Hydrogen peroxide dependent cis-dihydroxylation of benzoate by fully oxidized benzoate 1,2-dioxygenase

Rieske dioxygenases catalyze the reductive activation of O2 for the formation of cis-dihydrodiols from unactivated aromatic compounds. It is known that O2 is activated at a mononuclear non-heme iron site utilizing electrons supplied by a nearby Rieske iron sulfur cluster. However, it is controversial whether the reactive species is an Fe(III)-(hydro)peroxo or an Fe(II)-(hydro)peroxo (or electronically equivalent species formed by breaking the O-O bond). Here it is shown that benzoate 1,2 dioxygenase oxygenase component (BZDO) prepared in a form with the Rieske cluster oxidized and the mononuclear iron in the Fe(III) state can utilize H2O2 as a source of reduced oxygen to form the correct cis-dihydrodiol product from benzoate. The reaction approaches stoichiometric yield relative to the mononuclear Fe(III) concentration, being limited to a single turnover by inefficient product release from the Fe(III)-product complex. EPR and M?ssbauer studies show that the iron remains ferric throughout this single turnover "peroxide shunt" reaction. These results strongly support Fe(III)-(hydro)peroxo (or Fe(V)-oxo-hydroxo) as the reactive species because there is no source of additional reducing equivalents to form the Fe(II)-(hydro)peroxo state. This conclusion could be further tested in the case of BZDO because the peroxide shunt occurs very slowly compared with normal turnover, allowing the reactive intermediate to be trapped for spectroscopic analysis. We attribute the slow reaction rate to a forced change in the normally strict order of the substrate binding and enzyme reduction steps that regulate the catalytic cycle. The reactive intermediate is a high-spin ferric species exhibiting an unusual negative zero field splitting and other EPR and M?ssbauer spectroscopic properties reminiscent of previously characterized side-on-bound peroxide adducts of Fe(III) model complexes. If the species in BZDO is a similar adduct, its isomer shift is most consistent with an Fe(III)-hydroperoxo reactive state.     

Quinolinic acid — Iron(II) complexes: Slow autoxidation,but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti³⁺/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.     

Quinolinic acid-iron(ii) complexes: slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti³⁺/hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-D-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-d-glucamine dithiocarbamate

Iron(II)-dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)-dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and -25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)-dtcs and iron(II)-mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)-dtcs and -mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6-3.2) x 10(4) and (0.7-1.1) x 10(5) M(-1) s(-1), respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)-dtcs and iron(II)-mgd by hydrogen peroxide is significantly slower (ca. 10-15 M(-1) s(-1)). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)-dtcs and iron(III)-mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M(-1) s(-1), respectively.     

Esr Spin Trapping Studies Into of Nature of the Oxidizing Species Formed in the Fenton Reaction: Pitfalls Associated With of Use Of 5,5-Dimethyl-1-Pyrroline-n-Oxide in the Detection of the Hydroxyl Radical

Several investigators have challenged the widely held view that the hydroxyl radical is the primary oxidant formed in the reaction between the ferrous ion and hydrogen peroxide. In recent studies, using the ESR spin trapping technique, Yamazaki and Piette found that the stoichiometry of oxidant formation in the reaction between Fe²⁺ and H₂O₂ often shows a marked deviation from the expected value of 1:1 (I. Yamazaki and L. H. Piette (1990) J. Am. Chem. Soc. 113, 7588-7593). In order to account for these observations, it was suggested that additional oxidizing species are formed, such as the ferryl ion (FeO²⁺), particularly when iron is present at high concentration and chelated to EDTA.

In this paper it is shown that secondary reactions, involving the redox cycling of iron and the oxidation of the hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by iron, operate under the reaction conditions employed by Yamazaki and Piette. Consequently, the stoichiometry of oxidant formation can be rationalized without the need to envisage the formation of oxidizing species other than the hydroxyl radical. It is also demonstrated that the iron(III) complex of DETAPAC can react directly with DMPO to form the DMPO hydroxyl radical adduct (DMPO/OH) in the absence of hydrogen peroxide. Therefore, to avoid the formation of (DMPO/OH) as an artefact, it is suggested that DETAPAC should not be used as a reagent to inactivate containating adventitious iron in experiments using DMPO.     

