Towards characterization of photo-excited electron transfer and catalysis in natural and artificial systems using XFELs.

The ultra-bright femtosecond X-ray pulses provided by X-ray Free Electron Lasers (XFELs) open capabilities for studying the structure and dynamics of a wide variety of biological and inorganic systems beyond what is possible at synchrotron sources. Although the structure and chemistry at the catalytic sites have been studied intensively in both biological and inorganic systems, a full understanding of the atomic-scale chemistry requires new approaches beyond the steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure at ambient conditions, while overcoming X-ray damage to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by using the intense and ultra-short femtosecond X-ray pulses from an XFEL, where sample is probed before it is damaged. We have developed methodology for simultaneously collecting X-ray diffraction data and X-ray emission spectra, using an energy dispersive spectrometer, at ambient conditions, and used this approach to study the room temperature structure and intermediate states of the photosynthetic water oxidizing metallo-protein, photosystem II. Moreover, we have also used this setup to simultaneously collect the X-ray emission spectra from multiple metals to follow the ultrafast dynamics of light-induced charge transfer between multiple metal sites. A Mn-Ti containing system was studied at an XFEL to demonstrate the efficacy and potential of this method.

Photoelectrochemistry of a photosystem I - Ferredoxin construct on ITO electrodes.

In this study, photobioelectrodes based on a ferredoxin-modified photosystem I (PSI-Fd) from Thermosynechococcus vestitus have been prepared and characterized regarding the direct electron transfer between PSI-Fd and the electrode. The modified PSI with the covalently linked ferredoxin (Fd) on its stromal side has been immobilized on indium-tin-oxide (ITO) electrodes with a 3-dimensional inverse-opal structure. Compared to native PSI, a lower photocurrent and a lower onset potential of the cathodic photocurrent have been observed. This can be mainly attributed to a different adsorption behavior of the PSI-Fd-construct onto the 3D ITO. However, the overall behavior is rather similar to PSI. First experiments have been performed for applying this PSI-Fd photobioelectrode for enzyme-driven NADPH generation. By coupling the electrode system with ferredoxin-NADP+-reductase (FNR), first hints for the usage of photoelectrons for biosynthesis have been collected by verifying NADPH generation.

"Invisible" Detergents Enable a Reliable Determination of Solution Structures of Native Photosystems by Small-Angle Neutron Scattering.

Photosystems I (PSI) and II (PSII) are pigment-protein complexes capable of performing the light-induced charge separation necessary to convert solar energy into a biochemically storable form, an essential step in photosynthesis. Small-angle neutron scattering (SANS) is unique in providing structural information on PSI and PSII in solution under nearly physiological conditions without the need for crystallization or temperature decrease. We show that the reliability of the solution structure critically depends on proper contrast matching of the detergent belt surrounding the protein. Especially, specifically deuterated ("invisible") detergents are shown to be properly matched out in SANS experiments by a direct, quantitative comparison with conventional matching strategies. In contrast, protonated detergents necessarily exhibit incomplete matching so that related SANS results systematically overestimate the size of the membrane protein under study. While the solution structures obtained are close to corresponding high-resolution structures, we show that temperature and solution state lead to individual structural differences compared with high-resolution structures. We attribute these differences to the presence of a manifold of conformational substates accessible by protein dynamics under physiological conditions.

Current limits of structural biology: The transient interaction between cytochrome c 6 and photosystem I.

Trimeric photosystem I from the cyanobacterium Thermosynechococcus elongatus (TePSI) is an intrinsic membrane protein, which converts solar energy into electrical energy by oxidizing the soluble redox mediator cytochrome c 6 (Cyt c 6 ) and reducing ferredoxin. Here, we use cryo-electron microscopy and small angle neutron scattering (SANS) to characterize the transient binding of Cyt c 6 to TePSI. The structure of TePSI cross-linked to Cyt c 6 was solved at a resolution of 2.9 Å and shows additional cofactors as well as side chain density for 84% of the peptide chain of subunit PsaK, revealing a hydrophobic, membrane intrinsic loop that enables binding of associated proteins. Due to the poor binding specificity, Cyt c 6 could not be localized with certainty in our cryo-EM analysis. SANS measurements confirm that Cyt c 6 does not bind to TePSI at protein concentrations comparable to those for cross-linking. However, SANS data indicate a complex formation between TePSI and the non-native mitochondrial cytochrome from horse heart (Cyt c HH ). Our study pinpoints the difficulty of identifying very small binding partners (less than 5% of the overall size) in EM structures when binding affinities are poor. We relate our results to well resolved co-structures with known binding affinities and recommend confirmatory methods for complexes with K M values higher than 20 µM.

Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus.

Binding of herbicides to photosystem II inhibits the electron transfer from Q(A) to Q(B) due to competition of herbicides with plastoquinone bound at the Q(B) site. We investigated herbicide binding to monomeric and dimeric photosystem II core complexes (PSIIcc) isolated from Thermosynechococcus elongatus by a combination of different methods (isothermal titration and differential scanning calorimetry, CD spectroscopy and measurements of the oxygen evolution) yielding binding constants, enthalpies and stoichiometries for various herbicides as well as information regarding stabilization/destabilization of the complex. Herbicide binding to detergent-solubilized PSIIcc can be described by a model of single independent binding sites present on this important membrane protein. Interestingly, binding stoichiometries herbicide:PSIIcc are lower than 1:1 and vary depending on the herbicide under study. Strong binding herbicides such as terbutryn stabilize PSIIcc in thermal unfolding experiments and endothermically binding herbicides like ioxynil probably cause large structural changes accompanied with the binding process as shown by differential scanning calorimetry experiments of the unfolding reaction of PSIIcc monomer in the presence of ioxynil. In addition we studied the occupancy of the Q(B) sites with plastoquinone (PQ9) by measuring flash induced fluorescence relaxation yielding a possible explanation for the deviations of herbicide binding from a 1:1 herbicide/binding site model.

Purification, characterisation and crystallisation of photosystem II from Thermosynechococcus elongatus cultivated in a new type of photobioreactor.

The thermophilic cyanobacterium Thermosynechococcus elongatus was cultivated under controlled growth conditions using a new type of photobioreactor, allowing us to optimise growth conditions and the biomass yield. A fast large-scale purification method for monomeric and dimeric photosystem II (PSII) solubilized from thylakoid membranes of this cyanobacterium was developed using fast protein liquid chromatography (FPLC). The obtained PSII core complexes (PSIIcc) were analysed for their pigment stoichiometry, photochemical and oxygen evolution activities, as well as lipid and detergent composition. Thirty-six chlorophyll a (Chla), 2 pheophytin a (Pheoa), 9+/- 1 beta-carotene (Car), 2.9+/-0.8 plastoquinone 9 (PQ9) and 3.8+/-0.5 Mn were found per active centre. For the monomeric and dimeric PSIIcc, 18 and 20 lipid as well as 145 and 220 detergent molecules were found in the detergent shell, respectively. The monomeric and dimeric complexes showed high oxygen evolution activity with 1/4 O(2) released per 37-38 Chla and flash in the best cases. Crystals were obtained from dimeric PSIIcc by a micro-batch method. They diffract synchrotron X-rays to a maximum resolution of 2.9-A, resulting in complete data sets of 3.2 A resolution.

Synthesis and kinetic study of transition state analogs for ribonuclease T1.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.

First photosystem II crystals capable of water oxidation.

Oxygen evolution and proton release of crystallised photosystem II core complexes isolated from Synechococcus elongatus were measured. The yields show that the crystals themselves are capable of highly active water oxidation. This opens the possibility for the structural analysis of the outstanding water-oxidising apparatus.

Static and dynamic studies of the potential-sensitive membrane probe RH421 in dimyristoylphosphatidylcholine vesicles.

