Chemical changes in the photoreceptor outer segments due to iron induced oxidative stress: analysis by Fourier transform infrared (FT-IR) microspectroscopy.

Oxidative stress is thought to be an important pathogenic mechanism in many diseases of the retina. The purpose of this study was to investigate the chemical changes that are present in the photoreceptor outer segments of the retina following exposure to oxidative stress. Fourier transform infrared (FT-IR) microspectroscopy enables the characterization and semi-quantitation of chemical functional groups in microscopic regions of tissue sections. This technique was used to evaluate the chemical changes in the outer segments following exposure to ferrous sulfate, which promotes oxidative tissue damage. A reduction of C=C-H and C=O functional groups was observed in the outer segments of iron-injected eyes compared to vehicle-injected eyes at 3 days following injection, which is prior to major histological changes that occur by 7 days. These functional groups are found in docosahexaenoic acid (DHA), which is present at a high concentration in the outer segments. DHA contains a series of six cis-conjugated double bonds, which are vulnerable to free radical attack, and the reduction of these unsaturation group absorptions suggests that DHA was degraded and/or removed from the outer segments. An unexpected finding was that several other chemical functional groups increased in concentration over time in the outer segments of vehicle-injected eyes compared to non-injected eyes. These increases generally did not include C=C-H or C=O, which suggests that either DHA was being degraded while other organic molecules were being concentrated, or that production of DHA failed to be upregulated in vehicle-injected eyes. In summary, there was a loss of both C=C-H and C=O functional group concentrations in the outer segments of iron-injected eyes, and there was an increased concentration of several other chemical functional groups following trauma induced by vehicle injection.     

Non-classical hydrogen bonds in interleukins: the role of C-H...O interactions

The role of classical hydrogen bonds in the structural stability of biological macro-molecules is well understood. In the present study, we explore the influence of C-H...O interactions in relation to other environmental preferences in interleukins. Main chain-main chain interactions are predominant. Pro residues might stabilize helices and strands by C-H...O H-bonds in interleukins. Majority of the C-H...O interacting residues were solvent exposed. 62% of C-H...O interactions was long-range interactions. The results presented in this study might be useful for structural stability studies in interleukins.     

Enzymatic cross-linking of beta-lactoglobulin: conformational properties using FTIR spectroscopy

In this study, we use FTIR spectroscopy to probe the conformational changes of beta-lactoglobulin (beta-LG)-the main constituent of whey proteins-as subjected to enzymatic cross-linking by transglutaminase. We investigate both the amide I region (1600-1700 cm(-1)) and the C-H stretching region (2800-3100 cm(-1)). In the amide I region, spectra of denatured conformations of beta-LG, known to be necessary for cross-linking, differ according to the denaturation procedure, i.e., chemical or thermal treatment. Denaturation by chemical denaturants, dithiothreitol (DTT) or beta-mercaptoethanol, show no effect on the alpha-helix, while shifting the monomer dimer equilibrium toward higher monomer concentration. On the other hand, denaturing by thermal treatment dissociates the beta-sheets in the native structure, leading to new intermolecular beta-sheets being formed. Preheated then enzyme cross-linked beta-LG molecules show very similar spectra in the amide I region to the molecules with no cross-linking, indicating minimal effects of the cross-links on the carbonyl stretching mode. However, chemically denatured (using beta-mercaptoethanol) then enzyme cross-linked beta-LG molecules show noticeable diminution in the alpha-helix band and formation of strong hydrogen-bonded intermolecular beta-sheets. In the C-H stretching region, preheated then enzyme cross-linked beta-LG molecules exhibit a different degree of exposure of aliphatic amino acids due to the enzyme action. The same behavior is observed for DTT-treated then enzyme cross-linked beta-LG molecules. Generally, the changes in the C-H stretching region clearly indicate that hydrophobic interactions are altered upon enzymatic cross-linking.     

