RESUMEN
We have sequenced the chloroplast genome of red spruce (Picea rubens) for the first time using the single-end, short-reads (44 bp) Illumina sequences, assembled and functionally annotated it, and identified simple sequence repeats (SSRs). The contigs were assembled using SOAPdenovo2 following the retrieval of chloroplast genome sequences using the black spruce (Picea mariana) chloroplast genome as the reference. The assembled genome length was 122,115 bp (gaps included). Comparatively, the P. rubens chloroplast genome reported here may be considered a near-complete draft. Global genome alignment and phylogenetic analysis based on the whole chloroplast genome sequences of Picea rubens and 10 other Picea species revealed high sequence synteny and conservation among 11 Picea species and phylogenetic relationships consistent with their known classical interrelationships and published molecular phylogeny. The P. rubens chloroplast genome sequence showed the highest similarity with that of P. mariana and the lowest with that of P. sitchensis. We have annotated 107 genes including 69 protein-coding genes, 28 tRNAs, 4 rRNAs, few pseudogenes, identified 42 SSRs, and successfully designed primers for 26 SSRs. Mononucleotide A/T repeats were the most common followed by dinucleotide AT repeats. A similar pattern of microsatellite repeats occurrence was found in the chloroplast genomes of 11 Picea species.
Asunto(s)
Genoma del Cloroplasto , Picea , Picea/genética , Filogenia , Repeticiones de Microsatélite/genética , Sintenía , Anotación de Secuencia MolecularRESUMEN
The axial bonding ability of aluminum(III) porphyrin (AlPor) has been exploited to synthesize the vertically linked dyad 'aluminum(III) porphyrin-gold(III) porphyrin' (AlPor-Ph-AuPor(+)) and the two corresponding self-assembled triads 'tetrathiafulvalene-aluminum(III) porphyrin-gold(III) porphyrin' (TTF-pyâAlPor-Ph-AuPor(+) and TTF-Ph-pyâAlPor-Ph-AuPor(+)). The unique topology of these triads provides an excellent opportunity to investigate the sequential electron transfer in the perpendicular direction to the AlPor plane where the AlPor acts as a photosensitizer and primary electron donor while the AuPor and TTF serve as an electron acceptor and donor, respectively. The ground state properties of the dyad and triad suggest that there are no direct intramolecular interactions between the oppositely disposed AuPor and TTF units of the triad. However, the NMR and UV-visible absorption studies of the dyad reveal intermolecular interactions in non-coordinating solvents due to the coordination of counterion PF6(-) to the Al center of AlPor. Steady-state and femtosecond transient absorption studies of the dyad show that the lowest excited singlet state of AlPor ((1)AlPor*) is strongly quenched by ultrafast electron transfer to AuPor(+) with a time constant of 3.16 ps. The resulting charge separated state (AlPor(+)Ë-AuPorË) decays to ground state biexponentially with time constants of 27.26 and 2557 ps. Analogously, upon photo-excitation the triads also produce the same primary radical pair (AlPor(+)Ë-AuPorË). However, the formed radical pair is further involved in a rapid hole transfer from AlPor(+)Ë to TTF to form a stable final radical pair TTF(+)Ë-AlPor-AuPorË. The lifetime of the charge separated state exhibits an increase from 27.26 ps in AlPor-Ph-AuPor to 1393 ps in TTF-pyâAlPor-Ph-AuPor(+) and 1484 ps in TTF-Ph-pyâAlPor-Ph-AuPor(+). These results reveal successful charge stabilization in the self-assembled supramolecular reaction center mimics constructed via the axial linkage strategy.
