RESUMEN
James Barber, known to colleagues and friends as Jim, passed away in January 2020 after a long battle against cancer. During his long and distinguished career in photosynthesis research, Jim made many outstanding contributions with the pinnacle achieving his dream of determining the first detailed structure of the Mn cluster involved in photosynthetic water oxidation. Here, colleagues and friends remember Jim and reflect upon his scientific career and the impact he had on their lives and the scientific community.
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Fotosíntesis , Agua , Humanos , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , Agua/químicaRESUMEN
The electronic properties of carotenoid molecules underlie their multiple functions throughout biology, and tuning of these properties by their in vivo locus is of vital importance in a number of cases. This is exemplified by photosynthetic carotenoids, which perform both light-harvesting and photoprotective roles essential to the photosynthetic process. However, despite a large number of scientific studies performed in this field, the mechanism(s) used to modulate the electronic properties of carotenoids remain elusive. We have chosen two specific cases, the two ß-carotene molecules in photosystem II reaction centers and the two luteins in the major photosystem II light-harvesting complex, to investigate how such a tuning of their electronic structure may occur. Indeed, in each case, identical molecular species in the same protein are seen to exhibit different electronic properties (most notably, shifted absorption peaks). We assess which molecular parameters are responsible for this in vivo tuning process and attempt to assign it to specific molecular events imposed by their binding pockets.
Asunto(s)
Complejos de Proteína Captadores de Luz/química , Luteína/química , Complejo de Proteína del Fotosistema II/química , beta Caroteno/química , Luz , Fotosíntesis , Unión Proteica , Conformación Proteica , Solventes , Espectrofotometría Ultravioleta , Espectrometría Raman , Spinacia oleracea/enzimología , TemperaturaRESUMEN
In this review, I outline the indirect evidence for the formation of singlet oxygen ((1)O(2)) obtained from experiments with the isolated PSII reaction center complex. I also review the methods we used to measure singlet oxygen directly, including luminescence at 1,270 nm, both steady state and time resolved. Other methods we used were histidine-catalyzed molecular oxygen uptake (enabling (1)O(2) yield measurements), and dye bleaching and difference absorption spectroscopy to identify where quenchers of (1)O(2) can access this toxic species. We also demonstrated the protective behavior of carotenoids bound within Chl-protein complexes which bring about a substantial amount of (1)O(2) quenching within the reaction center complex. Finally, I describe how these techniques have been used and expanded in research on photoinhibition and on the role of (1)O(2) as a signaling molecule in instigating cellular responses to various stress factors. I also discuss the current views on the role of (1)O(2) as a signaling molecule and the distance it might be able to travel within cells.
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Fotosíntesis/efectos de la radiación , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Plantas/metabolismo , Oxígeno Singlete/metabolismo , beta Caroteno/metabolismo , Clorofila/metabolismo , Transporte de Electrón , Transferencia de Energía , Luz , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/efectos de la radiación , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/efectos de la radiación , Plantas/efectos de la radiación , Unión Proteica , Transducción de Señal , Oxígeno Singlete/análisis , Estrés FisiológicoRESUMEN
Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.
Asunto(s)
Benzoquinonas/metabolismo , Cianobacterias , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Vitamina K 1/metabolismo , Cianobacterias/química , Cianobacterias/enzimología , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Cinética , Oxidación-Reducción , Proteolisis , Análisis Espectral , Synechocystis/enzimología , Synechocystis/crecimiento & desarrollo , Synechocystis/metabolismo , Factores de Tiempo , Vitamina K 1/químicaRESUMEN
Research on mutant tobacco plants shows that a pigment called ß-carotene is not necessary for photosynthesis.
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Clorofila , beta Caroteno , Fotosíntesis , Nicotiana/metabolismo , beta Caroteno/metabolismoRESUMEN
Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740(+)-P740) and (F(A/B)(-)-F(A/B)) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740(+)A(1)(-)-P740A(1)) and ((3)P740-P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A(1)(-)), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450+/-10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000+/-4000 M(-1) cm(-1) at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1 : to approximately 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.
