RESUMEN
Nonphotochemical quenching (NPQ) is a crucial mechanism for fine-tuning light harvesting and protecting the photosystem II (PSII) reaction centres from excess light energy in plants and algae. This process is regulated by photoprotective proteins LHCSR1, LHCSR3, and PsbS in green algae, such as Chlamydomonas reinhardtii. The det1-2 phot mutant, which overexpresses these photoprotective proteins, resulting in a significantly higher NPQ response, has been recently discovered in C. reinhardtii. Here, we analysed the physiological impact of this response on algal cells and found that det1-2 phot was capable of efficient growth under high light intensities, where wild-type (WT) cells were unable to survive. The mutant exhibited a smaller PSII cross-section in the dark and showed a detachment of the peripheral light-harvesting complex II (LHCII) antenna in the NPQ state, as suggested by a rise in the chlorophyll fluorescence parameter of photochemical quenching in the dark (qPd > 1). Furthermore, fluorescence decay-associated spectra demonstrated a decreased excitation pressure on PSII, with excess energy being directed toward PSI. The amount of LHCSR1, LHCSR3, and PsbS in the mutant correlated with the magnitude of the protective NPQ response. Overall, the study suggests the mechanism by which the overexpression of photoprotective proteins in det1-2 phot brings about an efficient and effective photoprotective response, enabling the mutant to grow and survive under high light intensities that would otherwise be lethal for WT cells.
Asunto(s)
Chlamydomonas reinhardtii , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Luz , Tilacoides/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Clorofila/metabolismo , FotosíntesisRESUMEN
In green plants, photosystem I (PSI) and photosystem II (PSII) bind to their respective light-harvesting complexes (LHCI and LHCII) to form the PSI-LHCI supercomplex and the PSII-LHCII supercomplex, respectively. These supercomplexes further form megacomplexes, like PSI-PSII and PSII-PSII in Arabidopsis (Arabidopsis thaliana) and spinach to modulate their light-harvesting properties, but not in the green alga Chlamydomonas reinhardtii. Here, we fractionated and characterized the stable rice PSI-PSII megacomplex. The delayed fluorescence from PSI (lifetime â¼25 ns) indicated energy transfer capabilities between the two photosystems (energy spillover) in the rice PSI-PSII megacomplex. Fluorescence lifetime analysis revealed that the slow PSII to PSI energy transfer component was more dominant in the rice PSI-PSII supercomplexes than in Arabidopsis ones, suggesting that PSI and PSII in rice form a megacomplex not directly but through LHCII molecule(s), which was further confirmed by the negatively stained electron microscopy analysis. Our results suggest species diversity in the formation and stability of photosystem megacomplexes, and the stable PSI-PSII supercomplex in rice may reflect its structural adaptation.
Asunto(s)
Arabidopsis , Oryza , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Oryza/metabolismo , Arabidopsis/metabolismo , Complejos de Proteína Captadores de Luz/metabolismoRESUMEN
As a ubiquitous picophytoplankton in the ocean and an early-branching green alga, Ostreococcus tauri is a model prasinophyte species for studying the functional evolution of the light-harvesting systems in photosynthesis. Here, we report the structure and function of the O. tauri photosystem I (PSI) supercomplex in low light conditions, where it expands its photon-absorbing capacity by assembling with the light-harvesting complexes I (LHCI) and a prasinophyte-specific light-harvesting complex (Lhcp). The architecture of the supercomplex exhibits hybrid features of the plant-type and the green algal-type PSI supercomplexes, consisting of a PSI core, an Lhca1-Lhca4-Lhca2-Lhca3 belt attached on one side and an Lhca5-Lhca6 heterodimer associated on the other side between PsaG and PsaH. Interestingly, nine Lhcp subunits, including one Lhcp1 monomer with a phosphorylated amino-terminal threonine and eight Lhcp2 monomers, oligomerize into three trimers and associate with PSI on the third side between Lhca6 and PsaK. The Lhcp1 phosphorylation and the light-harvesting capacity of PSI were subjected to reversible photoacclimation, suggesting that the formation of OtPSI-LHCI-Lhcp supercomplex is likely due to a phosphorylation-dependent mechanism induced by changes in light intensity. Notably, this supercomplex did not exhibit far-red peaks in the 77 K fluorescence spectra, which is possibly due to the weak coupling of the chlorophyll a603-a609 pair in OtLhca1-4.
