Far-Red Absorbing Rhodopsins, Insights From Heterodimeric Rhodopsin-Cyclases

The recently discovered Rhodopsin-cyclases from Chytridiomycota fungi show completely unexpected properties for microbial rhodopsins. These photoreceptors function exclusively as heterodimers, with the two subunits that have very different retinal chromophores. Among them is the bimodal photoswitchable Neorhodopsin (NeoR), which exhibits a near-infrared absorbing, highly fluorescent state. These are features that have never been described for any retinal photoreceptor. Here these properties are discussed in the context of color-tuning approaches of retinal chromophores, which have been extensively studied since the discovery of the first microbial rhodopsin, bacteriorhodopsin, in 1971 (Oesterhelt et al., Nature New Biology, 1971, 233 (39), 149–152). Further a brief review about the concept of heterodimerization is given, which is widely present in class III cyclases but is unknown for rhodopsins. NIR-sensitive retinal chromophores have greatly expanded our understanding of the spectral range of natural retinal photoreceptors and provide a novel perspective for the development of optogenetic tools.Peer Reviewe

Modeling of variant copies of subunit D1 in the structure of photosystem II from Thermosynechococcus elongatus

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.In the cyanobacterium Thermosynechococcus elongatus BP-1, living in hot springs, the light environment directly regulates expression of genes that encode key components of the photosynthetic multi-subunit protein-pigment complex photosystem II (PSII). Light is not only essential as an energy source to power photosynthesis, but leads to formation of aggressive radicals which induce severe damage of protein subunits and organic cofactors. Photosynthetic organisms develop several protection mechanisms against this photo-damage, such as the differential expression of genes coding for the reaction center subunit D1 in PSII. Testing the expression of the three different genes (psbAI, psbAII, psbAIII) coding for D1 in T. elongatus under culture conditions used for preparing the material used in crystallization of PSII showed that under these conditions only subunit PsbA1 is present. However, exposure to high-light intensity induced partial replacement of PsbA1 with PsbA3. Modeling of the variant amino acids of the three different D1 copies in the 3.0 Å resolution crystal structure of PSII revealed that most of them are in the direct vicinity to redox-active cofactors of the electron transfer chain. Possible structural and mechanistic consequences for electron transfer are discussed.DFG, SFB 498, Protein-Kofaktor-Wechselwirkungen in biologischen ProzessenEC/FP6/516510/EU/Linking molecular genetics and bio-mimetic chemistry - a multidisciplinary approach to achieve renewable hydrogen production/SOLAR-

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Aufreinigung, Kristallisation und Strukturanalyse des monomeren Photosystem II core Komplexes aus Thermosynechococcus elongatus

