RESUMO
Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.
Assuntos
Serina C-Palmitoiltransferase , Serina , Sphingobacterium , Domínio Catalítico , Cristalização , Medição da Troca de Deutério , Elétrons , Hidrogênio/metabolismo , Palmitoil Coenzima A/metabolismo , Serina/análogos & derivados , Serina/metabolismo , Serina C-Palmitoiltransferase/química , Serina C-Palmitoiltransferase/metabolismo , Sphingobacterium/enzimologia , Sphingobacterium/metabolismo , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Esfingosina/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis, which catalyzes the pyridoxal-5'-phosphate-dependent decarboxylative condensation reaction of l-serine (l-Ser) and palmitoyl-CoA (PalCoA) to form 3-ketodihydrosphingosine called long chain base (LCB). SPT is also able to metabolize l-alanine (l-Ala) and glycine (Gly), albeit with much lower efficiency. Human SPT is a membrane-bound large protein complex containing SPTLC1/SPTLC2 heterodimer as the core subunits, and it is known that mutations of the SPTLC1/SPTLC2 genes increase the formation of deoxy-type of LCBs derived from l-Ala and Gly to cause some neurodegenerative diseases. In order to study the substrate recognition of SPT, we examined the reactivity of Sphingobacterium multivorum SPT on various amino acids in the presence of PalCoA. The S. multivorum SPT could convert not only l-Ala and Gly but also l-homoserine, in addition to l-Ser, into the corresponding LCBs. Furthermore, we obtained high-quality crystals of the ligand-free form and the binary complexes with a series of amino acids, including a nonproductive amino acid, l-threonine, and determined the structures at 1.40 to 1.55 Å resolutions. The S. multivorum SPT accommodated various amino acid substrates through subtle rearrangements of the active-site amino acid residues and water molecules. It was also suggested that non-active-site residues mutated in the human SPT genes might indirectly influence the substrate specificity by affecting the hydrogen-bonding networks involving the bound substrate, water molecules, and amino acid residues in the active site of this enzyme. Collectively, our results highlight SPT structural features affecting substrate specificity for this stage of sphingolipid biosynthesis.
Assuntos
Serina C-Palmitoiltransferase , Sphingobacterium , Humanos , Palmitoil Coenzima A/química , Palmitoil Coenzima A/metabolismo , Serina/química , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Sphingobacterium/enzimologia , Esfingolipídeos/metabolismo , Especificidade por SubstratoRESUMO
Cyanobacterial photosynthetic apparatus efficiently capture sunlight, and the energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. PSII is a unique membrane protein complex that photo-catalyzes oxidation of water and majorly contains photosynthetic pigments of chlorophyll a and carotenoids. In the present study, the ultrafast energy transfer and charge separation dynamics of PSII from a thermophilic cyanobacterium Thermosynechococcus vulcanus were reinvestigated by femtosecond pump-probe spectroscopic measurements under low temperature and weak intensity excitation condition. The results imply the two possible models of the energy transfers and subsequent charge separation in PSII. One is the previously suggested "transfer-to-trapped limit" model. Another model suggests that the energy transfers from core CP43 and CP47 antennas to the primary electron donor ChlD1 with time-constants of 0.71 ps and 3.28 ps at 140 K (0.17 and 1.33 ps at 296 K), respectively and that the pheophytin anion (PheoD1-) is generated with the time-constant of 43.0 ps at 140 K (14.8 ps at 296 K) upon excitation into the Qy band of chlorophyll a at 670 nm. The secondary electron transfer to quinone QA: PheoD1-QA â PheoD1QA- is observed with the time-constant of 650 ps only at 296 K. On the other hand, an inefficient ß-carotene â chlorophyll a energy transfer (33%) occurred after excitation to the S2 state of ß-carotene at 500 nm. Instead, the carotenoid triplet state appeared in an ultrafast timescale after excitation at 500 nm.
