Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

Nitration activates tyrosine toward reaction with the hydrated electron

3-Nitrotyrosine has been reported as an important biomarker of oxidative stress that may play a role in a variety of diseases. In this work, transient UV-visible absorption spectra and kinetics observed during the reaction of the hydrated electron, e(aq)(-), with 3-nitrotyrosine and derivatives thereof were investigated. The absorption spectra show characteristics of aromatic nitro anion radicals. The absorptivity of radical anion product at 300 nm is estimated to be (1.0 ± 0.2) × 10(4) M(-1) cm(-1) at pH 7.3. The rate constants determined for the reaction of e(aq)(-) with 3-nitrotyrosine, N-acetyl-3-nitrotyrosine ethyl ester and glycylnitrotyrosylglycine at neutral pH (3.0 ± 0.3) × 10(10) M(-1) s(-1), (2.9 ± 0.2) × 10(10) M(-1) s(-1) and (1.9 ± 0.2) × 10(10) M(-1) s(-1), respectively, approach the diffusion-control limit and are almost two orders of magnitude higher than those for the reactions with tyrosine and tyrosine-containing peptides. The magnitude of the rate constants supports reaction of e(aq)(-) at the nitro group, and the product absorbance at 300 nm is consistent with formation of the nitro anion radical. The pH dependence of the second-order rate constant for e(aq)(-) decay (720 nm) in the presence of 3-nitrotyrosine shows a decrease with increasing pH, consistent with unfavorable electrostatic interactions. The pH dependence of the second-order rate constant for formation of radical anion (300 nm) product suggests that deprotonation of the amino group slows the rate, which indicates that deamination to form the 1-carboxy-2-(4-hydroxy-3-nitrophenyl)ethyl radical occurs. We conclude that the presence of the nitro group activates tyrosine and derivatives toward reaction with e(aq)(-) and can affect the redox chemistry of biomolecules exposed to oxidative stress.     

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O2 half-saturation pressure, measured at 25 degrees C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 degrees C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -- deltalog p1/2 (O2)/deltapH = 0.36 mol H+/mol O2 and amplitude of the Bohr-effect curve deltalogp1/2 (O2) = 0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O2 affinities at 25 degrees C which follows the order: proto less than meso less than deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with gparallel = 2.035 and by the low-field shift of gparallel. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with gparallel - 2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O2 affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in gparallel. The correlation of the cobalt hyperfine constants of the oxy forms with the O2 affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

Oxygenation and EPR spectral properties of Aplysia myoglobins containing cobaltous porphyrins.

Cobalt myoglobins (Aplysia) have been reconstituted from apo-myoglobin (Aplysia) and proto-, meso-, and deutero-cobalt porphyrins. Each of them showed the 30--60 times lower oxygen affinity than those of the corresponding cobalt myoglobins (Sperm whale). Kinetic investigation of their oxygenation by the temperature-junp relaxation technique showed that the low oxygen affinity of cobalt myoglobin (Aplysia) is due to a large dissociation rate constant. the electron paramagnetic resonance (EPR) spectrum of oxy cobalt myoglobin (Aplysia) is affected by the replacement of H2O with D2O, suggesting a possible interaction between the bound oxygen and the neighboring hydrogen atom. A low temperature photodissociation study showed that the product of photolysis of oxy cobalt myoglobin (Aplysia) gives an EPR spectrum different from that of the deoxy-cobalt myoglobin (Aplysia) and from that of the photolysed form of oxy-cobalt myogloin (Sperm whale). These observations suggest that in oxy-cobalt myoglobin (Aplysia) the bound oxygen might interact with amino acid adjacent to it, but the interaction is weaker than that in oxy cobalt myoglobin (Sperm whale).     

