Ortho-para spin isomers of the protons in the methylene group--possible implications for protein structure

The two hydrogen atoms attached to the carbon in the methylene group are of two different spin configurations, similar to those in the case of water: ortho, where the two proton spins are parallel to each other, and para, where they are antiparallel. The ortho configuration has three degenerate states, while the para configuration is singular, leading to a statistical ratio of these isomers 3:1 ortho/para. Such spin isomers are present in glycine and most chiral amino acids where they may induce broadening of structural zones, a possibility which remains to be assessed. The implications of this neglected possibility could be far-reaching, in particular with respect to protein structure and the origins of biochirality.     

Spin-dependent absorption of water molecules

The effects of spin state of water molecules on its absorption on lyophilized DNA, lysozyme and some inorganic sorbents were studied. It was shown that the absorption rates of ortho and para water from vapor differ noticeably. The para isomer binding with preparations is distinctly faster than that of the ortho isomer in all cases. Clear-cut distinction in the sorption kinetics is determined by the difference in quantum statistics for spin isomers, which in its turn can give rise to remarkable differences in physico-chemical properties of ortho and para water. This finding opens a wide field of activity in studying fundamental and applied problems relating to the role of the spin state of water molecules in physics, chemistry, biology and medicine.     

Spin isomeric selectivity of water molecules upon DNA hydration

Four-photon coherent scattering of laser radiation was used to study the influence of DNA on the content of quasi-free ortho and para isomers of water molecules in its aqueous solution. It was shown that the concentration of quasi-free molecules that form the rotational spectrum of spin isomers increases considerably in the hydration shell of the DNA molecule as compared with pure water. The increase in the concentration of spin isomers occurs disproportionally. In the presence of DNA, the intensity of the rotational spectrum of ortho isomers is on the average much greater than that of para isomers. It was also demonstrated that the character of hydration and the ortho/para ratio change noticeably upon DNA denaturation, which may be evidence of changes in preferable solvation of DNA during its denaturation. The data obtained allowed us to assume that the stability of different biologically important states of macromolecules can be changed by varying the relative concentration of water spin isomers in solution.     

Ortho/para spin-isomers of H2O molecules as a factor responsible for formation of two structural motifs in water

According to the latest results obtained by small-angle X-ray scattering and X-ray spectroscopy, it was suggested that water on a nanometer scale represents a fluctuating mixture of clusters with tetrahedral structure and a subphase with partially broken hydrogen bonds, whereas the nuclear configuration of the H₂O molecule corresponds to single tetrahedral coordination. The basic reason of such structural partition is not clear until now. Here we show that it can be associated with existence of two nuclear H₂O spin isomers that have different probability to be in one or the other subphase. The para molecule can transfer an excess of its rotational energy to the environment, up to complete stopping of rotation because its rotational quantum number J = 0 in the basic state. This property is favorable for formation of clusters with closed H-bonds. Ortho molecules with odd-numbered J states lack this property and thus should be predominantly present in the locations with impaired bonds.     

Effect of quantum differences of ortho and para H2O spin-isomers on water properties: Biophysical aspect

Quantum differences of ortho/para H₂O spin-isomers are considered as a key factor that determines water property taking into account the ortho/para conversion and the unbalanced (1: 1) ortho/para ratio in water. The biophysical mechanism of jump in the permeability of erythrocytes through a microcapillary at 36.6°C in water-based physiological solution and at 37.4°C in heavy water is proposed and discussed.     

Structural basis of the genotoxicity of nitrosatable phenols and derivatives present in smoked food products

The CASE methodology for studying structure-activity relationships has been applied to investigating the basis of the genotoxicity of phenols and derivatives following exposure to nitrous acid. The structural features identified include availability of positions para or ortho to the hydroxyl groups, that one meta position must remain unoccupied and one ortho or para position must be unsubstituted as well. The analyses revealed that genotoxicity is dependent upon the ease of formation of the active phenyldiazonium intermediate and is influenced only secondarily by the nature of the genotoxicant or its ease of entry into the cell. With this data base, CASE predicts the genotoxicity, following nitrosation, of a number of agents, including serotonin, acetaminophen, and of some naturally-occurring pesticides present in edible plants.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.     