Assuntos
Cianobactérias , beta Caroteno , Clorofila A , beta Caroteno/metabolismo , Análise Espectral , Transporte de Elétrons , Cianobactérias/metabolismo , Carotenoides/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Clorofila/metabolismo , ThermosynechococcusRESUMO
Cyanobacterial photosynthetic systems efficiently capture sunlight using the pigment-protein megacomplexes, phycobilisome (PBS). The energy is subsequently transferred to photosystem I (PSI) and II (PSII), to produce electrochemical potentials. In the present study, we performed picosecond (ps) time-resolved fluorescence and femtosecond (fs) pump-probe spectroscopies on the intact PBS from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, to reveal excitation energy transfer dynamics in PBS. The photophysical properties of the intact PBS were well characterized by spectroscopic measurements covering wide temporal range from femtoseconds to nanoseconds. The ps fluorescence measurements excited at 570 nm, corresponding to the higher energy of the phycocyanin (PC) absorption band, demonstrated the excitation energy transfer from the PC rods to the allophycocyanin (APC) core complex as well as the energy transfer in the APC core complex. Then, the fs pump-probe measurements revealed the detailed energy transfer dynamics in the PC rods taking place in an ultrafast time scale. The results obtained in this study provide the full picture of the funnel-type excitation energy transfer with rate constants of (0.57 ps)-1 â (7.3 ps)-1 â (53 ps)-1 â (180 ps)-1 â (1800 ps)-1.
Assuntos
Transferência de Energia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Ficobilissomas/metabolismo , Espectrometria de Fluorescência , Thermosynechococcus/metabolismoRESUMO
The development of techniques capable of using membrane proteins in a surfactant-free aqueous buffer is an attractive research area, and it should be elucidated for various membrane protein studies. To this end, we examined a method using new solubilization surfactants that do not detach from membrane protein surfaces once bound. The designed solubilization surfactants, DKDKC12K-PAn (n = 5, 7, and 18), consist of two parts: one is the lipopeptide-based solubilization surfactant part, DKDKC12K, fand the other is the covalently connected linear polyacrylamide (PA) chain with different Mw values of 5, 7, or 18 kDa. Intermolecular interactions between the PA chains in DKDKC12K-PAn concentrated on the surfaces of membrane proteins via amphiphilic binding of the DKDKC12K part to the integral membrane domain was observed. Therefore, DKDKC12K-PAn (n = 5, 7, and 18) could maintain a bound state even after removal of the unbound by ultrafiltration or gel-filtration chromatography. We used photosystem I (PSI) from Thermosynecoccus vulcanus as a representative to assess the impacts of new surfactants on the solubilized membrane protein structure and functions. Based on the maintenance of unique photophysical properties of PSI, we evaluated the ability of DKDKC12K-PAn (n = 5, 7, and 18) as a new solubilization surfactant.
Assuntos
Resinas Acrílicas/química , Soluções Tampão , Proteínas de Membrana/química , Polímeros/química , Tensoativos/química , Fenômenos Químicos , Técnicas de Química Sintética , Concentração de Íons de Hidrogênio , Estrutura Molecular , Tamanho da Partícula , Polímeros/síntese química , Solubilidade , Tensoativos/síntese químicaRESUMO
In this study, we improved the hydrogen production efficiency by combining photosystem I with an artificial light harvesting dye, Lumogen Red. In the reaction system, Lumogen Red allows light absorption and energy transfer to photosystem I by Förster resonance energy transfer; therefore, the Pt nanoparticles act as active sites for hydrogen generation.
RESUMO
Photosystem II (PSII) catalyzes light-induced water splitting, leading to the evolution of molecular oxygen indispensible for life on the earth. The crystal structure of PSII from cyanobacteria has been solved at an atomic level, but the structure of eukaryotic PSII has not been analyzed. Because eukaryotic PSII possesses additional subunits not found in cyanobacterial PSII, it is important to solve the structure of eukaryotic PSII to elucidate their detailed functions, as well as evolutionary relationships. Here we report the structure of PSII from a red alga Cyanidium caldarium at 2.76 Å resolution, which revealed the structure and interaction sites of PsbQ', a unique, fourth extrinsic protein required for stabilizing the oxygen-evolving complex in the lumenal surface of PSII. The PsbQ' subunit was found to be located underneath CP43 in the vicinity of PsbV, and its structure is characterized by a bundle of four up-down helices arranged in a similar way to those of cyanobacterial and higher plant PsbQ, although helices I and II of PsbQ' were kinked relative to its higher plant counterpart because of its interactions with CP43. Furthermore, two novel transmembrane helices were found in the red algal PSII that are not present in cyanobacterial PSII; one of these helices may correspond to PsbW found only in eukaryotic PSII. The present results represent the first crystal structure of PSII from eukaryotic oxygenic organisms, which were discussed in comparison with the structure of cyanobacterial PSII.
Assuntos
Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Rodófitas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Complexo de Proteína do Fotossistema II/ultraestrutura , Proteínas de Plantas/ultraestrutura , Conformação Proteica , Multimerização Proteica , Alinhamento de SequênciaRESUMO
The oxygen-evolving complex (OEC) forms the heart of photosystem II (PSII) in photosynthesis. The crystal structure of PSII from Thermosynechococcus vulcanus has been reported at a resolution of 1.9 Å and at an averaged X-ray dose of 0.43 MGy. The OEC structure is suggested to be partially reduced to Mn(II) by EXAFS and DFT computational studies. Recently, the "radiation-damage-free" structures have been published at 1.95 Å resolution using XFEL, but reports continued to appear that the OEC is reduced to the S0-state of the Kok cycle. To elucidate much more precise structure of the OEC, in this study two structures were determined at extremely low X-ray doses of 0.03 and 0.12 MGy using conventional synchrotron radiation source. The results indicated that the X-ray reduction effects on the OEC were very small in the low dose region below 0.12 MGy, that is, a threshold existed for the OEC structural changes caused by X-ray exposure. The OEC structures of the two identical monomers in the crystal were clearly different under the threshold of the radiation dose, although the surrounding polypeptide frameworks of PSII were the same. The assumption that the OECs in the crystal were in the dark-stable S1-state of the Kok cycle should be re-evaluated.
RESUMO
Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 Å. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 Å. From our electron density map, we located all of the metal atoms of the Mn(4)CaO(5) cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn(4)CaO(5) cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.
Assuntos
Cianobactérias/química , Modelos Moleculares , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/química , Sítios de Ligação , Clorofila/química , Cristalização , Lipídeos/química , Plastoquinona/química , Estrutura Terciária de Proteína , beta Caroteno/químicaRESUMO
In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.
Assuntos
Reparo do DNA , Recombinação Homóloga , Recombinases Rec A/química , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Biocatálise , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Ligação Proteica , Conformação Proteica , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Serina Endopeptidases/metabolismoRESUMO
Adenosine diphosphate ribose pyrophosphatase (ADPRase), a member of the Nudix family proteins, catalyzes the metal-induced and concerted general acid-base hydrolysis of ADP ribose (ADPR) into AMP and ribose-5'-phosphate (R5P). The ADPR-hydrolysis reaction of ADPRase from Thermus thermophilus HB8 (TtADPRase) requires divalent metal cations such as Mn(2+), Zn(2+), or Mg(2+) as cofactors. Here, we report the reaction pathway observed in the catalytic center of TtADPRase, based on cryo-trapping X-ray crystallography at atomic resolutions around 1.0 Å using Mn(2+) as the reaction trigger, which was soaked into TtADPRase-ADPR binary complex crystals. Integrating 11 structures along the reaction timeline, five reaction states of TtADPRase were assigned, which were ADPRase alone (E), the ADPRase-ADPR binary complex (ES), two ADPRase-ADPR-Mn(2+) reaction intermediates (ESM, ESMM), and the postreaction state (E'). Two Mn(2+) ions were inserted consecutively into the catalytic center of the ES-state and ligated by Glu86 and Glu82, which are highly conserved among the Nudix family, in the ESM- and ESMM-states. The ADPR-hydrolysis reaction was characterized by electrostatic, proximity, and orientation effects, and by preferential binding for the transition state. A new reaction mechanism is proposed, which differs from previous ones suggested from structure analyses with nonhydrolyzable substrate analogues or point-mutated ADPRases.
Assuntos
Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Manganês/química , Manganês/metabolismo , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The development of additional extraction surfactants for membrane proteins is necessary for membrane protein research, since optimal combinations for the successful extraction of target membrane proteins from biological membranes that minimize protein denaturation are hard to predict. In particular, those that have a unique basal molecular framework are quite attractive and highly desired in this research field. In this study, we successfully constructed a new extraction surfactant for membrane proteins, NPDGC12KK, from the peptide-gemini-surfactant (PG-surfactant) molecular framework. The PG-surfactant is a U-shaped lipopeptide scaffold, consisting of a short linker peptide (-X-) between two long alkyl-chain-modified Cys residues and a peripheral peptide (Y-) at the N-terminal side of long alkyl-chain-modified Cys residues. Using photosystem I (PSI) and photosystem II (PSII) derived from Thermosynecoccus vulcanus as representative membrane proteins, we evaluated whether NPDGC12KK could solubilize membrane proteins while maintaining structure and functions. Neither the membrane integral domain nor the cytoplasmic domain of PSI and PSII suffered any damage upon the use of NPDGC12KK based on detailed photophysical measurements. Using thylakoid membranes of T. vulcanus as a representative biological membrane sample, we performed experiments to extract membrane proteins, such as PSI and PSII. Based on the extraction efficiency and maintenance of protein supramolecular structure established using clear native-PAGE analyses, we proved that NPDGC12KK functions as a novel class of peptide-containing extraction surfactants for membrane proteins.