A CON-based NMR assignment strategy for pro-rich intrinsically disordered proteins with low signal dispersion: the C-terminal domain of histone H1.0 as a case study

The C-terminal domain of histone H1.0 (C-H1.0) is involved in DNA binding and is a main determinant of the chromatin condensing properties of histone H1.0. Phosphorylation at the (S/T)-P-X-(K/R) motifs affects DNA binding and is crucial for regulation of C-H1.0 function. Since C-H1.0 is an intrinsically disordered domain, solution NMR is an excellent approach to characterize the effect of phosphorylation on the structural and dynamic properties of C-H1.0. However, its very repetitive, low-amino acid-diverse and Pro-rich sequence, together with the low signal dispersion observed at the ¹H–¹⁵N HSQC spectra of both non- and tri-phosphorylated C-H1.0 preclude the use of standard ¹H-detected assignment strategies. We have achieved an essentially complete assignment of the heavy backbone atoms (¹⁵N, ¹³C′ and ¹³C_α), as well as ¹H^N and ¹³C_β nuclei, of non- and tri-phosphorylated C-H1.0 by applying a novel ¹³C-detected CON-based strategy. No C-H1.0 region with a clear secondary structure tendency was detected by chemical shift analyses, confirming at residue level that C-H1.0 is disordered in aqueous solution. Phosphorylation only affected the chemical shifts of phosphorylated Thr’s, and their adjacent residues. Heteronuclear {¹H}–¹⁵N NOEs were also essentially equal in the non- and tri-phosphorylated states. Hence, structural tendencies and dynamic properties of C-H1.0 free in aqueous solution are unmodified by phosphorylation. We propose that the assignment strategy used for C-H1.0, which is based on the acquisition of only a few 3D spectra, is an excellent choice for short-lived intrinsically disordered proteins with repetitive sequences.     

Progress towards the easier use of P450 enzymes

The cytochrome P450 enzymes (P450s or CYPs) form a large family of heme proteins involved in drug metabolism and in the biosynthesis of steroids, lipids, vitamins and natural products. Their remarkable ability to catalyze the insertion of oxygen into non-activated C-H bonds has attracted the interest of chemists for several decades. Very few chemical methods exist that directly hydroxylate aliphatic or aromatic C-H bonds, and most of them are not selective or of limited scope. Biocatalysts such as P450s represent a promising alternative: however, their applications have been limited by substrate specificity, low activity, poor stability and the need for cofactors. This review covers the attempts to overcome these limitations using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization.     

Enzymatic C-H activation by metal-superoxo intermediates

The mechanisms of four enzymes that initiate oxidation of their substrates by using mid-valent metal-superoxo intermediates, rather than the more frequently described high-valent iron-oxo complexes, to cleave relatively strong C-H bonds have come into focus in the past several years. In two of these reactions, the alternative manifold for O2 and C-H activation enables unique four-electron oxidation reactions, thus significantly augmenting Nature's arsenal for transformation of aliphatic carbon compounds. General principles of this alternative manifold, including common kinetic characteristics and thermodynamic limitations, are emerging. Recent, combined experimental and computational studies on other systems have shown how a more thorough understanding of the structures of the metal-superoxo intermediates and the mechanisms by which they cleave C-H bonds might be achieved.     

Mechanistic insights into C-H activation from radical clock chemistry: oxidation of substituted methylcyclopropanes catalyzed by soluble methane monooxygenase from Methylosinus trichosporium OB3b

The soluble methane monooxygenase (MMO) system isolated from Methylosinus trichosporium OB3b catalyzes the adventitious oxidation of alkyl substituted methylcyclopropanes. If the chemical mechanism of C-H activation by MMO involves formation of a radical or carbocation intermediate at the methyl C-H of these 'radical clock' substrates, then cyclopropyl ring opened alcohols may appear in the product mixture due to rearrangement of the intermediate. The lifetime of radical intermediates can be determined from known rearrangement rate constants, k(r). Rearrangement was observed during the oxidation of 1,1,2,2-tetramethylcyclopropane (k(r)=1.7-17. 5x10(8) s(-1), 30 degrees C) but not for cis- or trans-1, 2-dimethylcyclopropane (k(r)=1.2-6.4x10(8) s(-1), 30 degrees C) or the very fast radical clock, trans-2-phenylmethylcyclopropane (k(r)=3.4x10(11) s(-1), 30 degrees C). The results show that the occurrence of rearranged products fails to correlate with either the chemical nature of the C-H bond being broken, which is very similar for all of the methylcyclopropanes studied here, or the magnitude of the radical k(r) value. This study suggests that the steric properties of the substrate play an important role in determining the outcome of the reaction. Substrates with bulky substituents near the C-H bond that is attacked appear to yield intermediates with sufficient lifetimes to rearrange. In contrast, substrates with less steric bulk are postulated to be able to approach the reactive oxygen species in the MMO active site more closely so that intermediates are either rapidly quenched or undergo subsequent interaction with the dinuclear iron cluster of MMO that prevents rearrangement.     