Regular oscillatory behavior of aqueous solutions of CuII salts related to effects on equilibrium dynamics of ortho/para hydrogen spin isomers of water

Cell surface and growth-related NADH oxidases with protein disulfide-thiol interchange activity, ECTO-NOX, exhibit copper-dependent, clock-related, temperature-independent and entrainable patterns of regular oscillations in the rate of oxidation of NAD(P)H as do aqueous solutions of copper salts. Because of time scale similarities, a basis for the oscillatory patterns in nuclear spin orientations of the hydrogen atoms of the copper-associated water was sought. Extended X-ray absorption fine structure (EXAFS) measurements at 9302 eV on pure water were periodic with a ca. 3.5 min peak to peak separation. Decomposition fits revealed 5 unequally spaced maxima similar to those observed previously for Cu(II)Cl(2) to generate a period length of about 18 min. With D(2)O, the period length was proportionately increased by 30% to 24 min. The redox potential of water and of D(2)O also oscillated with 18 and 24 min period lengths, respectively. Measurements in the middle infrared spectral region above a water sample surface revealed apparent oscillations in the two alternative orientations of the nuclear spins (ortho and para) of the hydrogen atoms of the water or D(2)O with 5 unequally spaced maxima and respective period lengths of 18 and 24 min. Thus, the time keeping oscillations of ECTO-NOX proteins appear to reflect the equilibrium dynamics of ortho-para hydrogen atom spin ratios of water where the presence of metal cations such as Cu(II) in solution determine period length.     

Density functional theory calculations for resveratrol

The calculations based on the density functional theory (DFT) have been used to study the structure-activity of resveratrol in the chain reaction of autooxidation. According to the geometry obtained by using a B3LYP/6-31G**, the HOMO, LUMO of resveratrol and the spin density, the single electron distribution of the 4'- and 5-radical of resveratrol were calculated, it was found that resveratrol is a potential antioxidant. The 4'-hydroxyl group of resveratrol is more reactive than 3- and 5-positions because of the resonance effects. The dominant structure of the resveratrol radicals is a semiquinone structure which determines the stability of radicals, and the unpaired electron is mainly distributed to the O-atom and its ortho and para positions. The antioxidant activity of resveratrol is related to the spin density and the unpaired electron distribution of the O-atom.     

DNA cleavage by hydroxy-salicylidene-ethylendiamine-iron complexes.

Bis(hydroxy)salen.Fe complexes were designed as self-activated chemical nucleases. The presence of a hy-droxyl group on the two salicylidene moieties serve to form a hydroquinone system cooperating with the iron redox system to facilitate spontaneous formation of free radicals. We compared the DNA binding and cleaving properties of the ortho -, meta- and para -(bishydroxy) salen.Fe complexes with that of the corresponding chelate lacking the hydroxyl groups. DNA melting temperature studies indicated that the para complex exhibits the highest affinity for DNA. In addition, this para compound was considerably more potent at cleaving supercoiled plasmid DNA than the regio-isomeric ortho - and meta -hydroxy-salen.Fe complexes, even in the absence of a reducing agent, such as dithiothreitol used to activate the metal complex. The DNA cleaving activity of the para isomer is both time and concentration dependent and the complexed iron atom is absolutely essential for the sequence uniform cleavage of DNA. From a mechanistic point of view, electron spin resonance measurements suggest that DNA contributes positively to the activation of the semi-quinone system and the production of ligand radical species responsible for subsequent strand scission in the absence of a reducing agent. The para -hydroxy-salen.Fe complex has been used for detecting sequence-specific drug-DNA interactions. Specific binding of Hoechst 33258 to AT sequences and chromomycin to GC sequences were shown. The para -bis(hydroxy)salen.Fe derivative complements the tool box of footprinting reagents which can be utilised to produce efficient cleavage of DNA.     

Free radical production by hydroxy-salen manganese complexes studied by ESR and XANES

Three salen-Mn(II) complexes bearing hydroxyl groups in either the ortho, para or meta positions have been synthesized and the structures of the metal complexes and their potential to produce free radicals investigated by electron spin resonance (ESR) and X-ray absorption near edge structures (XANES) spectroscopy. All three compounds were shown to generate a high level of superoxide anions in dimethyl sulfoxide (DMSO) solution. The production of oxygen radicals results from a one electron process oxidation of Mn(II) species leading to the formation Mn(III) redox state species, as revealed by a higher XANES edge energy of 2.7 eV. The formation of superoxide anion was characterized by ESR, both directly and via the use of a spin-trapping method. Under reductive condition in the presence of ascorbic acid, the reduction of Mn(III) to Mn(II) leads to the production of hydroxyl radicals by the ortho and para compounds. The efficient production O(2)*- by such salen-Mn complexes could be useful to evaluate the scavenging properties of antioxidant molecules.     