The dynamics of the potential-sensitive styryl dye RH421 in dimyristoylphosphatidylcholine vesicles have been investigated above and below the main phase transition temperature using iodine-laser temperature-jump relaxation spectrophotometry and time-resolved fluorescence lifetime measurements. Equilibrium fluorescence titrations have shown that the affinity of the dye for the membrane is much higher in the liquid-crystalline state than in the gel state. The interaction can be described by either a partition or a binding model and a theory is presented providing a relation between these two approaches. In the liquid-crystalline state bound dye exhibits steady-state fluorescence relaxation processes in the submicrosecond and millisecond time range following a temperature jump. Time-resolved fluorescence measurements show a variation in the fluorescence lifetime across the emission spectrum, suggesting an excited-state process occurring on the subnanosecond time scale. These processes are most likely related to dye and/or lipid reorientation following the temperature jump or excitation pulse. Temperature-dependent changes in the fluorescence excitation spectrum of bound dye suggest that the dye exists in at least two different sites within the membrane.

Interaction of the fluorescent probe RH421 with ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments.

Fluorescence titrations have shown that the voltage-sensitive probe RH421 interacts with the water-soluble protein ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments. The probe exhibits significantly different fluorescence excitation spectra in pure lipid and pure protein environments. Experiments with a range of polyamino acids showed interactions of the probe with tyrosine, lysine and arginine residues. At saturating RH421 concentrations (> or = microM) the probe quenches 60-75% of the total tryptophan fluorescence of the Na+,K(+)-ATPase preparation. Inhibition of the hydrolytic activity of the Na+,K(+)-ATPase occurs at RH421 concentrations in the micromolar range. This may be due to a probe-induced change in membrane fluidity. The sensitivity of the probe towards conformational changes of the Na+,K(+)-ATPase decreases hyperbolically as one increases the probe concentration. The decrease in sensitivity correlates well with association of the probe in the vicinity of membrane protein, as measured by tryptophan quenching. These results have important practical consequences for the application of RH421 as a voltage indicator in membrane preparations. Based on these and previously reported results, the fluorescent response of RH421 to the ATP-induced conformational change of the Na+,K+-ATPase is consistent with either a redistribution of dye from the liquid-crystalline lipid matrix into the vicinity of membrane protein or a reorganisation of the lipids surrounding the protein into a more rigid structure caused by the conformational change of the protein.

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage sensitivity of the fluorescent probe RH421 in a model membrane system.

The voltage sensitivity of the fluorescent styrylpyridinium dye RH421 has been investigated in dimyristoylphosphatidylcholine vesicles by inducing an intramembrane electric field through the binding of the hydrophobic ion tetraphenylborate (TPB). To assess the probability of electrochromic and solvatochromic mechanisms for the dye response, the ground-state dipole moment of the dye in chloroform solution was determined from dielectric constant measurements to be 12 (+/- 1) Debye, and the change in dipole moment upon excitation was calculated from measurements of the Stokes shift in solvents of varying polarity to be 25 (+/- 11) Debye. As well as causing absorbance and fluorescence changes of membrane-bound dye, the TPB-induced electrical field was found to reduce significantly the pKa of the dye. The pH at which experiments are carried out is, thus, an important factor in determining the amplitude of the voltage-induced absorbance and fluorescence changes. The observed absorbance changes induced by the field are inconsistent with a pure electrochromic mechanism. A reorientation/solvatochromic mechanism, whereby the electrical field reorients the dye molecules so that they experience a change in polarity of their lipid environment is likely to make a significant contribution to both the spectral changes and to the field effect on the acid-base properties of the dye.

Excited-state dynamics in photosystem II: insights from the x-ray crystal structure.

The heart of oxygenic photosynthesis is photosystem II (PSII), a multisubunit protein complex that uses solar energy to drive the splitting of water and production of molecular oxygen. The effectiveness of the photochemical reaction center of PSII depends on the efficient transfer of excitation energy from the surrounding antenna chlorophylls. A kinetic model for PSII, based on the x-ray crystal structure coordinates of 37 antenna and reaction center pigment molecules, allows us to map the major energy transfer routes from the antenna chlorophylls to the reaction center chromophores. The model shows that energy transfer to the reaction center is slow compared with the rate of primary electron transport and depends on a few bridging chlorophyll molecules. This unexpected energetic isolation of the reaction center in PSII is similar to that found in the bacterial photosystem, conflicts with the established view of the photophysics of PSII, and may be a functional requirement for primary photochemistry in photosynthesis. In addition, the model predicts a value for the intrinsic photochemical rate constant that is 4 times that found in bacterial reaction centers.