Modeling tyrosinase activity. Effect of ligand topology on aromatic ring hydroxylation: an overview

Synthetic modeling of tyrosinase (o-phenol ring hydroxylation) has emerged as a novel class of successful biomimetic studies. It is a well-established fact that the reaction of dioxygen with copper(I) complexes of m-xylyl-based ligands generate putative copper-oxygen intermediate species such as side-on peroxo {CuII2(mu-O2)}2+ [in some cases bis-oxo {CuIII2(mu-O)2}2+ in equilibrium with isomeric side-on peroxo], due to oxygen activation. Electrophilic attack of such species brings about monooxygenase activity by incorporating one of the oxygens to m-xylyl ring of the ligand and the other oxygen is reduced to hydroxide ion. The goal of this review is to provide a concise overview of the present day knowledge in this field of research to emphasize the important role the designed ligands play in eliciting the desired tyrosinase-like chemistry.     

Fe(3+)-eta(2)-peroxo species in superoxide reductase from Treponema pallidum. Comparison with Desulfoarculus baarsii

Superoxide reductases (SORs) are superoxide (O2-)-detoxifying enzymes that catalyse the reduction of O2- into hydrogen peroxide. Three different classes of SOR have been reported on the basis of the presence or not of an additional N-terminal domain. They all share a similar active site, with an unusual non-heme Fe atom coordinated by four equatorial histidines and one axial cysteine residues. Crucial catalytic reaction intermediates of SOR are purported to be Fe(3+)-(hydro)peroxo species. Using resonance Raman spectroscopy, we compared the vibrational properties of the Fe3+ active site of two different classes of SOR, from Desulfoarculus baarsii and Treponema pallidum, along with their ferrocyanide and their peroxo complexes. In both species, rapid treatment with H2O2 results in the stabilization of a side-on high spin Fe(3+)-(eta(2)-OO) peroxo species. Comparison of these two peroxo species reveals significant differences in vibrational frequencies and bond strengths of the Fe-O2 (weaker) and O-O (stronger) bonds for the T. pallidum enzyme. Thus, the two peroxo adducts in these two SORs have different stabilities which are also seen to be correlated with differences in the Fe-S coordination strengths as gauged by the Fe-S vibrational frequencies. This was interpreted from structural variations in the two active sites, resulting in differences in the electron donating properties of the trans cysteine ligand. Our results suggest that the structural differences observed in the active site of different classes of SORs should be a determining factor for the rate of release of the iron-peroxo intermediate during enzymatic turnover.     

Redox cycling of iron complexes of N-(dithiocarboxy)sarcosine and N-methyl-d-glucamine dithiocarbamate

Iron(II)–dithiocarbamate complexes are used to trap nitrogen monoxide in biological samples, and the resulting nitrosyliron(II)–dithiocarbamate is detected and quantified by ESR. As the chemical properties of these compounds have been little studied, we investigated whether iron dithiocarbamate complexes can redox cycle. The electrode potentials of iron complexes of N-(dithiocarboxy)sarcosine (dtcs) and N-methyl-d-glucamine dithiocarbamate (mgd) are 56 and −25 mV at pH 7.4, respectively, as measured by cyclic voltammetry. The autoxidation and Fenton reaction of iron(II)–dtcs and iron(II)–mgd were studied by stopped-flow spectrophotometry with both iron(II) complexes and dioxygen or hydrogen peroxide in excess. In the case of excess iron(II)–dtcs and –mgd complexes, the rate constants of the autoxidation and the Fenton reaction are (1.6–3.2) × 10⁴ and (0.7–1.1) × 10⁵ M⁻¹ s⁻¹, respectively. In the presence of nitrogen monoxide, the oxidation of iron(II)–dtcs and iron(II)–mgd by hydrogen peroxide is significantly slower (ca. 10–15 M⁻¹ s⁻¹). The physiological reductants ascorbate, cysteine, and glutathione efficiently reduce iron(III)–dtcs and iron(III)–mgd. Therefore, iron bound to dtcs and mgd can redox cycle between iron(II) and iron(III). The ligands dtcs and mgd are slowly oxidized by hydrogen peroxide with rate constants of 5.0 and 3.8 M⁻¹ s⁻¹, respectively.     

Studies on Host-selective Toxins Produced by a Pathotype of Alternaria citri Causing Brown Spot Disease of Mandarins

Two host-selective pathotoxins, ACTG-toxins A and B, and four related but less active toxins, C, D, E, and F, were isolated from the culture broth of Alternaria citri, a fungus that produces brown spot disease of Dancy tangerine (Citrus reticulata) and other mandarin cultivars. The basic common structural features of these toxins were the presence of a six-membered group bonded, via a methylene group, to a five-membered ring having an alkenyl substituent (Fig. 1). For Toxins A, B, C and F, the six-membered ring had an enolizable β-diketo group. For Toxin C, the five-membered ring was a tetrahydrofuran group. For Toxins D and E, an additional dihydropyran ring was formed by dehydration between a tertiary hydroxyl on the cyclopentene ring and an enolic hydroxyl group on the cyclohexane ring, and also the presence of a terminal hydroxymethyl group on the alkenyl substituent, instead of the methyl groups in Toxins A, B and C. In Toxin F, the terminal group was a formyl.     

