Magnetic interactions in the catalyst used by nature to split water: a DFT plus U multiscale study on the Mn4CaO5 core in photosystem II

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The electron–proton bottleneck of photosynthetic oxygen evolution

Photosynthesis fuels life on Earth by storing solar energy in chemical form. Today’s oxygen-rich atmosphere has resulted from the splitting of water at the protein-bound manganese cluster of photosystem II during photosynthesis. Formation of molecular oxygen starts from a state with four accumulated electron holes, the S4 state—which was postulated half a century ago1 and remains largely uncharacterized. Here we resolve this key stage of photosynthetic O2 formation and its crucial mechanistic role. We tracked 230,000 excitation cycles of dark-adapted photosystems with microsecond infrared spectroscopy. Combining these results with computational chemistry reveals that a crucial proton vacancy is initally created through gated sidechain deprotonation. Subsequently, a reactive oxygen radical is formed in a single-electron, multi-proton transfer event. This is the slowest step in photosynthetic O2 formation, with a moderate energetic barrier and marked entropic slowdown. We identify the S4 state as the oxygen-radical state; its formation is followed by fast O–O bonding and O2 release. In conjunction with previous breakthroughs in experimental and computational investigations, a compelling atomistic picture of photosynthetic O2 formation emerges. Our results provide insights into a biological process that is likely to have occurred unchanged for the past three billion years, which we expect to support the knowledge-based design of artificial water-splitting systems

A molecular dynamics-guided mutagenesis identifies two aspartic acid residues involved in the pH-dependent activity of OG-OXIDASE 1

During the infection, plant cells secrete different OG-oxidase (OGOX) paralogs, defense flavoproteins that oxidize the oligogalacturonides (OGs), homogalacturonan fragments released from the plant cell wall that act as Damage Associated Molecular Patterns. OGOX-mediated oxidation inactivates their elicitor nature, but on the other hand makes OGs less hydrolysable by microbial endo-polygalacturonases (PGs). Among the different plant defense responses, apoplastic alkalinization can further reduce the degrading potential of PGs by boosting the oxidizing activity of OGOXs. Accordingly, the different OGOXs so far characterized showed an optimal activity at pH values greater than 8. Here, an approach of molecular dynamics (MD)-guided mutagenesis succeeded in identifying the amino acids responsible for the pH dependent activity of OGOX1 from Arabidopsis thaliana. MD simulations indicated that in alkaline conditions (pH 8.5), the residues Asp325 and Asp344 are engaged in the formation of two salt bridges with Arg327 and Lys415, respectively, at the rim of enzyme active site. According to MD analysis, the presence of such ionic bonds modulates the size and flexibility of the cavity used to accommodate the OGs, in turn affecting the activity of OGOX1. Based on functional properties of the site-directed mutants OGOX1.D325A and OGOX.D344A, we demonstrated that Asp325 and Asp344 are major determinants of the alkaline-dependent activity of OGOX1

Interaction Pattern of Arg 62 in the A-Pocket of Differentially Disease-Associated HLA-B27 Subtypes Suggests Distinct TCR Binding Modes

The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype

Nanodynamik und Charakterisierung der Protonierungszustände von Haupthistokompatibilitätskomplexen und Thioredoxinen