Asunto(s)
Clorofila/química , Cianobacterias/química , Complejo de Proteína del Fotosistema I/química , Análisis Espectral/métodos , Oxidación-Reducción , TemperaturaRESUMEN
We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P(+)Q(-), between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (Q(A)). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, Chl(D1). It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.
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Clorofila/metabolismo , Cianobacterias/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Clorofila A , Cianobacterias/química , FotoquímicaRESUMEN
Photosystems I and II convert solar energy into the chemical energy that powers life. Chlorophyll a photochemistry, using red light (680 to 700 nm), is near universal and is considered to define the energy "red limit" of oxygenic photosynthesis. We present biophysical studies on the photosystems from a cyanobacterium grown in far-red light (750 nm). The few long-wavelength chlorophylls present are well resolved from each other and from the majority pigment, chlorophyll a. Charge separation in photosystem I and II uses chlorophyll f at 745 nm and chlorophyll f (or d) at 727 nm, respectively. Each photosystem has a few even longer-wavelength chlorophylls f that collect light and pass excitation energy uphill to the photochemically active pigments. These photosystems function beyond the red limit using far-red pigments in only a few key positions.
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Clorofila/análogos & derivados , Cianobacterias/efectos de la radiación , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema I/efectos de la radiación , Complejo de Proteína del Fotosistema II/efectos de la radiación , Clorofila/química , Clorofila/efectos de la radiación , Clorofila A , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismo , Luz , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema II/químicaRESUMEN
The triplet states in plant photosystem II (PS II), 3P680, and from chlorophyll a, 3Chl a, in organic solution have been investigated using pulse ENDOR combined with repetitive laser excitation at cryogenic temperature with the aim to obtain their hyperfine (hf) structure. The large zero field splitting (ZFS) tensor of 3P680 enabled orientation selection via the electron spin resonance (EPR) field setting along the ZFS tensor axes. ENDOR spectra have been obtained for the first time also for the in-plane X- and Y-orientations of the ZFS tensor. This allowed a full determination of the hf-tensors of the three methine protons and one methyl group of 3P680. Based on the orientations of the axes of these hf-tensors, a unique orientation of the axes of the ZFS tensor of 3P680 in the Chl a molecular frame was obtained. These data serve as a structural basis for determining the orientation of 3P680 in the PS II protein complex by EPR on single crystals (see M. Kammel et al. in this issue). The data obtained represent the first complete set of the larger hf-tensors of the triplet state 3P680. They reflect the spin density distribution both in the highest occupied (HOMO) and lowest unoccupied (LUMO) orbitals. The data clearly confirm that 3P680 is a monomeric Chl a species at low temperature (T=10 K) used, as has been proposed earlier based on D- and E-values obtained from EPR and optically detected magnetic resonance (ODMR) studies. Comparison with the hf data for the cation and anion radicals of Chl a indicates a redistribution of spin densities in particular for the LUMO orbital of the triplet states. The electron spin distribution in the LUMO orbital is of special interest since it harbours the excited electron in the excited P680 singlet state, from which light-induced electron transfer proceeds. Observed shifts of hf couplings from individual nuclei of 3P680 as compared with 3Chl a in organic solution are of special interest, since they indicate specific protein interactions, e.g. hydrogen bonding, which might be used in future studies for assigning 3P680 to a particular chlorophyll molecule in PS II.
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Clorofila/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Clorofila A , Espectroscopía de Resonancia por Spin del Electrón , Complejos de Proteína Captadores de Luz , Complejo de Proteína del Fotosistema II , Spinacia oleracea/químicaRESUMEN
Fluorescence line-narrowing and resonance Raman properties of various chlorophylls have been measured in organic solvents. Resonance Raman spectroscopy is already a well-established method for the study of photochemical reactions in the various pigment-protein complexes involved in photosynthesis, while fluorescence line-narrowing is still an emerging technique for such systems. Interpretation of these vibrational spectra requires accurate comparative data on the pure isolated pigments. By comparing three different chlorophylls, a, b, and d, which have different substituents on the porphyrin ring, the various spectral lines associated with vinyl and formyl groups on the X and Y electronic axes could be distinguished. The difference between five- and six-coordination of the central Mg atom in FT-Raman spectra was determined by varying the organic solvent used. These chlorophylls are important in photosynthesis: all three in light-harvesting and energy transfer and, in the case of a and d, also in electron transfer. The assignment of spectral bands which we provide here, along with the description of their behavior with respect to the conformation and state of interaction of the pigment molecule, constitutes an essential step if these vibrational techniques are to be exploited to their full potential.
