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Iron-sulfur clusters are inorganic cofactors found in many proteins involved in fundamental biological processes including DNA processing. The prokaryotic DNA repair enzyme PhrB, a member of the protein family of cryptochromes and photolyases, carries a four-iron-four-sulfur cluster [4Fe4S] in addition to the catalytic cofactor flavin adenine dinucleotide (FAD) and a second pigment 6,7-dimethyl-8-ribityllumazine (DMRL). The light-induced redox reactions of this multi-cofactor protein complex were recently shown as two interdependent photoreductions of FAD and DMRL mediated by the [4Fe4S] cluster functioning as an electron cache to hold a fine balance of electrons. Here, we apply the more traditional temperature-scan cryo-trapping technique in protein crystallography and the newly developed technology ofin situserial Laue diffraction at room temperature. These diffraction methods in dynamic crystallography enable us to capture strong signals of electron density changes in the [4Fe4S] cluster that depict quantized electronic movements. The mixed valence layers of the [4Fe4S] cluster due to spin coupling and their dynamic responses to light illumination are observed directly in our difference maps between its redox states. These direct observations of the quantum effects in a protein bound iron-sulfur cluster have thus opened a window into the mechanistic understanding of metal clusters in biological systems.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. In plants, phytochromes play a central role in growth and differentiation during the entire life cycle. Phytochromes of plants and other groups of archaeplastida have a common evolutionary origin in prokaryotes, but the exact prokaryotic origin is as yet uncertain. Two possibilities are presently discussed: either, archaeplastidal phytochromes arose from the last eukaryotic common ancestor (LECA) or they arose from the cyanobacterial endosymbiont that gave rise to plastids. We first constructed standard phylogenetic trees based on N-terminal protein sequences of the chromophore module. As usual, variation of algorithms and parameters led to different trees. A relationship between cyanobacteria and archaeplastida was observed in 7 out of 36 trees. The lack of consistency between results obtained from variation of parameters of tree constructions reflects the uncertainty of archaeplastidal origin. To gain more information about a possible cyanobacterial and archaeplastidal relationship, we performed phylogenetic studies based on the amino acids that line the chromophore pockets. These amino acids are highly conserved and could provide more accurate information about long evolutionary time scales, but the reduction of traits could also lead to insignificant results. From 30 selected chromophore-binding amino acids, 6 were invariant. The subsequent studies were thus based on the information dependent on 24 or fewer amino acid positions. Again, multiple trees were constructed to get information about the robustness of relationships. The very low number of information-containing traits resulted in low bootstrap values and many indistinguishable leaves. However, the major groups fungi, bacteria, cyanobacteria, and plants remained united. Without exception, cyanobacteria and archaeplastida were always closely linked. In this respect, the results were more robust than those of the classic approach, based on long contiguous sequences. We therefore consider cyanobacteria as the most likely origin of archaeplastidal phytochromes.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome‐dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme‐derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C‐terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000‐fold higher than the affinity of the holoprotein, suggesting a “kiss‐and‐go” mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi., Characterization of fungal heme oxygenases suggests a role for the mitochondrial outer membrane in chromophore production for light‐sensing phytochromes.

True to its name, light-harvesting complex II (LHC II) harvests light energy for photosystem II (PS II). However, LHC II can stray, harvesting light energy for photosystem I (PS I) instead. Cryo-electron microscopy (cryo-EM) now shows how this mobile antenna becomes so attached to its new partner.

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.

Background and aims This study examined differences in personality, psychological distress, and stress coping in inflammatory bowel disease (IBD) depending on type of disease and disease activity. We compared patients suffering from Crohn’s disease (CD) and ulcerative colitis (UC) with controls. While the literature is replete with distinctive features of the pathogenesis of IBD, the specific differences in psychological impairments are not well studied. Methods In this German national multicenter study, participants were recruited from 32 centers. Two hundred ninety-seven questionnaires were included, delivering vast information on disease status and psychological well-being based on validated instruments with a total of 285 variables. Results CD patients were more affected by psychological impairments than patients suffering from UC or controls. Importantly, patients with active CD scored higher in neuroticism (p Conclusions We identified specific aspects of psychological impairment in IBD depending on disease and disease activity. Our results underscore the need for psychological support and treatment particularly in active CD.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg2+ . We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA-binding site are essential for the effect of Mg2+ . Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+ . In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron-sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron-sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment.

Phormidium lacuna, a filamentous cyanobacterium without heterocysts, can be transformed by natural transformation. These filaments are motile on agar and other surfaces and display rapid lateral movements in liquid culture. Furthermore, they exhibit phototactic response under vertical illumination in Petri dishes. We generated mutants in which a KanR resistance cassette was integrated in the phytochrome gene cphA and in various genes of the type IV pilin apparatus. pilM, pilN, pilQ and pilT mutants were defective in all three responses, indicating that type IV pili are involved in all three kinds of motility. Rapid movements of wild type in liquid culture requires an extracellular matrix with type IV pili as central player. pilB mutants are only partially blocked in their responses. pilB is the proposed ATPase for expelling of the filament. In the mutant, this function could be overtaken by an alternative protein, like pilT, which regularly mediates retraction of pili. The cphA mutant revealed a significantly reduced phototactic response towards red light. We assume that together with other photoreceptors, CphA regulates the phototactic response by down regulation of surface attachment.

