RESUMEN
Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.
Asunto(s)
Oxígeno , Complejo de Proteína del Fotosistema II , Biocatálisis/efectos de la radiación , Calcio/metabolismo , Cristalografía , Transporte de Electrón/efectos de la radiación , Electrones , Manganeso/metabolismo , Oxidación-Reducción/efectos de la radiación , Oxígeno/química , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Complejo de Proteína del Fotosistema II/efectos de la radiación , Protones , Factores de Tiempo , Tirosina/metabolismo , Agua/química , Agua/metabolismoRESUMEN
Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ß-barrel across the time window of our measurements.
Asunto(s)
Colorantes , Proteínas , Cristalografía , Estructura Secundaria de ProteínaRESUMEN
Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.
Asunto(s)
Manganeso , Muramidasa , Muramidasa/química , Manganeso/química , Cristalografía por Rayos X , Porosidad , Complejos de Coordinación/química , Modelos Moleculares , Animales , Monóxido de Carbono/química , Factores de Tiempo , PollosRESUMEN
Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.
Asunto(s)
Proteínas Arqueales , Reparación del ADN , Desoxirribodipirimidina Fotoliasa , Methanosarcina , Dímeros de Pirimidina , Proteínas Arqueales/química , Catálisis , Cristalografía/métodos , Desoxirribodipirimidina Fotoliasa/química , ADN/química , ADN/efectos de la radiación , Methanosarcina/enzimología , Conformación Proteica , Dímeros de Pirimidina/química , Rayos UltravioletaRESUMEN
One of the primary objectives in chemistry research is to observe atomic motions during reactions in real time. Although X-ray free-electron lasers (XFELs) have facilitated the capture of reaction intermediates using time-resolved serial femtosecond crystallography (TR-SFX), only a few natural photoactive proteins have been investigated using this method, mostly due to the lack of suitable phototriggers. Here we report the genetic encoding of a xanthone amino acid (FXO), as an efficient phototrigger, into a rationally designed human liver fatty-acid binding protein mutant (termed XOM), which undergoes photo-induced C-H bond transformation with high selectivity and quantum efficiency. We solved the structures of XOM before and 10-300 ns after flash illumination, at 1.55-1.70 Å resolutions, and captured the elusive excited-state intermediates responsible for precise C-H bond activation. We expect that most redox enzymes can now be investigated by TR-SFX, using our method, to reveal reaction intermediates key for their efficiency and selectivity.
Asunto(s)
Electrones , Rayos Láser , Cristalografía por Rayos X , Humanos , Proteínas , Rayos XRESUMEN
Serial crystallography is a rapidly growing method that can yield structural insights from microcrystals that were previously considered to be too small to be useful in conventional X-ray crystallography. Here, conditions for growing microcrystals of the photosynthetic reaction centre of Blastochloris viridis within a lipidic cubic phase (LCP) crystallization matrix that employ a seeding protocol utilizing detergent-grown crystals with a different crystal packing are described. LCP microcrystals diffracted to 2.25â Å resolution when exposed to XFEL radiation, which is an improvement of 0.15â Å over previous microcrystal forms. Ubiquinone was incorporated into the LCP crystallization media and the resulting electron density within the mobile QB pocket is comparable to that of other cofactors within the structure. As such, LCP microcrystallization conditions will facilitate time-resolved diffraction studies of electron-transfer reactions to the mobile quinone, potentially allowing the observation of structural changes associated with the two electron-transfer reactions leading to complete reduction of the ubiquinone ligand.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética , Cristalización , Cristalografía por Rayos X , Lípidos/química , Proteínas de la Membrana/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , UbiquinonaRESUMEN
Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FADâ¢- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FADâ¢-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Protones , Arginina/metabolismo , Cristalografía , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Transporte de Electrón , Electrones , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Flavinas , Oxidación-ReducciónRESUMEN
Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the "quartet core") exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein-coupled receptors and will help the design of biased ligands for optimized therapeutics.
RESUMEN
Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alterations that occurred upon redox change at pH 5.7 in Asp51, the magnesium-containing cluster, haem groups and helix X, which provide important structural evidence for the H-pathway proton-pumping proposal, also occur at pH 7.3. These structural alterations were restricted to a local region of CcO; no domain movement was detected, nor were significant structural alterations detected in peripheral regions at either pH value. These observations indicate that the small and precise structural alterations that occur over the course of the reaction cycle are not affected by pH change, and that isolated CcO precisely performs proton pumping via the H-pathway over a wide pH range. Because the pH is not uniform across the molecular surface of CcO, the fact that the overall structure of CcO is not affected by pH changes ensures the high enzymatic efficiency of this protein in the mitochondria.
Asunto(s)
Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Animales , Bovinos , Concentración de Iones de Hidrógeno , Ligandos , Oxidación-Reducción , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Difracción de Rayos X/métodosRESUMEN
Cytochrome c oxidase (CcO) couples proton pumping to O2 reduction. Its enzymatic activity depends sensitively on pH over a wide range. However, owing to difficulty in crystallizing this protein, X-ray structure analyses of bovine CcO aimed at understanding its reaction mechanism have been conducted using crystals prepared at pH 5.7, which is significantly lower than that in the cell. Here, oxidized CcO at pH 7.3 was crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77â Å resolution. No structural differences between crystals obtained at the neutral pH and the acidic pH were detected within the molecules. On the other hand, some differences in intermolecular interactions were detected between the two types of crystal. The influence of pH on the molecular surface is likely to contribute to the pH dependency of the aerobic oxidation of ferrocytochrome c.