pH Dependence of oxy and deoxy cobalt-substituted leghemoglobin from soybean

In leghemoglobin a, which is the major hemoglobin component in soybean root nodules, the haem iron has been replaced by cobalt. The electron spin resonance (ESR) of frozen solutions of the cobalt-substituted leghemoglobin has been studied at 77 K in the deoxy and oxy forms respectively. Both ligation states exhibit rhombic g tensors. The hyperfine constants of ⁵⁹Co, ¹⁴N-imidazole (residue of the proximal histidine) and ¹⁴N-pyrroles are determined for the three principal directions of the g tensor. Both, the oxy and the deoxy state exhibit pH-dependent changes of the hyperfine structures. For oxy cobalt leghemoglobin a quantitative analysis of the pH titration and of the ESR parameters of the low and high-pH forms respectively are performed. The interconversion of the low and the high-pH forms is controlled by a proton-dissociating group with pK=6.4 which is most probably the distal histidine. g tensors and hyperfine constants are compared with those described for oxy cobalt myoglobin crystal spectra [34] allowing assignments of the low and high-pH species of leghemoglobin to stereoelectronic structures with non-equivalent and equivalent dioxygen atoms respectively. Hydrogen-bonding of the distal histidine with dioxygen favours the structure with equivalent oxygen atoms. The pH dependence of the deoxy form is interpreted as interaction of the proximal imidazole with the central cobalt atom.     

One-electron reduction of S-nitrosothiols in aqueous medium

One-electron reduction of S-nitrosothiols (RSNO) has been studied using radiolytically produced reducing entity, the hydrated electron (e(aq)(-)), in aqueous medium. Both kinetics of the reaction and the mechanistic aspects of the decomposition of S-nitroso derivatives of glutathione, L-cysteine, N-acetyl-L-cysteine, N-acetyl-D,L-penicillamine, N-acetylcysteamine, L-cysteine methyl ester, and D,L-penicillamine have been investigated at neutral and acidic pH. The second-order rate constants of the reaction of e(aq)(-) with RSNOs were determined using a pulse radiolysis technique and were found to be diffusion controlled (10(10) dm(3) mol(-1) s(-1)) at neutral pH. The product analysis using HPLC, fluorimetry, and MS revealed the formation of thiol and nitric oxide as the major end products. It is therefore proposed that one-electron reduction of RSNO leads to the liberation of NO. There is no intermediacy of a thiyl radical as in the case of oxidation reactions of RSNOs. The radical anion of RSNO (RSN(*)O(-)) is proposed as a possible intermediate. The overall reaction could be written as RSNO + e(aq)(-) --H+--> RSH + (*)NO.     

Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen.

The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.     

Mechanism of radiation-induced reactions in aqueous solution of coumarin-3-carboxylic acid: effects of concentration, gas and additive on fluorescent product yield

The radiation-induced reactions of a water-soluble coumarin derivative, coumarin-3-carboxyl acid (C3CA), have been investigated in aqueous solutions by pulse radiolysis with a 35 MeV electron beam, final product analysis following (60)Co γ-irradiations and deterministic model simulations. Pulse radiolysis revealed that C3CA reacted with both hydroxyl radicals ((?)OH) and hydrated electrons (e(-) (aq)) with near diffusion-controlled rate constants of 6.8 × 10(9) and 2.1 × 10(10) M(-1) s(-1), respectively. The reactivity of C3CA towards O(2)(? -) was not confirmed by pulse radiolysis. Production of the fluorescent molecule, 7-hydroxy-coumarin-3-carboxylic acid (7OH-C3CA), was confirmed by final product analysis with a fluorescence spectrometer coupled to a high performance liquid chromatography (HPLC) system. Production yields of 7OH-C3CA following (60)Co γ-irradiations depended on the irradiation conditions and ranged from 0.025 to 0.18 (100 eV) (-1). Yield varied with saturating gas, additive and C3CA concentration, implying the presence of at least two pathways capable of providing 7OH-C3CA as a stable product following the scavenging reaction of C3CA with (?)OH, including a peroxidation/elimination sequence and a disproportionation pathway. A reaction mechanism for the two pathways was proposed and incorporated into a deterministic simulation, showing that the mechanism can explain experimentally measured 7OH-C3CA yields with a constant conversion factor of 4.7% from (?)OH scavenging to 7OH-C3CA production, unless t-BuOH was added.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Kinetic studies on the cupric ion oxidation of sheep hemoglobin