The soil bacterium and plant pathogen Agrobacterium fabrum C58 has two phytochrome photoreceptors, Agp1 and Agp2. We found that plant infection and tumor induction by A. fabrum is down-regulated by light and that phytochrome knockout mutants of A. fabrum have diminished infection rates. The regulation pattern of infection matches with that of bacterial conjugation reported earlier, suggesting similar regulatory mechanisms. In the regulation of conjugation and plant infection, phytochromes are active in darkness. This is a major difference to plant phytochromes, which are typically active after irradiation. We also found that propagation and motility were affected in agp1− and agp2− knockout mutants, although propagation was not always affected by light. The regulatory patterns can partially but not completely be explained by modulated histidine kinase activities of Agp1 and Agp2. In a mass spectrometry-based proteomic study, 24 proteins were different between light and dark grown A. fabrum, whereas 382 proteins differed between wild type and phytochrome knockout mutants, pointing again to light independent roles of Agp1 and Agp2.

The soil bacterium Agrobacterium fabrum C58 infects plants by a unique DNA transfer mechanism. A. fabrum has two phytochrome photoreceptors, Agp1 and Agp2. We found that DNA transfer into plants by A. fabrum is down regulated by light and that phytochrome knockout mutants have diminished DNA transfer rates. The regulation pattern matches with that of bacterial conjugation reported earlier. Growth, swimming and interbacterial competition were also affected in phytochrome knockout mutants, although these effects were often not affected by light. We can thus distinguish between light-regulated and light-independent phytochrome responses. In microarray studies, transcription of only 4 genes was affected by light, indicating that most light responses are regulated post-transcriptionally. In a mass spectrometery-based proteomic study, 24 proteins were different between light and dark grown bacteria, whereas 382 proteins differed between wild type and phytochrome knockout mutants, pointing again to light-dependent and light-independent roles of Agp1 and Agp2.

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6-4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7-dimethyl-8-ribityllumazine (DMRL) and a cubane-type Fe-S cluster. Tyr424 of PhrB is part of the DNA-binding site and could provide an electron link to the Fe-S cluster. The PhrBY424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrBI51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrBY424F revealed a water network extending to His366, which are part of the lesion-binding site. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrBI51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.

The adaptation of microorganisms to different temperatures is an advantage in habitats with steadily changing conditions and raises the question about temperature sensing. Here we show that in the filamentous fungus Aspergillus nidulans, the hybrid histidine kinase TcsB and phytochrome are involved in temperature-induced gene transcription. Temperature-activated phytochrome fed the signal into the HOG MAP kinase pathway. There is evidence that the photoreceptor phytochrome fulfills a temperature sensory role in plants and bacteria. The effects in plants are based on dark reversion from the active form of phytochrome, Pfr, to the inactive form, Pr. Elevated temperature leads to higher dark reversion rates, and hence, temperature sensing depends on light. In A. nidulans and in Alternaria alternata, the temperature response was light-independent. In order to understand the primary temperature response of phytochrome, we performed spectral analyses of recombinant FphA from both fungi. Spectral properties after heat stress resembled the spectrum of free biliverdin, suggesting conformational changes and a softening of the binding pocket of phytochrome, possibly mimicking photoactivation. We propose a novel function for fungal phytochrome as temperature sensor.

