bacteria capable of performing oxygenic photosynthesis and are major contributors to the global carbon and nitrogen cycles with dioxygen (O 2 ) being released as a byproduct. Cyanobacteria are widely distributed and well adapted to changing environments, including stress conditions such as high light illuminations, extreme temperatures, heavy metals, etc.1,2 Light drives the photosynthetic light reactions and the production of the reducing power (NADPH) and ATP that are used in the Calvin−Benson cycle of carbon fixation.3,4 All photosynthetic organisms have evolved a light-harvesting antenna complex (LHC) system to effectively capture solar energy and feed it to the reaction centers (RCs).5−7 However, when light energy collected by LHCs exceeds the capability of downstream assimilation, the overexcited pigments in LHCs and RCs will inevitably produce reactive oxygen species (ROS) that are damaging and even lethal for photosynthetic organisms.8,9 In cyanobacteria, the orange carotenoid protein (OCP) helps the cyanobacterial LHC (phycobilisomes, PBS) dissipate excess energy as heat, a process generally called nonphotochemical fluorescence quenching (NPQ), which is a major mechanism to protect most cyanobacteria from photodamage under high-light conditions.10,11 OCP, a 35 kDa water-soluble protein, consists of two domains (N-terminal domain, NTD, and C-terminal domain, CTD) encompassing a photoresponsive pigment, a keto © 2019 American Chemical Society

Received: May 7, 2019 Revised: June 12, 2019 Published: June 17, 2019 3109

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115

Article

Biochemistry

Figure 1. Chemoconversion of OCPO to OCPR in the presence of copper ion, ascorbate. (A) OCP incubated with Cu2+ and Cu2+ + AA under blue light illumination for 5 min or in dark conditions. (B) OCP in the presence of AA in dark or blue light for 10 min. The green dash dotted line overlaps with the blue line in panels A and B. Inset: Color changes of OCP under the corresponding conditions. BL, blue light; m, minute, AA, ascorbate.

the supernatant using HisTrap HP affinity chromatography and further purified by HiTrap Q HP ion exchange chromatography (Sigma, St. Louis, MO, United States).14,35 PBS Isolation. The PBSs were isolated from wild-type (WT) Synechocystis sp. PCC 6803 according to the methods described by Glazer36 and Gwizdala et al.16 with minor modifications. Two liters of culture cells were harvested and resuspended in 0.8 M pH 7.5 potassium phosphate buffer (KP).37 The cells were broken by passing twice through a French Press at a pressure of 20 000 psi. The broken cells were incubated with Triton X-100 at a final concentration of 1% (v/ v) for 30 min with gentle shaking at room temperature. The cell debris and unbroken cells were removed by centrifugation at 20 000 rpm using a SS-34 rotor (Sorvall Evolution RC) at 23 °C for 30 min. The supernatant was loaded onto a sucrose gradient with 2.0, 1.0, 0.75, 0.5, and 0.25 M sucrose solutions in 0.8 M KP buffer in a SW 32 Ti centrifuge tube (Beckman Coulter, Indianapolis, IN, United States). The sucrose gradient was spun at 32 000 rpm using a SW 32 Ti rotor at 23 °C overnight. The blue band between the 2.0 and 1.0 M sucrose layers was collected. FRP Purification. The FRP protein was purified from E. coli cells that overexpress a C-terminally His6-tagged FRP protein cloned from Synechocystis sp. PCC 6803, as previously described.38 Absorption Spectroscopy. The absorption spectra of the OCP samples were collected on a UV2510PC Shimadzu UV− vis spectrophotometer (Kyoto, Japan). OCP and PBS Reconstitution and Fluorescence Spectroscopy. Fluorescence analysis was performed using a Cary Eclipse Fluorescence Spectrophotometer (Mulgrave, Australia) at room temperature. PBS emission spectra were performed in a 1 cm path length cuvette with excitation at 580 nm. Fluorescence quenching was induced by 5 min of blue light illumination (1000 μmol photons m−2·s−1, 482 nm peak, 25.7 nm full width at half-maximum (fwhm)) using a custombuilt LED lighting system (Figure S4). Chemically induced fluorescence quenching by Cu+ was performed in a double buffering system in the presence of 1 mM sodium thiosulfate. All detections of the Cu+ related activation and recovery were operated in an anaerobic chamber. The concentration of PBS used in all the experiments is 0.013 μM. The concentrations of PBS, OCP, and FRP were calculated based on the absorbance using extinction coefficients εγ=280 nm = 15 220 M−1 cm−1 for FRP, εγ=495 nm = 63 000 M−1 cm−1 for OCP, and εγ=622 nm = 42 660 mM−1 cm−1 for PBS.16,39