Radicals from "Good's" buffers

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.     

Enzymatic properties of human hemalbumin.

The binding of hemin to the primary site of human serum albumin (HSA) has been reinvestigated using UV-Vis, CD and NMR techniques. The major fraction of bound hemin contains a five-coordinated high-spin iron(III) center, but a minor fraction of the metal appears to be in a six-coordinated, low-spin state, where a 'distal' residue, possibly a second histidine residue, completes the coordination sphere. The reduced, iron(II) form of the adduct contains six-coordinated low-spin heme. The distal residue hinders the access to the iron(III) center of hemin-HSA to small anionic ligands like azide and cyanide and destabilizes the binding of neutral diatomics like dioxygen and carbon monoxide to the iron(II) form. In spite of these limitations, the hemin-HSA complex promotes hydrogen peroxide activation processes that bear the characteristics of enzymatic reactions and may have biological relevance. The complex is in fact capable of catalyzing peroxidative reactions on phenolic compounds related to tyrosine and hydrogen peroxide dismutation. Kinetic and mechanistic studies confirm that the low efficiency with which peroxidative processes occur depends on the limited rate of the reaction between hydrogen peroxide and the iron(III) center, to form the active species, and by the competitive peroxide degradation reaction.     

Synthesis of 1-homoaustraline

The 1,3-dipolar cycloaddition of a five-membered cyclic nitrone derived from malic acid and unsaturated D-threo-hexonolactone leads to a single adduct, which was transformed into 1-homoaustraline via a reaction sequence involving rearrangement of the six-membered lactone ring into the five-membered one, removal of the terminal carbon atom from the sugar chain, reduction of the lactone fragment into triol, protection of primary hydroxy groups, mesylation of the secondary one, cleavage of the N-O bond, and the intramolecular alkylation of the nitrogen atom.     

Theoretical investigation of the ring opening process of verdoheme to biliverdin in the presence of dioxygen

The conversion of ferrous verdoheme to ferric biliverdin in the presence of O₂ was investigated using the B3LYP method. Both 6-31G and 6-31G (d) basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Three possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In the first route oxygen and reducing electron were employed. In this path formation of ferrous verdoheme-O₂ complex was followed by the addition of one electron to the ferrous-oxycomplex to produce ferric peroxide intermediate. The ferric peroxide intermediate experienced an intramolecular nucleophilic attack to the most positive position at 5-oxo carbons on the ring to form a closed ring biliverdin. Subsequently the ring opening process took place and the iron (III) biliverdin complex was formed. Closed ring iron biliverdin intermediate and open ring iron biliverdin formed as a product of verdoheme cleavage were respectively 13.20 and 32.70 kcal mol⁻¹ more stable than ferric peroxide intermediate. Barrier energy for conversion of ferric peroxide to closed ring Fe (III) biliverdin and from the latter to Fe (III) biliverdin were respectively 8.67 and 3.35 kcal mol⁻¹. In this path spin ground states are doublet except for iron (III) biliverdin in which spin state is quartet. In the second path a ferrous-O₂ complex was formed and, without going to a one electron reduction process, nucleophilic attack of iron superoxide complex took place followed by the formation of iron (III) biliverdin. This path is thermodynamically and kinetically less favorable than the first one. In addition, iron hydro peroxy complex or direct attack of O₂ to macrocycle to form an isoporphyrin type intermediate have shown energy surfaces less favorable than aforementioned routes.     

Proximal ligand control of heme iron coordination structure and reactivity with hydrogen peroxide: investigations of the myoglobin cavity mutant H93G with unnatural oxygen donor proximal ligands

The role of the proximal heme iron ligand in activation of hydrogen peroxide and control of spin state and coordination number in heme proteins is not yet well understood. Although there are several examples of amino acid sidechains with oxygen atoms which can act as potential heme iron ligands, the occurrence of protein-derived oxygen donor ligation in natural protein systems is quite rare. The sperm whale myoglobin cavity mutant H93G Mb (D. Barrick, Biochemistry 33 (1994) 6546) has its proximal histidine ligand replaced by glycine, a mutation which leaves an open cavity capable of accommodation of a variety of unnatural potential proximal ligands. This provides a convenient system for studying ligand-protein interactions. