Radicals produced by gamma-irradiation of hyperquenched glassy water containing 2'-deoxyguanosine-5'-monophosphate.

Hyperquenched glassy water (HGW) has been suggested as the best model for liquid water, to be used in low-temperature studies of indirect radiation effects on dissolved biomolecules (Bednarek et al. J. Am. Chem. Soc. 1996, 118, 9387). In the present work, these effects are examined by X-band electron spin resonance spectroscopy (ESR) in gamma-irradiated HGW matrix containing 2'-deoxyguanosine-5'-monophosphate. Analysis of the complex ESR spectra indicates that, in addition to OH(*) and HO2(*) radicals generated by water radiolysis, three species are trapped at 77 K:(i) G(C8)H(*) radical, the H-adduct to the double bond at C8; (ii) G(- *) radical anion, the product of electron scavenging by the aromatic ring of the base; and (iii) dR(-H)(*) radicals formed by H abstraction from the sugar moiety, predominantly at the C'5 position. We discuss the yields of the radicals, their thermal stability and transformations, as well as the effect of photobleaching. This study confirms our earlier suggestion that in HGW the H atom addition/abstraction products are created at 77 K in competition with HO2(*) radicals, in a concerted process following ionization of water molecule at L-type defect sites of the H-bonded matrix. The lack of OH(*) reactivity toward the solute suggests that the H-bonded structure in HGW is much more effective in recombining OH(*) radicals than that of aqueous glasses obtained from highly concentrated electrolyte solutions. Furthermore, complementary experiments for the neat matrix have provided evidence that HO2(*) radicals are not the product of H atom reaction with molecular oxygen, possibly generated by ultrasounds used in the process of sample preparation.

Raman spectroscopic study of hydrogen ordered ice XIII and of its reversible phase transition to disordered ice V.

Raman spectra of recovered ordered H(2)O (D(2)O) ice XIII doped with 0.01 M HCl (DCl) recorded in vacuo at 80 K are reported in the range 3600-200 cm(-1). The bands are assigned to the various types of modes on the basis of isotope ratios. On thermal cycling between 80 and 120 K, the reversible phase transition to disordered ice V is observed. The remarkable effect of HCl (DCl) on orientational ordering in ice V and its phase transition to ordered ice XIII, first reported in a powder neutron diffraction study of DCl doped D(2)O ice V (C. G. Salzmann, P. G. Radaelli, A. Hallbrucker, E. Mayer, J. L. Finney, Science, 2006, 311, 1758), is demonstrated by Raman spectroscopy and discussed. The dopants KOH and HF have only a minor effect on hydrogen ordering in ice V, as shown by the Raman spectra.

The preparation and structures of hydrogen ordered phases of ice.

Two hydrogen ordered phases of ice were prepared by cooling the hydrogen disordered ices V and XII under pressure. Previous attempts to unlock the geometrical frustration in hydrogen-bonded structures have focused on doping with potassium hydroxide and have had success in partially increasing the hydrogen ordering in hexagonal ice I (ice Ih). By doping ices V and XII with hydrochloric acid, we have prepared ice XIII and ice XIV, and we analyzed their structures by powder neutron diffraction. The use of hydrogen chloride to release geometrical frustration opens up the possibility of completing the phase diagram of ice.

Isobaric annealing of high-density amorphous ice between 0.3 and 1.9 GPa: in situ density values and structural changes.

We report in situ density values of amorphous ice obtained between 0.3 and 1.9 GPa and 144 to 183 K. Starting from high-density amorphous ice made by pressure-amorphizing hexagonal ice at 77 K, samples were heated at a constant pressure until crystallization to high-pressure ices occurred. Densities of amorphous ice were calculated from those of high-pressure ice mixtures and the volume change on crystallization. In the density versus pressure plot a pronounced change of slope occurs at approximately 0.8 GPa, with a slope of 0.21 g cm(-3) GPa(-1) below 0.8 GPa and a slope of 0.10 g cm(-3) GPa(-1) above 0.8 GPa. Both X-ray diffractograms and Raman spectra of recovered samples show that major structural changes occur up to approximately 0.8 GPa, developing towards those of very high-density amorphous ice reported by (T. Loerting, C. Salzmann, I. Kohl, E. Mayer and A. Hallbrucker, Phys. Chem. Chem. Phys., 2001, 3, 5355) and that further increase of pressure has only a minor effect. In addition, the effect of annealing temperature (T(A)) at a given pressure on the structural changes was studied by Raman spectra of recovered samples in the coupled O-H and decoupled O-D stretching band region: at 0.5 GPa structural changes are observed between approximately 100-116 K, at 1.17 GPa between approximately 121-130 K. Further increase of T(A) or of annealing time has no effect, thus indicating that the samples are fully relaxed. We conclude that mainly irreversible structural changes between 0.3 to approximately 0.8 GPa lead to the pronounced increase in density, whereas above approximately 0.8 GPa the density increase is dominated to a large extent by reversible elastic compression. These results seem consistent with simulation studies by (R. Martonàk, D. Donadio and M. Parrinello, J. Chem. Phys., 2005, 122, 134501) where substantial reconstruction of the topology of the hydrogen bonded network and changes in the ring statistics from e.g. mainly six-membered to mainly nine-membered rings were observed on pressure increase up to 0.9 GPa and further pressure increase had little effect.