Asunto(s)
Aluminio/química , Oro/química , Compuestos Heterocíclicos/química , Metaloporfirinas/química , Biomimética , Transporte de Electrón , Modelos Moleculares , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/químicaRESUMEN
Photosystem II (PSII) catalyzes the oxidation of water in the conversion of light energy into chemical energy in photosynthesis. Water delivery and oxygen removal from the oxygen evolving complex (OEC), buried deep within PSII, are critical requirements to facilitate the reaction and minimize reactive oxygen damage. It has often been assumed that water and oxygen travel through separate channels within PSII, as demonstrated in cytochrome c oxidase. This study describes all-atom molecular dynamics simulations of PSII designed to investigate channels by fully characterizing the distribution and permeation of both water and oxygen. Interestingly, most channels found in PSII were permeable to both oxygen and water, however individual channels exhibited different energetic barriers for the two solutes. Several routes for oxygen diffusion within PSII with low energy permeation barriers were found, ensuring its fast removal from the OEC. In contrast, all routes for water showed significant energy barriers, corresponding to a much slower permeation rate for water through PSII. Two major factors were responsible for this selectivity: (1) hydrogen bonds between water and channel amino acids, and (2) steric restraints. Our results reveal the presence of a shared network of channels in PSII optimized to both facilitate the quick removal of oxygen and effectively restrict the water supply to the OEC to help stabilize and protect it from small water soluble inhibitors.
Asunto(s)
Metabolismo Energético , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Agua/metabolismo , Simulación de Dinámica Molecular , Oxidación-Reducción , Permeabilidad , Complejo de Proteína del Fotosistema II/química , Conformación ProteicaRESUMEN
Two self-assembled supramolecular donor-acceptor triads consisting of Al(III) porphyrin (AlPor) with axially bound naphthalenediimide (NDI) as an acceptor and tetrathiafulvalene (TTF) as a secondary donor are reported. In the triads, the NDI and TTF units are attached to Al(III) on opposite faces of the porphyrin, through covalent and coordination bonds, respectively. Fluorescence studies show that the lowest excited singlet state of the porphyrin is quenched through electron transfer to NDI and hole transfer to TTF. In dichloromethane hole transfer to TTF dominates, whereas in benzonitrile (BN) electron transfer to NDI is the main quenching pathway. In the nematic phase of the liquid crystalline solvent 4-(n-pentyl)-4'-cyanobiphenyl (5CB), a spin-polarized transient EPR spectrum that is readily assigned to the weakly coupled radical pair TTF(.+)NDI(.-) is obtained. The initial polarization pattern indicates that the charge separation occurs through the singlet channel and that singlet-triplet mixing occurs in the primary radical pair. At later time the polarization pattern inverts as a result of depopulation of the states with singlet character by recombination to the ground state. The singlet lifetime of TTF(.+)NDI(.-) is estimated to be 200-300 ns, whereas the triplet lifetime in the approximately 350 mT magnetic field of the X-band EPR spectrometer is about 10 µs. In contrast, in dichloromethane and BN the lifetime of the charge separation is <10 ns.
Asunto(s)
Aluminio/química , Fulerenos/química , Compuestos Heterocíclicos/química , Imidas/química , Metaloporfirinas/química , Naftalenos/química , Porfirinas/química , Transporte de Electrón , Microscopía de Polarización , Estructura MolecularRESUMEN
Chloride binding in photosystem II (PSII) is essential for photosynthetic water oxidation. However, the functional roles of chloride and possible binding sites, during oxygen evolution, remain controversial. This paper examines the functions of chloride based on its binding site revealed in the X-ray crystal structure of PSII at 1.9 Å resolution. We find that chloride depletion induces formation of a salt bridge between D2-K317 and D1-D61 that could suppress the transfer of protons to the lumen.
Asunto(s)
Cloruros/química , Cloruros/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Oxígeno/metabolismo , Protones , Relación Estructura-ActividadRESUMEN
The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or "site." Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment-proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment-protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna-Mathews-Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.