This study examined differences in personality, psychological distress, and stress coping in inflammatory bowel disease (IBD) depending on type of disease and disease activity. We compared patients suffering from Crohn's disease (CD) and ulcerative colitis (UC) with controls. While the literature is replete with distinctive features of the pathogenesis of IBD, the specific differences in psychological impairments are not well studied. In this German national multicenter study, participants were recruited from 32 centers. Two hundred ninety-seven questionnaires were included, delivering vast information on disease status and psychological well-being based on validated instruments with a total of 285 variables. CD patients were more affected by psychological impairments than patients suffering from UC or controls. Importantly, patients with active CD scored higher in neuroticism (p 0.01), psychological distress (p 0.001) and maladaptive stress coping (escape, p = 0.03; rumination, p 0.03), but less need for social support (p = 0.001) than controls. In contrast, patients suffering from active UC showed psychological distress (p 0.04) and maladaptive coping (avoidance, p 0.03; escape, p = 0.01). Patients in remission seemed to be less affected. In particular, patients with UC in remission were not inflicted by psychological impairments. The group of CD patients in remission however, showed insecurity (p 0.01) and paranoid ideation (p = 0.04). We identified specific aspects of psychological impairment in IBD depending on disease and disease activity. Our results underscore the need for psychological support and treatment particularly in active CD. Diese Studie untersuchte Unterschiede in der Persönlichkeit, psychische Belastung und Stressbewältigung bei chronisch-entzündlichen Darmerkrankungen (CED) in Abhängigkeit von der Art der Erkrankung und der Krankheitsaktivität. Wir verglichen Patienten mit Morbus Crohn (CD) und Colitis ulcerosa (UC) mit Kontrollgruppen. Während die Literatur zu charakteristischen Merkmalen der Pathogenese von IBD zahlreich ist, sind die spezifischen Unterschiede in den psychischen Beeinträchtigungen nicht ausreichend untersucht. In dieser deutschen nationalen Multicenterstudie wurden Teilnehmer aus 32 Zentren rekrutiert. Es wurden 297 Fragebögen mit umfassenden Informationen über den Krankheitsstatus und das psychische Wohlbefinden auf der Grundlage validierter psychosozialer Instrumente mit insgesamt 285 Items evaluiert. CD-Patienten waren stärker von psychischen Beeinträchtigungen betroffen als Patienten mit UC oder Kontrollen. Wichtig ist, dass Patienten mit aktiver CD in den Bereichen Neurotizismus (p 0,01), psychische Belastung (p 0,001) und maladaptive Stressbewältigung (p 0,03) höhere Werte, aber weniger Bedarf für soziale Unterstützung (p = 0,001) als Kontrollen aufwiesen. Im Gegensatz dazu zeigten Patienten, die an aktiver UC litten, psychische Belastung (p 0,04) und maladaptive Coping (Vermeidung, p 0,03; Flucht, p = 0,01). Patienten in Remission schienen weniger betroffen zu sein. Die Gruppe der CD-Patienten in Remission zeigte jedoch Unsicherheit (p 0,01). Wir identifizierten spezifische Aspekte der psychischen Beeinträchtigung bei IBD abhängig von Krankheit und Krankheitsaktivität. Unsere Ergebnisse untermauern den Bedarf an psychologischer Unterstützung und Behandlung, insbesondere bei aktiver CD.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. In theAgrobacterium(6-4) photolyase PhrB, the repair of DNA with UV-induced (6-4) pyrimidin dimers is stimulated by Mg2+. We show that Mg2+is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH-chromophore to the DNA lesion. Two highly conserved Asp residues close to the DNA binding site are essential for the Mg2+effect. Simulations showed that two Mg2+bind to the region around these residues. DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+. Here, the structurally overlapping region contains no Asp but positively charged Lys or Arg. During evolution, charge stabilization and DNA binding by Mg2+was therefore replaced by a positive amino acid. We argue that this transition has evolved in a freshwater environment. Prokaryotic (6-4) photolyases usually contain an FeS cluster. DNA repair of a cyanobacterial member of this group which is missing the FeS cluster was also found to be stimulated by Mg2+.

Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors., Phytochromes are photoreceptors that are present in plants, bacteria and fungi. Here the authors present crystal structures of the phytochrome Agp2 from Agrobacterium fabrum in the parent Pfr state as well as a functional Meta-F intermediate and discuss mechanistic implications for photoconversion.

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.

Two-component signal transduction systems (TCSs) consist of sensor histidine kinases and response regulators. TCSs mediate adaptation to environmental changes in bacteria, plants, fungi and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all known cyanobacteria and as chloroplast sensor kinase in eukaryotic algae and plants. Sodium ions have been shown to inhibit the autophosphorylation activity of Hik2 that precedes phosphoryl transfer to response regulators, but the mechanism of inhibition has not been determined. We report on the mechanism of Hik2 activation and inactivation probed by chemical cross-linking and size exclusion chromatography together with direct visualisation of the kinase using negative-stain transmission electron microscopy of single particles. We show that the functional form of Hik2 is a higher-order oligomer such as a hexamer or octamer. Increased NaCl concentration converts the active hexamer into an inactive tetramer. The action of NaCl appears to be confined to the Hik2 kinase domain.

FAD photoreduction mechanism by different aromatic residues in a phylogenetically ancient photolyase., Cryptochromes and photolyases form a flavoprotein family in which the FAD chromophore undergoes light induced changes of its redox state. During this process, termed photoreduction, electrons flow from the surface via conserved amino acid residues to FAD. The bacterial (6–4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Mutagenesis studies have identified Trp342 and Trp390 as essential for charge transfer. Trp342 is located at the periphery of PhrB while Trp390 connects Trp342 and Tyr391. The role of Tyr391, which lies between Trp390 and FAD, is however unclear as its replacement by phenylalanine did not block photoreduction. Experiments reported here, which replace Tyr391 by Ala, show that photoreduction is blocked, underlining the relevance of Tyr/Phe at position 391 and indicating that charge transfer occurs via the triad 391-390-342. This raises the question, why PhrB positions a tyrosine at this location, having a less favourable ionisation potential than tryptophan, which occurs at this position in many proteins of the photolyase/cryptochrome family. Tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings. Since replacement of Tyr391 by Trp resulted in loss of FAD and DMRL chromophores, electron transfer cannot be studied experimentally in this mutant, but calculations on a mutant model suggest that Trp might participate in the electron transfer cascade. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad. Our results suggest that evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent.