One and two dimensional 1H and 13C high resolution NMR investigation of lariat ethers and their alkali metal ionic complexes: a more tangible evidence for the presence of less common C-H***O hydrogen bonds

The possible existence of less common hydrogen bonds in three lariat ethers and their alkali-metal ionic complexes have been investigated with one- and two-dimensional (1D and 2D) proton and carbon-13 high resolution liquid state NMR spectroscopy. The occurrence of hydrogen-bonding induced by the addition of metal ions has been identified with the observation of indirect dipolar coupling between the coupling partners involved in the hydrogen-bonding. The addition of metal ions, moreover, causes appreciable change of chemical shift of several protons and carbons. The chemical shift change depends on the ion radius, larger ions causing smaller change. Moreover, the change of chemical shift is in coincidence with the occurrence of hydrogen-bonding. The values of the coupling constants have been obtained for each of these hydrogen bonds and were used for evaluating the hydrogen-bond strength. An intriguing and surprising observation is that a C-H***O hydrogen bond identified in solution by this work was not found in the previous study with X-ray diffraction or other methods.     

Quantum-chemical analysis of C-H...O and C-H...N interactions in RNA base pairs--H-bond versus anti-H-bond pattern

Geometries and interaction energies of unusual UU and AA base pairs with one standard hydrogen bond (H-bond) and additional C-H...O or C-H...N contacts have been determined by quantum-chemical methods taking into account electron correlation. Whereas the C-H bond length in the UU C-H...O contact increases upon complex formation (H-bond pattern), the C-H bond of the AA C-H....N interaction is shortened (anti-H-bond pattern). The same properties are found for model complexes between U or A and formaldehyde that have intermolecular C-H...acceptor contacts but no standard H-bonds. Both the C-H...acceptor H-bond and anti-H-bond interactions are attractive. A possible influence of the donor CH group charge distribution on the interaction pattern is discussed.     

Probing the relative timing of hydrogen abstraction steps in the flavocytochrome b2 reaction with primary and solvent deuterium isotope effects and mutant enzymes

Flavocytochrome b(2) catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate O-H and C-H bonds by the wild-type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The (D)V(max) and (D)(V/K(lactate)) values are both 3.0 with the wild-type enzyme at pH 7.5 and 25 degrees C, increasing to about 3.6 with the flavin domain and increasing further to about 4.5 with the D282N enzyme. Under these conditions, the (D20)V(max) values are 1.38, 1.18, and 0.98 for the wild-type enzyme, the flavin domain, and the D282N enzyme, respectively; the (D20)(V/K(lactate)) values are 0.9, 0.44, and 1.0, respectively. The (D)k(red) value is 5.4 for the wild-type enzyme and 3.5 for the flavin domain, whereas the solvent isotope effect on this kinetic parameter is 1.0 for both enzymes. The V(max) values for the wild-type enzyme and the flavin domain are 32 and 15% limited by viscosity, respectively. In contrast, the V/K(lactate) value for the flavin domain increases about 2-fold at moderate concentrations of glycerol. The data are consistent with a minimal chemical mechanism in which the lactate hydroxyl proton is not in flight in the transition state for C-H bond cleavage and there is an internal equilibrium involving the lactate-bound enzyme prior to C-H bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium.     

Observation of a unique pattern of bifurcated hydrogen bonds in the crystal structures of the N-glycoprotein linkage region models

Elucidation of the intra- and intermolecular carbohydrate-protein interactions would greatly contribute toward obtaining a better understanding of the structure-function correlations of the protein-linked glycans. The weak interactions involving C-H...O have recently been attracting immense attention in the domain of biomolecular recognition. However, there has been no report so far on the occurrence of C-H...O hydrogen bonds in the crystal structures of models and analogs of N-glycoproteins. We present herein an analysis of C-H...O interactions in the crystal structures of all N-glycoprotein linkage region models and analogs. The study reveals a cooperative network of bifurcated hydrogen bonds consisting of N-H...O and C-H...O interactions seen uniquely for the models. The cooperative network consists of two antiparallel chains of bifurcated hydrogen bonds, one involving N1-H, C2'-H and O1' of the aglycon moiety and the other involving N2-H, C1-H and O1' of the sugar. Such bifurcated hydrogen bonds between the core glycan and protein are likely to play an important role in the folding and stabilization of proteins.     