The oxidation of sheep hemoglobin, in both the oxygenated and deoxygenated forms, by cuprous ions have been studied by spectrophotometric and stopped-flow techniques. Mixing of both the oxy and deoxy forms with excess Cu²⁺ leads to the rapid oxidation of the iron atoms of all four of the hem groups of the tetrameric protein, followed by the slow formation of hemichromes (low spin Fe^III forms of hemoglobin). Stopped-flow studies show that the oxidations follow simple monophasic kinetics with second-order rate constants of 65 and 310 M^?1 sec^?1 for the oxy and deoxy forms, respectively. Variable temperature studies yield Arrhenius activation energies of 43 for the oxy form and 113 kJ mole^?1 for the deoxy form. For each form of the protein the activation energy is very similar to the activation enthalpy. While the deoxy form is characterized by an activation energy and enthalpy that is more than twice the corresponding value in the oxy form. The activation entropies show highly significant differences being ?128 e.u. and 136 e.u. at 25°C for the oxy and deoxy forms, respectively.     

Substrate stereochemistry of 2-methyl-branched-chain acyl-CoA dehydrogenase: elimination of one hydrogen each from (pro-2S)-methyl and alpha-methine of isobutyryl-CoA

Dehydrogenation of (2S)-[3-13C]isobutyryl-CoA was carried out in vitro using 2-methyl-branched-chain acyl-CoA dehydrogenase purified from rat liver mitochondria. The product was subsequently hydrated by the addition of bovine crotonase. The resulting 3-hydroxyisobutyric acid was predominantly enriched with 13C at the beta-hydroxy position as determined as a methyl ester using electron ionization-gas chromatography-mass spectroscopy. This finding indicates that isobutyryl-CoA is dehydrogenated stereospecifically at the (pro-2S)-methyl and alpha-methine groups to form methacrylyl-CoA, which is later hydrated with the addition of hydrogen on the same side of the molecule from which it was subtracted to produce 3-hydroxyisobutyryl-CoA.     

One-electron reduction of the oxyform of 2,4-diacetyldeuterocytochrome P-450cam

The reaction of the hydrated electron with a ferrous oxygenated form of modified cytochrome P-450cam, containing 2,4-diacetyldeuteroheme, was investigated by the use of pulse radiolysis. The ferrous oxygenated form of this enzyme was reduced by hydrated electrons to form the product, which exhibits absorption maximum at 470 and 370 nm. From the spectrum obtained, the oxidation state of the product is discussed in relation to the higher oxidation states of chloroperoxidase.     

On the basics of carbohydrate-metal chemistry: complexes of palladium(II) with hydroxyaldehyde and -ketone hydrates

In this work, we present novel complexes of hydroxyaldehydes and ketones with palladium(II). The compounds are studied by two-dimensional NMR spectroscopy in solution (COSY, HMQC, HMBC). Glycolaldehyde, d-glyceraldehyde, glyoxal and 2,4-O-ethylidene-d-erythrose were used as aldehydes, d-erythrulose was used as an alpha-hydroxyketone. Although different species are present in solution, only the hydrated forms of the aldehydes can coordinate to the metal centre. These complexes are stable at 4 degrees C in aqueous solution. The crystal structure of a complex formed by mesoxalic acid and palladium(II) is reported and shows coordination by both hydroxy groups of the hydrated ketone.     

A stopped-flow study of the cupric ion oxidation of adult-human haemoglobin.

The addition of excess Cu2+ to adult human haemoglobin leads to the production of alpha 2(2+) beta 2(3+), in both the oxy and deoxy forms of the protein. Stopped-flow studies of the oxidation process yields apparent second-order rate constants of 196M-1 X S-1 and 41M-1 X S-1 for the deoxy and oxy forms respectively. The rate of the deoxy-form oxidation is linearly dependent on [Cu2+], whereas that of the oxy form is rate-limited above 2 mM to 0.11 S-1. Arrhenius activation energies of the two processes are almost identical at 91 kJ X mol-1, as are the activation enthalpies of 89 kJ X mol-1. The activation entropies show small differences, being 31 entropy units and 48 entropy units for the oxy and deoxy forms respectively. ATP and glycerate 2,3-bisphosphate at saturating concentrations do not affect the rate of oxidation of the oxy form, but halve the rate found for the deoxy form. These data are discussed in terms of the previously proposed mechanism of oxidation in which slow Cu2+ binding is followed by rapid electron transfer.     