The (6-4) photolyases use blue light to reverse UV-induced (6-4) photoproducts in DNA. This (6-4) photorepair was thought to be restricted to eukaryotes. Here we report a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens , and propose that (6-4) photolyases are broadly distributed in prokaryotes. The crystal structure of photolyase related protein B (PhrB) at 1.45 Å resolution suggests a DNA binding mode different from that of the eukaryotic counterparts. A His-His-X-X-Arg motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. The PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore and a [4Fe-4S] cluster bound to the catalytic domain. A significant part of the Fe-S fold strikingly resembles that of the large subunit of eukaryotic and archaeal primases, suggesting that the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. Our study presents a unique prokaryotic (6-4) photolyase and proposes that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family.

Agrobacterium fabrum is a widely used model bacterium for gene transfer from pro- to eukaryote, for genetics and metabolism. The phytochrome system of Agrobacterium, encompassing the two phytochromes Agp1 and Agp2, has provided deep insight into phytochrome action in a bacterial organism. This review summarizes recent results on phytochrome evolution, phytochrome regulation of conjugation and plant infection and biochemical studies including the crystal structure of Agp1-PCM, the photosensory core module of Agp1.

Die (6-4) Photolyasen der bakteriellen Cryptochrom/Photolyase (BCP) -Gruppe gehoren zu einer Familie von Flavoproteinen, die als Reparaturenzyme fur UV-B-induzierte DNA-Lasionen (Photolyasen) oder als Blaulicht-Photorezeptoren (Cryptochrome) fungieren. Die (6-4) BCP-Proteine kommen ausschlieslich in Prokaryoten vor. In dieser Arbeit wurden 3 Mitglieder der (6-4) BCP-Gruppe, PhrB von Agrobacterium fabrum, CryB von Rhodobacter sphaeroides und Proma-PL von Prochlorococcus marinus mit einer eukaryotischen (6-4) Photolyase von Ostreococcus tauri OtCPF1 und einem Mitglied der Klasse III CPD Photolyasen PhrA von Agrobacterium fabrum verglichen. Es zeigte sich, dass die DNA-Reparatur-Effizienz von (6-4) BCP-Proteinen durch Mg2+ oder andere zweiwertige Kationen stimuliert wird, wahrend bei OtCPF1 und PhrA keine Wirkung von zweiwertigen Kationen beobachtet wurde. Der Einfluss zweiwertiger Kationen auf die Photoreduktion bei verschiedenen Photolyasen wurde ebenfalls untersucht. Die Photoreduktion von PhrB wurde durch Mg2+ negativ beeinflusst, wahrend bei PhrA Mg2+ einen stimulierenden Effekt hatte. Es stellte sich klar heraus, dass die Abhangigkeit von Mg2+ bei der DNA-Reparatur der (6-4) BCP und nicht bei der Photoreduktion zu suchen ist. Die veranlasste uns zu der Annahme, dass Mg2+ die DNA-Bindung und -Reparatur in (6-4) BCP-Proteinen beeinflusst. Zusammen mit den Strukturdaten und der Sequenzanalyse fanden wir eine vorgeschlagene Mg2+ Bindungsposition neben der DNA-Lasion und modifizierten die beiden betreffenden Aminosauren durch ortsgerichtete Mutagenese. Der Effekt von Mg2+ ging fur beide Mutanten verloren, wahrend die Basisreparaturaktivitat ohne Mg2+ von der Mutation nicht beeinflusst wurde. Vermutlich fordert Mg2+ die Bindung der (6-4) Lasion und erhoht die Elektronen-Affinitat des Substrats. Auserdem wird die Barriere fur Elektronentransfer verringert, wodurch und die Reaktion reibungsloser verlauft. Ich untersuchte auch die Reparatureffizienz fur verschiedene Langen einzelstrangiger und doppelstrangiger DNA. Je langer die DNA war, desto schneller war die Reparatur. Bei einer Lange der DNA von 12 nt oder mehr anderte sich allerdings mit Zunahme der Lange die Geschwindigkeit der Reparatur nicht mehr. In dieser Arbeit werden auch die Mutanten PhrB-Y424F und PhrB-I51W vorgestellt. Tyr424 von PhrB ist Teil der DNA-Bindungsstelle und konnte eine Elektronenverbindung zum Fe-S-Cluster bilden. Die PhrB-Y424F-Mutante zeigte eine starke Verringerung der Bindung von Lasions-DNA und DNA-Reparatur. Die Mutante PhrB-I51W ist durch den Verlust des DMRL-Chromophors, reduzierte Photoreduktion und reduzierte DNA-Reparaturkapazitat gekennzeichnet. Die Kristallstrukturen zeigen eine hohe Ubereinstimmung mit der Wildtyp-Struktur, somit beeinflussen Mutationen nur lokale Proteinumgebungen. Die Photoreduktion von PhrB sich unterscheidet von dem typischen Muster, da die dem FAD benachbarte Aminosaure der Elektronenkaskade ein Tyrosin (Tyr391) ist, wahrend Photolyasen und Cryptochrome anderer Gruppen ein Tryptophan als direkten Elektronendonor von FAD besitzen. In einer Mutante, in der Tyr391 durch Tryptophan ersetzt wurde, ging der Cofaktor-FAD verloren und die PhrB-Struktur war instabil. Trp342 und Trp390 sind fur den Ladungstransfer essentiell sind. Trp342 befindet sich an der Peripherie von PhrB. Die Rolle von Tyr391, die zwischen Trp390 und FAD liegt, war jedoch unklar, da der Ersatz durch Phenylalanin die Photoreduktion nicht blockierte. Bei der Substitution von Tyr391 durch Ala wurde die Photoreduktion blockiert, was darauf hindeutet, dass Tyr391 ein Teil der Elektronentransferkette ist und zeigt, dass der Ladungstransfer uber die Triade 342-391-390 erfolgt. Diese Ergebnisse deuten auf ein tunneling (Elektronentransfer ohne Ladungsanderung) zwischen Trp390 und FAD hin.

Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 A resolution and of a surface-engineered mutant of this PCM at 1.85 A resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion.

Structural studies of active states of the visual pigment rhodopsin, a G protein-coupled receptor, have previously been limited to apoprotein or opsin forms that do not contain the agonist all-trans-retinal. Two groups now report structures that reveal more details of the transformations involved in rhodopsin activation. Choe et al. solve the X-ray crystal structure of the metarhodopsin II intermediate of the photoreceptor rhodopsin, and Standfuss et al. determine the structure of a constitutively active mutant of rhodopsin bound to a peptide derived from the C-terminus of the G protein transducin. Here the X-ray crystal structure of the metarhodopsin II intermediate of the photoreceptor rhodopsin is solved. Comparison of this structure with previously published structures enabled the proposal of how retinal translocation and rotation induce the conformational changes observed in this structure. G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ)1. The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein2. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit3,4,5. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal’s high concentration of opsin in an active conformation (Ops*)6,7 may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 A and 2.85 A crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

The hydrogenase maturation factor HypF1 is a truncated but functional version of the HypF protein. HypF is known to be involved in the supply of the CN(-) ligands of the active site of [NiFe]-hydrogenases, utilizing carbamoyl phosphate as a substrate. The first crystallization and preliminary X-ray studies of HypF1 from Ralstonia eutropha H16 are reported here. Crystals of HypF1 (394 amino acids, 40.7 kDa) were obtained by the sitting-drop vapour-diffusion technique using sodium formate as a precipitant. The crystals belonged to space group I222, with unit-cell parameters a = 79.7, b = 91.6, c = 107.2 A. Complete X-ray diffraction data sets were collected at 100 K from native crystals and from a platinum derivative to a maximum resolution of 1.65 A.

A coleopteran triosephosphate isomerase (TIM) from Tenebrio molitor (yellow mealworm beetle) was recombinantly expressed in Escherichia coli and characterized with respect to thermal stability, kinetic parameters and oligomeric state. The enzyme was successfully crystallized and the structure determined by X-ray analysis to 2.0 A resolution. This is the first example of an invertebrate TIM. We compare structural features with known structures of TIMs from microorganisms, plants and vertebrates, and discuss the utility of the Tenebrio TIM sequence, together with several newly sequenced insect TIMs, for molecular phylogenetic analysis.

Introduction The pathogenesis of Crohn's disease is unclear, but an abnormal immune response seems to play an important role. This study investigated whether serum tryptase could be shown in Crohn's disease as a marker of disease. Materials and methods Mast cell tryptase was detected in sera of 48 patients with active Crohn's disease, 31 patients with inactive Crohn's disease, 17 patients with mastocytosis, and 50 controls. Tryptase detection was carried out by UniCap System. Tryptase levels are given as U/I x m 2 body surface area to overcome variations of body weight, height, etc. between malnourished and normal persons. Results Serum tryptase levels (U/I x m 2 body surface area) of controls were 2.4 ± 1.0, of patients with Crohn's disease 2.5 ± 2.0. In contrast, serum tryptase values were significantly increased in untreated patients with mastocytosis (21.19 ± 18.55). Discussion Mast cell tryptase is not elevated in sera of Crohn's disease. It might thus be speculated that this highly mast cell associated mediator might only contribute to local symptoms of Crohn's disease such as diarrhea, abdominal pain, etc., but not to its systemic inflammatory effects (Th 1 cytokine pattern). Tryptase may be well used for the screening of patients with mastocytosis.

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein-the carboxy terminus of the alpha-subunit (GalphaCT)-stabilizes Ops*. Here we present the 3.2 A crystal structure of the bovine Ops*-GalphaCT peptide complex. GalphaCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7-helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an alpha-helical conformation in GalphaCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)(5,6)F motifs. On the basis of the Ops*-GalphaCT structure and known conformational changes in Galpha, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.

Agrobacterium tumefaciens is a model organism which delivers genes into plants and induces plant tumors. We study the photobiology of A .tumefaciens, which contains two phytochromes, Agp1 and Agp2,...

