Binding of aromatic donor molecules to lactoperoxidase: proton NMR and optical difference spectroscopic studies

The interaction of aromatic donor molecules with lactoperoxidase (LPO) was studied using 1H-NMR and optical difference spectroscopy techniques. pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with pKa of 6.1) which is presumably a distal histidine. Dissociation constants evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements were found to be an order of magnitude larger than those for binding to horse radish peroxidase (HRP), indicating relatively weak binding of the donors to LPO. The dissociation constants evaluated in presence of excess of I- and SCN- showed a considerable increase in their values, indicating that the iodide and thiocyanate ions compete for binding at the same site. The dissociation constant of the substrate binding was, however, not affected by cyanide binding to the ferric centre of LPO. All these results indicate that the organic substrates bind to LPO away from the ferric center. Comparison of the dissociation constants between the different substrates suggested that hydrogen bonding of the donors with the distal histidine amino acid, and hydrophobic interaction between the donors and the active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the LPO-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative and relatively low compared to those for binding to HRP. The distances of the substrate protons from the ferric center were found to be in the range 9.4-11.1 A which are 2-3 A larger than those reported for the HRP-substrate complexes. These structural informations suggest that the heme in LPO may be more deeply buried in the heme crevice than that in the HRP.     

Kinetic studies on oxidation of aromatic donor molecules by horseradish peroxidase and lactoperoxidase

Based on kinetic evidence, it has been shown for the first time that the mode of binding of aromatic donor molecules is similar in horseradish peroxidase and lactoperoxidase; also that the nature of the heme plays an important role in the reaction with hydrogen peroxide, and has no effect on the reaction of the intermediate compound II with aromatic substrates.     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

Distinct heme-substrate interactions of lactoperoxidase probed by resonance Raman spectroscopy: difference between animal and plant peroxidases

Resonance Raman scattering from cow milk lactoperoxidase (LPO) and its complexes with various electron donors and inhibitors was investigated. The Raman spectrum of LPO is strikingly close to that of hog intestinal peroxidase but distinctly dissimilar to that of horseradish peroxidase (HRP). The v10 frequency suggested the six-coordinate high-spin structure of heme for native LPO in contrast with the five-coordinate high-spin structure for HRP. For the v10 band, benzohydroxamic acid caused a frequency shift with HRP but not with LPO. Guaiacol, o-toluidine, and histidine brought about a frequency shift of the v4 mode for LPO but not for HRP. The frequency shift was restored upon removal of the substrate or inhibitor by dialysis. The down shift of the v4 frequency is considered to represent an appreciable donation of electrons from the substrate or inhibitor to the porphyrin LUMO and thus their direct interaction with the heme group. From the relative intensity of the shifted and unshifted v4 lines, the dissociation constant was determined to be Kd = 52 mM for guaiacol and Kd = 87 mM for histidine at pH 7.4. The binding of histidine was relatively retarded in the presence of sulfate anion (Kd = 150 mM for 0.53 M sulfate present), and imidazole alone yielded no frequency shift, indicating the binding of the carboxyl group of histidine to the protein cationic site on one hand and a weak charge-transfer interaction between the imidazole group and the heme group on the other.     

Interaction of lactoperoxidase with thiols and diiodotyrosine.

Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.     

Interaction between external medium and haem pocket in myoglobin probed by low-temperature optical spectroscopy

The visible absorption spectra of carbonmonoxymyoglobin in the temperature range 300 to 20 K are reported and compared with the analogous spectra of carbonomonoxyhaemoglobin. The temperature dependence of the zeroth, first and second moment of the observed bands is analysed to obtain information on the local dynamics in the proximity of the haem. Contrary to haemoglobin, the first moment of the observed bands in myoglobin is markedly affected by the solvent composition and its value saturates at temperatures at which the solvent undergoes the glass transition. These data indicate that solvent properties influence the haem pocket stereodynamics in myoglobin; moreover, the different behaviour between myoglobin and haemoglobin suggests that the process should involve the surfaces that are buried in the haemoglobin tetramer and exposed to the solvent in myoglobin, and/or the different protein compressibility.     

Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics.