Asunto(s)
Proteínas Bacterianas/fisiología , Bacterioclorofilas/fisiología , Complejos de Proteína Captadores de Luz/fisiología , Simulación de Dinámica Molecular , Transferencia de Energía , Electricidad EstáticaRESUMEN
The CaMn(4) cluster of the oxygen-evolving complex (OEC) of photosynthesis catalyzes the light-driven splitting of water into molecular oxygen, protons, and electrons. The OEC is buried within photosystem II (PSII), a multisubunit integral membrane protein complex, and water must find its way to the CaMn(4) cluster by moving through protein. Channels for water entrance, and proton and oxygen exit, have previously been proposed following the analysis of cavities found within X-ray structures of PSII. However, these analyses do not account for the dynamic motion of proteins and cannot track the movement of water within PSII. To study water dynamics in PSII, we performed molecular dynamics simulations and developed a novel approach for the visualization of water diffusion within protein based on a streamline tracing algorithm used in fluid dynamics and diffusion tensor imaging. We identified a system of branching pathways of water diffusion in PSII leading to the OEC that connect to a number of distinct entrance points on the lumenal surface. We observed transient changes in the connections between channels and entrance points that served to moderate both the flow of water near the OEC and the exchange of water inside and outside of the protein. Water flow was significantly altered in simulations lacking the OEC which were characterized by a simpler and wider channel with only two openings, consistent with the creation of an ion channel that allows entry of Mn(2+), Ca(2+), and Cl(-) as required for construction of the CaMn(4) cluster.
Asunto(s)
Simulación de Dinámica Molecular , Complejo de Proteína del Fotosistema II/química , Movimientos del Agua , Agua/química , Algoritmos , Cristalografía por Rayos X , Difusión , Oxígeno/química , Complejo de Proteína del Fotosistema II/análisis , Proteínas de Plantas/química , Agua/análisisRESUMEN
Squalene-hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.
RESUMEN
The oxygen-evolving complex (OEC) of photosystem II (PSII) is a unique Mn4O5Ca cluster that catalyzes water oxidation via four photoactivated electron transfer steps. As the protein influence on the redox and protonation chemistry of the OEC remains an open question, we present a classical valence model of the OEC that allows the redox state of each Mn and the protonation state of bridging µ-oxos and terminal waters to remain in equilibrium with the PSII protein throughout the redox cycle. We find that the last bridging oxygen loses its proton during the transition from S0 to S1. Two possible S2 states are found depending on the OEC geometry: S2 has Mn4(IV) with a proton lost from a terminal water (W1) trapped by the nearby D1-D61 if O5 is closer to Mn4, or Mn1(IV), with partial deprotonation of D1-H337 and D1-E329 if O5 is closer to Mn1. In S3, the OEC is Mn4(IV) with W2 deprotonated. The estimated OEC Em's range from +0.7 to +1.3 V, enabling oxidation by P680(+), the primary electron donor in PSII. In chloride-depleted PSII, the proton release increases during the S1 to S2 transition, leaving the OEC unable to properly advance through the water-splitting cycle.
Asunto(s)
Oxígeno/química , Complejo de Proteína del Fotosistema II/química , Protones , Teoría Cuántica , Calcio/química , Calcio/metabolismo , Transporte de Electrón , Electrones , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Photosystem II (PSII) of photosynthesis has the unique ability to photochemically oxidize water. Recently an engineered bacterioferritin photochemical 'reaction centre' (BFR-RC) using a zinc chlorin pigment (ZnCe6) in place of its native heme has been shown to photo-oxidize bound manganese ions through a tyrosine residue, thus mimicking two of the key reactions on the electron donor side of PSII. To understand the mechanism of tyrosine oxidation in BFR-RCs, and explore the possibility of water oxidation in such a system we have built an atomic-level model of the BFR-RC using ONIOM methodology. We studied the influence of axial ligands and carboxyl groups on the oxidation potential of ZnCe6 using DFT theory, and finally calculated the shift of the redox potential of ZnCe6 in the BFR-RC protein using the multi-conformational molecular mechanics-Poisson-Boltzmann approach. According to our calculations, the redox potential for the first oxidation of ZnCe6 in the BRF-RC protein is only 0.57 V, too low to oxidize tyrosine. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. We discuss the possibilities for modifying the BFR-RC to achieve this goal.