Quantum-Chemical Analysis of C-H…O and C-H…N Interactions in RNA Base Pairs—H-Bond Versus Anti-H-Bond Pattern

Abstract

Geometries and interaction energies of unusual UU and AA base pairs with one standard hydrogen bond (H-bond) and additional C-H…O or C-H…N contacts have been determined by quantum-chemical methods taking into account electron correlation. Whereas the C-H bond length in the UU C-H…O contact increases upon complex formation (H-bond pattern), the C-H bond of the AA C-H….N interaction is shortened (anti-H-bond pattern). The same properties are found for model complexes between U or A and formaldehyde that have intermolecular C-H…acceptor contacts but no standard H-bonds. Both the C-H…acceptor H-bond and anti-H-bond interactions are attractive. A possible influence of the donor CH group charge distribution on the interaction pattern is discussed.     

13C-N.M.R.-spectroscopic investigation of agarose oligomers

A complete, unambiguous assignment of all of the ¹³C-n.m.r.-spectral signals of agarose oligomers produced by enzymic hydrolysis has been achieved. The ¹J¹³C-H coupling constants are reported, and the chemical shifts and coupling constants of both the agarose polymer and oligomers are compared.     

A neutral molecule in a cation-binding site: specific binding of a PEG-SH to acetylcholinesterase from Torpedo californica

The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.     

Approaches towards the stabilization of hemiaminal function at ornithine unit of mulundocandin

Semisynthetic modifications at position-12 (ornithine-5-position, hemiaminal function) of mulundocandin were carried out to improve its chemical stability. New carbon-carbon (C-C) and carbon-hydrogen (C-H) linkage at hemiaminal function -12 has been achieved. Lewis acid catalyzed introduction of electron rich aryl group at position-12 of mulundocandin is developed. Synthesized mulundocandin analogues were evaluated for their chemical stability and antifungal activity against C. albicans and A. fumigatus.     

Computer study of intramolecular ordering in octadecatriene chains with cis-double bonds

Computer simulations of isolated unperturbed hydrocarbon molecules of C18:3 with methylene-interrupted cis double bonds were carried out using the Monte Carlo method based on the continuum model. A molecule-fixed coordinate system (with the axes along the principal axes of inertia of each molecule conformation) was used. The orientation distribution functions rho and order parameters S for C-H and C-C bonds relative to the maximum molecule span axis were calculated. It was shown that the widths of functions rho (factor of bond "fluctuations") are dependent on the chemical structure and position of the segment, fluctuations increase from the centre of the chain towards the terminals, all things being equal. The orientation distributions rho of C-H bonds flanking the double bond are the most narrow, the functions rho of CH2-groups located between two double bonds are the widest. It turned out that order parameter -SCH profiles of isolated chains of C18:3 include both the positive and negative values. The parameter SCC odd-even effect in unsaturated molecules of such structure changes the sign between double bonds.     

Free radical generation in the toxicity of inhaled mineral particles: the role of iron speciation at the surface of asbestos and silica

Free radical generation at the particle/biological fluid interface is one of the chemical processes that contributes to pathogenicity. In order to investigate the role played by iron, fibres of crocidolite asbestos have been modified by thermal treatments to alter their surface iron content. Two radical mechanisms, HO* from H2O2 and cleavage of a C-H bond, which are both active on the original fibres, have been tested on the modified fibres. C-H cleavage is dependent on Fe(II) abundance and location and is suppressed by surface oxidation while HO* release appears independent of the oxidation state of iron. Quartz specimens with different levels of iron impurities have been tested in a similar manner. A commercially available quartz (Min-U-Sil 5) containing trace levels of iron is also active in both tests, but reactivity is not fully suppressed by treatment with desferrioxamine, which should remove/inactivate iron. The radical yield attained is close to the level produced by a pure quartz dust, suggesting the presence of active sites other than iron. Ascorbic acid reacts with both crocidolite and quartz, with subsequent depletion of the level of antioxidant defences when particle deposition occurs in the lung lining layer. Following treatment with ascorbic acid the radical yield increases with quartz, but decreases with asbestos. Selective removal of iron and silicon from the surface may account for the differences in behaviour of the two particulates.     

