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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.

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.

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 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.

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.

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.

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.

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.

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.

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.

Photolyases are flavoproteins that repair UV-damaged DNA using the energy of sunlight. The flavin adenine dinucleotide (FAD) chromophores of photolyases and the related cryptochromes are bound to the chromophore pocket in a U-shaped conformation. Often, a second cofactor serves as antenna chromophore in photolyases. This can be methenyltetrahydrofolate (MTHF), 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), FAD, or flavin mononucleotide (FMN). Two kinds of DNA lesions are produced by UV light, cyclobutan-pyrimidine dimers (CPDs) or (6-4) photoproducts. These lesions are repaired by two different types of photolyases, namely the CPDs and (6-4) photolyases. Recently, a new phylogenetic group of the photolyase cryptochrome family termed iron–sulfur bacterial cryptochromes/photolyases (FeS-BCP) was discovered. Two members of this group, CryB from Rhodobacter sphaeroides and ‘photolyase related protein’ (PhrB) from Agrobacterium tumefaciens, carry a new antenna chromophore, 6,7-dimethyl 8- ribityl-lumazin (DMRL), and incorporate an 4Fe-4S cluster. PhrB acts as a (6-4) photolyase with a repair activity that is higher for double-stranded than for single-stranded DNA. There are presently more than 900 prokaryotic PhrB homologs in the database in which the characteristic cysteine residues for incorporation of the Fe-S cluster and other key amino acids are conserved. This new group of (6-4) photolyases with an Fe-S cluster is thus widely distributed among prokaryotes. Unlike in other photolyases, the loop connecting the N-terminal and the C-terminal domains of PhrB interacts with the DNA lesion. A C-terminal extension, which could have a regulatory function, interacts with the DNA. The comparison between PhrB and eukaryotic (6-4) photolyases highlights a conserved His residue which is located next to the DNA lesion. This residue serves probably as proton donor/acceptor during the repair cycle. 3D Structure Overall structure of the (6-4) photolyase PhrB (PDB code 4DJA). FAD is drawn in yellow, DMRL in magenta, and the FeS cluster in yellow/brown. (Figure prepared with PyMOL.1) Keywords: UV light; DNA repair; photorepair; DNA lesion; photolyase (evolution); photoreduction; bacteria; Agrobacterium tumefaciens

The amylose fraction of starch occurs in double-helical A- and B-amyloses and the single-helical V-amylose. The latter contains a channel-like central cavity that is able to include molecules, “iodine’s blue” being the best-known representative. Molecular models of these amylose forms have been deduced by solid state 13 C cross-polarization/magic angle spinning NMR and by x-ray fiber and electron diffraction combined with computer-aided modeling. They remain uncertain, however, as no structure at atomic resolution is available. We report here the crystal structure of a hydrated cycloamylose containing 26 glucose residues (cyclomaltohexaicosaose, CA26), which has been determined by real/reciprocal space recycling starting from randomly positioned atoms or from an oriented diglucose fragment. This structure provides conclusive evidence for the structure of V-amylose, as the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by twofold rotational pseudosymmetry. In the V-helices, all glucose residues are in syn orientation, forming systematic interglucose O(3) n ⋅⋅⋅O(2) n +l and O(6) n ⋅⋅⋅O(2) n +6 /O(3) n +6 hydrogen bonds; the central cavities of the V-helices are filled by disordered water molecules. The folding of the CA26 macrocycle is characterized by typical “band-flips” in which diametrically opposed glucose residues are in anti rather than in the common syn orientation, this conformation being stabilized by interglucose three-center hydrogen bonds with O(3) n as donor and O(5) n +l , O(6) n +l as acceptors. The structure of CA26 permitted construction of an idealized V-amylose helix, and the band-flip motif explains why V-amylose crystallizes readily and may be packed tightly in seeds.

An improved electron density map of photosystem I from Synechococcus elongatus calculated at 4-A resolution for the first time reveals a second phylloquinone molecule and thereby completes the set of cofactors constituting the electron transfer system of this iron-sulfur type photosynthetic reaction center: six chlorophyll a, two phylloquinones, and three Fe4S4 clusters. The location of the newly identified phylloquinone pair, the individual plane orientations of these molecules, and the resulting distances to other cofactors of the electron transfer system are discussed and compared with those determined by magnetic resonance techniques.

