RESUMEN
Photosystem II starts the photosynthetic electron transport chain that converts solar energy into chemical energy and thus sustains life on Earth. It catalyzes two chemical reactions: water oxidation to molecular oxygen and plastoquinone reduction. Coupling of electron and proton transfer is crucial for efficiency; however, the molecular basis of these processes remains speculative owing to uncertain water binding sites and the lack of experimentally determined hydrogen positions. We thus collected high-resolution cryo-electron microscopy data of fully hydrated photosystem II from the thermophilic cyanobacterium Thermosynechococcus vestitus to a final resolution of 1.71 angstroms. The structure reveals several previously undetected partially occupied water binding sites and more than half of the hydrogen and proton positions. This clarifies the pathways of substrate water binding and plastoquinone B protonation.
Asunto(s)
Hidrógeno , Complejo de Proteína del Fotosistema II , Protones , Thermosynechococcus , Agua , Sitios de Unión , Microscopía por Crioelectrón , Transporte de Electrón , Hidrógeno/química , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/ultraestructura , Complejo de Proteína del Fotosistema II/metabolismo , Plastoquinona/metabolismo , Plastoquinona/química , Thermosynechococcus/enzimología , Agua/químicaRESUMEN
The influence of poly(ethylene glycol) (PEG) polymers H-(O-CH2-CH2)p-OH with different average molecular sizes p on the micelle formation of n-alkyl-ß-D-maltoside detergents with the number of carbon atoms in the alkyl chain ranging from 10 to 12 is investigated with the aim to learn more about the detergent behavior under conditions suitable for the crystallization of the photosynthetic pigment-protein complex photosystem II. PEG is shown to increase the critical micelle concentration (CMC) of all three detergents in the crystallization buffer in a way that the free energy of micelle formation increases linearly with the concentration of oxyethylene units (O-CH2-CH2) irrespective of the actual molecular weight of the polymer. The CMC shift is modeled by assuming for simplicity that it is dominated by the interaction between PEG and detergent monomers and is interpreted in terms of an increase of the transfer free energy of a methylene group of the alkyl chain by 0.2 kJ mol-1 per 1 mol L-1 increase of the concentration of oxyethylene units at 298 K. Implications of this effect for the solubilization and crystallization of protein-detergent complexes as well as detergent extraction from crystals are discussed.
RESUMEN
The water oxidation reaction in photosystem II (PS II) produces most of the molecular oxygen in the atmosphere, which sustains life on Earth, and in this process releases four electrons and four protons that drive the downstream process of CO2 fixation in the photosynthetic apparatus. The catalytic center of PS II is an oxygen-bridged Mn4Ca complex (Mn4CaO5) which is progressively oxidized upon the absorption of light by the chlorophyll of the PS II reaction center, and the accumulation of four oxidative equivalents in the catalytic center results in the oxidation of two waters to dioxygen in the last step. The recent emergence of X-ray free-electron lasers (XFELs) with intense femtosecond X-ray pulses has opened up opportunities to visualize this reaction in PS II as it proceeds through the catalytic cycle. In this review, we summarize our recent studies of the catalytic reaction in PS II by following the structural changes along the reaction pathway via room-temperature X-ray crystallography using XFELs. The evolution of the electron density changes at the Mn complex reveals notable structural changes, including the insertion of OX from a new water molecule, which disappears on completion of the reaction, implicating it in the O-O bond formation reaction. We were also able to follow the structural dynamics of the protein coordinating with the catalytic complex and of channels within the protein that are important for substrate and product transport, revealing well orchestrated conformational changes in response to the electronic changes at the Mn4Ca cluster.
RESUMEN
One of the reasons for the high efficiency and selectivity of biological catalysts arise from their ability to control the pathways of substrates and products using protein channels, and by modulating the transport in the channels using the interaction with the protein residues and the water/hydrogen-bonding network. This process is clearly demonstrated in Photosystem II (PS II), where its light-driven water oxidation reaction catalyzed by the Mn4CaO5 cluster occurs deep inside the protein complex and thus requires the transport of two water molecules to and four protons from the metal center to the bulk water. Based on the recent advances in structural studies of PS II from X-ray crystallography and cryo-electron microscopy, in this review we compare the channels that have been proposed to facilitate this mass transport in cyanobacteria, red and green algae, diatoms, and higher plants. The three major channels (O1, O4, and Cl1 channels) are present in all species investigated; however, some differences exist in the reported structures that arise from the different composition and arrangement of membrane extrinsic subunits between the species. Among the three channels, the Cl1 channel, including the proton gate, is the most conserved among all photosynthetic species. We also found at least one branch for the O1 channel in all organisms, extending all the way from Ca/O1 via the 'water wheel' to the lumen. However, the extending path after the water wheel varies between most species. The O4 channel is, like the Cl1 channel, highly conserved among all species while having different orientations at the end of the path near the bulk. The comparison suggests that the previously proposed functionality of the channels in T. vestitus (Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Hussein et al., Nat Commun 12:6531, 2021) is conserved through the species, i.e. the O1-like channel is used for substrate water intake, and the tighter Cl1 and O4 channels for proton release. The comparison does not eliminate the potential role of O4 channel as a water intake channel. However, the highly ordered hydrogen-bonded water wire connected to the Mn4CaO5 cluster via the O4 may strongly suggest that it functions in proton release, especially during the S0 â S1 transition (Saito et al., Nat Commun 6:8488, 2015; Kern et al., Nature 563:421-425, 2018; Ibrahim et al., Proc Natl Acad Sci USA 117:12624-12635, 2020; Sakashita et al., Phys Chem Chem Phys 22:15831-15841, 2020; Hussein et al., Nat Commun 12:6531, 2021).