Assuntos
Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Tensoativos/química , Fracionamento Químico/métodos , Cisteína/química , Lipopeptídeos/química , Micelas , Peptídeos/química , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema II/química , Engenharia de Proteínas/métodos , Espectrometria de Fluorescência , Synechocystis/química , Tilacoides/químicaRESUMO
The development of artificial photosynthesis has focused on the efficient coupling of reaction at photoanode and cathode, wherein the production of hydrogen (or energy carriers) is coupled to the electrons derived from water-splitting reactions. The natural photosystem II (PSII) complex splits water efficiently using light energy. The PSII complex is a large pigment-protein complex (20 nm in diameter) containing a manganese cluster. A new photoanodic device was constructed incorporating stable PSII purified from a cyanobacterium Thermosynechococcus vulcanus through immobilization within 20 or 50 nm nanopores contained in porous glass plates (PGPs). PSII in the nanopores retained its native structure and high photoinduced water splitting activity. The photocatalytic rate (turnover frequency) of PSII in PGP was enhanced 11-fold compared to that in solution, yielding a rate of 50-300 mol e(-)/(mol PSII·s) with 2,6-dichloroindophenol (DCIP) as an electron acceptor. The PGP system realized high local concentrations of PSII and DCIP to enhance the collisional reactions in nanotubes with low disturbance of light penetration. The system allows direct visualization/determination of the reaction inside the nanotubes, which contributes to optimize the local reaction condition. The PSII/PGP device will substantively contribute to the construction of artificial photosynthesis using water as the ultimate electron source.
Assuntos
2,6-Dicloroindofenol/química , Proteínas de Bactérias/química , Cianobactérias/enzimologia , Vidro/química , Nanoporos , Oxigênio/química , Complexo de Proteína do Fotossistema II/química , PorosidadeRESUMO
The electron density map of the 3D crystal of Photosystem II from Thermosynechococcus vulcanus with a 1.9 Šresolution (PDB: 3ARC ) exhibits, in the two monomers in the asymmetric unit cell, an, until now, unidentified and uninterpreted strong difference in electron density centered at a distance of around 1.5 Šfrom the nitrogen Nδ of the imidazole ring of D2-His336. By MALDI-TOF/MS upon tryptic digestion, it is shown that ~20-30% of the fragments containing the D2-His336 residue of Photosystem II from both Thermosynechococcus vulcanus and Thermosynechococcus elongatus bear an extra mass of +16 Da. Such an extra mass likely corresponds to an unprecedented post-translational or chemical hydroxyl modification of histidine.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Histidina/metabolismo , Radical Hidroxila/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Cianobactérias/química , Cianobactérias/genética , Histidina/química , Histidina/genética , Modelos Moleculares , Peso Molecular , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genéticaRESUMO
The crystal structure of Photosystem II (PSII) analyzed at a resolution of 1.9 Å revealed deformations of chlorin rings in the chlorophylls for the first time. We investigated the degrees of chlorin ring deformation and factors that contributed to them in the PSII crystal structure, using a normal-coordinate structural decomposition procedure. The out-of-plane distortion of the P(D1) chlorin ring can be described predominantly by a large "doming mode" arising from the axial ligand, D1-His198, as well as the chlorophyll side chains and PSII protein environment. In contrast, the deformation of P(D2) was caused by a "saddling mode" arising from the D2-Trp191 ring and the doming mode arising from D2-His197. Large ruffling modes, which were reported to lower the redox potential in heme proteins, were observed in P(D1) and Chl(D1), but not in P(D2) and Chl(D2). Furthermore, as P(D1) possessed the largest doming mode among the reaction center chlorophylls, the corresponding bacteriochlorophyll P(L) possessed the largest doming mode in bacterial photosynthetic reaction centers. However, the majority of the redox potential shift in the protein environment was determined by the electrostatic environment. The difference in the chlorin ring deformation appears to directly refer to the difference in "the local steric protein environment" rather than the redox potential value in PSII.