Human lysosomal arylsulfatase A (ASA) is a prototype member of the sulfatase family. These enzymes require the posttranslational oxidation of the -CH2SH group of a conserved cysteine to an aldehyde, yielding a formylglycine. Without this modification sulfatases are catalytically inactive, as revealed by a lysosomal storage disorder known as multiple sulfatase deficiency. The 2.1 A resolution X-ray crystal structure shows an ASA homooctamer composed of a tetramer of dimers, (alpha 2)4. The alpha/beta fold of the monomer has significant structural analogy to another hydrolytic enzyme, the alkaline phosphatase, and superposition of these two structures shows that the active centers are located in largely identical positions. The functionally essential formylglycine is located in a positively charged pocket and acts as ligand to an octahedrally coordinated metal ion interpreted as Mg2+. The electron density at the formylglycine suggests the presence of a 2-fold disordered aldehyde group with the possible contribution of an aldehyde hydrate, -CH(OH)2, with gem-hydroxyl groups. In the proposed catalytic mechanism, the aldehyde accepts a water molecule to form a hydrate. One of the two hydroxyl groups hydrolyzes the substrate sulfate ester via a transesterification step, resulting in a covalent intermediate. The second hydroxyl serves to eliminate sulfate under inversion of configuration through C-O cleavage and reformation of the aldehyde. This study provides the structural basis for understanding a novel mechanism of ester hydrolysis and explains the functional importance of the unusually modified amino acid.

An improved structural model of the photosystem I complex from the thermophilic cyanobacterium Synechococcus elongatus is described at 4 A resolution. This represents the most complete model of a photosystem presently available, uniting both a photosynthetic reaction centre domain and a core antenna system. Most constituent elements of the electron transfer system have been located and their relative centre-to-centre distances determined at an accuracy of approximately 1 A. These include three pseudosymmetric pairs of Chla and three iron-sulphur centres, FX, FA and FB. The first pair, a Chla dimer, has been assigned to the primary electron donor P700. One or both Chla of the second pair, eC2 and eC'2, presumably functionally link P700 to the corresponding Chla of the third pair, eC3 and eC'3, which is assumed to constitute the spectroscopically-identified primary electron acceptor(s), A0, of PSI. A likely location of the subsequent phylloquinone electron acceptor, QK, in relation to the properties of the spectroscopically identified electron acceptor A1 is discussed. The positions of a total of 89 Chla, 83 of which constitute the core antenna system, are presented. The maximal centre-to-centre distance between antenna Chla is < or = 16 A; 81 Chla are grouped into four clusters comprising 21, 23, 17 and 20 Chla, respectively. Two "connecting" Chla are positioned to structurally (and possibly functionally) link the Chla of the core antenna to those of the electron transfer system. Thus the second and third Chla pairs of the electron transfer system may have a dual function both in energy transfer and electron transport. A total of 34 transmembrane and nine surface alpha-helices have been identified and assigned to the 11 subunits of the PSI complex. The connectivity of the nine C-terminal (seven transmembrane, two "surface") alpha-helices of each of the large core subunits PsaA and PsaB is described. The assignment of the amino acid sequence to the transmembrane alpha-helices is proposed and likely residues involved in co-ordinating the Chla of the electron transfer system discussed.

The 4 A X-ray structure model of trimeric photosystem I of the cyanobacterium Synechococcus elongatus reveals 31 transmembrane, nine surface and three stromal alpha-helices per monomer, assigned to the 11 protein subunits: PsaA and PsaB are related by a pseudo two-fold axis normal to the membrane plane, along which the electron transfer pigments are arranged. 65 antenna chlorophyll a (Chl a) molecules separated by < or = 16 A form an oval, clustered net continuous with the electron transfer chain through the second and third Chl a pairs of the electron transfer system. This suggests a dual role for these Chl a both in excitation energy and electron transfer. The architecture of the protein core indicates quinone and iron-sulphur type reaction centres to have a common ancestor.

Photosystem I of Synechococcus elongatus was crystallized showing maximal diffraction to 4 A resolution (Witt et al. (1988) Ber. Bunsenges. Phys. Chem. 92, 1503–1506). The crystal structure has been determined at a resolution of 6 A (Kraus et al. (1993) Nature 361, 326–331). Based on this structure, docking sites for plastocyanin and ferredoxin are proposed. The arrangement of helices is analysed in terms of hydrophobicity plots and homology to the reaction center of purple bacteria. A possible coordination of the primary donor P700 by the helices in subunits A and B is suggested based on the relative three-dimensional arrangement of the helices.