Asunto(s)
Complejo de Proteína del Fotosistema II , Protones , Complejo de Proteína del Fotosistema II/metabolismo , Agua/metabolismo , Microscopía por Crioelectrón , Oxidación-ReducciónRESUMEN
In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn4CaO5 cluster first stores four oxidizing equivalents, the S0 to S4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O-O bond formation chemistry1-3. Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok's photosynthetic water oxidation cycle, the S3â[S4]âS0 transition where O2 is formed and Kok's water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn4CaO5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom Ox, which was introduced as a bridging ligand between Ca and Mn1 during the S2âS3 transition4-6, disappears or relocates in parallel with Yz reduction starting at approximately 700 µs after the third flash. The onset of O2 evolution, as indicated by the shortening of the Mn1-Mn4 distance, occurs at around 1,200 µs, signifying the presence of a reduced intermediate, possibly a bound peroxide.
Asunto(s)
Oxígeno , Fotosíntesis , Complejo de Proteína del Fotosistema II , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Protones , Agua/química , Agua/metabolismo , Manganeso/química , Manganeso/metabolismo , Calcio/química , Calcio/metabolismo , Peróxidos/metabolismoRESUMEN
The critical micelle concentration (CMC) of nonionic detergents is defined as the breaking point in the monomer concentration as a function of the total detergent concentration, identified by setting the third derivate of this function to zero. Combined with a mass action model for micelle formation, this definition yields analytic formulae for the concentration ratio of monomers to total detergent at the CMC and the relationship between the CMC and the free energy of micellization g mic. The theoretical breaking point is shown to coincide with the breaking point of the experimental titration curve, if the fluorescence enhancement of 8-anilino-1-naphthalene-sulfonic acid (ANS) or a similar probe dye is used to monitor micelle formation. Application to a series of n-alkyl-ß-d-maltosides with the number of carbon atoms in the alkyl chain ranging from 8 to 12 demonstrates the good performance of a molecular thermodynamic model, in which the free energy of micellization is given by g mic = σΦ + g pack + g st. In this model, σ is a fit parameter with the dimension of surface tension, Φ represents the change in area of hydrophobic molecular surfaces in contact with the aqueous phase, and g pack and g st are contributions, respectively, from alkyl chain packing in the micelle interior and steric repulsion of detergent head groups. The analysis of experimental data from different sources shows that varying experimental conditions such as co-solutes in the aqueous phase can be accounted for by adapting only σ, if the co-solutes do not bind to the detergent to an appreciable extent. The model is considered a good compromise between theory and practicability to be applied in the context of in vitro investigations of membrane proteins.
RESUMEN
One restriction for biohybrid photovoltaics is the limited conversion of green light by most natural photoactive components. The present study aims to fill the green gap of photosystem I (PSI) with covalently linked fluorophores, ATTO 590 and ATTO 532. Photobiocathodes are prepared by combining a 20 µm thick 3D indium tin oxide (ITO) structure with these constructs to enhance the photocurrent density compared to setups based on native PSI. To this end, two electron transfer mechanisms, with and without a mediator, are studied to evaluate differences in the behavior of the constructs. Wavelength-dependent measurements confirm the influence of the additional fluorophores on the photocurrent. The performance is significantly increased for all modifications compared to native PSI when cytochrome c is present as a redox-mediator. The photocurrent almost doubles from -32.5 to up to -60.9 µA cm-2. For mediator-less photobiocathodes, interestingly, drastic differences appear between the constructs made with various dyes. While the turnover frequency (TOF) is doubled to 10 e-/PSI/s for PSI-ATTO590 on the 3D ITO compared to the reference specimen, the photocurrents are slightly smaller since the PSI-ATTO590 coverage is low. In contrast, the PSI-ATTO532 construct performs exceptionally well. The TOF increases to 31 e-/PSI/s, and a photocurrent of -47.0 µA cm-2 is obtained. This current is a factor of 6 better than the reference made with native PSI in direct electron transfer mode and sets a new record for mediator-free photobioelectrodes combining 3D electrode structures and light-converting biocomponents.