Amino acid residues interacting with both the bound quinone and coenzyme, pyrroloquinoline quinone, in Escherichia coli membrane-bound glucose dehydrogenase

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.     

Bohr-effect and pH-dependence of electron spin resonance spectra of a cobalt-substituted monomeric insect haemoglobin

The monomeric haemoglobin IV from Chironomus thummi thummi (CTT IV) exhibits an alkaline Bohr-effect and therefore it is an allosteric protein. By substitution of the haem iron for cobalt the O₂ half-saturation pressure, measured at 25 C, increases 250-fold. The Bohr-effect is not affected by the replacement of the central atom. The parameters of the Bohr-effect of cobalt CTT IV for 25 C are: inflection point of the Bohr-effect curve at pH 7.1, number of Bohr protons -log p_1/2 (O₂)/gDpH=0.36 mol H⁺/mol O₂ and amplitude of the Bohr-effect curve log p_1/2 (O₂)=0.84. The substitution of protoporphyrin for mesoporphyrin causes a 10 nm blue-shift of the visible absorption maxima in both, the native and the cobalt-substituted forms of CTT IV. Furthermore, the replacement of vinyl groups by ethyl groups at position 2 and 4 of the porphyrin system leads to an increase of O₂ affinities at 25 C which follows the order: proto < meso < deutero for iron and cobalt CTT IV, respectively. Again, the Bohr-effect is not affected by the replacement of protoporphyrin for mesoporphyrin or deuteroporphyrin. The electron spin resonance (ESR) spectra of both, deoxy cobalt proto- and deoxy cobalt meso-CTT IV, are independent of pH. The stronger electron-withdrawing effect by protoporphyrin is reflected by the decrease of the cobalt hyperfine constants coinciding with g=2.035 and by the low-field shift of g. The ESR spectra of oxy cobalt proto- and oxy cobalt meso-CTT IV are dependent of pH. The cobalt hyperfine constants coinciding with g=2.078 increase during transition from low to high pH. The pH-induced ESR spectral changes correlate with the alkaline Bohr-effect. Therefore, the two O₂ affinity states can be assigned to the low-pH and high-pH ESR spectral species. The low-pH form (low-affinity state) is characterized by a smaller, the high-pH form (high-affinity state) by a larger cobalt hyperfine constant in g. The correlation of the cobalt hyperfine constants of the oxy forms with the O₂ affinities is discussed for several monomeric haemoglobins. The Co-O-O bond angle in cobalt oxy CTT IV is characterized by an ozonoid type of binding geometry and varies little during the pH-induced conformation transition. Due to the lack of the distal histidine in CTT IV no additional interaction via hydrogen-bonding with dioxygen is possible; this is reflected by the cobalt hyperfine constants.     

One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis

The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.     

Synthesis and analytical investigation of C‐terminally modified peptide aldehydes and ketone: application to oxime ligation

C‐terminally modified peptides aldehyde (glycinal and alpha‐oxo aldehyde peptides) and ketone (pyruvic acid‐containing peptide) were synthesised to get new insights into the mechanism of acido‐catalysed oxime ligation. Their tetrahedral hydrated forms were investigated in solution and in the gas phase, using NMR and in‐source collision‐induced dissociation mass spectrometry, respectively, and the kinetics of the oximation reactions followed using analytical HPLC. The results obtained confirmed that the first step of the oximation reaction was the limiting step for the pyruvic acid‐containing peptides because of the steric effect and of the carbon angular strain of the ketone. The second step is the determining step for the aldehyde peptides because the basicity of the oxygen of the hydroxyl function of the tetrahedral form is greater for glycinal than for alpha‐oxo aldehyde. These data strongly suggest that the hydrated form of the aldehyde partner has to be considered when oxime reactions are performed in aqueous buffer. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Probing the interactions of the solvated electron with DNA by molecular dynamics simulations: II. bromodeoxyuridine-thymidine mismatched DNA