Asunto(s)
Chlorophyta , Complejo de Proteína del Fotosistema I , Complejo de Proteína del Fotosistema I/química , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Clorofila , Fotosíntesis , Chlorophyta/metabolismoRESUMEN
Major light-harvesting complex (LHCII) trimers in plants induce the thermal dissipation of absorbed excitation energy against photooxidative damage under excess light conditions. LHCII trimers in green algae have been thought to be incapable of energy dissipation without additional quencher proteins, although LHCIIs in plants and green algae are homologous. In this study, we investigated the energy-dissipative capabilities of four distinct types of LHCII trimers isolated from the model green alga Chlamydomonas reinhardtii using spectroscopic analysis. Our results revealed that the LHCII trimers possessing LHCII type II (LHCBM5) and LHCII type IV (LHCBM1) had efficient energy-dissipative capabilities, whereas LHCII type I (LHCBM3/4/6/8/9) and type III (LHCBM2/7) did not. On the basis of the amino acid sequences of LHCBM5 and LHCBM1 compared with the other LHCBMs, we propose that positively charged extra N-terminal amino acid residues mediate the interactions between LHCII trimers to form energy-dissipative states.
Asunto(s)
Chlamydomonas reinhardtii/química , Complejo de Proteína del Fotosistema II/metabolismo , Chlamydomonas reinhardtii/metabolismo , Procesos Fotoquímicos , Complejo de Proteína del Fotosistema II/químicaRESUMEN
The spectrally silent photoreaction of a blue light sensor protein YcgF, composed of the N-terminal BLUF domain and the C-terminal EAL domain, was investigated by the time-resolved transient grating method. Comparing photoinduced reactions of full-length YcgF with that of the BLUF-linker construct, it was found that a major conformation change after photoinduced dimerization is predominantly localized on the EAL domain. Furthermore, the photoinduced conformational change displayed significant temperature dependence. This result is explained by an equilibrium of reactive and nonreactive YcgF species, with the population of photoreactive species decreasing as the temperature is lowered in the dark state. We consider that the dimer form is the nonreactive species and it is the dominant species at lower temperatures. The temperature sensitivity of the photoreaction of YcgF suggests that this protein could have a biological function as a temperature sensor as well as behaving as a light sensor.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Luz , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Procesos Fotoquímicos , Temperatura , Oscuridad , Multimerización de Proteína/efectos de la radiación , Estructura Cuaternaria de ProteínaRESUMEN
Dahl salt-sensitive (Dahl-S) rats, serving as a model of hereditary hypertension, were used to examine the effect of mannooligosaccharides (MOS) on blood pressure. Dahl-S rats were induced to develop hypertension by administering them with a 1.25% salt solution ad libitum. In a 10-wk experimental period, the Dahl-S control and MOS groups developed and maintained significantly higher blood pressure than the Dahl salt-resistant normal control group. The MOS group showed a significantly lower blood pressure than the Dahl-S control group after 5-wk of treatment (p<0.05). In addition, the serum aldosterone level of the MOS group significantly decreased (p<0.05). The findings of this study using a model of hypertensive rats suggest that MOS are able to suppress an elevation in blood pressure.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Café/química , Hipertensión/prevención & control , Mananos/farmacología , Oligosacáridos/farmacología , Aldosterona/sangre , Animales , Peso Corporal/efectos de los fármacos , Dieta/métodos , Modelos Animales de Enfermedad , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Masculino , Mananos/administración & dosificación , Oligosacáridos/administración & dosificación , Ratas , Ratas Endogámicas Dahl , Cloruro de Sodio/administración & dosificación , Factores de TiempoRESUMEN
The photochemical reaction dynamics of YcgF, a BLUF protein, were investigated by the pulsed laser-induced transient grating (TG) technique. The TG signal showed three reaction time constants: 2.7 micros, 13 micros, and 2 ms. The fastest was tentatively attributed to relaxation of the excited triplet state of the chromophore, flavin adenine dinucleotide (FAD), and the others represented conformational changes of the protein. The TG signal provided clear evidence that the diffusion coefficient (D) of the photoproduct (3.8x10(-11) m2 s-1) was significantly less than that of the reactant (8.3x10(-11) m2 s-1), with a time constant of 2 ms at a protein concentration of 700 microM. Interestingly, the rate constant increased in proportion to the concentration of the protein, indicating that protein dimerization was one of the main reactions occurring after photoexcitation. The significant reduction in D indicates that a conformational change leading to an increase in interactions with water molecules occurs upon formation of the signaling state. The 13 mus dynamics was attributed to the conformational change that induced transient dimerization. This conformational change might be an essential process for the creation of the signaling state. A detailed scheme for the photochemical reaction of YcgF is proposed.