stability and conformational dynamics of OCP and may be involved in the regulation of the activation and relaxation of OCP in vivo. However, it is still unclear whether other factors are also involved in the regulation of in vivo photoreception. Copper is an essential micronutrient metal for photosynthesis and respiration. A copper ion can accept and donate a single electron, cycling between Cu+ and Cu2+, which makes it an ideal cofactor for redox active enzymes such as cytochrome c oxidase, plastocyanin, and Cu/Zn-superoxide dismutase, among others.24 Photosynthetic organisms have a higher copper requirement compared with the nonphotosynthetic organisms. However, copper in excess is also toxic to photosynthetic organisms.25,26 Copper, even at low concentrations, is a powerful inhibitor of photosynthesis.27 It is reported that excess copper can induce NPQ in both algae and plants, reducing fluorescence intensity and PSΙΙ activity.27−31 It is assumed that the LHC of PSΙΙ is the primary site of copper inhibition in plants.31 In cyanobacteria, high copper ion concentrations can also significantly reduce the fluorescence intensity.32,33 We recently reported that photoactivation of OCP in the presence of Cu2+ results in OCP locked in its red state and prevents its relaxation back to the inactive, orange form, which potentially increases NPQ effects.34 The molecular mechanism of such a chemical process, however, remains unclear. In this work, we tested effects of copper ion in its different redox states on the conversion of the two forms of OCPs in the presence or absence of another redox active component, ascorbate, which ubiquitously exists in living organisms. Our results of OCP conversion under different chemical conditions, and more importantly, the functional studies using reconstituted OCP−PBS, suggest that copper could play an important role in OCP-related NPQ in cyanobacteria in a redox-dependent manner and be part of copper homeostasis.

■

MATERIALS AND METHODS OCP Isolation. C-terminally His6-tagged OCP strain of cyanobacteria Synechocystis sp. PCC 6803 was cultured in BG11 at 30 °C under continuous fluorescence illumination (30 μmol photons m−2·s−1).35 Briefly, the cells were centrifuged at 6000 rpm using a SLC 6000 rotor (Sorvall Evolution RC, Thermo Scientific, Massachusetts, MC, United States) for 10 min and were resuspended in 20 mM pH 7.5 Tris-HCl buffer. Then, the cells were lysed by two passages through a French Press. The unbroken cells, cell debris, and membrane were removed using Beckman Coulter ultracentrifuge Ti 45 (Beckman Coulter, Indianapolis, IN, United States) at 45 000 rpm for 30 min. The OCP protein was purified from 3110

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115

Article

Biochemistry

Figure 2. Chemoconversion of OCPO to OCPR in the presence of Cu+ without light illumination. (A) OCP treated with different concentrations of Cu+ under anaerobic conditions (30 min). (B) The kinetics of OCPO to OCPR conversion under different concentrations of Cu+. The samples were detected in MOPS buffer (20 mM, pH 7.5). Inset: color changes of OCP after treatments; m, minute.