1-Arylimino-pyrazole Betaines, Pyrazoles and 1,2,4-Triazines from 3-Arylazo-propenoic Acid N-Chloramides Reaction of 3-arylazo-propenoic acid amides 5 with NaOCl yields N-chloroamides 6 which give under alkaline conditions 1,2,4-triazines 9 and 10 (products of a Hofmann degradation). This reaction competes with an intramolecular electrophilic amination and a 1,2-shift reaction giving 1-arylimino-pyrazole betaines 11 and 4-chloroimino-pyrazolones 13. Exchange of the halogen in 11 with nucleophiles leads to further betaines 12. Bis-pyrazole 14 and 1,2,3-triazoles 15 are available from 13b. The X-ray structure analysis of compound 11b is discussed.

X-ray structure analysis shows that the monomer of trimeric photosystem I (PS I) of Synechococcus sp. consists of a catalytic domain and a smaller domain that connects the monomers. The 4Fe–4S clusters FX,FA and FB, 28 α-helices and 45 chlorophyll a molecules were located. The two large subunits of PS I are represented by nine a-helices each; they are related by a local 2-fold rotation axis passing through Fx. Electron densities close to this axis are interpreted as carriers of the electron transfer chain.

Ring Transformations of Pyrazolinones via Azoolefins – a New Way to the 1,2,3-Thiadiazol-System Elektrophilic attack of thiocyanate/bromine to azoenamine (2a) yields thiocyanato compound 3a which cyclizes into 1,2,3-thiadiazolium-salt (5a) on treatment with perchloric acid. 2-Halo-3-arylazo-propenamides (8) form directly thiadiazolium-salts (11) with KSCN/HOAc. The addition of N- or O-nucleophiles to 11 occurs in the 5-position to form 1,2,3-thiadiazolines (13).

In preliminary work, a millimeter-wave vector network analyzer is used to analyze pectate lyase and a sequenced concentration of glucose solutions. Data reveal concentration-dependant spectra for glucose and Ca2+-sensitive spectra for pectate lyase.

X-ray structure analysis shows that the monomer of trimeric photosystem I (PS I) of Synechococcus sp. consists of a catalytic domain and a smaller domain that connects the monomers. The 4Fe–4S clusters FX,FA and FB, 28 α-helices and 45 chlorophyll a molecules were located. The two large subunits of PS I are represented by nine a-helices each; they are related by a local 2-fold rotation axis passing through Fx. Electron densities close to this axis are interpreted as carriers of the electron transfer chain.

This chapter describes the data accumulated in the last decade regarding the specific function of lipids in oxygenic photosynthesis, based on crystal structures of at least 3.0 A resolution of the main photosynthetic membrane protein—pigment complexes, photosystem I, photosystem II and cytochrome b 6 f. Comparisons with other structures of membrane protein complexes like the bacterial reaction center and the external antenna system from the plant light harvesting complexes II reveal the functional versatility of integral lipids. A detailed structural description of the membrane protein complexes pinpoints the various interactions of integral lipids between protein and pigments (e.g., chlorophylls, carotenoids, quinones) and gives a deep insight into their functional roles. A particular focus in this chapter is on the lipid-filled plastoquinone exchange cavities in photosystem II and cytochrome b 6 f. The differences in extent and lipophilic character of these cavities will be discussed in the light of the resulting plastoqui-none/plastoquinol exchange mechanism. An exceptional feature of PS II is the water splitting reaction enabled by the Mn4Ca cluster. This results in the release of protons to the lumenal aqueous phase, release of electrons to a chain of acceptors, which provides metabolically available reduction equivalents, and release of dioxygen to the atmosphere. The high content of lipids in the interior of photosystem II will be correlated with possible diffusion pathways of the dioxygen and the turnover of the D1 protein, necessary to counteract the photodamage occurring within photosystem II. More structural details of integral lipids derived from higher resolution data from these remarkable membrane protein complexes in combination with data from mutant and/or spectroscopic studies will lead to extended functional insights in the future.

The X-ray structure of the Fab fragment from the anti-c-myc antibody 9E10 was determined both as complex with its epitope peptide and for the free Fab. In the complex, two Fab molecules adopt an unusual head to head orientation with the epitope peptide arranged between them. In contrast, the free Fab forms a dimer with different orientation. In the Fab/peptide complex the peptide is bound to one of the two Fabs at the "back" of its extended CDR H3, in a cleft with CDR H1, thus forming a short, three-stranded antiparallel beta-sheet. The N- and C-terminal parts of the peptide are also in contact with the neighboring Fab fragment. Comparison between the CDR H3s of the two Fab molecules in complex with the peptide and those from the free Fab reveals high flexibility of this loop. This structural feature is in line with thermodynamic data from isothermic titration calorimetry.