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Clorofila/química , Fluorescencia , Fotosíntesis , Espectrometría de Fluorescencia , Espectrometría RamanRESUMEN
We studied the charge recombination characteristics of Photosystem II (PSII) redox components in whole cells of the chlorophyll (Chl) d-dominated cyanobacterium, Acaryochloris marina, by flash-induced chlorophyll fluorescence and thermoluminescence measurements. Flash-induced chlorophyll fluorescence decay was retarded in the mus and ms time ranges and accelerated in the s time range in Acaryochloris marina relative to that in the Chl a-containing cyanobacterium, Synechocystis PCC 6803. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea, which blocks the Q(B) site, the relaxation of fluorescence decay arising from S(2)Q(A)(-) recombination was somewhat faster in Acaryochloris marina than in Synechocystis PCC 6803. Thermoluminescence intensity of the so called B band, arising from the recombination of the S(2)Q(B)(-) charge separated state, was enhanced significantly (2.5 fold) on the basis of equal amounts of PSII in Acaryochloris marina as compared with Synechocystis 6803. Our data show that the energetics of charge recombination is modified in Acaryochloris marina leading to a approximately 15 meV decrease of the free energy gap between the Q(A) and Q(B) acceptors. In addition, the total free energy gap between the ground state and the excited state of the reaction center chlorophyll is at least approximately 25-30 meV smaller in Acaryochloris marina, suggesting that the primary donor species cannot consist entirely of Chl a in Acaryochloris marina, and there is a contribution from Chl d as well.
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Clorofila/fisiología , Cianobacterias/fisiología , Fluorescencia , Complejo de Proteína del Fotosistema II/fisiología , Secuencia de Aminoácidos , Clorofila A , Datos de Secuencia Molecular , Fotosíntesis , TemperaturaRESUMEN
We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P(740)) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319-13323), the midpoint potential of the electron donor P(740) has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P(740), via the cytochrome b(6)f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b(6)f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems.
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Clorofila/química , Cianobacterias/metabolismo , Complejo de Citocromo b6f/química , Complejo de Proteína del Fotosistema I , Bioquímica/métodos , Electrones , Cinética , Luz , Modelos Biológicos , Oxidación-Reducción , Espectrometría de Fluorescencia/métodos , Termodinámica , Tilacoides/metabolismo , Factores de TiempoRESUMEN
The photosystem II reaction centre of all oxygenic organisms is subject to photodamage by high light i.e. photoinhibition. In this review I discuss the reasons for the inevitable and unpreventable oxidative damage that occurs in photosystem II and the way in which beta-carotene bound to the reaction centre significantly mitigates this damage. Recent X-ray structures of the photosystem II core complex (reaction centre plus the inner antenna complexes) have revealed the binding sites of some of the carotenoids known to be bound to the complex. In the light of these X-ray structures and their known biophysical properties it is thus possible to identify the two beta-carotenes present in the photosystem II reaction centre. The two carotenes are both bound to the D2 protein and this positioning is discussed in relation to their ability to act as quenchers of singlet oxygen, generated via the triplet state of the primary electron donor. It is proposed that their location on the D2 polypeptide means there is more oxidative damage to the D1 protein and that this underlies the fact that this latter protein is continuously re-synthesised, at a far greater rate than any other protein involved in photosynthesis. The relevance of a cycle of electrons around photosystem II, via cytochrome b(559), in order to re-reduce the beta-carotenes when they are oxidised and hence restore their ability to quench singlet oxygen, is also discussed.
