Structural role of histidine residues in NAD(P)-glutamate dehydrogenase from the bovine liver

Photooxidation of bovine liver glutamate dehydrogenase (GDH, EC 1.4.1.3) in the presence of methylene blue at a low light intensity occurs in two stages. At the first stage, the duration of which depends on temperature and dye concentration, a slight activation is observed simultaneously with the oxidation of two histidine residues. At the second stage, the inactivation is concomitant with the oxidation of three histidine and one tryptophan residues. The inactivation is a first order reaction (k = 3,22 X 10(-2) min-1) and is correlated with changes in the circular dichroism spectra. These data testify to the structural role of histidine residues in the GDH molecule. The kinetic behaviour of GDH during its modification with diethylpyrocarbonate (DEP) depends on pH and the reagent concentration. Four histidine residues undergo carbethoxylation at pH 6.0 and 7.5, but the modification rate is much higher at pH 7.5. At low DEP concentrations, a remarkable activation is observed with a simultaneous modification of one histidine residue, which is independent of pH. At high DEP concentrations, a rapid inactivation takes place at pH 7.5. Treatment of the carbethoxylated inactive enzyme with hydroxylamine results in the deacylation of histidine residues without any noticeable reactivation. The data on the combined effect of DEP and pyridoxal-5'-phosphate suggest that GDH inactivation by DEP at pH 7.5 is a result of modification of an essential epsilon-NH2 group of lysine-126.     

Regulation of peroxo-bridge formation between cobalt(II)-carnosine complexes by histidine: Possible involvement in polycythemia

The mechanisms by which histidine stabilizes the cobalt(II)-carnosine complex from oxidation to cobalt(III) in aqueous solution are investigated with ¹H-nmr, laser Raman, and Fourier transform-infrared spectroscopy. Histidine has at least three effects on the cobalt(II)-carnosine complex. First, over the concentration range of at least 5 to 250 mM, histidine stabilizes the cobalt(II)-carnosine complex from oxidation by excluding solvent molecules from the equatorial coordination positions. Second, at the upper end of this concentration range, histidine reduces the strained nonplanarity of the equatorial coordination positions around the cobalt(II) ion that results from tridentate chelation by carnosine. Bidentate ligation by histidine causes the carnosine to bind as a bidentate ligand also. Third, bidentate ligation of two carnosine molecules to the equatorial coordination positions of Co(II) ion places the β-alanyl residues inthe vicinity of the two axial coordination positions and thereby inhibits the binding of molecular oxygen. Substitution of a molecule of histidine for one of these two carnosine molecules makes an axial coordination position available for binding oxygen. The first two effects are expected to stabilize the cobalt(II) ion from rapid oxidation, whereas the third effect is expected to give long-term stability of the peroxo-bridged complex. Since bidentate ligation of histidine is favored over monodentate ligation only when the concentration of Co(II) ion is not limiting and is inhibited by high concentrations of carnosine in the same solution, the results presented provide a possible explanation for the observation that the stability of the Co(II) complexes toward oxidation and their ability to bind molecular oxygen depend on both the relative and absolute concentrations of Co(II) ion, carnosine, and histidine in solution. Furthermore, these results provide additional support to the suggestion that the high activity of carnosinase in kidney is involved in part in regulation of the oxygen sensor in this organ.     

Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 μM Cu²⁺ binds 28.0 ± 3.3 Cu ions per LDL particle (mean ± SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.     

Photooxidation of human serum albumin and its complex with bilirubin.

Irradiation with visible light of human serum albumin in aqueous solution at pH 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole O2, and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole O2. Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment.     

Circular dichroism studies of complexes of sequential histidine polypeptides with methyl orange

The induced circular dichroism (c.d.) spectra of poly(l-histidine) and sequential histidine polypeptide-dye complexes were measured. Two dichroic bands associated with the blue shifted absorption shoulder of methyl orange at around 370 nm were observed by complex formation between the polypeptides and the dye. Induced c.d. arose from the dye bound to the polypeptide in random coil structure, the optimal pH being 4.1. Added sodium chloride decreases the intensity of the induced c.d. Intramolecular interaction was assumed from the relationship between the concentrations of the polypeptide-dye complex and the intensities of the induced c.d. The intensity of the induced c.d. decreases with increasing distance between the intramolecular histidine residues. The induced c.d. spectrum of the poly(l-histidine)-dye complex shows the irreversible thermal change when heating.     