RESUMEN
Ever since the discovery that Mn was required for oxygen evolution in plants by Pirson in 1937 and the period-four oscillation in flash-induced oxygen evolution by Joliot and Kok in the 1970s, understanding of this process has advanced enormously using state-of-the-art methods. The most recent in this series of innovative techniques was the introduction of X-ray free-electron lasers (XFELs) a decade ago, which led to another quantum leap in the understanding in this field, by enabling operando X-ray structural and X-ray spectroscopy studies at room temperature. This review summarizes the current understanding of the structure of Photosystem II (PS II) and its catalytic centre, the Mn4 CaO5 complex, in the intermediate Si (i = 0-4)-states of the Kok cycle, obtained using XFELs.
Asunto(s)
Fotosíntesis , Agua , Agua/química , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/metabolismo , Rayos Láser , Oxígeno/químicaRESUMEN
Synthetic materials can be combined with biological components in many ways. One example that provides scientists with multiple challenges is a photobioelectrode that converts sunlight into electrons in a biohybrid approach. In the present study several key parameters are evaluated concerning their influence on the direct electron transfer from a 3D indium tin oxide (ITO) electrode material to photosystem I (PSI) as a light-harvesting biomolecule. In contrast to previous investigations, no mediating molecule is added to shuttle the electrons to the luminal side of PSI. Thus, this setup is less complex than foregoing ones. The solution composition drastically influences the interaction of PSI with the ITO surface. Here, the application of higher buffer concentrations and the addition of salts are advantageous, whereas the nature of the buffer ions plays a minor role. The artificial electrode material's thickness is adjustable since a spin-coating procedure is used for preparation. With a 30 µm thick structure and immobilized PSI cathodic photocurrents up to 10.1 µA cm-2 are obtained at 100 mW cm-2 illumination intensity and an applied potential of -0.1V vs. Ag/AgCl. Over a period of three days the photobioelectrodes are illuminated for a total of 90 min and stored between the measurements at ambient temperature. The stability of the setup is noteworthy as still about 90% of the photocurrent is retained. The photocathode described here offers many positive features, including a high onset potential for the photocurrent starting sligthly above the redox potentail of P700, and applicability in a wide pH range from pH 5 to 8.
Asunto(s)
Técnicas Biosensibles , Complejo de Proteína del Fotosistema I , Transporte de Electrón , Electrones , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismoRESUMEN
In the last ten years, x-ray free-electron lasers (XFELs) have been successfully employed to characterize metalloproteins at room temperature using various techniques including x-ray diffraction, scattering, and spectroscopy. The approach has been to outrun the radiation damage by using femtosecond (fs) x-ray pulses. An example of an important and damage sensitive active metal center is the Mn4CaO5 cluster in photosystem II (PS II), the catalytic site of photosynthetic water oxidation. The combination of serial femtosecond x-ray crystallography and Kß x-ray emission spectroscopy (XES) has proven to be a powerful multimodal approach for simultaneously probing the overall protein structure and the electronic state of the Mn4CaO5 cluster throughout the catalytic (Kok) cycle. As the observed spectral changes in the Mn4CaO5 cluster are very subtle, it is critical to consider the potential effects of the intense XFEL pulses on the Kß XES signal. We report here a systematic study of the effects of XFEL peak power, beam focus, and dose on the Mn Kß1,3 XES spectra in PS II over a wide range of pulse parameters collected over seven different experimental runs using both microcrystal and solution PS II samples. Our findings show that for beam intensities ranging from â¼5 × 1015 to 5 × 1017 W/cm2 at a pulse length of â¼35 fs, the spectral effects are small compared to those observed between S-states in the Kok cycle. Our results provide a benchmark for other XFEL-based XES studies on metalloproteins, confirming the viability of this approach.
RESUMEN
Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons.
Asunto(s)
Complejo de Proteína del Fotosistema II/metabolismo , Enlace de Hidrógeno , Complejo de Proteína del Fotosistema II/genética , Protones , AguaRESUMEN
Photosystem I (PS I) has a symmetric structure with two highly similar branches of pigments at the center that are involved in electron transfer, but shows very different efficiency along the two branches. We have determined the structure of cyanobacterial PS I at room temperature (RT) using femtosecond X-ray pulses from an X-ray free electron laser (XFEL) that shows a clear expansion of the entire protein complex in the direction of the membrane plane, when compared to previous cryogenic structures. This trend was observed by complementary datasets taken at multiple XFEL beamlines. In the RT structure of PS I, we also observe conformational differences between the two branches in the reaction center around the secondary electron acceptors A1A and A1B. The π-stacked Phe residues are rotated with a more parallel orientation in the A-branch and an almost perpendicular confirmation in the B-branch, and the symmetry breaking PsaB-Trp673 is tilted and further away from A1A. These changes increase the asymmetry between the branches and may provide insights into the preferential directionality of electron transfer.