Asunto(s)
Proteínas de Escherichia coli/química , Hidrolasas Diéster Fosfóricas/química , Difusión , Dimerización , Cinética , Modelos Químicos , Fotoquímica , Conformación Proteica , Análisis Espectral/métodosRESUMEN
Lhl4 encodes a distant relative of light-harvesting Chl-a/b proteins in the green alga Chlamydomonas reinhardtii. Lhl4 mRNA markedly accumulated within 30 min after illumination and in proportion to the light intensity up to a fluence rate much higher than that required for photosynthesis. The high intensity light (HL)-induced accumulation of Lhl4 mRNA required continuous illumination, and the mRNA level rapidly decreased when the cells were placed in the dark. HL only slightly stabilized the mRNA, suggesting that the HL-induced expression of the Lhl4 gene is primarily regulated at the level of transcription. Blue light was more effective for inducing Lhl4 gene expression than green or red light, and far-red light had no effect. The action spectrum for Lhl4 gene expression was examined at wavelengths between 325 and 775 nm using the Okazaki Large Spectrograph. The obtained spectrum showed a distinct peak in the blue region (450 nm) and a shoulder in the UV-A region (375 nm). The curve in the spectrum rose steeply in the short wavelength UV region. In addition, we observed two minor peaks in the green (575 nm) and the red (675 nm) regions. The action spectrum suggests that a blue/UV-A light photoreceptor with a flavin-based chromophore participates in the HL response of Lhl4 gene expression. However, the hypersensitivity to near UV-B light suggests the involvement of an unidentified UV light perception system in the expression of the Lhl4 gene.
Asunto(s)
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efectos de la radiación , Regulación de la Expresión Génica , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Animales , Células Cultivadas , Chlamydomonas reinhardtii/fisiología , Oscuridad , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Estabilidad del ARN/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Changes in structural coupling between the Mn cluster and a putative histidine ligand during the S-state cycling of the oxygen-evolving complex (OEC) have been detected directly by Fourier transform infrared (FTIR) spectroscopy in photosystem (PS) II core particles from the cyanobacterium Synechocystis sp. PCC6803, in which histidine residues were selectively labeled with l-[(15)N(3)]histidine. The bands sensitive to the histidine-specific isotope labeling appeared at 1120-1090 cm(-)(1) in the spectra induced upon the first-, second-, and fourth-flash illumination, for the S(2)/S(1), S(3)/S(2), and S(1)/S(0) differences, at similar frequencies with different sign and/or intensity depending on the respective S-state transitions. However, no distinctive band was observed in the third-flash induced spectrum for the S(0)/S(3) difference. The results indicate that a single histidine residue coupled with the structural changes of the OEC during the S-state cycling is responsible for the observed histidine bands, in which the histidine modes changed during the S(0)-to-S(1) transition are reversed upon the S(1)-to-S(2) and S(2)-to-S(3) transitions. The 1186(+)/1178(-) cm(-)(1) bands affected by l-[(15)N(3)]histidine labeling were observed only for the S(2)/S(1) difference, but those affected by universal (15)N labeling appeared prominently showing a clear S-state dependency. Possible origins of these bands and changes in the histidine modes during the S-state cycling are discussed.