■

RESULTS AND DISCUSSION Cu2+ and Ascorbate Induced OCPO to OCPR Conversion in Darkness. We define OCP chemoconversion as a process that OCPO is converted to OCPR by a chemical treatment independent of light illumination.23 The chemoconversion of OCPO to OCPR was tested in the dark. In Figure 1A, the spectra of the samples #2, 3, and 5 were recorded right after 5 min of blue light illumination (light strength = 1000 μmol photons m−2·s−1), and their spectra following 10 min of dark relaxation are shown in Figure 1B. Photoactivated OCP (Figure 1A, #2, control) relaxes quickly to its orange form with its characteristic absorption peaks of 475 and 495 nm (Figure 1B, #1). In the presence of 50 μM Cu2+ after photoactivation (Figure 1A, #3) and dark relaxation (Figure 1B, #3), however, the absorption spectrum of OCP showed observable changes: first, broad absorption values from 350 to 700 nm. Absorptions beyond 400 nm may indicate aggregation-induced diffraction of OCP protein after illumination in the presence of Cu2+. Second, the peaks at either 475 or 495 nm of the Cu2+containing sample became less distinctive, and the absorption beyond 500 nm increased, as represented by A495/A510 values. The A495/A510 decreased from 1.24 of OCPO to 1.04 of copper locked OCP (Figure 1A, #2 and Figure 1B, #3) vs 0.82 of OCP R (Figure 1A, #2). Visually, copper locked OCP R demonstrated a similar color as photoactivated OCPR (Figure 1B, inset), indicative of locked OCPR in its red form, and could not relax back to OCPO in darkness. This is consistent with our previous OCPR report.34 It is known that Cu2+ can be reduced to Cu+ by light.40,41 It is also known that L-ascorbic acid or ascorbate, a ubiquitous reductant in photosynthetic organisms, is essential for detoxification of superoxide and hydrogen peroxide in chloroplasts and cyanobacteria as well. Additionally, previous studies indicate that ascorbate can reduce Cu2+ to Cu+.41 Prompted by this scenario, we performed ascorbate addition experiments. In the presence of 100 μM ascorbate after light illumination (Figure 1A, #5) and dark relaxation (Figure 1B, #5), OCP + Cu2+ solution showed significant color changes by comparison with the experiment of OCP + Cu2+ + light: complete disappearance of the 475 nm peak and shifting of the 495 nm peak to 508 nm, indicative of a complete conversion of OCPO to OCPR. Interestingly, mixing ascorbate with OCPO + Cu2+ solution, which originally was orange in the dark, instantly results in red OCP independent of light (Figure 1A and B, #4, blue line). Please note the absorption spectrum of #4 completely overlaps with that of #5 (green dash dot). However, samples with additions of either ascorbate or Cu2+

alone did not have any significant changes on OCP absorption peaks at either 475 or 495 nm region (Figure S1A and B). At tested concentrations, neither the ascorbate nor the Cu2+ solution showed significant absorption peaks at 475, 495, or 508 nm (Figure S1C). Cu+ Spontaneously Drives OCP Coloration. Ascorbate can reduce Cu2+ to Cu+.41 However, Cu+ is unstable in aqueous solutions: it is easily oxidized to Cu2+ and immediately disproportions into Cu2+ and Cu0 in liquid. The oxidation of thiosulfate ([S2O3]2−) by oxygen in liquid is known to be very slow, and [S2O3]2− can quickly reduce Cu2+ into Cu+.42 For these reasons, in our following experiments, [S2O3]2− was used as a ligand to stabilize Cu+. Please note that no effects of [S2O3]2− on OCP were observed (Figure 2A, #2). To further avoid aerial oxygen effects on the chemicals that were used in our experiments, we performed spectrophotometry measurements in an anaerobic chamber flushed with N2 and H2 with installed palladium catalyst. After incubation with increasing concentrations of Cu+ in darkness for 30 min, the OCP protein showed progressive color changes: disappearance of the 475 nm peak and shifting of the 495 nm peak toward longer wavelengths (508 nm) (Figure 2A). Visual differences are also presented and are consistent with our measurements (Figure 2A, inset). The kinetics of OCP chemoconversion under different Cu+ concentrations were also recorded in a time range of 2340 s monitored at 550 nm, which has been used for monitoring OCP color changes (Figure 2B).16,34 Cu+ has high affinity for thiol and thioether groups and the sulfur donors, including cysteine (C), histidine (H), and methionine (M).43 We hesitate to name OCP as a copperbinding protein because OCP does not seem to possess a common CXXC (X is any amino acid) Cu+-binding motif.44,45 However, the NTD of the OCP protein is rich in methionine residues (Figure S2). The methionine thioether group lacks the electrostatic group component of cysteine thiolate; consequently, the methionine residue has a weaker Cu+ affinity compared with that of cysteine.43 The methionine-rich sites enhance their selectivity for Cu+, and the methionine residues are more difficult to oxidize than the CXXC motifs. By combining Cu+ results and those of Cu2+ plus ascorbate experiment and Cu2+ plus light (photoreduction) as well, it seems that in our experiment, it is Cu+ rather than Cu2+ that specifically converts OCPO into OCPR and locks OCP in its red form in darkness (Figure 2); metal ions such as Fe2+ or Mn2+ do not have any influence on the OCP chemoconversion and relaxation under either dark or illuminated conditions.34 All of these results imply formation of an OCPR−Cu+ complex 3111