The X-ray crystal structure of the complex of salicylhydroxamic acid (SHA) with Arthromyces ramosus peroxidase (ARP) has been determined at 1.9 A resolution. The position of SHA in the active site of ARP is similar to that of the complex of benzhydroxamic acid (BHA) with ARP [Itakura, H., et al. (1997) FEBS Lett. 412, 107-110]. The aromatic ring of SHA binds to a hydrophobic region at the opening of the distal pocket, and the hydroxamic acid moiety forms hydrogen bonds with the His56, Arg52, and Pro154 residues but is not asscoiated with the heme iron. X-ray analyses of ARP-resorcinol and ARP-p-cresol complexes failed to identify the aromatic donor molecules, most likely due to the very low affinities of these aromatic donors for ARP. Therefore, we examined the locations of these and other aromatic donors on ARP by the molecular dynamics method and found that the benzene rings are trapped similarly by hydrophobic interactions with the Ala92, Pro156, Leu192, and Phe230 residues at the entrance of the heme pocket, but the dihedral angles between the benzene rings and the heme plane vary from donor to donor. The distances between the heme iron and protons of SHA and resorcinol are similar to those obtained by NMR relaxation. Although SHA and BHA are usually considered potent inhibitors for peroxidase, they were found to reduce compound I and compound II of ARP and horseradish peroxidase C in the same manner as p-cresol and resorcinol. The aforementioned spatial relationships of these aromatic donors to the heme iron in ARP are discussed with respect to the quantum chemical mechanism of electron transfer in peroxidase reactions.     

Thermal perturbation difference spectra and D2O vs H2O isothermal difference spectra of protein-model aromatic chromophores.

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D₂O and H₂O spectra of these chromophores. For phenols approximately equal Δ? values are obtained in both types of spectra, but for their methyl ethers Δ? values of D₂O vs H₂O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D₂O or H₂O. For phenols in D₂O vs H₂O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D₂O and H₂O. The refractive index difference between D₂O and H₂O was shown to be a minor contribution by means of experiments in which D₂O was at 5 dgC and H₂O at 47 dgC, conditions of equal refractive index (Na_D). D₂O vs H₂O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D₂O vs H₂O spectra of proteins are discussed.     

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.     

Interaction of lactoperoxidase with enzymes and immunoglobulins in bovine milk

The interaction of lactoperoxidase with lysozyme and ribonuclease as well as immunoglobulins from cow milk has been investigated. As gel filtration and enzyme kinetics experiments have shown, the lactoperoxidase was slightly activated by complexing to lysozyme, while IgA and IgM were inhibitory for the peroxidase. Oh the other hand, IgG and ribonuclease had no effect on the enzyme activity although the latter did form a complex with the lactoperoxidase. The interaction between the lysozyme and lactoperoxidase appears to be rather specific since the alteration of the lactoperoxidase sugar moiety by periodate oxidation, prevented the formation of the lactoperoxidase-lysozyme complex.     

Cellular functions of cholesterol probed with optical biosensors

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.     

Mechanical properties of plant cell walls probed by relaxation spectra

Transformants and mutants with altered cell wall composition are expected to display a biomechanical phenotype due to the structural role of the cell wall. It is often quite difficult, however, to distinguish the mechanical behavior of a mutant's or transformant's cell walls from that of the wild type. This may be due to the plant's ability to compensate for the wall modification or because the biophysical method that is often employed, determination of simple elastic modulus and breakstrength, lacks the resolving power necessary for detecting subtle mechanical phenotypes. Here, we apply a method, determination of relaxation spectra, which probes, and can separate, the viscoelastic properties of different cell wall components (i.e. those properties that depend on the elastic behavior of load-bearing wall polymers combined with viscous interactions between them). A computer program, BayesRelax, that deduces relaxation spectra from appropriate rheological measurements is presented and made accessible through a Web interface. BayesRelax models the cell wall as a continuum of relaxing elements, and the ability of the method to resolve small differences in cell wall mechanical properties is demonstrated using tuber tissue from wild-type and transgenic potatoes (Solanum tuberosum) that differ in rhamnogalacturonan I side chain structure.     

Glass transition of deoxymyoglobin probed by optical absorption spectroscopy

We have measured the absorption spectrum of horse deoxymyoglobin in glycerol-water mixture around 430 nm in the 130 - 320 K temperature range. The observed asymmetric spectral shape of the Soret band was analyzed using a configuration-coordinate model. The results support the idea that myoglobin is liquid-like at physiological temperatures, but is glass-like below about 250 K. The equilibrium position of the iron atom in the heme group in the electronic excited state was estimated from the determined parameter values.     