The structure of a complex of the anti-cholera toxin antibody TE33 Fab (fragment antibody) with the D-peptide vpGsqhyds was solved to 1.78 A resolution. The D-peptide was derived from the linear L-peptide epitope VPGSQHIDS by a stepwise transformation. Despite the very similar amino acid sequence—the only difference is a tyrosine residue in position 7—there are marked differences in the individual positions with respect to their contribution to the peptide overall affinity as ascertained by a complete substitutional analysis. This is reflected by the X-ray structure of the TE33 Fab/D-peptide complex where there is an inverted orientation of the D-peptide as compared with the known structure of a corresponding complex containing the epitope L-peptide, with the side chains establishing different contacts within the binding site of TE33. The D- and L-peptide affinities are comparable and the surface areas buried by complex formation are almost the same. Thus the antibody TE33 provides a typical example for polyspecific binding behavior of IgG family antibodies. Copyright © 2007 John Wiley & Sons, Ltd.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in anti-oxidative defense, sperm development, and cerebral embryogenesis. Among GPx-isoforms, GPx4 is unique because of its capability to reduce complex lipid hydroperoxides and its tendency toward polymerization, but the structural basis for these properties remained unclear. To address this, we solved the crystal structure of the catalytically active U46C mutant of human GPx4 to 1.55 A resolution. X-ray data indicated a monomeric protein consisting of four alpha-helices and seven beta-strands. GPx4 lacks a surface exposed loop domain, which appears to limit the accessibility of the active site of other GPx-isoforms, and these data may explain the broad substrate specificity of GPx4. The catalytic triad (C46, Q81, and W136) is localized at a flat impression of the protein surface extending into a surface exposed patch of basic amino acids (K48, K135, and R152) that also contains polar T139. Multiple mutations of the catalytic triad indicated its functional importance. Like the wild-type enzyme, the U46C mutant exhibits a strong tendency toward protein polymerization, which was prevented by reductants. Site-directed mutagenesis suggested involvement of the catalytic C46 and surface exposed C10 and C66 in polymer formation. In GPx4 crystals, these residues contact adjacent protein monomers.

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state. Fil: von Stetten, David. Technische Universität Berlin; Alemania Fil: Seibeck, Sven. Freie Universität Berlin.; Alemania Fil: Michael, Norbert. Freie Universität Berlin.; Alemania Fil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; Alemania Fil: Mroginski, Maria Andrea. Technische Universität Berlin; Alemania Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; Alemania Fil: Krauss, Norbert. Freie Universität Berlin.; Alemania Fil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; Alemania Fil: Hildebrandt, Peter. Technische Universität Berlin; Alemania Fil: Borucki, Berthold. Freie Universität Berlin.; Alemania Fil: Lamparter, Tilman. Freie Universität Berlin.; Alemania

金沢大学大学院自然科学研究科物質創成, 金沢大学理学部, Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14-C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5-C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5-C6 syn C15=C16 Z C14-C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14-C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5-C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.

The expression, purification, crystallization and preliminary X-ray diffraction studies of mouse centrin1 are reported. Centrins belong to a family of Ca{sup 2+}-binding EF-hand proteins that play a fundamental role in centrosome duplication and the function of cilia. To shed light on the structure–function relationship of these proteins, mouse centrin1 has been crystallized. The mouse centrin1 has been expressed in Escherichia coli as a GST-centrin fusion protein containing a thrombin protease cleavage site between the fusion partners. Two constructs with different linking-sequence lengths were expressed and purified. Thrombin cleavage yielded functional centrin1 and N-terminally extended centrin1 containing 25 additional residues upstream of its N-terminus. Only N-terminally extended centrin1 (MW ≃ 22 240 Da) could be crystallized at room temperature, using 20–25%(w/v) PEG 1500, 5–10%(v/v) ethylene glycol and 1–2%(v/v) dioxane. Crystals were suitable for X-ray analysis, diffracting to 2.9 A at 295 K using a rotating-anode X-ray source. They belong to space group C2, with unit-cell parameters a = 60.7, b = 59.6, c = 58.3 A, β = 109.4°. Assuming the asymmetric cell to be occupied by one centrin1 molecule of 22.2 kDa, the unit cell contains 45% solvent with a crystal volume per protein weight, V{sub M}, of 2.2 A{supmore » 3} Da{sup −1}.« less

Using structural information from recently published crystal structures of photosystems I and II, the processes of excitation energy transfer and electron transfer in oxygenic photosynthesis have been studied in great detail by experimental and theoretical methods. Although both systems share numerous common structural and functional features, efficiency and regulation are differently weighted in the individual processes that are involved in the transformation of light energy into chemical energy in the two complexes.