Assuntos
Complexo de Proteína do Fotossistema II/química , Porfirinas/química , Proteínas de Bactérias/química , Clorofila/química , Cristalografia por Raios X , Cianobactérias/química , Modelos Moleculares , Estrutura Molecular , Oxirredução , Eletricidade EstáticaRESUMO
PsbM and PsbI are two low molecular weight subunits of photosystem II (PSII), with PsbM being located in the center, and PsbI in the periphery, of the PSII dimer. In order to study the functions of these two subunits from a structural point of view, we crystallized and analyzed the crystal structure of PSII dimers from two mutants lacking either PsbM or PsbI. Our results confirmed the location of these two subunits in the current crystal structure, as well as their absence in the respective mutants. The relative contents of PSII dimers were found to be decreased in both mutants, with a concomitant increase in the amount of PSII monomers, suggesting a destabilization of PSII dimers in both of the mutants. On the other hand, the accumulation level of the overall PSII complexes in the two mutants was similar to that in the wild-type strain. Treatment of purified PSII dimers with lauryldimethylamine N-oxide at an elevated temperature preferentially disintegrated the dimers from the PsbM deletion mutant into monomers and CP43-less monomers, whereas no significant degradation of the dimers was observed from the PsbI deletion mutant. These results indicate that although both PsbM and PsbI are required for the efficient formation and stability of PSII dimers in vivo, they have different roles, namely, PsbM is required directly for the formation of dimers and its absence led to the instability of the dimers accumulated. On the other hand, PsbI is required in the assembly process of PSII dimers in vivo; once the dimers are formed, PsbI was no longer required for its stability.
Assuntos
Proteínas de Bactérias/química , Cianobactérias/genética , Mutagênese , Complexo de Proteína do Fotossistema II/química , Deleção de Sequência , Proteínas de Bactérias/genética , Cristalografia por Raios X , Dimerização , Complexo de Proteína do Fotossistema II/genética , Conformação ProteicaRESUMO
ADP-ribose pyrophosphatase-I from Thermus thermophilus HB8 (TtADPRase-I) prevents the intracellular accumulation of ADP-ribose by hydrolyzing it to AMP and ribose 5'-phosphate. To understand the catalytic mechanism of TtADPRase-I, it is necessary to investigate the role of glutamates and metal ions as well as the coordination of water molecules located at the active site. A macroseeding method was developed in order to obtain a large TtADPRase-I crystal which was suitable for a neutron diffraction study to provide structural information. Neutron and X-ray diffraction experiments were performed at room temperature using the same crystal. The crystal diffracted to 2.1 and 1.5 Å resolution in the neutron and X-ray diffraction experiments, respectively. The crystal belonged to the primitive space group P3(2)21, with unit-cell parameters a = b = 50.7, c = 119 Å.
Assuntos
Pirofosfatases/química , Thermus thermophilus/enzimologia , Cristalização , Difração de NêutronsRESUMO
The chloride ion, Cl(-), is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn(4)Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl(-) with a bromide ion (Br(-)) or an iodide ion (I(-)) in PSII and analyzed the crystal structures of PSII with Br(-) and I(-) substitutions. Substitution of Cl(-) with Br(-) did not inhibit oxygen evolution, whereas substitution of Cl(-) with I(-) completely inhibited oxygen evolution, indicating the efficient replacement of Cl(-) by I(-). PSII with Br(-) and I(-) substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of the Mn(4)Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn(4)Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl(-) anions are required to maintain the coordination structure of the Mn(4)Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.
Assuntos
Cloretos/química , Cloretos/metabolismo , Oxigênio/química , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Sítios de Ligação , Bromo/química , Bromo/metabolismo , Cristalografia por Raios X , Cianobactérias/enzimologia , Cianobactérias/genética , Iodo/química , Iodo/metabolismo , Modelos Moleculares , Complexo de Proteína do Fotossistema II/genética , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
Cyanobacteria, glaucophytes, and rhodophytes utilize giant, light-harvesting phycobilisomes (PBSs) for capturing solar energy and conveying it to photosynthetic reaction centers. PBSs are compositionally and structurally diverse, and exceedingly complex, all of which pose a challenge for a comprehensive understanding of their function. To date, three detailed architectures of PBSs by cryo-electron microscopy (cryo-EM) have been described: a hemiellipsoidal type, a block-type from rhodophytes, and a cyanobacterial hemidiscoidal-type. Here, we report cryo-EM structures of a pentacylindrical allophycocyanin core and phycocyanin-containing rod of a thermophilic cyanobacterial hemidiscoidal PBS. The structures define the spatial arrangement of protein subunits and chromophores, crucial for deciphering the energy transfer mechanism. They reveal how the pentacylindrical core is formed, identify key interactions between linker proteins and the bilin chromophores, and indicate pathways for unidirectional energy transfer.