Asunto(s)
Proteínas Bacterianas/química , Histidina/química , Oxígeno/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Complejo de Proteína del Fotosistema II/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Histidina/metabolismo , Ligandos , Isótopos de Nitrógeno/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Synechocystis/química , Synechocystis/metabolismoRESUMEN
A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at approximately 70% of the rate of control wild-type particles. The E189Q OEC showed typical S(2) state electron spin resonance signals, and the spin center distance between the S(2) state manganese cluster and the Y(D) (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S(2) state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S(1) to S(2) transition.
Asunto(s)
Manganeso/metabolismo , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Complejo de Proteína del Fotosistema II/genética , Unión Proteica , Synechocystis/enzimología , Synechocystis/genética , Synechocystis/metabolismoRESUMEN
In photosynthetic water oxidation, two water molecules are converted to an oxygen molecule through five reaction intermediates, designated S(n) (n = 0-4), at the catalytic Mn cluster of photosystem II. To understand the mechanism of water oxidation, changes in the chemical nature of the substrate water as well as the Mn cluster need to be defined during S-state cycling. Here, we report for the first time a complete set of Fourier transform infrared difference spectra during S-state cycling in the low-frequency (670-350 cm(-1)) region, in which interactions between the Mn cluster and its ligands can be detected directly, in PS II core particles from Thermosynechococcus elongatus. Furthermore, vibrations from oxygen and/or hydrogen derived from the substrate water and changes in them during S-state cycling were identified using multiplex isotope-labeled water, including H2(18)O, D2(16)O, and D2(18)O. Each water isotope affected the low-frequency S-state cycling spectra, characteristically. The bands sensitive only to (16)O/(18)O exchange were assigned to the modes from structures involving Mn and oxygen having no interactions with hydrogen, while the bands sensitive only to H/D exchange were assigned to modes from amino acid side chains and/or polypeptide backbones that associate with water hydrogen. The bands sensitive to both (16)O/(18)O and H/D exchanges were attributed to the structure involving Mn and oxygen structurally coupled with hydrogen in a direct or an indirect manner through hydrogen bonds. These bands include the changes of intermediate species derived from substrate water during the process of photosynthetic water oxidation.
Asunto(s)
Cianobacterias/química , Complejo de Proteína del Fotosistema II/química , Espectroscopía Infrarroja por Transformada de Fourier , Agua/química , Medición de Intercambio de Deuterio , Óxido de Deuterio/química , Hidrógeno/química , Oxidación-Reducción , Isótopos de Oxígeno , Fotólisis , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Changes in the chemical structure of alpha-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [(13)C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S(2)/S(1) FTIR difference spectrum were affected by selective (13)C labeling of the alpha-carboxylate of Ala with l-[1-(13)C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the alpha-methyl of Ala with l-[3-(13)C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH(3) deformation coupled with the alpha-carboxylate. The isotopic bands for the alpha-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S(1)-to-S(2) transition and to a lesser extent upon the S(2)-to-S(3) transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S(3)-to-S(0) transition. No obvious isotopic band appeared upon the S(0)-to-S(1) transition. These results indicate that the alpha-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S(1)-to-S(2) transition and reduced reversely upon the S(3)-to-S(0) transition but is not associated with manganese ion(s) oxidized during the S(0)-to-S(1) (and S(2)-to-S(3)) transition(s). Consistently, l-[1-(13)C]alanine labeling also induced spectral changes in the low frequency (670-350 cm(-1)) S(2)/S(1) FTIR difference spectrum.
