Peroxidase-catalyzed halide ion oxidation

The first complete mechanistic analysis of halide ion oxidation by a peroxidase was that of iodide oxidation by horseradish peroxidase. It was shown conclusively that a two-electron oxidation of iodide by compound I was occurring. This implied that oxygen atom transfer was occurring from compound I to iodide, forming hypoiodous acid, HOI. Searches were conducted for other two-electron oxidations. It was found that sulfite was oxidized by a two-electron mechanism. Nitrite and sulfoxides were not. If a competing substrate reduces some compound I to compound II by the usual one-electron route, then compound II will compete for available halide. Thus compound II oxidizes iodide to an iodine atom, I*, although at a slower rate than oxidation of I by compound I. An early hint that mammalian peroxidases were designed for halide ion oxidation was obtained in the reaction of lactoperoxidase compound II with iodide. The reaction was accelerated by excess iodide, indicating a co-operative effect. Among the heme peroxidases, only chloroperoxidase (for example from Caldariomyces fumago) and mammalian myeloperoxidase are able to oxidize chloride ion. There is not yet a consensus as to whether the chlorinating agent produced in a peroxidase-catalyzed reaction is hypochlorous acid (HOCl), enzyme-bound hypochlorous acid (either Fe-HOCl or X-HOCl where X is an amino acid residue), or molecular chlorine Cl2. A study of the nonenzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I2. It was impossible to tell which species because of the equilibria: [reaction: see text] The same considerations apply to product analysis of an enzyme-catalyzed reaction. Detection of molecular chlorine Cl2 does not prove it is the chlorinating species. If Cl2 is in equilibrium with HOCl then one cannot tell which (if either) is the chlorinating reagent. Examples will be shown of evidence that peroxidase-bound hypochlorous acid is the chlorinating agent. Also a recent clarification of the mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride along with accurate determination of the elementary rate constants will be discussed.     

Uptake of iodide in the marine haptophyte Isochrysis sp. (T.ISO) driven by iodide oxidation

Uptake of iodide was studied in the marine microalga Isochrysis sp. (isol. Haines, T.ISO) during short‐term incubations with radioactive iodide (¹²⁵I^?). Typical inhibitors of the sodium/iodide symporter (NIS) did not inhibit iodide uptake, suggesting that iodide is not taken up through this transport protein, as is the case in most vertebrate animals. Oxidation of iodide was found to be an essential step for its uptake by T.ISO and it seemed likely that hypoiodous acid (HOI) was the form of iodine taken up. Uptake of iodide was inhibited by the addition of thiourea and of other reducing agents, like L‐ascorbic acid, L‐glutathione and L‐cysteine and increased after the addition of oxidized forms of the transition metals Fe and Mn. The simultaneous addition of both hydrogen peroxide (H₂O₂) and a known iodide‐oxidizing myeloperoxidase (MPO) significantly increased iodine uptake, but the addition of H₂O₂ or MPO separately, had no effect on uptake. This confirms the observation that iodide is oxidized prior to uptake, but it puts into doubt the involvement of H₂O₂ excretion and membrane‐bound or extracellular haloperoxidase activity of T.ISO. The increase of iodide uptake by T.ISO upon Fe(III) addition suggests the nonenzymatic oxidation of iodide by Fe(III) in a redox reaction and subsequent influx of HOI. This is the first report on the mechanism of iodide uptake in a marine microalga.     

A newly discovered oxidant defence system and its involvement in the development of Aurelia aurita (Scyphozoa, Cnidaria): reactive oxygen species and elemental iodine control medusa formation