Many filamentous cyanobacteria produce specialized nitrogen-fixing cells called heterocysts, which are located at semiregular intervals along the filament with about 10 to 20 photosynthetic vegetative cells in between. Nitrogen fixation in these complex multicellular bacteria depends on metabolite exchange between the two cell types, with the heterocysts supplying combined-nitrogen compounds but dependent on the vegetative cells for photosynthetically produced carbon compounds. Here, we used a fluorescent tracer to probe intercellular metabolite exchange in the filamentous heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. We show that esculin, a fluorescent sucrose analog, is incorporated by a sucrose import system into the cytoplasm of Anabaena cells. The cytoplasmic esculin is rapidly and reversibly exchanged across vegetativevegetative and vegetative-heterocyst cell junctions. Our measurements reveal the kinetics of esculin exchange and also show that intercellular metabolic communication is lost in a significant fraction of older heterocysts. SepJ, FraC, and FraD are proteins located at the intercellular septa and are suggested to form structures analogous to gap junctions. We show that a sepJ fraC fraD triple mutant shows an altered septum structure with thinner septa but a denser peptidoglycan layer. Intercellular diffusion of esculin and fluorescein derivatives is impaired in this mutant, which also shows a greatly reduced frequency of nanopores in the intercellular septal cross walls. These findings suggest that FraC, FraD, and SepJ are important for the formation of junctional structures that constitute the major pathway for feeding heterocysts with sucrose. IMPORTANCE Anabaena and its relatives are filamentous cyanobacteria that exhibit a sophisticated form of prokaryotic multicellularity, with the formation of differentiated cell types, including normal photosynthetic cells and specialized nitrogen-fixing cells called heterocysts. The question of how heterocysts communicate and exchange metabolites with other cells in the filament is key to understanding this form of bacterial multicellularity. Here we provide the first information on the intercellular exchange of a physiologically important molecule, sucrose. We show that a fluorescent sucrose analog can be imported into the Anabaena cytoplasm by a sucrose import system. Once in the cytoplasm, it is rapidly and reversibly exchanged among all of the cells in the filament by diffusion across the septal junctions. Photosynthetically produced sucrose likely follows the same route from cytoplasm to cytoplasm. We identify some of the septal proteins involved in sucrose exchange, and our results indicate that these proteins form structures functionally analogous to metazoan gap junctions.

Current data clearly suggest that VCE has an important role in the diagnostic workup of complicated Cd, especially in those cases with suspected RCd or EATL. This is supported by the high yield of relevant pathological findings in distal parts of the small intestine, such as severe ulceration and mucosal infiltration, by VCE compared to conventional upper GI endoscopy. In this setting, VCE allows for an assessment of the extent of small bowel involvement, detection of overt though often small neoplasms and assists in planning further diagnostic procedures, especially push or double-balloon enteroscopy which are necessary to obtain specimens either to rule out or to confirm aberrant monoclonal T cell proliferation. Currently, those patients in whom RCd type II or EATL are excluded profit most from a thorough diagnostic workup, since most of them can be treated or cured pharmacologically. At present the prognosis of RCd type II or EATL is poor due to lack of effective therapies. However, treatments that effectively target the aberrant lymphocytes can be anticipated.