Asunto(s)
Alanina/química , Ácidos Carboxílicos/química , Oxígeno/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Synechocystis/metabolismo , Isótopos de Carbono , Estructura Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A free alpha-COO(-) in the C-terminal alanine-344 (Ala344) in the D1 protein of photosystem II is thought to be responsible for ligating the Mn cluster. The effects of the side group of the C-terminus of the D1 protein on the functional and structural properties of the oxygen-evolving complex (OEC) were comprehensively studied by replacing Ala344 with glycine (Gly), valine (Val), aspartate (Asp), or asparagine (Asn). All the mutants grew photoautotrophically under low-light conditions with lower O(2) evolution activity depending on the mutants when compared with the activity of the control wild type. The Gly-, Asp-, and Asn-substituted mutants did not grow under high-light conditions, while the Val-substituted mutant grew even under the high-light conditions. S(2)-state thermoluminescence bands appeared at slightly elevated temperatures when compared with those of the wild type in the Asp- and Gly-substituted mutants, but at almost normal temperatures in the Val- and Asn-substituted mutants. The oxygen-evolving core particles isolated from the mutants showed little change in protein composition. The Gly-, Asp-, and Asn-substituted core particles exhibited low-temperature electron spin resonance (ESR) spectra with reduced S(2) multiline and enhanced g = 4.1 ESR signals, while the Val-substituted particles showed a spectrum similar to that of the control particles. Mid-frequency Fourier transform infrared difference spectra showed distinctive changes in several bands arising from the putative carboxylate ligands for the Mn cluster in all substituted particles, but the bands for the putative C-terminal alpha-carboxylate did not seem to change in the substituted spectra. The changes induced by the Asp and Asn substitution resembled each other except for the amide I region, and showed some similarity to those induced by the Gly substitution in the symmetric carboxylate stretching region. The results were interpreted to mean that similar types of changes of the carboxylate ligands are induced by these substitutions. The band from a putative histidine ligand for the Mn cluster was similarly affected in the Gly-, Asp-, and Asn-substituted spectra, but not in the Val-substituted spectrum. Notably, marked changes in the amide I, amide II, and carboxylate bands were observed in the Val-substituted spectrum, which was different from the Gly-, Asp-, and Asn-substituted spectra. The results indicated that the structural perturbations induced by the Val substitution include large changes of the protein backbone and are considerably different from those induced by the other substitutions. Possible amino acid ligands participating in the changes deduced by Ala344 replacement in the D1 C-terminal and the effects of the changes of the side group on these ligands were considered on the basis of the available X-ray model of the OEC.
Asunto(s)
Manganeso/química , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/genética , Sustitución de Aminoácidos/genética , Espectroscopía de Resonancia por Spin del Electrón , Ligandos , Mediciones Luminiscentes , Oxidación-Reducción , Oxígeno/metabolismo , Fragmentos de Péptidos/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-Actividad , Synechocystis/química , Synechocystis/genética , Synechocystis/metabolismoRESUMEN
Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.
Asunto(s)
Cianobacterias/química , Cianobacterias/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Agua/química , Agua/metabolismo , Oxidación-Reducción , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The C-terminal alanine 344 (Ala-344) in the D1 protein of photosystem II is conserved in all of the organisms performing oxygenic photosynthesis. A free alpha-COO(-) of Ala-344 has been proposed to be responsible for ligating the Mn cluster. Here, we constructed a mutant having D1 in which D1-Ala-344 was replaced with glycine (Gly) in cyanobacterium Synechocystis sp. PCC 6803. The effects of this minimal change in the side group from methyl to hydrogen on the properties of the oxygen-evolving complex were comprehensively investigated using purified core particles. The mutant grew photoautotrophically, and little change was observed in the protein composition of the oxygen-evolving core particles. The Gly-substituted oxygen-evolving complex showed small but normal S(2) multiline and enhanced g = 4.1 electron spin resonance signals and S(2)-state thermoluminescence bands with slightly elevated peak temperature. The Gly substitution resulted in distinct but relatively small changes in a few bands arising from the putative carboxylate ligand for the Mn cluster in the mid-frequency (1800-1000 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. In contrast, the low frequency (670-350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was markedly changed by the substitution. The results indicate that the internal structure of the Mn cluster and/or the interaction between the Mn cluster and its ligand are considerably altered by a simple change in the side group, from methyl to hydrogen, at the C-terminal of the D1 protein.