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Luz , Complejo de Proteína del Fotosistema II/metabolismo , beta Caroteno/farmacología , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/efectos de la radiación , Modelos Moleculares , Oxidación-Reducción/efectos de los fármacos , Oxidación-Reducción/efectos de la radiación , Fotoquímica , Complejo de Proteína del Fotosistema II/química , Estructura Cuaternaria de ProteínaRESUMEN
Pigment-protein complexes enriched in photosystem II (PS II) have been isolated from the chlorophyll (Chl) d containing cyanobacterium, Acaryochloris marina. A small PS II-enriched particle, we call 'crude reaction centre', contained 20 Chl d, 0.5 Chl a and 1 redox active cytochrome b-559 per 2 pheophytin a, plus the D1 and D2 proteins. A larger PS II-enriched particle, we call 'core', additionally bound the antenna complexes, CP47 and CP43, and had a higher chlorophyll per pheophytin ratio. Pheophytin a could be photoreduced in the presence of a strong reductant, indicating that it is the primary electron acceptor in photosystem II of A. marina. A substoichiometric amount of Chl a (less than one chlorophyll a per 2 pheophytin a) strongly suggests that Chl a does not have an essential role in the photochemistry of PS II in this organism. We conclude that PS II, in A. marina, utilizes Chl d and not Chl a as primary electron donor and that the primary electron acceptor is one of two molecules of pheophytin a.
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Clorofila/metabolismo , Cianobacterias/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Clorofila/química , Cianobacterias/química , Citocromos b/química , Citocromos b/metabolismo , Oxidación-Reducción , Feofitinas/química , Feofitinas/metabolismo , Fotoquímica , Unión Proteica , Análisis Espectral , TemperaturaRESUMEN
During photosynthesis carotenoids normally serve as antenna pigments, transferring singlet excitation energy to chlorophyll, and preventing singlet oxygen production from chlorophyll triplet states, by rapid spin exchange and decay of the carotenoid triplet to the ground state. The presence of two beta-carotene molecules in the photosystem II reaction centre (RC) now seems well established, but they do not quench the triplet state of the primary electron-donor chlorophylls, which are known as P(680). The beta-carotenes cannot be close enough to P(680) for triplet quenching because that would also allow extremely fast electron transfer from beta-carotene to P(+)(680), preventing the oxidation of water. Their transfer of excitation energy to chlorophyll, though not very efficient, indicates close proximity to the chlorophylls ligated by histidine 118 towards the periphery of the two main RC polypeptides. The primary function of the beta-carotenes is probably the quenching of singlet oxygen produced after charge recombination to the triplet state of P(680). Only when electron donation from water is disturbed does beta-carotene become oxidized. One beta-carotene can mediate cyclic electron transfer via cytochrome b559. The other is probably destroyed upon oxidation, which might trigger a breakdown of the polypeptide that binds the cofactors that carry out charge separation.
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Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , beta Caroteno/metabolismo , Clorofila/química , Clorofila/metabolismo , Transporte de Electrón , Transferencia de Energía , Luz , Complejos de Proteína Captadores de Luz , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fotosíntesis , Complejo de Proteína del Fotosistema II , Conformación Proteica , beta Caroteno/químicaRESUMEN
We present a spectroscopic characterization of the two nonequivalent beta-carotene molecules in the photosystem II reaction center. Their electronic and vibrational properties exhibit significant differences, reflecting a somewhat different configuration for these two cofactors. Both carotenoid molecules are redox-active and can be oxidized by illumination of the reaction centers in the presence of an electron acceptor. The radical cation species show similar differences in their spectroscopic properties. The results are discussed in terms of the structure and unusual function of these carotenoids. In addition, the attribution of resonance Raman spectra of photosystem II preparations excited in the range 800-900 nm is discussed. Although contributions of chlorophyll cations cannot be formally ruled out, our results demonstrate that these spectra mainly arise from the cation radical species of the two carotenoids present in photosystem II reaction centers.