Because of the wide variations in the clinical presentation of celiac disease and because treatment exists that is effective in most cases, screening of the general population for celiac disease has been considered. There is still no evidence that patients who have symptom-free celiac disease are at increased risk of small intestinal lymphoma or other complications. Prevention of osteoporosis seems to be the strongest indicator for widespread screening today [22]. The major cause of failure to respond to a gluten-free diet is continuing ingestion of gluten, but other underlying diseases must be considered. Many different drugs (eg, anti-tumor necrosis factor [TNF]-alpha) have been used in patients who have RCD [23]. Steroid treatment has been reported to be effective even in patients who have underlying early EATL. Histologic recovery in patients who have celiac disease usually takes several months but can take up to 1 year, even if the patient remains on a strict gluten-free diet. Some patients report celiac-related symptoms for months after a single gluten intake. The definitions for RCD in literature vary. The authors consider the definition give by Daum and colleagues [24] suitable. They defined true RCD as villous atrophy with crypt hyperplasia and increased IELs persisting for more than 12 months in spite of a strict gluten-free diet. If a patient is not responding well to a gluten-free diet, three considerations are necessary: (1) the initial diagnosis of celiac disease must be reassessed;(2) the patient should be sent to a dietician to check for errors in diet or compliance problems, because problems with the gluten-free diet are the most important cause for persisting symptoms; (3) other reasons for persisting symptoms (eg, pancreatic insufficiency, irritable bowel syndrome, bacterial overgrowth, lymphocytic colitis, collagenous colitis, ulcerative jejunitis, protein-losing enteropathy,T-cell lymphoma, fructose intolerance, cavitating lymphadenopathy, and tropical sprue) should be considered. Other causes for villous atrophy are Crohn's disease, collagenous sprue, and autoimmune enteropathy. Abdulkarim and colleagues [25] examined 55 patients who had a diagnosis of nonresponsive celiac disease. He found that 6 did not have celiac disease, and25 still had some gluten ingestion.Tursi and colleagues [26] reported 15 patients who had celiac disease with persisting symptoms. Because histology improved in all patients after several months, RCD was excluded. Of the 15 patients, 10 had small intestinal bacterial overgrowth, 2 showed lactose malabsorption causing the described symptoms, 1 had mistakenly taken an antibiotic containing gluten, and 1 patient each had Giardia lamblia and Ascaris lumbricoides. Thus, other entities must be considered in patients who have celiac disease and ongoing symptoms. In a follow-up clinical trial, 158 patients who had celiac disease underwent follow-up small intestine biopsies within 2 years after starting a gluten-free diet. Eleven patients (7.0%) with persisting (partial) villous atrophy were considered to have RCD; 5 of them developed EATL [27].RCD type I is characterized by normal expression of T-cell antigens and polyclonal TCR gene rearrangement.RCD type II is characterized by an abnormal IEL phenotype with the expression of intracytoplasmic CD3e, surface CD103, and the lack of classic surface T-cell markers such as CD8, CD4, and TCR-alpha/beta. This clonal IEL population can be considered crypt IEL [24]. RCD II has a poor prognosis, which is a problem for therapy. Clonal TCR gene rearrangements and loss of T-cell antigens such as CD8 and TCR-beta in IELs may indicate the development of an EATL in patients who have RCD. The markers for an overt EATL are a positive stool blood test, increased lactate dehydrogenase, or beta2-microglobulin [24]. If an overt lymphoma is suspected, upper and lower endoscopy, an ear, nose, and throat work-up, CT scan, capsule endoscopy, and possibly double-balloon enteroscopy should be performed. Most reports of the difficulties in treating patients who have true RCE are casereports. Turner and colleagues [28] reported on an induction of remission by useof the anti-TNF-alpha antibody infliximab and maintenance with prednisoloneand azathioprine. Olaussen and colleagues [29] and Mandal and colleagues [30]tried a nonimmunogenic elemental diet. Gillet and colleagues [31] reported successful treatment of a patient who hadRCD using anti-TNF-alpha antibodies (infliximab) for induction and azathioprinefor maintenance. Maurino and colleagues [32] studied seven consecutive patients diagnosed ashaving refractory sprue and no response to oral or parenteral steroids. Aftertreatment with azathioprine (2 mg/kg/d) and oral prednisone (1 mg/kg/d), fivepatients had a complete clinical remission. Two patients who did not respond totreatment at any time died. Goerres and colleagues [33] described 18 patients who had RCD, 10 of whomhad type I RCD, and 8 of whom had type II RCD. Treatment consisted ofazathioprine combined with prednisone for 1 year. Consistent with reports byother investigators, the response rates in the two groups differed. Eight of the10 patients who had type I RCD had a histologic response. Seven of the eightpatients who had type II RCD died, and six of the eight developed a lymphoma. At present there is no effective treatment for type II RCD.Fig. 3 presents a proposed algorithm for monitoring patients who have ce-liac disease.

Bereits in den 1970er Jahren wurden Hamoclips als Zubehor fur die flexible gastroenterologische Endoskopie entwickelt. Kommerziell war der Hamoclip (HX-600 und HX-200, Olympus Endotherapie, Hamburg

The X-ray structure analysis of photosystem (PS) I single crystals showed that the primary electron donor P700 is a heterodimer formed by one chlorophyll (Chl) a and one Chl a′ [Nature 411 (2001) 909]. The electronic structure of the cation radical P700+ of the primary donor, which is created in the charge separation process, has been probed by semiempirical molecular orbital calculations including spin polarization effects (RHF-INDO/SP). The calculations, which were based on the X-ray structure, clearly show that P700 is a supermolecule formed by two chlorophyll species. They furthermore predict an asymmetrical charge and spin density distribution in favor of the monomeric Chl a half of this dimer in accordance with results from earlier EPR and ENDOR studies [J. Phys. Chem. B 105 (2000) 1225]. The stepwise inclusion of various electrostatic interactions of the dimer with its nearest surrounding (one threonine forming a hydrogen bond to the keto group of Chl a′ and two histidines liganding the Mg atoms of the two chlorophylls) leads to a systematic enhancement of this electronic asymmetry yielding a spin density ratio of almost 5:1 as also found experimentally. A large part of this value is caused by spin polarization effects. This result is only weakly affected by the electrostatic field of more remote amino acid residues and other pigment molecules (‘accessory’ Chl a molecules) present in PS I. A separate group of calculations involving local geometry optimizations by energy minimization techniques yields a further enhancement of the spin density asymmetry. A particularly strong effect is obtained by allowing for variations of the geometry of the vinyl groups on both chlorophylls of the P700 dimer. Theoretical results for individual isotropic proton and nitrogen hyperfine coupling constants, showing a satisfactory agreement with experimental findings, are also presented.