In this thesis the dynamics and the functions of two different classes of globular proteins, the major histocompatibility complex (MHC) proteins and the thioredoxin-like proteins, were investigated by molecular dynamics (MD) simulations. MHC proteins are molecules located on the surface of the cells where they bind and present antigen peptides to T-cell receptors (TCR). The recognition of a pathogen agent-derived peptide by a T-cell receptor induces an immune response against the antigen presenting cell that ultimately leads to killing of the infected cell. Here, the molecular mechanisms behind the immune recognition process were addressed by a comparative study of two structurally similar but differentially disease-associated MHC class I molecules, the ankylosing spondylitis (AS) associated HLA-B*2705 and the non-AS associated HLA-B*2709, in complex with various viral- and self-derived peptides. The strength of peptide-binding to these MHC molecules, that was shown for other complexes to be related to the immunogenicity of the antigen, was found to be modulated by a peptide- and subtype-dependent protonation pattern of MHC key residues within the peptide-binding groove. This result was corroborated by fluorescence depolarization experiments. MD simulations of MHC-peptide complexes (pMHC) revealed an increased mobility of the MHC α1-helix that is involved in the T-cell receptor recognition for the disease-associated HLA-B*2705 subtype when compared to HLA-B*2709. Based on these results, a decisive role of conformational flexibility of MHC class I molecules in the context of recognition by TCR is suggested. The increased flexibility of HLA-B*2705 may further counteract a proper selection of self-reactive T-cells during T-cell maturation. The in silico studies of pMHC complexes additionally allowed to identify Arg62 of HLA-B27 as a key residue for TCR binding to the antigen. Functional experiments of the MHC wildtype and of Arg62 mutants supported this finding. Subtype-dependent differences in the peptide recognition further suggested distinct TCR binding modes for the two investigated HLA-B27 subtypes. The second class of proteins investigated in this thesis, the thioredoxin-like proteins, are enzymes catalyzing the formation, reduction, and isomerization of disulfide bonds in substrate proteins. The molecular basis of their catalytic function was investigated in this work choosing the DsbL protein as a model system. DsbL is the most oxidizing thioredoxin-like protein known to date. Combining MD simulations and Poisson-Boltzmann-based pKa calculations, clear evidence for a proton shuffling between a cysteine and a lysine in the active site of the DsbL protein is reported. This proton shuffling is proposed to be essential for the catalytic mechanism of this enzyme and the correspondingly proposed mechanism is probably conferrable to other members of the thioredoxin family. The results further suggest a functional role of hydration entropy of thioredoxin active site groups. The developed methodology enables the study of proton transfer reactions in proteins on the nanosecond and nanometer scale by combining classical mechanics- and electrostatics-based methods.In dieser Arbeit wurde die Dynamik und die Funktion zweier unterschiedlicher Klassen von Proteinen, den Haupthistokompatibilitätskomplexen (MHC) sowie von thioredoxin-ähnlichen Proteinen, in Moleküldynamik(MD)-Simulationen untersucht. MHC Proteine sind auf der Oberfläche von Zellen lokalisiert, wo sie gebundene Antigenpeptide den T-Zell Rezeptoren (TCR) präsentieren. Die Erkennung eines Antigens durch einen T-Zell Rezeptor induziert eine Immunantwort gegen die antigenpräsentierende Zelle, die schließlich zur Tötung der Zelle führt. Hier wurden in einer vergleichenden Studie von zwei differentiell mit der Autoimmunerkrankung Spondylitis ankylosans assoziierten peptidbeladenen MHC Klasse I Molekülen die molekularen Mechanismen der Immunerkennung adressiert. Als Untersuchungsobjekt dienten der krankheitsassoziierte HLA-B*2705 Subtyp sowie der nicht-assoziierte HLA-B*2709 Subtyp im Komplex sowohl mit einem viralen Peptid als auch mit unterschiedlichen Selbstpeptiden. Frühere Studien zeigten, daß die Affinität der Peptidbindung an MHC Moleküle mit der immunogenen Aktivität des Antigens korreliert. Für HLA-B*2705 sowie HLA-B*2709 wurde eine Modulation der Bindungsaffinität aufgrund einer peptid- und subtypabhängigen Protonierung von Schlüsselresiduen innerhalb der Peptidbindungsstelle der MHC Moleküle gefunden. Die vorhergesagten unterschiedlichen Bindungsaffinitäten konnten durch Fluoreszenzdepolarisationsexperimente untermauert werden. MD Simulationen der MHC-Peptid (pMHC) Komplexe zeigten eine erhöhte Mobilität der der α1-Helix für HLA-B*2705 im Vergleich zu HLA-B*2709. Dieser Helix kommt eine tragende Rolle in der Erkennung durch T-Zell Rezeptoren zu. Basierend auf den Ergebnissen für die MHC Dynamik wird vorgeschlagen, daß die konformationelle Flexibilität der MHC Klasse I Moleküle entscheidend für die Erkennung durch TCR ist. Die erhöhte Flexibilität von HLA-B*2705 könnte zudem der Selektion von selbst-reaktiven T-Zellen während der T-Zell-Reifung entgegenwirken. Die in silico Untersuchung von pMHC Komplexen erlaubte ferner die Identifikation von Arg62 als wichtiger Residue für die T-Zell-Bindung zum Antigen. Funktionelle Studien des MHC Wildtyps sowie von Arg62-Mutanten unterstützten dieses Ergebnis. Gemessene subtypabhängige Unterschiede in der Peptiderkennung führten zudem zu der Vorhersage unterschiedlicher Bindungsmoden des TCR an die untersuchten HLA-B27 Subtypen. Als zweite Klasse von Proteinen wurden thioredoxinähnliche Proteine untersucht. Diese Enzyme katalysieren die Bildung, Reduktion und die Isomerisierung von Disulfidbrücken in Substratproteinen. In dieser Arbeit wurde die molekulare Basis ihrer katalytischen Funktion am Beispiel des DsbL Proteins analysiert. DsbL hat die größte Oxidationskraft unter den zur Zeit bekannten thioredoxinähnlichen Proteinen. Die Kombination von MD Simulationen mit Poisson-Boltzmann basierten pKa Berechnungen ergab klare Indizien für einen Protonenaustausch zwischen einem Cystein und einem Lysin im Aktiven Zentrum des DsbL. Dieser Protonenaustausch wird als essentiell für den katalytischen Mechanismus dieses Enzyms vorgeschlagen und ist vermutlich übertragbar auf andere Mitglieder der Thioredoxinfamilie. Die Ergebnisse legen ferner eine funktionelle Bedeutung der Hydratationsentropie für das Aktive Zentrum nahe. Die in diesem Projekt entwickelte Methodik mit einer Kombination von Methoden der klassischen Mechanik und Elektrostatik erlaubt die computergestützte Untersuchung von Protonentransferprozessen auf der Nanosekunden- und Nanometerskala