Electrochemical behaviour of proteins at graphite electrodes. II. Electrooxidation of amino acids

Electrochemical oxidation of L,alpha-amino acids at a paraffin-wax impregnated spectroscopic graphite electrode (WISGE) was studied by means of linear sweep, cyclic, phase-sensitive alternating current and differential pulse voltammetric techniques. It was found that out of the amino acids usually occurring in proteins only tyrosine, tryptophan, histidine, cystine, cysteine and methionine were oxidized at the WISGE. At relatively low concentrations of amino acids (up to ca. 2 x 10(-4) M) the electrode process in which the amino acids are oxidized at the WISGE has the characteristics of an irreversible reaction controlled by diffusion. Coulometric measurements showed that oxidation of tyrosine and tryptophan at the WISGE, i.e. of amino acids which are responsible for the oxidizability of proteins at graphite electrodes, is a two-electron process. At higher concentrations of tyrosine-and tryptophan (above ca. 2 x 10(-4) M) adsorption of the oxidation product of these amino adds was demonstrated.     

Effect of nitrous oxide and methionine on hepatic S-adenosylmethionine levels and in vivo histidine oxidation in rats.

Nitrous oxide (N2O) decreased in vivo oxidation of histidine in rats fed a basal diet marginally deficient in methionine, although hepatic levels of S-adenosylmethionine (AdoMet) were not significantly altered. Excess dietary methionine increased hepatic levels of AdoMet and increased histidine oxidation. However, it did not protect histidine oxidation when the rats were treated with N2O. Parenteral administration of methionine greatly increased hepatic levels of AdoMet and increased histidine oxidation in normal and N2O treated rats. This indicates that when hepatic levels of AdoMet are greatly elevated by administration of methionine, N2O does not affect in vivo histidine oxidation.     

Oligomeric state of lipocalin-1 (LCN1) by multiangle laser light scattering and fluorescence anisotropy decay

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 microM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 microM-1 mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein.     

Thermoluminescence evidence for light-induced oxidation of tyrosine and histidine residues in manganese-depleted photosystem II particles.

In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) abolishes the TL band appearing around -55 degrees C (TL-55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around -30 degrees C (TL-30). NBD treatment also abolishes the EPR signal IIfast of oxidized tyrosine donor, Yz, and inhibits the electron transport from diphenylcarbazide to 2,6-dichlorophenol-indophenol. It is concluded that the TL-55 and TL-30 bands can be assigned to oxidized tyrosine (Yz+) and histidine (His+) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680+.     

The formation of ferritin from apoferritin. Inhibition and metal ion-binding studies

Inhibition by Zn(2+) of iron uptake by apoferritin at very low substrate concentrations is shown to be competitive. It is proposed that Zn(2+) competes with Fe(2+) for sites on the protein at which the oxidation of Fe(2+) is catalysed. Interpretation of titration data suggests there are two independent classes of binding site for Zn(2+) and several other cations. Sites in one such class are probably on the external surface of the apoferritin molecule. The catalytic binding sites are presumed to be internal and may involve histidine or possibly cysteine as ligands.     

Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.     

The activation of rabbit muscle, liver, and kidney fructose bisphosphatases by histidine and citrate

Purified fructose 1,6-bisphosphatases from rabbit muscle, liver, and kidney require a metal chelator for optimal activity at neutral pH. This requirement is satisfied by physiological concentrations of histidine and citrate, and at pH 7 their effects are additive. In the presence of both histidine and citrate the optimum activity is shifted from about pH 8 to pH 7.2, and the activity is greater than that obtained with optimal concentrations of EDTA. Carnosine, anserine, and 1-methyl histidine are also effective, but only at much higher concentrations, while 3-methyl histidine is effective in the same concentration range as is histidine. Isocitrate can replace citrate. The results suggest that fructose bisphosphatases possess distinct binding sites for divalent cations (Mg²⁺ or Mn²⁺) and also for histidine and citrate complexes of these cations.     

Metabolic responses of folic acid and related compounds to thyroxine in rats

This study deals with the effects of thyroidectomy and feeding thyroid powder on histidine and folic acid metabolism. Normal rats maintained on a soy protein diet, low in methionine but supplemented with vitamin B-12, oxidize approx. 10% of an injected dose of [2-¹⁴C]histidine in 3 h and excrete low levels of formiminoglutamic acid. Addition of methionine increases histidine oxidation to approx. 20%. The feeding of thyroid powder or the injection of high levels of thyroxine decreases histidine oxidation and increases formiminoglutamic acid excretion. Surgical thyroidectomy at weaning increases histidine oxidation to approx. 45% and, thus, resembles the effect of methionine in promoting histidine oxidation and decreasing formiminoglutamic acid excretion. The feeding of methionine to the thyroidectomized animal further increases histidine oxidation to 65%. The distribution of folate forms in the liver was determined by column chromatography following administration of a dose of tritiated folic acid. In the normal animal, tetrahydrofolate accounts for 38% of the total folate present. The feeding of methionine increases this to 48%, which is consistent with the observed increase in histidine metabolism. Thyroidectomy increases the percentage of tetrahydrofolate to 63% and the feeding of methionine further increases it to 68%. The percentage of tetrahydrofolate relative to total folate is in proportion to the observed rate of histidine metabolism. The action of thyroidectomy in increasing histidine oxidation may be accounted for by its effect in increasing the proportion of tetrahydrofolate.     