Der in der Thylakoidmembran vorkommene Photosystem II-core Komplex (PSIIcc) ist eines der Schlüsselenzyme der oxygenen Photosynthese. Er katalysiert die lichtinduzierte Oxidation von Wasser. Bislang waren die zur Aufklärung der Funktionsweise notwendigen und mittels Röngtenkristallographie gewonnenen Strukturinformationen auf die Verwendung der homodimeren Form des PSIIcc beschränkt. In dieser Arbeit wird erstmalig die Kristallisation und Strukturanalyse des monomeren PSIIcc aus dem thermophilen Cyanobaterium Thermosynechococcus elongatus beschrieben. Aufgrund eines verbesserten Präparations-Protokolls konnten hochreine, monomere PSIIcc erhalten werden, die eine hohe Sauerstoffentwicklungsaktivität aufweisen. Das PSIIcc Monomer weist alle 20 Protein-Untereinheiten auf, die in vollständigen, funktionellen Komplexen vorkommen. Mittels dynamischer Lichtstreuung konnte die Homogenität der PSIIcc Monomer Fraktion nachgewiesen werden. Erste Proteinkristalle konnten gezüchtet werden, die zur orthorhombischen Raumgruppe C2221 gehören und ein PSIIcc Monomer in der asymmetrischen Einheit aufweisen. Die Analyse der Röntgen-Diffraktionsdaten erlaubte es, ein erstes Strukturmodell des PSIIcc Monomers bei einer Auflösung von 3.6 Å zu erstellen. Das Modell beinhaltet 19 Protein-Untereinheiten, 35 Chlorophyll a- und zwei Pheophytin a-Moleküle, zwei Häm-Gruppen, das Nicht-Häm Eisen, elf Karotinoide, das primäre Plastochinon QA, 22 Lipide und den Mn4Ca Cluster des Wasser-oxidierenden Komplexes (WOC). Erwartungsgemäß ist die Struktur des PSIIcc Monomers im Wesentlichen identisch mit der des dimeren Komplexes. Dennoch bilden die Strukturdaten eine neue Grundlage, um die Rolle der Lipide und Protein-Untereinheiten im Zusammenhang mit der Oligomerisierung des PSIIcc und dem Austausch des D1-Proteins zu diskutieren. Erstmalig konnte eine deutlich andere Kristall-Packung des PSIIcc erhalten werden, die folgende Vorzüge aufweist: (I) geringere Anisotropie der Röntgen-Diffraktion (Abhängig-keit der Auflösung von der Kristallorientierung), (II) die Reduzierung der Orientierungen des PSIIcc innerhalb der Einheitszelle und (III) die senkrechte Orientierung der Membran-Normalen zu einer Kristallachse. Punkt (I) ist ein Vorteil für die Verbesserung der Röntgen-Diffraktionsdaten, während (II) und (III) die Eignung von orientierungsanhängigen, spektro-skopischen Methoden erhöht. Letzteres eröffnet neue Möglichkeiten die Struktur und Funktionsweise des WOC aufzuklären. Erste Messungen bestätigen die Anwendbarkeit von polarisierter Röntgenabsorptions-Spektroskopie auf diese neue Kristallform. Die Akzeptorseite des PSIIcc ist das Wirkungsziel vieler kommerziell genutzter Herbizide, jedoch fehlen bislang direkte Informationen über die Interaktion von Herbiziden mit PSIIcc. In dieser Arbeit konnte, mittels Kristallen des dimeren PSIIcc, ein erstes Struktur-modell von an PSIIcc gebundenen Terbutryn bei einer Auflösung von 3.4 Å erhalten werden. Dieses Modell belegt, dass Triazin-Derivate in gleicher Art an den PSIIcc binden, wie an das Reaktionszentrum aus Purpurbakterien. Bindungsstudien zeigen eine Abhängigkeit der Terbutryn-Bindung vom funktionellen Zustand der Akzeptorseite des PSIIcc. Des Weiteren weisen erste Strukturdaten eines Herbizids der Harnstoff-Klasse (Chlorbromuron) bei 3.7 Å Auflösung, auf zwei unterschiedliche Bindungskonformationen dieses Herbizids hin. An der Donorseite konnte eine Halogenid-Bindungsstelle im Abstand von 6.4 Å zum Mn4Ca Cluster lokalisiert werden. Dazu wurden Kristalle des dimeren PSIIcc verwendet, bei denen das natürlich vorkommende Chlorid durch Bromid funktionell ersetzt wurde. Diese Ergebnisse bestätigen, dass Bromid (und somit auch Chlorid) kein direkter Ligand des Mn4Ca Clusters ist, trotz der funktionellen Rolle bei der Wasser-Oxidation. Röngtenspektroskopische Untersuchungen weisen auf eine Stöchiometrie von einem Bromid pro Mn4Ca Cluster hin. Aufgrund der Bromid-Position am Eingang zweier vermeintlicher Protonenkanäle ist eine Funktion im Zusammenhang mit dem Proton-Transfer naheliegend.The membrane-embedded Photosystem II core complex (PSIIcc) is one of the key enzymes in oxygenic photosynthesis and uses light energy to oxidize water. To understand the functional mode of this enzyme knowledge of its molecular structure is necessary. So far, the information about the spatial structure of PSIIcc obtained from X-ray crystallography has been restricted to the homodimeric form of the PSIIcc from thermophilic cyanobacteria. This work describes the first crystallization and structural analysis of the monomeric PSIIcc isolated from the thermophilic cyanobacteria Thermosynechococcus elongatus. Based on an improved preparation protocol, it was possible to obtain a highly pure fraction of PSIIcc monomer with high oxygen evolution activity. The PSIIcc monomer consists of all 20 protein subunits known to be present in the complete functional complex. Dynamic light scattering on this PSIIcc monomer fraction revealed the homogeneity needed for protein crystallization. First protein crystals could be obtained, which belong to the space group C2221 and contain one monomer per asymmetric unit. The X-ray diffraction data derived from these crystals yielded a first structural model of the PSIIcc monomer at a resolution of 3.6 Å. This model includes the assignment of 19 protein subunits, 35 chlorophylls, two pheophytins, two heme groups, the non-heme iron, eleven carotenoids, the primary plastoquinone QA, 22 lipids and the Mn4Ca cluster of the water-oxidizing complex (WOC). As expected, the overall structure of the PSIIcc monomer is essentially identical to its dimeric counterpart. Nevertheless, this structure provides a new basis for the discussion of the role of lipids and protein subunits in the oligomerization of PSIIcc and the assembly/disassembly during the repair cycle of photodamaged subunit D1. For the first time, a significantly different packing of PSIIcc could be obtained that features the following advantages: (I) lower anisotropy of the X-ray diffraction (dependence of resolution on crystal orientation), (II) a reduced number of orientations of PSIIcc within the unit cell and (III) the orientation of the membrane normal perpendicular to one of the crystallographic axes. Point (I) is crucial for further improving the quality of X-ray diffraction data, whereas (II) and (III) make this crystal form more suitable for orientation-dependent spectroscopy. This opens novel possibilities to elucidate the structure and function of the WOC. Preliminary measurements of polarized X-ray absorption spectroscopy confirmed the applicability of this method on the new crystal form. The acceptor side of PSIIcc is the target for many commercially used herbicides, but direct information about herbicide interactions with PSIIcc is lacking. In this work, the first structural model of a triazine-type herbicide bound to PSIIcc at a resolution of 3.4 Å could be obtained by crystallizing PSIIcc dimer in the presence of terbutryn. The structure reveals a similar binding mode of triazines to the QB site of PSIIcc to that observed for the reaction center of purple bacteria. The analysis of terbutryn-binding to PSIIcc using isothermal titration calorimetry and fluorescence spectroscopy suggests that herbicide binding is affected by the functional state of the acceptor side. Furthermore, initial structural data on the binding of a urea-type herbicide (chlorbromuron) to PSIIcc dimer at 3.7 Å resolution suggests two different binding conformations of this herbicide at the QB site. On the donor side, one halide binding site could be located 6.4 Å away from the Mn4Ca cluster by using crystals of PSIIcc dimer, in which the naturally occurring chloride was functionally replaced by bromide. This result confirmed the hypothesis that bromide (and concomitantly chloride) is not a direct ligand of the Mn4Ca cluster, despite its role in the proper functioning of water oxidation. The stoichiometry of one bromide per reaction center could be further supported by X-ray spectroscopy. Due to the location of the halide at the entrance of two putative proton channels, a functional role in proton transfer during the catalytic cycle is likely