In Aurelia aurita, applied iodine induces medusa formation (strobilation). This process also occurs when the temperature is lowered. This was found to increase oxidative stress resulting in an increased production of iodine from iodide. One polyp produces several medusae (initially termed ephyrae) starting at the polyp's oral end. The spreading of strobilation down the body column is controlled by a feedback loop: ephyra anlagen decrease the tyrosine content in adjacent polyp tissue by producing melanin from tyrosine. Endogenous tyrosine is able to remove iodine by forming iodiferous tyrosine compounds. The reduced level of tyrosine causes the ephyra-polyp-border to move towards the basal end of the former polyp. We argue that an oxidant defence system may exist which makes use of iodide and tyrosine. Like other marine invertebrates, polyps of Aurelia contain iodide ions. Inevitably produced peroxides oxidise iodide into iodine. The danger to be harmed by iodine is strongly decreased by endogenous tyrosine which reacts with iodine to form iodiferous tyrosine compounds including thyroxin. Both substances together, iodide and tyrosine, form an efficient oxidant defence system which shields the tissue against damage by reactive oxygen species. In the course of evolution (from a species at the basis of the animal kingdom like Aurelia to a highly evolved species like man) the waste product thyroxin (indicating a high metabolic rate) has developed into a hormone which controls the metabolic rate.     

Peroxidase-catalyzed halide ion oxidation

Abstract

The first complete mechanistic analysis of halide ion oxidation by a peroxidase was that of iodide oxidation by horseradish peroxidase. It was shown conclusively that a two-electron oxidation of iodide by compound I was occurring. This implied that oxygen atom transfer was occurring from compound I to iodide, forming hypoiodous acid, HOI. Searches were conducted for other two-electron oxidations. It was found that sulfite was oxidized by a two-electron mechanism. Nitrite and sulfoxides were not. If a competing substrate reduces some compound I to compound II by the usual one-electron route, then compound II will compete for available halide. Thus compound II oxidizes iodide to an iodine atom, I^·, although at a slower rate than oxidation of I^- by compound I. An early hint that mammalian peroxidases were designed for halide ion oxidation was obtained in the reaction of lactoperoxidase compound II with iodide. The reaction was accelerated by excess iodide, indicating a co-operative effect. Among the heme peroxidases, only chloroperoxidase (for example from Caldariomyces fumago) and mammalian myeloperoxidase are able to oxidize chloride ion. There is not yet a consensus as to whether the chlorinating agent produced in a peroxidase-catalyzed reaction is hypochlorous acid (HOCl), enzyme-bound hypochlorous acid (either Fe–HOCl or X–HOCl where X is an amino acid residue), or molecular chlorine Cl₂. A study of the non-enzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I₂. It was impossible to tell which species because of the equilibria:

I₂+H₂O=HOI+I^-+H⁺</ p>

I^-+I₂=I₃^-

The same considerations apply to product analysis of an enzyme-catalyzed reaction. Detection of molecular chlorine Cl₂ does not prove it is the chlorinating species. If Cl₂ is in equilibrium with HOCl then one cannot tell which (if either) is the chlorinating reagent. Examples will be shown of evidence that peroxidase-bound hypochlorous acid is the chlorinating agent. Also a recent clarification of the mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride along with accurate determination of the elementary rate constants will be discussed.     

The Ozone-Iodine-Chlorate Clock Reaction

This work presents a new clock reaction based on ozone, iodine, and chlorate that differs from the known chlorate-iodine clock reaction because it does not require UV light. The induction period for this new clock reaction depends inversely on the initial concentrations of ozone, chlorate, and perchloric acid but is independent of the initial iodine concentration. The proposed mechanism considers the reaction of ozone and iodide to form HOI, which is a key species for producing non-linear autocatalytic behavior. The novelty of this system lies in the presence of ozone, whose participation has never been observed in complex systems such as clock or oscillating reactions. Thus, the autocatalysis demonstrated in this new clock reaction should open the possibility for a new family of oscillating reactions.     

Chemical and enzymatic modification of proteins in the 30 S ribosome of Escherichia coli

Methods are described for the iodination of ribosomal proteins by iodine monochloride and potassium iodide and bovine lactoperoxidase. Ribosomes that were maximally iodinated did not synthesize polyphenylalanine. About one-half of the tyrosine residues could be iodinated with iodine monochloride in the intact ribosome with no change in the sedimentation properties of the particle. When proteins were extracted and dissolved in 5 m-urea, all of the tyrosine residues could be iodinated with iodine monoehloride.     

Iodinated phospholipids and the iodination of proteins of dog thyroid gland in vitro

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.     