Mutagenicity test of dyes used in cosmetics with the Salmonella/mammalian-microsome test.

37 dyes including 3 anthraquinone, 22 azo; 5 xanthene, 5 fluorandiol, and 2 thioindigo dyes, were tested for mutagenic potential with the Salmonella/mammalian-microsome test. Two frame-shift histidine mutants (TA1537 and TA98) and two base-pair substituted histidine mutants (TA1535 and TA100) of Salmonella typhimurium were employed. Both the spot test and the plate-incorporation assay indicated that one azo dye, D&C Orange No. 17, was mutagenic with three of the bacterial test strains. The mutagenic response of D&C Orange No. 17 was depressed by the addition of the microsomal fractions from rat livers. Of the chemicals used to synthesize D&C Orange No; 17 was depressed by the addition of the microsomal fractions from rat livers. Of the chemicals used to synthesize D&C Orange No. 17, beta-naphthol was not mutagenic but 2,4-dinitroaniline was mutagenic to the same Salmonella strains as D&C Orange No. 17 . Dimethyl sulfoxide extracts of lipsticks of similar formula but without D&C Orange No. 17 were tested in the plate incorporation assay. Only those containing D&C Orange No. 17 were mutagenic and the dye was mutagenic at concentrations consumed in normal daily use.     

Chemiluminescence activated by rhodamine G in plasma in the presence of divalent iron ions

Rhodamine G was found to activate blood plasma chemiluminescence induced by ferrous ions. The dye in concentrations 300-500 mole/l increased chemiluminescence by an order of magnitude. The luminescence was inhibited by histidine and SOD. A conclusion may be drawn that the mechanism of the activated ferrous chemiluminescence with rhodamine G was related to superoxide anion-radicals and singlet oxygen.     

Histidyl-transfer ribonucleic acid synthetase mutants requiring a high internal pool of histidine for growth

Mutants that require histidine due to an altered structural gene for the histidyl-transfer ribonucleic acid synthetase (hisS) have been isolated by a general selection for histidine-requiring strains in which the mutation producing histidine auxotrophy is unlinked to the histidine operon. One of the mutants has been shown to require an abnormally high internal histidine pool for growth owing to an altered synthetase that is unstable at low histidine concentrations. It is difficult to determine accurately the K(m) for histidine of the synthetase enzyme from the mutant because of the instability of the enzyme at limiting histidine concentrations; however, a histidine K(m) value has been estimated that is approximately 100 times higher than the histidine K(m) of the wild-type enzyme. For the mutant strains to achieve the high internal pool of histidine required for growth, all the systems that transport histidine from the growth medium must be functioning to capacity. Amino acids that interfere with histidine transport strongly inhibit the growth of the mutants. The mutants have been useful in providing a selective genetic marker for transductional mapping in the hisS region. The mutants are discussed as representative of a general class of curable mutants that have an altered enzyme with poor affinity for a substrate or coenzyme.     

The analogous mechanisms of enzymatic inactivation induced by ascorbate and superoxide in the presence of copper

The mechanism of enzymatic inactivation of purified and membrane-bound acetylcholine esterase by ascorbate and copper was investigated. While the exposure of the enzyme to either ascorbate or copper did not cause enzymatic inactivation, the incubation of the enzyme with a combination of both ascorbate and copper resulted in a loss in acetylcholine esterase activity, which was time dependent. The enzymatic inactivation required either molecular oxygen or hydrogen peroxide under anaerobic conditions. Scavengers of hydroxyl radicals at concentrations of up to 100 mM did not provide protection to acetylcholine esterase. Only mannitol at very high concentrations (above 1 M) efficiently prevented the inactivation of the enzyme. The kinetics of the aerobic oxidation of reduced ascorbate in the presence of acetylcholine esterase and copper closely followed the rate of enzyme inactivation. Addition of the chelating agents EDTA and diethylenetriaminepentaacetic acid prevented both the oxidation of ascorbate and the inactivation of the enzyme. In the presence of low concentrations of histidine (0.5-2.0 mM), which forms high affinity complexes with copper, the rate of ascorbate oxidation was similar to that recorded in its absence. On the other hand, no enzyme inactivation was indicated in the presence of histidine. Low temperature EPR measurements have demonstrated the binding of copper to the enzyme, and have shown the reduction of the cupric enzyme to the corresponding cuprous complex. In view of these results, a general "site-specific" mechanism for biological damage can be offered, in which copper(II) ions are bound to enzymes or other biological macromolecules. Ascorbate plays a dual role: it reduces the cupric complex to the corresponding cuprous state and serves as a source for H2O2, which, in turn, reacts with the reduced copper complex, in a Fenton reaction. In this reaction, secondary hydroxyl radicals are site specifically formed, and react preferentially with the protein, at the site of their formation, causing its inactivation. This mechanism is analogous to that previously proposed (Samuni, A., Chevion, M., and Czapski, G. (1981) J. Biol. Chem. 256, 12632-12635) for the enhancement of the biological damage caused by superoxide in the presence of copper.     