The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions

Cyanobacterial photosystem II at 2.9-A resolution and the role of quinones, lipids, channels and chloride

Photosystem II (PSII) is a large homodimeric protein-cofactor complex located in the photosynthetic thylakoid membrane that acts as light-driven water:plastoquinone oxidoreductase. The crystal structure of PSII from Thermosynechococcus elongatus at 2.9-A resolution allowed the unambiguous assignment of all 20 protein subunits and complete modeling of all 35 chlorophyll a molecules and 12 carotenoid molecules, 25 integral lipids and 1 chloride ion per monomer. The presence of a third plastoquinone Q(C) and a second plastoquinone-transfer channel, which were not observed before, suggests mechanisms for plastoquinol-plastoquinone exchange, and we calculated other possible water or dioxygen and proton channels. Putative oxygen positions obtained from a Xenon derivative indicate a role for lipids in oxygen diffusion to the cytoplasmic side of PSII. The chloride position suggests a role in proton-transfer reactions because it is bound through a putative water molecule to the Mn(4)Ca cluster at a distance of 6.5 A and is close to two possible proton channels

Probing the accessibility of the Mn(4)Ca cluster in photosystem II:Channels calculation, noble gas derivatization, and cocrystallization with DMSO

Using the 2.9 A resolution structure of the membrane-intrinsic protein-cofactor complex photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus, we calculated and characterized nine possible substrate/product channels leading to/away from the Mn(4)Ca cluster, where water is oxidized to dioxygen, protons, and electrons. Five narrow channels could function in proton transport, assuming that no large structural changes are associated with water oxidation. Four wider channels could serve to supply water to or remove oxygen from the Mn(4)Ca cluster. One of them might be regulated by conformational changes of Lys134 in subunit PsbU. Data analyses of Kr derivatized crystals and complexes with dimethyl sulfoxide (DMSO) confirm the accessibility of the proposed dioxygen channels to other molecules. Results from Xe derivatization suggest that the lipid clusters within PSII could serve as a drain for oxygen because of their predominant hydrophobic character and mediate dioxygen release from the lumen

The femtosecond-to-second photochemistry of red-shifted fast-closing anion channelrhodopsin PsACR1

Anion channelrhodopsins (ACRs) are of great interest due to their ability to inhibit electrical signaling in optogenetic experiments. The photochemistry of ACRs is currently poorly understood and an improved understanding would be beneficial for rational design of ACRs with modified properties. Activation/deactivation of ACRs involves a series of photoreactions ranging from femtoseconds to seconds, thus real-time observation is essential to comprehend the full complexity of the photochemical processes. Here we investigate the photocycle of an ACR from Proteomonas sulcata (PsACR1), which is valuable for optogenetic applications due to the red-shifted absorption and action spectra compared to the prototype ACRs from Guillardia theta: GtACR1 and GtACR2, and the fast channel closing properties. From femto-to-submillisecond transient absorption spectroscopy, flash photolysis, and point mutations of acidic residues near the retinal Schiff base (RSB), E64, and D230, we found that the photoisomerization occurs in similar to 500 fs independent of the protonation state of E64. Notably, E64 is involved in the rearrangement of the hydrogen-bond network near the RSB after photoisomerization. Furthermore, we suggest that E64 works as a primary proton acceptor during deprotonation of the RSB as has been proposed for GtACR1. Our findings allow for a deeper understanding of the photochemistry on the activation/deactivation of ACRs