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115

Article

Biochemistry

Figure 3. PBS fluorescence quenching measurement. (A) Fluorescence quenching of PBS induced by OCP under blue light (5 min), Cu2+, or Cu2+ + AA. Inset, PBS−OCPR−Cu+ complex isolation and Western blot analysis. (B) The conversion of OCPO to OCPR in the presence of Cu2+ and AA under different concentrations of potassium phosphate buffer (KP)37 in darkness. The concentration of PBS is 0.013 μM; the ratio of OCP to PBS is 40:1. m, minute; AA, ascorbate.

Figure 4. PBS fluorescence quenching induced by OCPR−Cu+ and fluorescence recovery examinations. The ratio of OCP to PBS is 20:1 (A) and 40:1 (B), respectively. (C) Cu+ has no effects on PBS fluorescence (control experiment for panel B). (D) PBS fluorescence quenching recovery assisted by FRP in the presence or absence of Cu+ under dark conditions. The ratio of PBS:OCP:FRP is 1:40:80. BL, blue light; m, minute.

fraction (Figure 3A, inset, lane 2). It seems, however, that 0.8 M KP buffer essentially used for keeping PBS structure and function intact almost completely abolished the function of ascorbate that reduces Cu2+ to Cu+ and consequently blocked the formation of OCPR−Cu+ complex (Figure 3B), which may interpret the less significant fluorescence quenching as that shown in Figure S3B. The PBS fluorescence intensity decreased with time but reduced only about 12% after 30 min incubation (Figure S3B, orange line) with 100 μM Cu2+, 1 mM ascorbate, and OCP in 0.8 M KP buffer. Please note that ascorbate has no significant effects on PBS fluorescence changes (Figure S3A). Our data also suggested that ascorbate− Cu2+-induced OCPO to OCPR conversion is ionic-strengthdependent (Figure 3B). Thus, it is not possible to study the copper-activated OCPR-induced PBS fluorescence quenching in the presence of KP buffer, ascorbate and Cu2+ in darkness. Chemoconverted OCPR-Mediated PBS Fluorescence Quenching. Cu+ alone can induce chemoconversion of OCP, forming OCPR−Cu+. We are particularly interested in whether OCPR−Cu+ can bind PBS and induce PBS fluorescence quenching. PBS fluorescence intensity decreased about 7%

in our study (Figures 1 and 2). We hypothesize that under copper stress conditions, the methionine-rich sites in the NTD would bind Cu+, which induces OCP conformational changes, converts OCPO to OCPR, and keeps OCP in its red form. To some extent, copper ions from either deactivated copper binding proteins or other stress conditions could be bound to OCP. OCP-Mediated PBS Fluorescence Quenching Analysis in the Presence of Cu2+ and Ascorbate. Without light illumination, mixing of Cu2+ and OCPO and PBS did not induce fluorescence quenching (Figure 3A, blue line). However, after illumination under blue light for 5 min, the fluorescence intensity of reconstituted solution, originally containing Cu2+, OCPO, and PBS, decreased more than 80% (Figure 3A, green line), and fluorescence recovery upon dark incubation was completely abolished. By combing the data (Figures 1 and 2) that, upon illumination, OCP can be locked in its red form by forming the OCPR−Cu+ complex, we hypothesize that copper-locked OCPR can bind PBS and induce PBS fluorescence quenching. Indeed, Western blot analysis indicated the presence of OCP in the isolated PBS 3112

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115

Article

Biochemistry

Figure 5. A working model of OCPR−Cu+ involved photoprotection under copper stressed conditions. The cellular free Cu2+ could be reduced to Cu+ by light or cellular reducing substance (1). Cu+ binds and converts OCPO to OCPR (2) or directly bands to the photoactivated OCPR (3), the OCP protein is locked in the red form. OCPR−Cu+ complex binding to PBS allophycocyanin (APC) induces fluorescence quenching. The copperlocked OCPR could prolong the PBS fluorescence quenching process even in the presence of FRP, reducing the energy flow to the reaction center and protecting the photosystems from photodamage.