Asunto(s)
Complejo de Proteína del Fotosistema I/química , Vitamina K 1/química , Cristalografía por Rayos X , Fotosíntesis , Estructura Terciaria de Proteína , Temperatura , ThermosynechococcusRESUMEN
We describe the development of biohybrid electrodes constructed via combination of electrospun (e-spun) 3D indium tin oxide (ITO) with the trimeric supercomplex photosystem I and the small electrochemically active protein cytochrome c (cyt c). The developed 3D surface of ITO has been created by electrospinning of a mixture of polyelthylene oxide (PEO) and ITO nanoparticles onto ITO glass slides followed by a subsequent elimination of PEO by sintering the composite. Whereas the photosystem I alone shows only small photocurrents at these 3D electrodes, the co-immobilization of cyt c to the e-spun 3D ITO results in well-defined photoelectrochemical signals. The scaling of thickness of the 3D ITO layers by controlling the time (10 min and 60 min) of electrospinning results in enhancement of the photocurrent. Several performance parameters of the electrode have been analyzed for different illumination intensities.
Asunto(s)
Técnicas Biosensibles/métodos , Electrodos , Complejo de Proteína del Fotosistema I/química , Thermosynechococcus/metabolismoRESUMEN
Photobioelectrodes represent one of the examples where artificial materials are combined with biological entities to undertake semi-artificial photosynthesis. Here, an approach is described that uses reduced graphene oxide (rGO) as an electrode material. This classical 2D material is used to construct a three-dimensional structure by a template-based approach combined with a simple spin-coating process during preparation. Inspired by this novel material and photosystem I (PSI), a biophotovoltaic electrode is being designed and investigated. Both direct electron transfer to PSI and mediated electron transfer via cytochrome c from horse heart as redox protein can be confirmed. Electrode preparation and protein immobilization have been optimized. The performance can be upscaled by adjusting the thickness of the 3D electrode using different numbers of spin-coating steps during preparation. Thus, photocurrents up to â¼14 µA/cm2 are measured for 12 spin-coated layers of rGO corresponding to a turnover frequency of 30 e- PSI-1 s-1 and external quantum efficiency (EQE) of 0.07% at a thickness of about 15 µm. Operational stability has been analyzed for several days. Particularly, the performance at low illumination intensities is very promising (1.39 µA/cm2 at 0.1 mW/cm2 and -0.15 V vs Ag/AgCl; EQE 6.8%).
Asunto(s)
Proteínas Bacterianas/química , Electrodos , Grafito/química , Complejo de Proteína del Fotosistema I/química , Animales , Proteínas Bacterianas/efectos de la radiación , Citocromos c/química , Electricidad , Caballos , Proteínas Inmovilizadas/química , Luz , Complejo de Proteína del Fotosistema I/efectos de la radiación , Thermosynechococcus/enzimologíaRESUMEN
Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent-protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-ß-d-maltoside (ßDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D2O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2-10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 Å. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by X-ray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D2O provide information about the size and shape of the whole detergent-dPSIIcc. The maximum position of P(r) function increases to 68 Å, i.e., it is about 6 Å larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the ßDM-dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 Å. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II-detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the ßDM-dPSIIcc.
Asunto(s)
Detergentes , Complejo de Proteína del Fotosistema II , Cristalización , Difracción de Neutrones , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 â S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 â S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a "water wheel"-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of â¼350 µs) during the S2 â S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
Asunto(s)
Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Hidrógeno/metabolismo , Magnesio/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Fotones , Complejo de Proteína del Fotosistema II/química , Quinonas/metabolismo , Agua/metabolismoRESUMEN
Photosystem II (PSII) is a membrane-spanning, multi-subunit pigment-protein complex responsible for the oxidation of water and the reduction of plastoquinone in oxygenic photosynthesis. In the present review, the recent explosive increase in available structural information about the PSII core complex based on X-ray crystallography and cryo-electron microscopy is described at a level of detail that is suitable for a future structure-based analysis of light-harvesting processes. This description includes a proposal for a consistent numbering scheme of protein-bound pigment cofactors across species. The structural survey is complemented by an overview of the state of affairs in structure-based modeling of excitation energy transfer in the PSII core complex with emphasis on electrostatic computations, optical properties of the reaction center, the assignment of long-wavelength chlorophylls, and energy trapping mechanisms.
Asunto(s)
Luz , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/efectos de la radiación , Cristalografía por Rayos X , Modelos Moleculares , Complejo de Proteína del Fotosistema II/metabolismo , Conformación ProteicaRESUMEN
Photosystemâ II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn4 CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pKa values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pKa values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.