Asunto(s)
Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Ferritinas/metabolismo , Metaloporfirinas/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Porfirinas/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Clorofilidas , Grupo Citocromo b/química , Ferritinas/química , Concentración de Iones de Hidrógeno , Ligandos , Metaloporfirinas/química , Modelos Moleculares , Conformación Molecular , Método de Montecarlo , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , Porfirinas/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Soluciones , Tirosina/química , Tirosina/metabolismoRESUMEN
The influence of electrostatic interactions on the free energy of proton coupled electron transfer in biomimetic oxomanganese complexes inspired by the oxygen-evolving complex (OEC) of photosystem II (PSII) are investigated. The reported study introduces an enhanced multiconformer continuum electrostatics (MCCE) model, parametrized at the density functional theory (DFT) level with a classical valence model for the oxomanganese core. The calculated pKa's and oxidation midpoint potentials (E(m)'s) match experimental values for eight complexes, indicating that purely electrostatic contributions account for most of the observed couplings between deprotonation and oxidation state transitions. We focus on pKa's of terminal water ligands in [Mn(II/III)(H2O)6](2+/3+) (1), [Mn(III)(P)(H2O)2](3-) (2, P = 5,10,15,20-tetrakis(2,6-dichloro-3-sulfonatophenyl)porphyrinato), [Mn2(IV,IV)(µ-O)2(terpy)2(H2O)2](4+) (3, terpy = 2,2':6',2â³-terpyridine), and [Mn3(IV,IV,IV)(µ-O)4(phen)4(H2O)2](4+) (4, phen = 1,10-phenanthroline) and the pKa's of µ-oxo bridges and Mn E(m)'s in [Mn2(µ-O)2(bpy)4] (5, bpy = 2,2'-bipyridyl), [Mn2(µ-O)2(salpn)2] (6, salpn = N,N'-bis(salicylidene)-1,3-propanediamine), [Mn2(µ-O)2(3,5-di(Cl)-salpn)2] (7), and [Mn2(µ-O)2(3,5-di(NO2)-salpn)2] (8). The analysis of complexes 6-8 highlights the strong coupling between electron and proton transfers, with any Mn oxidation lowering the pKa of an oxo bridge by 10.5 ± 0.9 pH units. The model also accounts for changes in the E(m)'s by ligand substituents, such as found in complexes 6-8, due to the electron withdrawing Cl (7) and NO2 (8). The reported study provides the foundation for analysis of electrostatic effects in other oxomanganese complexes and metalloenzymes, where proton coupled electron transfer plays a fundamental role in redox-leveling mechanisms.
Asunto(s)
Materiales Biomiméticos/química , Manganeso/química , Oxígeno/química , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Electricidad Estática , Transporte de Electrón , Ligandos , Modelos Moleculares , Conformación Molecular , Compuestos Organometálicos/química , Oxidación-Reducción , Teoría Cuántica , Solventes/química , Termodinámica , Agua/químicaRESUMEN
Conversion of light energy in photosynthesis is extremely fast and efficient, and understanding the nature of this complex photophysical process is challenging. This review describes current progress in understanding molecular mechanisms of light harvesting and charge separation in photosystem II (PSII). Breakthroughs in X-ray crystallography have allowed the development and testing of more detailed kinetic models than have previously been possible. However, due to the complexity of the light conversion processes, satisfactory descriptions remain elusive. Recent advances point out the importance of variations in the photochemical properties of PSII in situ in different thylakoid membrane regions as well as the advantages of combining sophisticated time-resolved spectroscopic experiments with atomic level computational modeling which includes the effects of molecular dynamics.