Liquid-like relaxation in hyperquenched water at < or = 140 K.

Micrometre-sized water droplets were hyperquenched on a solid substrate held at selected temperatures between 150 and 77 K. These samples were characterized by differential scanning calorimetry (DSC) and X-ray diffraction. 140 K is the upper temperature limit to obtain mainly amorphous samples on deposition within 16-37 min. DSC scans of glassy water prepared at 140 K exhibit on heating an endothermic step assignable to glass --> liquid transition, with an onset temperature (T(g)) of 136 +/- 2 K on heating at 30 K min(-1). For T(g) of approximately 136 K, water relaxes during deposition at 140 K for 16 min, moving towards metastable equilibrium. The apparent increase in heat capacity (deltaC(p)) depends, for a given rate of heating, on the rate of prior cooling, and a so-called overshoot develops. 140 K deposits cooled at a rate of 5, 2 or 0.2 K min(-1) show on subsequent reheating at a rate of 30 K min(-1) deltaC(p) values of 0.7, 1.1 and 1.7 J K(-1) mol(-1). This is consistent with liquid-like relaxation at 140 K, and it indicates that different limiting structures are obtained. When these 140 K deposits are in addition annealed at 130 K for 90 min, after slow-cooling at 5, 2 or 0.2 K min(-1), their deltaC(p) values on subsequent reheating are similar to those of hyperquenched glassy water (HGW) deposits made at 77 K and annealed at 130 K. Thus, the previous deltaC(p) value of 1.6 J K(-1) mol(-1) obtained with glassy water samples annealed at 130 K (A. Hallbrucker, E. Mayer and G. P. Johari, Philos. Mag. B, 1989, 60, 179) must be an upper-bound limit because it contains a contribution from an overshoot. The T(g) value of 140 K deposits, which had relaxed during deposition towards metastable equilibrium, is within experimental error the same as that of 140 K deposits annealed in addition at 130 K. This contradicts Yue and Angell's (Y. Yue and C. Angell, Nature, 2004, 427, 717) claim for assigning the endothermic step to a sub-T(g) peak or a "shadow" T(g). Our new data further support the proposed fragile-to-strong transition on cooling liquid water from ambient temperature into the deeply supercooled and glassy state. We also describe in detail experimental aspects to obtain HGW specimens, show the ultrastructure of the deposits using electron microscopy, and discuss the mechanism of our hyperquenching method.

Water Behaviour: glass transition in hyperquenched water?

It has been unclear whether amorphous glassy water heated to around 140-150 K remains glassy until it crystallizes or whether instead it turns into a supercooled and very viscous liquid. Yue and Angell compare the behaviour of glassy water under these conditions to that of hyperquenched inorganic glasses, and claim that water stays glassy as it heats up to its crystallization point; they also find a 'hidden' glass-to-liquid transition at about 169 K. Here we use differential scanning calorimetry (DSC) heating to show that hyperquenched water deposited at 140 K behaves as an ultraviscous liquid, the limiting structure of which depends on the cooling rate--as predicted by theoretical analysis of the liquid-to-glass transition. Our findings are consistent with a glass-to-liquid transition-onset temperature (T(g)) in the region of 136 K (refs 3,4), and they indicate that measurements of the liquid's properties may clarify the anomalous properties of supercooled water.

Stepwise induced fit in the pico- to nanosecond time scale governs the complexation of the even-skipped transcriptional repressor homeodomain to DNA.

Induced fit effects in the complex of a DNA decamer with two even-skipped transcriptional repressor homeodomain molecules were investigated by means of molecular dynamics simulations. Dynamics of these effects are found to be in the time scale from pico- to nanoseconds. First steps are made by the fast-moving DNA backbone phosphates, which upon binding change their B(I)/B(II) substate distribution. Further rearrangements in the DNA double helix induced upon complexation, like bending of the helix axis, changes of the minor groove width, and of different helical parameters, are slower and occur within a few nanoseconds. The flexibility of the DNA, especially of its backbone, seems thereby to play an important role for specific DNA ligand recognition.

The conformer substates of nonoriented B-type DNA in double helical poly(dG-dC).

A nonoriented hydrated film of poly(dG-dC) with ?20 water molecules per nucleotide (called B* by Loprete and Hartman (Biochem. 32, 4077-4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)(2) dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on B(I)-to-B(II) interconversion in the classical B-DNA forms are very similar to those in the B*-form, and that the spectroscopic differences between the B(I) and B(II) features from classical B-DNA and those from the modified B*-form are minor. Nonexponential kinetics of the B(I)-->B(II) transition in the B*-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (approximately equal to 30 versus approximately equal to 10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is B(II)-->B(I) transition on cooling from 290 K and B(I)-->B(II) transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B*-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B*-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263-3274 (2002)).

PvuII-endonuclease induces structural alterations at the scissile phosphate group of its cognate DNA.