Study of the stability of aluminium trimeric clusters in aqueous solutions

Ab initio molecular dynamics (AIMD) based on density functional theory has been used to study small aluminium–oxygen complexes in water. Such Al–O clusters have been seen in several recent mass spectrometry studies. In this study, we have focused on trimeric Al–O clusters. The initial very compact trimeric Al–O structures opened up and formed linear Al–O chains. The typical Al–O coordination number in these chain structures was 5. We have performed long (up to 200 ps) AIMD simulations and these chain structures are stable on the nanosecond time scale. We have also studied the reactivity of the Al–O dimer and solvated Al. We found a formation path for the trimeric cluster, which has a action barrier (0.04 eV) and a reaction free energy of ? 0.55 eV. This suggests that the association of a dimer and a monomer Al–O species is fast and thermodynamically a very favourable process.     

ISC-like [2Fe–2S] ferredoxin (FdxB) dimer from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JCM 20004: structural and electron–nuclear double resonance characterization

The crystal structure of the ISC-like [2Fe–2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron–nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe³⁺ valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g _max direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe–2S] plane, whereas the g _int and g _min directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe–2S] proteins in different electron transport chains (e.g. adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe–2S] cluster under the influence of the protein environment around each iron site in general.     

A first-principles study of Ni<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript>Pd<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> (n = 1 − 5) clusters

A first-principle investigation of structures and properties of Ni _n Pd _n (n=1-5) clusters is presented. For this study, the linear combination of Gaussian-type orbitals auxiliary density functional theory (LCGTO-ADFT) method has been employed. In order to determine the lowest energy structures, several isomers in different spin multiplicities were studied, for each cluster size. Initial structures, for which successive geometry optimization was computed without any constrain, were taken along Born–Oppenheimer molecular dynamics (BOMD) trajectories. To discriminate between minima and transition state structures, harmonic frequency analyses were performed at the optimized structures. Ground state structures, bond lengths, harmonic frequencies, dissociation energy, ionization potential, electron affinity and spin density plots are presented. This work demonstrates, that the Pd atoms prefer to allocate on the surface of the cluster structures whose core is formed by the 3d TM atoms type. Moreover, it has been observed that the ground-state structure spin multiplicity increases as the system size grows. The results of this study contribute to gain insight into how structures and energy properties change with cluster size in bimetallic Pd-based alloys.     

Similarities between l-Phenylalanine and m-Fluorophenylalanine as Substrates for l-Phenylalanine Ammonia-Lyase

The ability of l -phenylalanine ammonia-lyase (E.C. 4.3.1.5) to metabolize dl -m-, dl -o- and dl -p-fluorophenylalanine in Avena sativa has been examined. Although all three amino acid analogues served as substrates for this enzyme, there was a marked difference in the behavior of the meta isomer from that of the para and ortho species. The Michaelis constant for the meta analogue was similar to that obtained for the natural substrate, l -phenylalanine, but distinct from the kinetic data for the para and ortho isomers. In addition, in vivo analyses demonstrated that both l -phenylalanine and the meta-fluoro-derivative served to protect against chlorogenic acid loss, whereas previous studies have shown that the para and ortho species depressed levels of this phenolic derivative. Finally, treatment of coleoptile apices with either the meta isomer or l -phenylalanine reversed dl -p-fluorophenylalanine stimulated growth and attendant reduction in chlorogenic acid content. These findings provide further clarification of the effects of fluorophenylalanines upon l -phenylalanine ammonia-lyase mediated biosynthesis of low molecular weight phenols in Avena.     

Nature of the Stacking of Nucleic Acid Bases in Water: a Monte Carlo Simulation

Abstract

The results of a Monte Carlo simulation of the hydration of uracil and thymine molecules, their stacked dimers and hydrogen-bonded base pairs are presented. Simulations have been performed in a cluster approximation. The semiempirical atom-atom potential functions have been used (cluster consisting of 200 water molecules). It has been shown that the stacking interactions of uracil and thymine molecules in water arise mainly due to the increase in the water-water interaction during the transition from monomers to dimer. It has been found out that stacked base associates are more preferable than base pairs in water. This preference is mainly due to the energetically more favourable structure of water around the stack.     

