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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.

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.

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.

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.

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.

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.

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 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.

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.

In order to understand elongator tRNA(Ser) and suppressor tRNA(Sec) identity elements, the respective acceptor-stem helices have been synthesized and crystallized in order to analyse and compare their structures in detail at high resolution. The synthesis, crystallization and preliminary X-ray diffraction results for a seven-base-pair tRNA(Ser) acceptor-stem helix are presented here. Diffraction data were collected to 1.8 A, applying synchrotron radiation and cryogenic cooling. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 36.14, b = 38.96, c = 30.81 A, beta = 110.69 degrees .

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

An improved electron density map of photosystem I (PSI) calculated at 4-A resolution yields a more detailed structural model of the stromal subunits PsaC, PsaD, and PsaE than previously reported. The NMR structure of the subunit PsaE of PSI from Synechococcus sp. PCC7002 (Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062) has been used as a model to interpret the region of the electron density map corresponding to this subunit. The spatial orientation with respect to other subunits is described as well as the possible interactions between the stromal subunits. A first model of PsaD consisting of a four-stranded beta-sheet and an alpha-helix is suggested, indicating that this subunit partly shields PsaC from the stromal side. In addition to the improvements on the stromal subunits, the structural model of the membrane-integral region of PSI is also extended. The current electron density map allows the identification of the N and C termini of the subunits PsaA and PsaB. The 11-transmembrane alpha-helices of these subunits can now be assigned uniquely to the hydrophobic segments identified by hydrophobicity analyses.

The central magnesium (Mg) atoms of natural occurring tetrapyrroles such as chlorophylls (Chls) and bacteriochlorophylls (BChls) are typically five-coordinated, a fact which leads to the formation of diastereoisomers if the Mg–ligand bond is stable on the time scale of the observation method. This possibility has only been briefly addressed before in a CD-study of BChl c aggregates [T.S. Balaban, A.R. Holzwarth, K. Schaffner, J. Mol. Struct. 349 (1995) 183]. On the basis of the chlorophyll–protein complex photosystem I (PSI), which has recently been characterized by single crystal crystallography [P. Jordan, P. Fromme, H.T. Witt, O. Klukas, W. Saenger, N. Kraus, Nature 411 (2001) 909], we find that chlorophyll a molecules are much more frequently bound by the protein matrix from one side (anti) than the other one (syn) in a ratio of 82:14, which corresponds to a significant ΔΔG value of 4.3 kJ/mol. Syn and anti denote the orientation of the Mg–ligand with respect to the 17-propionic acid esterified by phytol. Furthermore, by parallel sequence analysis we find that the binding sites for both syn and anti chlorophylls have been strongly conserved during evolution—a fact which stresses the nonrandom manner in which chlorophylls are bound by the apoprotein in antenna complexes, in order to exert efficiently their light harvesting function and energy funnelling. Most remarkably, all the syn chlorophylls are part of the inner core antenna system. Results from semiempirical quantum mechanical and detailed exciton coupling calculations allow us to speculate on the functional relevance of the diasteretopicity for PSI functioning.

Photosystem I (PS I) is a large membrane protein complex that catalyzes the first step of solar conversion, the light-induced transmembrane electron transfer, and generates reductants for CO2 assimilation. It consists of 12 different proteins and 127 cofactors that perform light capturing and electron transfer. The function of PS I includes inter-protein electron transfer between PS I and smaller soluble electron transfer proteins. The structure of PS I is discussed with respect to the potential docking sites for the soluble electron acceptors, ferredoxin/flavodoxin, at the stromal side and the soluble electron donors, cytochrome c6/plastocyanin, at the luminal side of the PS I complex. Furthermore, the potential interaction sites with the peripheral antenna proteins are discussed.

In Photosystem I (PS I) the terminal electron acceptors, FA and FB, are iron–sulfur (4Fe–4S) centers, which are bound to the stromal subunit PsaC. The orientation of PsaC is determined relative to the whole PS I complex (see Schubert, W.-D. et al. (1995) in From Light to Biosphere (Mathis, P. ed.), Vol. II, pp. 3–10, Kluwer) from which a molecular model for the structure of PsaC within PS I is derived. Two strategies are followed: (i) PS I single crystal EPR data on the orientation of the g tensors of both F−A and F−B relative to each other and relative to the crystal axes (see preceding paper) are used in conjuction with the central structural part of the bacterial 2 [Fe4S4] ferredoxins, the cysteine binding motifs of which are known to be homologous to those of PsaC; (ii) the same core structure is fitted into the intermediate resolution electron density map of PS I. The PsaC orientation obtained both ways agree well. The local twofold symmetry axis inherent to the ferredoxin model leaves a twofold ambiguity in the structural conclusion. Deviations from this C2-symmetry in the amino acid sequence of PsaC are analyzed with respect to observable properties which would resolve the remaining structural ambiguity. Arguments both for and against FA being the distal iron–sulfur center (to FX) are discussed.