recover PBS fluorescence induced by chemoconverted OCPR but in a less effective manner. This phenomenon could be functionally advantageous in a cell because by binding a toxic metal ion, i.e., copper ion in this case, active OCPR−Cu+ can extend its quenching function of PBS fluorescence for the cells when they are under stress conditions and when excitation energy needs to be dissipated by whatever means. A Working Model for Copper and OCP-Involved Cellular Detoxification and Photoprotection. Copper is one of the most toxic metal ions for photosynthetic organisms.24 It is assumed that there are essentially no free copper ions (uncomplexed) in the cytosol of either eukaryotic or prokaryotic cells under normal physiological conditions.46,47 Cells have a great challenge to manage the homeostasis of such heavy metals. Under stress conditions, the cellular leaky Cu2+ from deactivated copper binding proteins (regents) or extracellular sources can be reduced to Cu+ by either light or reducing agents found within the cytoplasm (Figure 5, 1).40,41 Cu+ then binds and converts OCPO to OCPR (Figure 5, 3) or directly binds to the photoactivated OCPR (Figure 5, 4), forming stable OCPR−Cu+ complex (Figure 2). OCPR−Cu+ then binds PBS that extends PBS fluorescence quenching, less effectively controlled by FRP (Figure 5). Given this model, we hypothesize that OCP has dual functions under copper stress conditions: binding (chelating) of Cu+ that is produced by cellular reductants such as ascorbate to alleviate copper toxicity and binding (OCPR−Cu+) to PBS, a light harvesting complex antenna in cyanobacteria, to dissipate excitation energy that if not otherwise properly managed could exacerbate any stressed cellular conditions. In summary, we successfully identified the functional form of the copper ion that could lock OCP in its active form by testing both ascorbate-induced reduction of cupric ion to cuprous ion and cuprous ion alone. Our functional studies indicate that OCPR−Cu+ indeed binds PBS and executes PBS fluorescence quenching in an extended manner less dependent on FRP interference. On the basis of previously published results and our current results, we propose an updated OCP function pathway interconnected with copper redox chemistry and a potential stress response strategy for cells under

after the PBS was incubated with OCP and Cu+ for 30 min at a ratio of 20:1 (OCP:PBS) in darkness (Figure 4A, green line). Interestingly, we noticed that fluorescence quenching progressively increased with time; the fluorescence intensity reached 37% of the original fluorescence intensity after 2 h of incubation (Figure 4A, purple line). If the ratio of OCP to PBS was raised to 40:1, the PBS fluorescence quenching was even higher: the fluorescence intensity decreased 18% and decreased 55% after 30 min (Figure 4B, green line) and 2 h of treatment (Figure 4B, purple line) in darkness, respectively, in contrast to negligible effects of PBS fluorescence intensity changes caused by Cu+ alone (Figure 4C). It should be noted that a higher ratio of OCP and PBS was repeatedly used in all reconstitution experiments because under 0.8 KP buffer, solution hydrophilicity is significantly reduced, and hydrophobic interactions between proteins are greatly encouraged to keep PBS integrity. Consequently, hydrophilic molecular diffusion coefficient and protein conformation associated physical processes are dramatically altered such as Cu+-induced OCPR and OCPR binding to PBS (Figure 4A and B). It was reported that PBS fluorescence quenching induced by illumination in the presence of OCP in 0.8 M KP buffer is stable for 24 h, and FRP has a negligible effect to recover the PBS fluorescence.16 This is in significant contrast to the situation that binding of OCPR to PBS is much weaker when 0.5 M KP buffer was used.16 So, in our following FRP addition experiment, 0.5 M KP buffer was used rather than 0.8 M KP buffer. FRP was added when the maximal OCP-induced PBS fluorescence quenching was achieved. For the non-Cu+ containing sample, FRP was added right after 5 min of illumination, while for the Cu+ containing sample, FRP was added after 2 h of incubation in the dark (Figure 4D). We found OCP-induced PBS fluorescence quenching in 0.5 M KP buffer recovered to 90% of the initial fluorescence by adding FRP, at a ratio of 1:40:80 (PBS:OCP:FRP), after 3.5 h of incubation in the dark (Figure 4D, from blue line to purple line). PBS fluorescence quenching induced by OCPR−Cu+ (without light), however, is recovered to 64% of the initial fluorescence by adding FRP in a similar setting (Figure 4D, from green line to yellow line). In other words, FRP does 3113