Rate constants studies of the dye-sensitized photoinactivation of lysozyme

Summary The rate constants for the photodynamic inactivation of hen egg-white lysozyme at different temperatures were studied. Arrhenius plots of the methylene blue sensitized photo-inactivation of lysozyme gave an experimental activation energy of 7.5 kcal/mol. The rate constants for the photodynamic inactivation of lysozyme in the presence of riboflavin decreased almost linearly in the temperature range 4–38° C. The photosensitized oxidation of lysozyme at –20° C in freezing and non-freezing solvents was possible only in the presence of riboflavin. The effect of dye concentration on the quantum yield and rate constant for the photodynamic inactivation of lysozyme was examined. The quantum yields were lower when the concentrations of methylene blue used were low, and increased on increasing dye concentration, getting to a maximum and then declined at higher dye concentrations. It was found that in the case of riboflavin sensitized photo-inactivation of lysozyme both the rate constant and the quantum yield increased as the dye concentration increased. No maximum was observed over the range of dye concentrations studied. A new mechanism is postulated for the photodynamic action of lysozyme in the presence of riboflavin.     

KCl:Dy phosphor for thermoluminescence dosimetry of ionizing radiation

The thermoluminescence (TL) characterizations of γ‐irradiated KCl:Dy phosphor for radiation dosimetry are reported. All phosphors were synthesized via a wet chemical route. Minimum fading of TL intensity is recorded in the prepared material. TL in samples containing different concentrations of Dy impurity was studied at different γ‐irradiation doses. Peak TL intensities varied sublinearly with γ‐ray dose in all samples, but were linear between 0.08 to 0.75 kGy for the KCl:Dy (0.1 mol%) sample. This material may be useful for dosimetry within this range of γ‐ray dose. TL peak height was found to be dependant on the concentration (0.05–0.5 mol%) of added Dy in the host. Copyright © 2012 John Wiley & Sons, Ltd.     

Apoplastic Ascorbate Does not Prevent the Oxidation of Fluorescent Amphiphilic Dyes by Ambient and Elevated Concentrations of Ozone in Leavesl

Cut leaves of spinach were infiltrated with solutions containingoxidation-sensitive fluorescent dyes. Excess solution was removedfrom the intercellular space by centrifugation. Fluorescenceof the dyes D494 and D283 started to decrease immediately afterthe onset of fumigation with ozone at concentrations similarto or not much higher than ambient concentrations in air onsunny days. Only part of ozone entering the leaves was interceptedby the dye. The major part was degraded by unspecified reactions.Photosynthesis was not inhibited while the introduced dye wasoxidized by ozone, showing that open stomata facilitated gasexchange after the introduction of dye into the leaf interior.Feeding of ascorbate to the leaves via the petiole failed toaffect the ozone-dependent decrease in fluorescence emissionfrom the dye. Likewise, infiltration of leaves by solutionscontaining dye and 10 mM ascorbate did not produce significantprotection of the dye against oxidation by ozone. However, suchprotection was observed in vitro, when solutions of dye andascorbate were bubbled with air containing ozone. Although thereis little doubt that apoplastic ascorbate occupies a centralposition in the antioxi-dative defense of leaf tissue, we aresurprised to find that it is much less effective than expectedto decrease the oxidation of fluorescent lipophilic probes whichhad been introduced into the leaf interior. The data suggestthat ascorbate is not a primary reductant of ozone in the apoplast.With a microscope-mounted CCD-camera connected to the gas exchangeequipment we obtained spatial information on the fluorescencesignal and present first results on an heterogeneous distributionof ozone action. (Received June 20, 1997; Accepted December 26, 1997)