Molecular orbital study of porphyrin-substrate interactions in cytochrome P450 catalysed aromatic hydroxylation of substituted anilines

The reaction mechanism for the primary reaction step of the hydroxylation of 3-fluoro-6-methylaniline, attacked at different positions (oxygen attack across a C-C bond and direct attack at positions para and ortho with respect to the NH(2)-group) catalysed by a high-valent ferryl-oxo porphyrin a(2u)-cation complex with H(3)CS(-) as an axial ligand, has been investigated on the basis of electronic structure calculations in local spin-density approximation. Non-repulsive potential curves are obtained only in cases of direct attack at the para- and ortho-positions with respect to NH(2), but not for epoxide formation. Comparing the potential curves for the hydroxylation at the positions para and ortho to the NH(2)-group, an attack at the para-position is more likely. The relative orientation of the substrate towards the porphyrin is essentially determined by the interaction between the substituents of the substrate and the porphyrin. Consequently, different geometrical orientations of the substrate are obtained for hydroxylation at the para- and ortho-positions. In both cases of direct attack the substrate plane is not parallel to the porphyrin plane. The decisive role of sulphur in the hydroxylation is demonstrated by the participation of the S(3p)-orbitals in all molecular orbitals involved in the reaction.     

A new view of water dynamics in immobilized proteins.

The inflection frequency of the deuteron magnetic relaxation dispersion from water in rotationally immobilized protein samples has recently been found to be essentially independent of temperature and protein structure. This remarkable invariance has been interpreted in terms of a universal residence time of 1 microseconds for protein-associated water molecules. We demonstrate here that this interpretation is an artifact of the conventional perturbation theory of spin relaxation, which is not valid for rotationally immobile proteins. Using a newly developed non-perturbative, stochastic theory of spin relaxation, we identify the apparent correlation time of 1 microseconds with the inverse of the nuclear quadrupole frequency, thus explaining its invariance. The observed dispersion profiles are consistent with a broad distribution of residence times, spanning the microseconds range. Furthermore, we argue that the deuteron dispersion is due to buried water molecules rather than to the traditional surface hydration previously invoked, and that the contribution from rapidly exchanging protein hydrogens cannot be neglected. The conclusions of the present work are also relevant to proton relaxation in immobilized protein samples and to magnetic resonance imaging of soft tissue.     

Bromo and chlorobiphenyl metabolism: gas chromatography mass spectrometric identification of urinary metabolites and the effects of structure on their rates of excretion

The identification of the hydroxylated rat urinary metabolites of the 2-, 3- and 4-chlorobiphenyls and 2-, 3- and 4-bromobiphenyls has been determined by gas chromatographic mass spectrometric analysis of their corresponding methyl ether derivatives. The electron impact fragmentation patterns of the bromotheoxybiphenyls and chloromethoxybiphenyls were used to assign the position of the methyoxyl group (ortho, meta or para to the biphenyl bond); the mass spectra of the corresponding [2H5]halobiphenyls confirmed the sites of the hydroxylation by distinguishing between the halophenyl and phenyl rings. The results illustrated that ring hydroxylation occurs predominantly at the para positions of the biphenyl nucleus and at sites which are ortho and para to the halogen substituents. 4,4'-Dimethoxyhalobiphenyls are major urinary metabolites of the 2- and 3-halobiphenyls and the rapid formation of these metabolites is illustrated in a time course study which monitors the urinary metabolites formed after the separate coadministration of the isomeric chlorobiphenyl and bromobiphenyl substrates to rats.     

Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: Application to HU protein from Bacillus stearothermophilus

A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment. In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra. Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence. The range of NOE-graph templates of secondary structure motifs, incorporating α-helices and β-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins. The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy. The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (¹³C, ¹⁵N) heteronuclear single quantum correlation spectrum of the (α + β) type protein HU from Bacillus stearothermophilus. The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU. In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the β-arm region (residues 53–76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the β-arm at Ala57 and Pro72, respectively. At these residues, both strands of the β-arm appear to flip inside out before continuing as a regular antiparallel β-sheet. © 1996 John Wiley & Sons, Inc.