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115

Article

Biochemistry

(9) Triantaphylides, C., and Havaux, M. (2009) Singlet oxygen in plants: production, detoxification and signaling. Trends Plant Sci. 14, 219−228. (10) Wilson, A., Ajlani, G., Verbavatz, J. M., Vass, I., Kerfeld, C. A., and Kirilovsky, D. (2006) A soluble carotenoid protein involved in phycobilisome-related energy dissipation in cyanobacteria. Plant Cell 18, 992−1007. (11) Melnicki, M. R., Leverenz, R. L., Sutter, M., Lopez-Igual, R., Wilson, A., Pawlowski, E. G., Perreau, F., Kirilovsky, D., and Kerfeld, C. A. (2016) Structure, Diversity, and Evolution of a New Family of Soluble Carotenoid-Binding Proteins in Cyanobacteria. Mol. Plant 9, 1379−1394. (12) Kirilovsky, D., and Kerfeld, C. A. (2013) The Orange Carotenoid Protein: a blue-green light photoactive protein. Photochem. Photobiol. Sci. 12, 1135−1143. (13) Polivka, T., Kerfeld, C. A., Pascher, T., and Sundstrom, V. (2005) Spectroscopic properties of the carotenoid 3′-hydroxyechinenone in the orange carotenoid protein from the cyanobacterium Arthrospira maxima. Biochemistry 44, 3994−4003. (14) Wilson, A., Punginelli, C., Gall, A., Bonetti, C., Alexandre, M., Routaboul, J. M., Kerfeld, C. A., van Grondelle, R., Robert, B., Kennis, J. T., and Kirilovsky, D. (2008) A photoactive carotenoid protein acting as light intensity sensor. Proc. Natl. Acad. Sci. U. S. A. 105, 12075−12080. (15) Jallet, D., Thurotte, A., Leverenz, R. L., Perreau, F., Kerfeld, C. A., and Kirilovsky, D. (2014) Specificity of the cyanobacterial orange carotenoid protein: influences of orange carotenoid protein and phycobilisome structures. Plant Physiol. 164, 790−804. (16) Gwizdala, M., Wilson, A., and Kirilovsky, D. (2011) In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803. Plant Cell 23, 2631−2643. (17) Stadnichuk, I. N., Yanyushin, M. F., Maksimov, E. G., Lukashev, E. P., Zharmukhamedov, S. K., Elanskaya, I. V., and Paschenko, V. Z. (2012) Site of non-photochemical quenching of the phycobilisome by orange carotenoid protein in the cyanobacterium Synechocystis sp. PCC 6803. Biochim. Biophys. Acta, Bioenerg. 1817, 1436−1445. (18) Squires, A. H., Dahlberg, P. D., Liu, H., Magdaong, N. C. M., Blankenship, R. E., and Moerner, W. E. (2019) Single-molecule trapping and spectroscopy reveals photophysical heterogeneity of phycobilisomes quenched by Orange Carotenoid Protein. Nat. Commun. 10, 1172. (19) Berera, R., van Stokkum, I. H., Gwizdala, M., Wilson, A., Kirilovsky, D., and van Grondelle, R. (2012) The photophysics of the orange carotenoid protein, a light-powered molecular switch. J. Phys. Chem. B 116, 2568−2574. (20) Boulay, C., Wilson, A., D’Haene, S., and Kirilovsky, D. (2010) Identification of a protein required for recovery of full antenna capacity in OCP-related photoprotective mechanism in cyanobacteria. Proc. Natl. Acad. Sci. U. S. A. 107, 11620−11625. (21) Thurotte, A., Bourcier de Carbon, C., Wilson, A., Talbot, L., Cot, S., Lopez-Igual, R., and Kirilovsky, D. (2017) The cyanobacterial Fluorescence Recovery Protein has two distinct activities: Orange Carotenoid Protein amino acids involved in FRP interaction. Biochim. Biophys. Acta, Bioenerg. 1858, 308−317. (22) Sluchanko, N. N., Slonimskiy, Y. B., Shirshin, E. A., Moldenhauer, M., Friedrich, T., and Maksimov, E. G. (2018) OCPFRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria. Nat. Commun. 9, 3869. (23) King, J. D., Liu, H., He, G., Orf, G. S., and Blankenship, R. E. (2014) Chemical activation of the cyanobacterial orange carotenoid protein. FEBS Lett. 588, 4561−4565. (24) Huertas, M. J., Lopez-Maury, L., Giner-Lamia, J., SanchezRiego, A. M., and Florencio, F. J. (2014) Metals in cyanobacteria: analysis of the copper, nickel, cobalt and arsenic homeostasis mechanisms. Life 4, 865−886. (25) Yruela, I. (2009) Copper in plants: acquisition, transport and interactions. Funct Plant Biol. 36, 409−430.