Studies on RNase T1 mutants affecting enzyme catalysis.

Using an Escherichia coli overproducing strain secreting Aspergillus oryzae RNase T1, we have constructed and characterized mutants where amino acid residues in the catalytic center have been substituted. The mutants are His40----Thr, Glu58----Asp, Glu58----Gln, His92----Ala and His92----Phe. His92----Ala and His92----Phe mutants are inactive. On the basis of their kcat/Km values, the mutants Glu58----Asp and Glu58----Gln show 10% and 7% residual activity, relative to wild-type RNase T1, whereas the His40----Thr mutant shows 2% activity. The effect of amino acid substitutions on the enzymatic activity of RNase T1 lends further support for a mechanism where Glu58 (possibly activated by His40 and His92 act as general base and acid respectively; this is discussed in terms of the known three-dimensional structure of the enzyme.

Modes of mononucleotide binding to ribonuclease T1.

The binding of the mononucleotide inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to genetically engineered ribonuclease T1 has been investigated by conventional inhibition kinetics, fluorimetric titrations, molecular modeling, and fast relaxation techniques. The fluorimetric titrations in conjunction with molecular modeling revealed that apart from the already known primary binding site, three to four additional sites are present on the enzyme's surface. The association constants obtained from the fluorimetric titrations and the temperature jump experiments range between 3.1 x 10(6) M-1 and 4.3 x 10(6) M-1, indicating that the binding of the mononucleotides to the specific binding site of ribonuclease T1 is at least one order of magnitude tighter than has been anticipated so far. The kinetics of binding are nearly diffusion controlled with a kon determined for 2'-GMP and 3'-GMP, as (5.0 +/- 0.5 x 10(9) and 6.1 +/- 0.5 x 10(9) M-1, s-1 and koff as 1.2 +/- 0.2 x 10(3) and 2.0 +/- 0.3 x 10(3) s-1, respectively. Molecular modeling studies indicate that all three nucleotides are able to bind via their phosphate group to a positively charged array of surface amino acids including His27, His40, Lys41, and most probably Lys25 without obvious stereochemical hindrance. We propose that RNA wraps around RNase T1 in a similar fashion via phosphate binding when enzymatic hydrolysis occurs.

Crystal structure of photosystem II from Synechococcus elongatus at 3.8 A resolution.

Oxygenic photosynthesis is the principal energy converter on earth. It is driven by photosystems I and II, two large protein-cofactor complexes located in the thylakoid membrane and acting in series. In photosystem II, water is oxidized; this event provides the overall process with the necessary electrons and protons, and the atmosphere with oxygen. To date, structural information on the architecture of the complex has been provided by electron microscopy of intact, active photosystem II at 15-30 A resolution, and by electron crystallography on two-dimensional crystals of D1-D2-CP47 photosystem II fragments without water oxidizing activity at 8 A resolution. Here we describe the X-ray structure of photosystem II on the basis of crystals fully active in water oxidation. The structure shows how protein subunits and cofactors are spatially organized. The larger subunits are assigned and the locations and orientations of the cofactors are defined. We also provide new information on the position, size and shape of the manganese cluster, which catalyzes water oxidation.

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: the tyrosine radical Y(D)(*).

Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y(D)(*) in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules/PS II; they are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four PS II dimers per unit cell. High-frequency EPR is used for enhancing the sensitivity of experiments performed on small single crystals as well as for increasing the spectral resolution of the g tensor components and of the different crystal sites. Magnitude and orientation of the g tensor of Y(D)(*) and related information on several proton hyperfine tensors are deduced from analysis of angular-dependent EPR spectra. The precise orientation of tyrosine Y(D)(*) in PS II is obtained as a first step in the EPR characterization of paramagnetic species in these single crystals.