The X-ray structure of Photosystem I (PS I) from Synechococcus elongatus was recently solved at 2.5A resolution (PDB entry 1JB0). It provides a structural model for the stromal subunits PsaC, PsaD and PsaE, which comprise the "stromal ridge" of PS I. In a separate set of studies the three-dimensional solution structures of the unbound, recombinant PsaC (PDB entry 1K0T) and PsaE (PDB entries 1PSF, 1QP2 and 1GXI) subunits were solved by NMR. The PsaC subunit of PS I is a small (9.3 kDa) protein that harbors binding sites for two [4Fe-4S] clusters F(A) and F(B), which are the terminal electron acceptors in PS I. Comparison of the PsaC structure in solution with that in the X-ray structure of PS I reveals significant differences between them which are summarized and evaluated here. Changes in the magnetic properties of [4Fe-4S] centers F(A) and F(B) are related to changes in the protein structure of PsaC, and they are further influenced by the presence of PsaD. Based on experimental evidence, three assembly stages are analyzed: PsaC(free), PsaC(only), PsaC(PS I). Unbound, recombinant PsaD, studied by NMR, has only a few elements of secondary structure and no stable three-dimensional structure in solution. When PsaD is bound in PS I, it has a well-defined three-dimensional structure. For PsaE the three-dimensional structure is very similar in solution and in the PS I-bound form, with the exception of two loop regions. We suggest that the changes in the structures of PsaC and PsaD are caused by the sequential formation of multiple networks of contacts between the polypeptides of the stromal ridge and between those polypeptides and the PsaA/PsaB core polypeptides. The three-dimensional structure of the C(2)-symmetric F(X)-binding loops on PsaA and PsaB were also analyzed and found to be significantly different from the binding sites of other proteins that contain interpolypeptide [4Fe-4S] clusters. The aim of this work is to relate contact information to structural changes in the proteins and to propose a model for the assembly of the stromal ridge of PS I based on this analysis.

The formation of a transient complex between photosystem I and ferredoxin is involved in the process of ferredoxin photoreduction in oxygenic photosynthetic organisms. Reduced ferredoxin is an essential redox intermediate involved in many assimilatory processes and is necessary for the reduction of NADP(+) to NADPH. Single crystals from a complex of photosystem I with ferredoxin were grown using PEG 400 and CaCl(2) as precipitation agents. The crystals diffract x-rays to a resolution of 7-8 A. The space group was determined to be orthorhombic with the unit cell dimensions a = 194 A, b = 208 A, and c = 354 A. The crystals contain photosystem I and ferredoxin in a 1:1 ratio. Electron paramagnetic resonance (EPR) measurements on these crystals are reported, where EPR signals of the three [4Fe-4S] clusters F(A), F(B), F(X), and the [2Fe-2S] cluster of ferredoxin were detected. From the EPR spectra observed at three particular orientations of the crystal in the magnetic field, the full orientation pattern of the F g-tensor was simulated. This simulation is consistent with the presence of 12 magnetically inequivalent F clusters per unit cell with the C(3) axis of the PSI trimers oriented at (23 degrees, 72 degrees, 77 degrees ) to the unit cell axes.

The structure of photosystem I from the thermophilic cyanobacterium Synechococcus elongatus has been recently resolved by x-ray crystallography to 2.5-A resolution. Besides the reaction center, photosystem I consists also of a core antenna containing 90 chlorophyll and 22 carotenoid molecules. It is their function to harvest solar energy and to transfer this energy to the reaction center (RC) where the excitation energy is converted into a charge separated state. Methods of steady-state optical spectroscopy such as absorption, linear, and circular dichroism have been applied to obtain information on the spectral properties of the complex, whereas transient absorption and fluorescence studies reported in the literature provide information on the dynamics of the excitation energy transfer. On the basis of the structure, the spectral properties and the energy transfer kinetics are simultaneously modeled by application of excitonic coupling theory to reveal relationships between structure and function. A spectral assignment of the 96 chlorophylls is suggested that allows us to reproduce both optical spectra and transfer and emission spectra and lifetimes of the photosystem I complex from S. elongatus. The model calculation allowed to study the influence of the following parameters on the excited state dynamics: the orientation factor, the heterogeneous site energies, the modifications arising from excitonic coupling (redistribution of oscillator strength, energetic splitting, reorientation of transition dipoles), and presence or absence of the linker cluster chlorophylls between antenna and reaction center. For the Förster radius and the intrinsic primary charge separation rate, the following values have been obtained: R(0) = 7.8 nm and k(CS) = 0.9 ps(-1). Variations of these parameters indicate that the excited state dynamics is neither pure trap limited, nor pure transfer (to-the-trap) limited but seems to be rather balanced.

The recently determined crystal structures of photosystems I and II at 2.5 A and 3.8 A resolution, respectively, have improved the structural basis for understanding the processes of light trapping, exciton transfer and electron transfer occurring in the primary steps of oxygenic photosynthesis. Understanding the assembly of the 12 protein subunits and 128 cofactors in photosystem I allows us to study the possible functions of the individual players in this protein-cofactor complex.