Asunto(s)
Alanina/metabolismo , Proteínas Bacterianas , Cianobacterias/enzimología , Glicina/metabolismo , Complejo de Proteína del Fotosistema II , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The sensor of blue-light using FAD (BLUF) domain is the flavin-binding fold categorized to a new class of blue-light sensing domain found in AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis, but little is known concerning the mechanism of blue-light perception. An open reading frame slr1694 in a cyanobacterium Synechocystis sp. PCC6803 encodes a protein possessing the BLUF domain. Here, a full-length Slr1694 protein retaining FAD was expressed and purified and found to be present as an oligomeric form (trimer or tetramer). Using the purified Slr1694, spectroscopic properties of Slr1694 were characterized. Slr1694 was found to show the same red-shift of flavin absorption and quenching of flavin fluorescence by illumination as those of AppA. These changes reversed in the dark although the rate of dark state regeneration was much faster in Slr1694 than AppA, indicating that Slr1694 is a blue-light receptor based on BLUF with the similar photocycle to that of AppA. The dark decay in D(2)O was nearly four times slower than in H(2)O. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was applied to examine the light-induced structure change of a chromophore and apo-protein with deuteration and universal (13)C and (15)N isotope labeling. The FTIR results indicate that light excitation induced distinct changes in the amide I modes of peptide backbone but relatively limited changes in flavin chromophore. Light excitation predominantly weakened the C(4)=O and C(2)=O bonding and strengthened the N1C10a and/or C4aN5 bonding, indicating formational changes of the isoalloxazine ring II and III of FAD but little formational change in the isoalloxazine ring I. The photocycle of the BLUF is unique in the sense that light excitation leads to the structural rearrangements of the protein moieties coupled with a minimum formational change of the chromophore.
Asunto(s)
Proteínas Bacterianas/química , Cianobacterias/química , Proteínas de Drosophila , Proteínas del Ojo , Flavina-Adenina Dinucleótido/química , Flavoproteínas/química , Luz , Células Fotorreceptoras de Invertebrados , Pliegue de Proteína , Proteínas Bacterianas/metabolismo , Isótopos de Carbono/metabolismo , Criptocromos , Cianobacterias/metabolismo , Medición de Intercambio de Deuterio , Flavina-Adenina Dinucleótido/metabolismo , Flavoproteínas/metabolismo , Isótopos de Nitrógeno/metabolismo , Unión Proteica , Receptores Acoplados a Proteínas G , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.
Asunto(s)
Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Isótopos de Carbono/metabolismo , Cianobacterias/química , Ferricianuros/química , Luz , Manganeso/química , Isótopos de Nitrógeno/metabolismo , Oxígeno/química , Isótopos de Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Illuminating of the Ca(2+)-depleted PS II in the S(2) state for a short period induced the doublet signal at g=2 with concomitant diminution of the multiline signal, both in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In the absence of DCMU, the doublet signal decayed (t(1/2) approximately 7 min) during subsequent dark incubation at 273 K and the multiline signal was regenerated to the original amplitude with the same kinetics of the doublet decay. In the presence of DCMU, the doublet signal decayed much faster (t(1/2) approximately 1 min) by charge recombination with Q(A)(-), while the time course of the multiline recovery was inherently identical with that observed in the absence of DCMU. A simple theoretical consideration indicates the direct conversion from the doublet-signal state to the multiline state with no intermediate state between them. Lengthy dark storage at 77 K led to disappearance of the DCMU-affected doublet signal and a Fe(2+)/Q(A)(-) electron spin resonance (ESR) signal, but no recovery of the multiline signal. Notably, the multiline signal was restored by subsequent dark incubation at 273 K. The charge recombination between Q(A)(-) and the doublet signal species led to a thermoluminescence band at 7 degrees C in a medium at pH 5.5. The peak position shifted to 17 degrees C at pH 7.0, presumably due to a pH-dependent change in the redox property of a donor-side radical species responsible for the doublet signal. Based on these results, redox events in the Ca(2+)-depleted PS II are discussed in contradistinction with the normal processes in oxygen-evolving PS II.