1H NMR investigation of the solution structure of substrate-free human heme oxygenase: comparison to the cyanide-inhibited, substrate-bound complex

1H NMR was used to investigate the molecular structure, and dynamic properties of soluble, recombinant, substrate-free human heme oxygenase (apohHO) on a comparative basis with similar studies on the substrate complex. Limited but crucial sequence-specific assignments identify five conserved secondary structural elements, and the detection of highly characteristic dipolar or H-bond interactions among these elements together with insignificant chemical shift differences confirm a strongly conserved folding topology of helices C-H relative to that of substrate complexes in either solution or the crystal. The correction of the chemical shifts for paramagnetic and porphyrin ring current influences in the paramagnetic substrate complex reveals that the strength of all but one of the numerous relatively robust H-bonds are conserved in apohHO, and similar ordered water molecules are located near these H-bond donors as observed in the substrate complexes. The unique and significant weakening of the Tyr(58) OH hydrogen bond to the catalytically critical Asp(140) carboxylate in apohHO is suggested to arise from the removal of the axial H-bond acceptor ligand rather than the loss of substrate. The interhelical positions of the conserved strong H-bonds argue for a structural role in maintaining a conserved structure for helices C-H upon loss of substrate. While the structure and H-bond network are largely conserved upon loss of substrate, the variably increased rate of NH lability dictates a significant loss of dynamic stability in the conserved structure, particularly near the distal helix F.     

Hydrogen exchange during cellulose synthesis distinguishes climatic and biochemical isotope fractionations in tree rings

The abundance of the hydrogen isotope deuterium (D) in tree rings is an attractive record of climate; however, use of this record has proved difficult so far, presumably because climatic and physiological influences on D abundance are difficult to distinguish. Using D labelling, we created a D gradient in trees. Leaf soluble sugars of relatively low D abundance entered cellulose synthesis in stems containing strongly D-labelled water. We used nuclear magnetic resonance (NMR) spectroscopy to quantify D in the C-H groups of leaf glucose and of tree-ring cellulose. Ratios of D abundances of individual C-H groups of leaf glucose depended only weakly on leaf D labelling, indicating that the D abundance pattern was determined by physiological influences. The D abundance pattern of tree-ring cellulose revealed C-H groups that exchanged strongly (C(2)-H) or weakly (C(6)-H2) with water during cellulose synthesis. We propose that strongly exchanging C-H groups of tree-ring cellulose adopt a climate signal stemming from the D abundance of source water. C-H groups that exchange weakly retain their D abundance established in leaf glucose, which reflects physiological influences. Combining both types of groups may allow simultaneous reconstruction of climate and physiology from tree rings.     

Microchemical analysis of retina layers in pigmented and albino rats by Fourier transform infrared microspectroscopy

Fourier transform infrared (FT-IR) microspectroscopy is a powerful technique that can be used to collect infrared spectra from microscopic regions of tissue sections. The infrared spectra are evaluated to chemically characterize the absorbing molecules. This technique can be applied to normal or diseased tissues. In the latter case, FT-IR microspectroscopy can reveal chemical changes that are associated with discrete regions of lesion sites, which can provide insights into the chemical mechanisms of disease processes. In the present study, FT-IR microspectroscopy was used to analyze sections of retina from normal (pigmented) and albino rats. The outer segments of retinas from pigmented animals were found to have unusually strong absorption values for C&z.dbnd6;C-H unsaturation and carbonyl functional groups. Docosahexaenoic acid (DHA), a major constituent of lipids in the outer segments, also had particularly high absorption values for these functional groups, which suggests that it is responsible for those enhanced absorption values. Absorbance values for the unsaturation and carbonyl functional groups were substantially reduced in the outer segments of retinas from albino animals. This finding, together with data from other studies on light-induced oxidative events in the retina, indicates a loss of DHA by a light-induced mechanism in albino animals. The outer nuclear layer had strong absorbance values for H-C-OH and P&z. dbnd6;O functional groups, which is likely due to the sugar phosphate backbone of DNA. The outer and inner plexiform layers were found to contain greater concentrations of CH(2) and C&z.dbnd6;O functional groups than the outer and inner nuclear layers, which is due to the high concentration of synaptic connections in the former layers. In summary, FT-IR microspectroscopy revealed a unique chemical profile in the outer segments compared to other retinal layers, and this profile was altered in albino animals.