Metabolism of 131I in relation to strobilation of Aurelia aurita L. (Scyphozoa)

The ¹³¹I isotope of iodine has been used to follow the uptake and metabolism of iodine during the process of strobilation in pre-conditioned polyps of Aurelia aurita L. Strobilating polyps accumulated free iodide from the media against a concentration gradient, the segmented portion of the polyps accumulating about three times as much as the basal portion. Almost all of the accumulated iodide appeared in the soluble portion of an acid-ethanol extract of polyp tissue as inorganic iodide. The time course of accumulation of iodine was not affected by previous exposure to either higher temperature or iodide. Inorganic iodide rather than organically bound iodine is thought to be the effective factor in the initiation of strobilation.     

Studies on mode of action of potassium iodide upon Sporotrichosis

Conclusion Growth inhibition ofSporotrichum schenckii was observed when potassium iodide or iodine-potassium iodide were added to Sabouraud's medium. The inhibition occurred also after the fungus was brought into contact with the compounds before inoculation. Iodine-potassium iodide was more effective on the inhibition than potassium iodide. The presence of I¹³¹ in association with the organism was demonstrated. The results suggest that the effect of iodine compounds on sporotrichosis is due to a direct fungicidal action of iodine.     

The mechanisms of methionine oxidation concomitant with hormone radioiodination comparative studies of various oxidants using a simple new method

The mechanism of oxidation of methionine concomitant with iodination was studied by ascending paper chromatography using $\text{l}\text{-[Me-}{}^{14}\text{C}\text{]}\text{methionine}$. The ability of the principal iodination reactants to oxidize free methionine was measured in order to predict in situ methionyl oxidation during radioiodination of peptides. Iodide, oxidant, and tyrosine were tested individually and in combination. Dilute chloramine-T and H₂O₂ effectively oxidized free methionine, whereas electric current at the low levels used for iodination did not. Whereas KI₃ did not cause significant methionine oxidation, iodine in statu nascendi was a potent oxidant. Oxidation caused by chemical oxidants was markedly reduced by the addition of iodide, whereas the opposite effect was seen with electric current. In our in vitro system, tyrosine inhibited the effect of the chemical oxidants to different degrees, in the absence of iodide. Under conditions simulating actual ‘mild’ radioiodination conditions, with added tyrosine, none of the oxidants studied caused methionine oxidation; the presence of tyrosine and iodide appeared to preclude the oxidation of methionine. Both excess chloramine-T and high electric current resulted in the formation of a new compound, possibly a sulphinic acid derivative of methionine. The important finding was that the release of free iodine and its uptake by tyrosine were the dominant factors to be considered in the prevention of methionine oxidation.Our new chromatographic technique for the estimation of methionine oxidation in peptides is based on the observed similarity between the reactivity of free methionine and that of accessible methionyl residues in peptides. The method is simple, sensitive and reproducible.     

Availability of iodide and iodate to spinach (Spinacia oleracea L.) in relation to total iodine in soil solution

A greenhouse pot experiment was carried out to investigate the availability of iodide and iodate to soil-grown spinach (Spinacia oleracea L.) in relation to total iodine concentration in soil solution. Four iodine concentrations (0, 0.5, 1, 2 mg kg⁻¹) for iodide (I⁻) and iodate (IO₃⁻) were used. Results showed that the biomass productions of spinach were not significantly affected by the addition of iodate and iodide to the soil, and that iodine concentrations in spinach plants on the basis of fresh weights increased with increasing addition of iodine. Iodine concentrations in tissues were much greater for plants grown with iodate than with iodide. In contrast to the iodide treatments, in iodate treatment leaves accounted for a larger fraction of the total plant iodine. The soil-to-leaf transfer factors (TF_leaf) for plants grown with iodate were about tenfold higher than those grown with iodide. Iodine concentrations in soil solution increased with increasing iodine additions to the soil irrespective of iodine species. However, total iodine in soil solution was generally higher for iodate treatments than iodide both in pots with and without spinach. According to these results, iodate can be considered as potential iodine fertilizer to increase iodine content in vegetables.     