(copper) stress conditions or light stress in general. The identification of structural binding domain(s) of copper on OCP remains a future research effort.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00409. Absorption spectra of OCP in the presence of Cu2+ or Lascorbate and control experiments, methionine and cysteine distribution in OCP (Synechocystis sp. PCC 6803), PBS fluorescence measurement in the presence of OCP, Cu2+, and L-ascorbate and control experiments, and LED design (PDF) Accession Codes

UniProt IDs: orange carotenoid-binding protein (P74102) and fluorescence recovery protein (P74103).

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Wenjing Lou: 0000-0002-7511-1915 Robert E. Blankenship: 0000-0003-0879-9489 Haijun Liu: 0000-0003-0537-0302 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was funded by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences, Photosynthetic Systems (PS) Program (Grant DE-FG02-07ER15902 to R.E.B. and H.L.). We thank members of the R. E. Blankenship group for collegial discussions, especially Drs. Jeremy D. King and Rafael Saer.

■

REFERENCES

(1) Murata, N., Takahashi, S., Nishiyama, Y., and Allakhverdiev, S. I. (2007) Photoinhibition of photosystem II under environmental stress. Biochim. Biophys. Acta, Bioenerg. 1767, 414−421. (2) Palenik, B. (2015) Molecular mechanisms by which marine phytoplankton respond to their dynamic chemical environment. Annual review of marine science 7, 325−340. (3) Michelet, L., Zaffagnini, M., Morisse, S., Sparla, F., Perez-Perez, M. E., Francia, F., Danon, A., Marchand, C. H., Fermani, S., Trost, P., and Lemaire, S. D. (2013) Redox regulation of the Calvin-Benson cycle: something old, something new. Frontiers in plant science 4, 470. (4) Welkie, D. G., Rubin, B. E., Diamond, S., Hood, R. D., Savage, D. F., and Golden, S. S. (2019) A Hard Day’s Night: Cyanobacteria in Diel Cycles. Trends Microbiol. 27, 231−242. (5) Sukenik, A., Bennett, J., and Falkowski, P. (1987) LightSaturated Photosynthesis - Limitation by Electron-Transport or Carbon Fixation. Biochim. Biophys. Acta, Bioenerg. 891, 205−215. (6) Ort, D. R., Zhu, X. G., and Melis, A. (2011) Optimizing Antenna Size to Maximize Photosynthetic Efficiency. Plant Physiol. 155, 79− 85. (7) Blankenship, R. E. (2014) Molecular Mechanisms of Photosynthesis, Blackwell Science, Oxford, UK. (8) Niyogi, K. K. (1999) Photoprotection revisited: Genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333−359. 3114