Optical spectra of lactoperoxidase as a function of solvent

The iron of lactoperoxidase is predominantly high-spin at ambient temperature. Optical spectra of lactoperoxidase indicate that the iron changes from high-spin to low-spin in the temperature range from room temperature to 20 K. The transformation is independent of whether the enzyme is in glycerol/water or solid sugar glass. Addition of the inhibitor benzohydroxamic acid increases the amount of the low-spin form, and again the transformation is independent of whether the protein is in an aqueous solution or a nearly anhydrous sugar. In contrast to lactoperoxidase, horseradish peroxidase remains high-spin over the temperature excursion in both solvents and with addition of benzohydroxamic acid. We conclude that details of the heme pocket of lactoperoxidase allow ligation changes with temperature that are dependent upon the apoprotein but independent of solvent fluctuations. At low pH, lactoperoxidase shows a solvent-dependent transition; the high-spin form is predominant in anhydrous sugar glass, but in the presence of water, the low-spin form is also present in abundance. The active site of lactoperoxidase is not as tightly constrained at low pH as at neutrality, though the enzyme is active over a wide pH range.     

Interaction of polynucleotides with aromatic hydrocarbons

DNA in its native and denatured form and yeast RNA complex individual aromatic hydrocarbon molecules but single-stranded poly A does not. The degree of complexing appears to depend on molecular dimensions; it is appreciable for phenanthrene, pyrene, and benzpyrene but very small or undetectible for coronene, tetracene, pentacene, and 20-methylcholanthrene.     

Blue-light-induced changes in Arabidopsis cryptochrome 1 probed by FTIR difference spectroscopy

Cryptochromes are blue-light photoreceptors that regulate a variety of responses in animals and plants, including circadian entrainment in Drosophila and photomorphogenesis in Arabidopsis. They comprise a photolyase homology region (PHR) of about 500 amino acids and a C-terminal extension of varying length. In the PHR domain, flavin adenine dinucleotide (FAD) is noncovalently bound. The presence of a second chromophore, such as methenyltetrahydrofolate, in animal and plant cryptochromes is still under debate. Arabidopsis cryptochrome 1 (CRY1) has been intensively studied with regard to function and interaction of the protein in vivo and in vitro. However, little is known about the pathway from light absorption to signal transduction on the molecular level. We investigated the full-length CRY1 protein by Fourier transform infrared (FTIR) and UV/vis difference spectroscopy. Starting from the fully oxidized state of the chromophore FAD, a neutral flavoprotein radical is formed upon illumination in the absence of any exogenous electron donor. A preliminary assignment of the chromophore bands is presented. The FTIR difference spectrum reveals only moderate changes in secondary structure of the apoprotein in response to the photoreduction of the chromophore. Deprotonation of an aspartic or glutamic acid, probably D396, accompanies radical formation, as is deduced from the negative band at 1734 cm(-)(1) in D(2)O. The main positive band at 1524 cm(-)(1) in the FTIR spectrum shows a strong shift to lower frequencies as compared to other flavoproteins. Together with the unusual blue-shift of the absorption in the visible range to 595 nm, this clearly distinguishes the radical form of CRY1 from those of structurally highly homologous DNA photolyases. As a consequence, the direct comparison of cryptochrome to photolyase in terms of photoreactivity and mechanism has to be made with caution.     

Heme structural perturbation of PEG-modified horseradish peroxidase C in aromatic organic solvents probed by optical absorption and resonance Raman dispersion spectroscopy

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B(1g) distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B(2g)-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP.     

Interaction of aflatoxin B1 with electron-donating organic molecules,including aromatic amino acids

The interaction of the carcinogenic mycotoxin, aflatoxin B₁, with some electrondonating organic compounds including aromatic hydrocarbons, dimethylaniline, and aromatic amino acids, was studied. Spectrophotometric analysis of aflatoxin B₁ revealed that hypochromicity in the absorption around 360 nm and hyperchromicity around 385 nm were induced by dimethylaniline, hexamethylbenzene, tryptophan, and imidazole. A similar shifting of aflatoxin B₁ absorption was observed in benzene, toluene, and xylene in the presence of ZnCl₂. The interaction of aflatoxin B₁ with polystyrene was observed in a biphasic system. The association constants of aflatoxin B₁: DMA⁴ (1:1) and of aflatoxin B₁: tryptophan (1:1) were found to be 0.64 and 22.6 liters per mole, respectively. The results suggest that charge-transfer interaction occurs between aflatoxin B₁ and these π-electron donors. Since the spectral changes on aflatoxin B₁ absorption induced by these π-electron donors are similar to those induced by nucleic acids and proteins, it is postulated that charge-transfer interaction also occurs between aflatoxin B₁ and these macromolecules. The role of such interaction in the biological activity of aflatoxin B₁ is discussed.