Effects of lodine in Various Formulations on the Growth of Barley and Pea Plants in Nutrient Solution Culture

The forms of iodine added to cultures of barley were potassiumiodide, potassium iodate, potassium periodate, and iodoaceticacid at iodine concentrations of 1.0 ppm and 10.0 ppm. Withpea, only iodide and iodate at 1.0 ppm iodine concentrationwere used. For both species, comparisons were made with culturesto which no iodine was added. In barley, growth was increased by 1.0 ppm iodine, the relativeeffectiveness of the different formulations being in the order:iodoacetic acid > iodide > iodate > periodate. With10.0 ppm, iodide and iodoacetic acid treatments gave reducedgrowth, iodate was without effect, and periodate enhanced growth. In pea, 1.0 ppm iodine was inhibitory, iodide being more toxicthan iodate. Analysis of dry matter showed iodine content according to treatmentto be in the order: iodide > iodoacetic acid > iodate> periodate     

Iodine uptake in Laminariales involves extracellular, haloperoxidase-mediated oxidation of iodide

Sporophytes of Laminaria digitata (L.) Lamour. were assayed for their content of accumulated iodine, which ranged from 0.4% of dry weight in adult plants up to 4.7% for young plantlets. Sporophyte tissue from Laminaria saccharina (L.) Lamour. and L. digitata took up iodide according to Michaelis-Menten kinetics. Hydrogen peroxide and various substances known to interfere with oxidative metabolism were shown to either inhibit or enhance the uptake of iodide, confirming that apoplastic oxidations play a key role in iodide uptake in Laminaria. Consistently, iodide uptake was triggered in L. saccharina protoplasts by incubation in the presence of hydrogen peroxide. Similarly, the uptake of iodide was enhanced in L. digitata gametophytes by addition of haloperoxidase, suggesting that this enzyme catalyses the oxidation of iodide by hydrogen peroxide and plays a key role in iodine uptake. Oxidative stress resulted in a marked efflux of the intracellular iodine. In both influx and efflux experiments, a marked proportion (10–30%) of the tracer was not accounted for, indicating volatilisation of iodine. The mechanism and possible functions of the accumulation of iodine by kelps are discussed. Received: 11 February 1998 / Accepted: 18 June 1998     

Selective Demonstration of Histidine

Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color.     

Selective Demonstration of Histidine

Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color.     

Mechanism of enzymatic and non-enzymatic tyrosine iodination. Inhibition by excess hydrogen peroxide and/or iodide

Non-enzymatic (I2-mediated) and lactoperoxidase-catalyzed iodination of tyrosine are inhibited by excess iodide (I-) and/or hydrogen peroxide (H2O2). This phenomenon is a consequence of the concentration-dependent dual role of I- and H2O2 in the iodinating system. I- and H2O2, in addition to their function as primary substrates of peroxidase, may act as alternative 'iodine acceptors' and therefore compete with tyrosine for the active iodinating agent, irrespective of whether this compound is an enzyme-associated iodinium cation (E X I delta +) or an equivalent oxidized iodine species (IOH, IC1, I2). The competitive reaction pathways resulting from excess I- and/or H2O2 in the iodination system are I2/I-3 generation and/or pseudo-catalatic degradation of H2O2, respectively. Our results also demonstrate that I2 (and alternative medium-dependent oxidized iodine species such as IOH and IC1) generated in the iodination system may play an important role as iodinating agent(s). They serve as a substitute for the enzyme-bound iodinium species (E X I delta +), if the prevailing I- concentration favours this pathway. The proposed mechanism of the various antagonistic and interactive reaction pathways is summarized in a scheme.     

The effect of iodide administration on hog thyroid gland and the composition of thyroglobulin and 27-S iodoprotein.