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115

Article

Biochemistry (26) Lopez-Maury, L., Giner-Lamia, J., and Florencio, F. J. (2012) Redox control of copper homeostasis in cyanobacteria. Plant Signaling Behav. 7, 1712−1714. (27) Mohanty, N., Vass, I., and Demeter, S. (1989) Copper Toxicity Affects Photosystem II Electron Transport at the Secondary Quinone Acceptor, Q(B). Plant Physiol. 90, 175−179. (28) Brown, M. T., and Newman, J. E. (2003) Physiological responses of Gracilariopsis longissima (S.G. Gmelin) Steentoft, L.M. Irvine and Farnham (Rhodophyceae) to sub-lethal copper concentrations. Aquat. Toxicol. 64, 201−213. (29) Nielsen, H. D., Brownlee, C., Coelho, S. M., and Brown, M. T. (2003) Inter-population differences in inherited copper tolerance involve photosynthetic adaptation and exclusion mechanisms in Fucus serratus. New Phytol. 160, 157−165. (30) Bhargava, P., Mishra, Y., Srivastava, A. K., Narayan, O. P., and Rai, L. C. (2008) Excess copper induces anoxygenic photosynthesis in Anabaena doliolum: a homology based proteomic assessment of its survival strategy. Photosynth. Res. 96, 61−74. (31) Lidon, F. C., Ramalho, J. C., and Henriques, F. S. (1993) Copper Inhibition of Rice Photosynthesis. J. Plant Physiol. 142, 12− 17. (32) Burgos, A., Maldonado, J., De Los Rios, A., Sole, A., and Esteve, I. (2013) Effect of copper and lead on two consortia of phototrophic microorganisms and their capacity to sequester metals. Aquat. Toxicol. 140−141, 324−336. (33) Seder-Colomina, M., Burgos, A., Maldonado, J., Sole, A., and Esteve, I. (2013) The effect of copper on different phototrophic microorganisms determined in vivo and at cellular level by confocal laser microscopy. Ecotoxicology 22, 199−205. (34) Liu, H., Lu, Y., Wolf, B., Saer, R., King, J. D., and Blankenship, R. E. (2018) Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion. Photosynth. Res. 135, 143−147. (35) Zhang, H., Liu, H., Niedzwiedzki, D. M., Prado, M., Jiang, J., Gross, M. L., and Blankenship, R. E. (2014) Molecular mechanism of photoactivation and structural location of the cyanobacterial orange carotenoid protein. Biochemistry 53, 13−19. (36) Glazer, A. N. (1988) Phycobiliproteins. Methods Enzymol. 167, 291−303. (37) Mkpong, O. E., Yan, H., Chism, G., and Sayre, R. T. (1990) Purification, Characterization, and Localization of Linamarase in Cassava. Plant Physiol. 93, 176−181. (38) Lu, Y., Liu, H., Saer, R. G., Zhang, H., Meyer, C. M., Li, V. L., Shi, L., King, J. D., Gross, M. L., and Blankenship, R. E. (2017) Native Mass Spectrometry Analysis of Oligomerization States of Fluorescence Recovery Protein and Orange Carotenoid Protein: Two Proteins Involved in the Cyanobacterial Photoprotection Cycle. Biochemistry 56, 160−166. (39) Wilson, A., Gwizdala, M., Mezzetti, A., Alexandre, M., Kerfeld, C. A., and Kirilovsky, D. (2012) The essential role of the N-terminal domain of the orange carotenoid protein in cyanobacterial photoprotection: importance of a positive charge for phycobilisome binding. Plant Cell 24, 1972−1983. (40) Tasdelen, M. A., and Yagci, Y. (2010) Light-induced copper(I)catalyzed click chemistry. Tetrahedron Lett. 51, 6945−6947. (41) Himo, F., Lovell, T., Hilgraf, R., Rostovtsev, V. V., Noodleman, L., Sharpless, K. B., and Fokin, V. V. (2005) Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates. J. Am. Chem. Soc. 127, 210−216. (42) Byerley, J. J., Fouda, S. A., and Rempel, G. L. (1975) Activation of Copper(Ii) Ammine Complexes by Molecular-Oxygen for Oxidation of Thiosulfate Ions. J. Chem. Soc., Dalton Trans., 1329− 1338. (43) Davis, A. V., and O’Halloran, T. V. (2008) A place for thioether chemistry in cellular copper ion recognition and trafficking. Nat. Chem. Biol. 4, 148−151. (44) Boal, A. K., and Rosenzweig, A. C. (2009) Structural Biology of Copper Trafficking. Chem. Rev. 109, 4760−4779.

(45) Badarau, A., Firbank, S. J., McCarthy, A. A., Banfield, M. J., and Dennison, C. (2010) Visualizing the metal-binding versatility of copper trafficking sites. Biochemistry 49, 7798−7810. (46) Rae, T. D., Schmidt, P. J., Pufahl, R. A., Culotta, V. C., and O’Halloran, T. V. (1999) Undetectable intracellular free copper: The requirement of a copper chaperone for superoxide dismutase. Science 284, 805−808. (47) Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C. E., O’Halloran, T. V., and Mondragon, A. (2003) Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383−1387.

3115

DOI: 10.1021/acs.biochem.9b00409 Biochemistry 2019, 58, 3109−3115