The interaction of solvated electrons with DNA results in various types of DNA lesions. The in vitro and in vivo sensitisation of DNA to -induced damage is achieved by incorporation of the electron-affinity radiosensitiser bromodeoxyuridine (BUdR) in place of thymidine. However, in DNA duplexes containing single-stranded regions (bulged BUdR-DNA), the type of lesion is different and the efficiency of damage is enhanced. In particular, DNA interstrand crosslinks (ICL) form at high efficiency in bulged DNA but are not detectable in completely duplex DNA. Knowledge about the processes and interactions leading to these differences is obscure. Previously, we addressed the problem by applying molecular modelling and molecular dynamics (MD) simulations to a system of normal (BUdR·A)-DNA and a hydrated electron, where the excess electron was modelled as a localised eˉ(H₂O)₆ anionic cluster. The goal of the present study was to apply the same MD simulation to a wobble system, containing a pyrimidine–pyrimidine mismatched base pair, BUdR·T. The results show an overall dynamic pattern similar to that of the motion around normal DNA. However, the number of configuration states when was particularly close to DNA is different. Moreover, in the (BUdR·T)-wobble DNA system, the electron frequently approaches the brominated strand, including BUdR, which was not observed with the normal (BUdR·A)-DNA. The structure and exchange of water at the sites of immobilisation near DNA were also characterised. The structural dynamics of the wobble DNA is prone to more extensive perturbations, including frequent formation of cross-strand (cs) interatomic contacts. The structural deviations correlated with approaching DNA from the major groove side, with sodium ions trapped deep in the minor groove. Altogether, the obtained results confirm and/or throw light on dynamic-structure determinants possibly responsible for the enhanced radiation damage of wobble DNA. Figure The structure of the tightly bound single water-layer between the DNA and the electron (Site-8, five H₂O molecules, bold capped sticks); the rest of the “second” shell waters (lines, in atom type colour) surround the ˉ(H₂O)₆ cluster (yellow, space fill). Orange dashed lines H-bonds; only one of the five molecules from the single H₂O layer mediates a single-step H-bond bridge with N7(A8); the other four present a network of two(three)-step H-bond bridges between DNA/ partner atoms     

Monte carlo simulations of site-specific radical attack to DNA bases

An atomistic biophysical model permitting the calculation of initial attacks to a 38-bp representation of B-DNA base moieties by water radicals is presented. This model is based on a previous radiation damage model developed by Aydogan et al. (Radiat. Res. 157, 38-44, 2002). Absolute efficiencies for radical attack to the 38-bp DNA molecule are calculated to be 41, 0.8 and 15% for hydroxyl radical ((.)OH), hydrogen radical (H(.)), and hydrated electron (e(aq))(,) respectively. Among the nucleobases, guanine is found to have the highest percentage (.)OH attack probability at 36%. Adenine, cytosine and thymine moieties have initial attack probabilities of 24, 18 and 22%, respectively. A systematic study is performed to investigate (.)OH attack probabilities at each specified attack site in four molecular models including free bases, single nucleotides, single base pairs, and the central eight base pairs of the 38-bp DNA molecule. Cytosine is the free base moiety for which the closest agreement is observed between the model prediction and the experimental data. The initial (.)OH attack probabilities for cytosine as the free base are calculated to be 72 and 28%, while experimental data are reported at 87 and 13% for the C5 and C6 positions on the base, respectively. In this study, we incorporated atomic charges to scale the site-specific (.)OH reaction rates at the individual atomic positions on the pyrimidine and purine bases. Future updates to the RIDNA model will include the use of electron densities to scale the reaction rates. With respect to reactions of the aqueous electron with DNA, a comparison of the initial distribution of electron attack sites calculated in this study and experimental results suggests an extremely rapid and extensive redistribution of the e(-)(aq) after their initial reactions with DNA.