We investigated the PvuII endonuclease with its cognate DNA by means of molecular dynamics simulations. Comparing the complexed DNA with a reference simulation of free DNA, we saw structural changes at the scissile phosphodiester bond. At this GpC step, the enzyme induces the highest twist and axial rise, inclination is increased and the minor groove widened. The distance between the scissile phosphate group and the phosphate group of the following thymine base is shortened significantly, indicating a substrate-assisted catalysis. A feasible reason for this vicinity is the catalytically important amino acid residue lysine 70, which bridges the free oxygen atoms of the successive phosphate groups. Due to this geometry, a compact reaction pocket is formed where a water molecule can be held, thus bringing the reaction partners for hydrolysis into contact. The O1-P-O2 angle of the scissile nucleotide is decreased, probably due to a complexation of the negative oxygen atoms through protein and solvent contacts.

Indirect readout of the trp-repressor-operator complex by B-DNA's backbone conformation transitions.

Although the trp-repressor-operator complex is one of the best studied transcriptional controlling systems, some questions regarding the specific recognition of the operator by the repressor remain. We performed a 2.35 ns long molecular dynamics simulation to clarify the influence of the two B-DNA backbone conformational substates B(I) and B(II) on complexation. The trp-repressor-operator is an ideal biological system for this study because experimental results have already figured out that the interaction between the internucleotide phosphates and the protein is essential for the formation of the high affinity complex. Our simulation supports these results, but more important it shows a strong correlation between the B(I)/B(II) phosphate substate and the number of interactions with this phosphate. In particular the B(I) <==> B(II) transitions occur synchronous to hydrogen bond breaking or formation. To the best of our knowledge, this was observed for the first time. Thus, we conclude that the sequence specific B(I)/B(II) behavior contributes via indirect readout to sequence specific recognition. These results have implication for the design of transcription-controlling drugs in view of the recently published influence of minor groove binders on the B(I)/B(II) pattern. The simulation also agrees with crystallographically observed hydration sites. This is consistent with experimental results and indicates the correctness of the model used.

Towards the experimental decomposition rate of carbonic acid (H2CO3) in aqueous solution.

Dry carbonic acid has recently been shown to be kinetically stable even at room temperature. Addition of water molecules reduces this stability significantly, and the decomposition (H2CO3 + nH2O --> (n+1)H2O + CO2) is extremely accelerated for n = 1, 2, 3. By including two water molecules, a reaction rate that is a factor of 3000 below the experimental one (10 s(-1)) at room temperature was found. In order to further remove the gap between experiment and theory, we increased the number of water molecules involved to 3 and took into consideration different mechanisms for thorough elucidation of the reaction. A mechanism whereby the reaction proceedes via a six-membered transition state turns out to be the most efficient one over the whole examined temperature range. The determined reaction rates approach experimental values in aqueous solution reasonably well; most especially, a significant increase in the rates in comparison to the decomposition reaction with fewer water molecules is found. Further agreement with experiment is found in the kinetic isotope effects (KIE) for the deuterated species. For water-free carbonic acid, the KIE (i.e., kH2CO3/kD2CO3) for the decomposition reaction is predicted to be 220 at 300 K, whereas it amounts to 2.2-3.0 for the investigated mechanisms including three water molecules. This result is therefore reasonably close to the experimental value of 2 (at 300 K). These KIEs are in much better accordance with the experiment than the KIE for decomposition with fewer water entities.

About the stability of sulfurous acid (H2SO3) and its dimer.

The characterization and isolation of sulfurous acid (H2SO3) have never been accomplished and thus still remain one of the greatest open challenges of inorganic chemistry. It is known that H2SO3 is thermodynamically unstable. In this study, however, we show that a Ci-symmetric dimer of sulfurous acid (H2SO3)2 is 3.5 kcal mol-1 more stable than its dissociation products SO2 and H2O at 77 K. Additionally, we have investigated the kinetic stability of the sulfurous acid monomer with respect to dissociation into SO2 and H2O and the kinetic isotope effect (KIE) on this reaction by transition-state theory. At 77 K, the half-life of H2SO3 is 15 x 10(9) years, but for the deuterated molecule (D2SO3) it increases to 7.9 x 10(26) years. At room temperature, the half-life of sulfurous acid is only 24 hours; however, a KIE of 3.2 x 10(4) increases it to a remarkable 90 years. Water is an efficient catalyst for the dissociation reaction since it reduces the reaction barrier tremendously. With the aid of two water molecules, one can observe a change in the reaction mechanism for sulfurous acid decomposition with increasing temperature. The most likely mechanism below 170 K is via an eight-membered transition-state ring; yet, above 170 K, a mechanism with a six-membered transition state ring becomes the predominant one. For deuterated sulfurous acid, this change in reaction mechanism can be observed at 120 K. Consequently, between 120 and 170 K, different predominant reaction mechanisms occur for the decomposition of normal and deuterated sulfurous acid when assisted by two water molecules. However, the much longer half-life of deuterated sulfurous acid and the stability of the sulfurous acid dimer at 77 K are encouraging for future synthesis and characterization under laboratory conditions.