The S-2 State of the Oxygen-Evolving Complex of Photosystem II Explored by QM/MM Dynamics: Spin Surfaces and Metastable States Suggest a Reaction Path Towards the S-3 State

Split and polish: Quantum mechanics/molecular mechanics simulations reveal the role of spin surfaces, kinetics, and thermodynamics on the interconversion between two structural models of the {Mn4CaO5} cluster (see picture) in the S2 state of the water-splitting Kok's cycle in photosystem-II. The results account for the temperature, illumination, and procedure dependence of historical EPR experiments and suggest a detailed pathway for the S2 to S3 transition. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Pathway for Mn-cluster oxidation by tyrosine-Z in the S2 state of photosystem II

Water oxidation in photosynthetic organisms occurs through the five intermediate steps S0-S4 of the Kok cycle in the oxygen evolving complex of photosystem II (PSII). Along the catalytic cycle, four electrons are subsequently removed from the Mn4CaO5 core by the nearby tyrosine Tyr-Z, which is in turn oxidized by the chlorophyll special pair P680, the photo-induced primary donor in PSII. Recently, two Mn4CaO5 conformations, consistent with the S2 state (namely, S2 A and S2 B models) were suggested to exist, perhaps playing a different role within the S 2-to-S3 transition. Here we report multiscale ab initio density functional theory plus U simulations revealing that upon such oxidation the relative thermodynamic stability of the two previously proposed geometries is reversed, the S2 B state becoming the leading conformation. In this latter state a proton coupled electron transfer is spontaneously observed at ∼100 fs at room temperature dynamics. Upon oxidation, the Mn cluster, which is tightly electronically coupled along dynamics to the Tyr-Z tyrosyl group, releases a proton from the nearby W1 water molecule to the close Asp-61 on the femtosecond timescale, thus undergoing a conformational transition increasing the available space for the subsequent coordination of an additional water molecule. The results can help to rationalize previous spectroscopic experiments and confirm, for the first time to our knowledge, that the water-splitting reaction has to proceed through the S2 B conformation, providing the basis for a structural model of the S3 state

Vibrational fingerprints of the Mn4CaO5 cluster in Photosystem II by mixed quantum-classical molecular dynamics

A detailed knowledge of the structures of the catalytic steps along the Kok-Joliot cycle of Photosystem II may help to understand the strategies adopted by this unique enzyme to achieve water oxidation. Vibrational spectroscopy has probed in the last decades the intermediate states of the catalytic cycle, although the interpretation of the data turned out to be often problematic. In the present work we use QM/MM molecular dynamics on the S-2 state to obtain the vibrational density of states at room temperature. To help the interpretation of the computational and experimental data we propose a decomposition of the Mn4CaO5 moiety into five separate parts, composed by "diamond" motifs involving four atoms. The spectral signatures arising from this analysis can be easily interpreted to assign experimentally known bands to specific molecular motions. In particular, we focused in the low frequency region of the vibrational spectrum of the S-2 state. We can therefore identify the observed bands around 600-620 cm(-1) as characteristic for the stretching vibrations involving Mn1-O1-Mn2 or Mn3-O5 moieties. (C) 2016 Elsevier B.V. All rights reserved

A Spotlight on the Compatibility between XFEL and Ab Initio Structures of the Oxygen Evolving Complex in Photosystem II

The Mn4CaO5 cluster of photosystem II promotes a crucial step in the oxygenic photosynthesis, namely, the water-splitting reaction. The structure of such cluster in the S-1 state of the Kok-Joliot's cycle has been recently resolved by femtosecond X-ray free-electron laser (XFEL) measurements. However, the XFEL results are characterized by appreciable discrepancies with previous X-ray diffraction (XRD), as well as with S-1 models based on ab initio calculations. We provide here a unifying picture based on a combined set of DFT-based structures and molecular dynamics simulations of the S-0 and S-1 states. Our findings indicate that the XFEL results cannot be interpreted on the grounds of a single structure. A combination of two S-1 stable isomers together with a minority contribution of the S-0 state is necessary to reproduce XFEL results within 0.16 angstrom

On the comparison between differential vibrational spectroscopy spectra and theoretical data in the carboxyl region of photosystem II

Understanding the structural modification experienced by the Mn4CaO5 oxygen‐evolving complex of photosystem II along the Kok‐Joliot's cycle has been a challenge for both theory and experiments since many decades. In particular, differential infrared spectroscopy was extensively used to probe the surroundings of the reaction center, to catch spectral changes between different S‐states along the catalytic cycle. Because of the complexity of the signals, only a limited quantity of identified peaks have been assigned so far, also because of the difficulty of a direct comparison with theoretical calculations. In the present work, we critically reconsider the comparison between differential vibrational spectroscopy and theoretical calculations performed on the structural models of the photosystem II active site and an inorganic structural mimic. Several factors are currently limiting the reliability of a quantitative comparison, such as intrinsic errors associated to theoretical methods, and most of all, the uncertainty attributed to the lack of knowledge about the localization of the underlying structural changes. Critical points in this comparison are extensively discussed. Comparing several computational data of differential S2/S1 infrared spectroscopy, we have identified weak and strong points in their interpretation when compared with experimental spectra