The effect of excess iodide on hog thyroid gland has been examined with regard to the change in the chemical composition of thyroglobulin and in the accumulation of 27-S iodoprotein by the in vivo treatment of hogs with iodide for various lengths of time. The iodine content of thyroglobulin was either unchanged by short term administration of excess iodide, or somewhat lowered. However, the iodine content as well as the total amount of thyroglobulin increased in the glands enlarged by prolonged treatment with iodide. The iodine highest reached 1.17% of the protein on an average. On the other hand, 27-S iodoprotein decreased and finally disappeared after the chronic treatment. Monoiodotyrosine and diiodotyrosine increased in parallel with the increase in the iodine content (0.15 to 1.17%) caused by the iodide treatment, while thyroxine increased but reached a plateau at the level of three residues per mole of thyroglobulin, and no change was observed even in the proteins with the higher iodine content than 0.75%. Proteolytic activity measured by amino acid release from the thyroid protein was depressed by the chronic treatment. On the other hand, the amount of iodocompound released by the autoproteolysis, which may reflect hormone secretion, increased, possibly because of the marked increase in the iodine content of thyroglobulin.     

Analytical Studies of Iodine in Food Substances

Four species of edible brown marine algae were fractionated by trichloroacetic acid precipitation and subsequent chromatography; and the iodine content of each fraction was determined. Quantitative determination of iodide form iodine was made possible by elimination of interfereing substances in cell extract using a weakly basic anion exchange resin column without drastic procedure.

Although the iodine content of each algae was of much diversity owing to species, the iodide form iodine varied between 83~85% of total iodine independent on total iodine content. The rest of the iodine was mostly found in trichloroacetic acid precipitate probably in protein bound form; and each algae contained little trichloroacetic acid soluble non iodide form of iodine, probably a low molecular organic form iodine.     

Hydrogen Peroxide-Dependent Uptake of Iodine by Marine Flavobacteriaceae Bacterium Strain C-21

The cells of the marine bacterium strain C-21, which is phylogenetically closely related to Arenibacter troitsensis, accumulate iodine in the presence of glucose and iodide (I⁻). In this study, the detailed mechanism of iodine uptake by C-21 was determined using a radioactive iodide tracer, ¹²⁵I⁻. In addition to glucose, oxygen and calcium ions were also required for the uptake of iodine. The uptake was not inhibited or was only partially inhibited by various metabolic inhibitors, whereas reducing agents and catalase strongly inhibited the uptake. When exogenous glucose oxidase was added to the cell suspension, enhanced uptake of iodine was observed. The uptake occurred even in the absence of glucose and oxygen if hydrogen peroxide was added to the cell suspension. Significant activity of glucose oxidase was found in the crude extracts of C-21, and it was located mainly in the membrane fraction. These findings indicate that hydrogen peroxide produced by glucose oxidase plays a key role in the uptake of iodine. Furthermore, enzymatic oxidation of iodide strongly stimulated iodine uptake in the absence of glucose. Based on these results, the mechanism was considered to consist of oxidation of iodide to hypoiodous acid by hydrogen peroxide, followed by passive translocation of this uncharged iodine species across the cell membrane. Interestingly, such a mechanism of iodine uptake is similar to that observed in iodine-accumulating marine algae.     

Sulfated tyrosines of thyroglobulin are involved in thyroid hormone synthesis.

Thyroid hormone synthesis is under the control of thyrotropin (TSH), which also regulates the sulfation of tyrosines in thyroglobulin (Tg). We hypothesized that sulfated tyrosine (Tyr[S]) might be involved in the hormonogenic process, since the consensus sequence required for tyrosine sulfation to occur was observed at the hormonogenic sites. Porcine thyrocytes, cultured with TSH but without iodide in the presence of [(35)S]sulfate, secreted Tg which was subjected to in vitro hormonosynthesis with increasing concentrations of iodide. A 63% consumption of Tyr[S] (1 residue) was observed at 40 atoms of iodine incorporated into Tg, corresponding to a 40% hormonosynthesis efficiency. In addition, hyposulfated Tg secreted by cells incubated with sodium chlorate was subjected to in vitro hormonosynthesis. With 0.5 Tyr[S] residue (31% of the initial content), the efficiency of the hormonosynthesis was 29%. In comparison, when hormonosynthesis was performed by cells, with only 0.25 Tyr[S] residue (16% of the initial content), the hormonosynthesis efficiency fell to 18%. These results show that there exists a close correlation between the sulfated tyrosine content of Tg and the production of thyroid hormones.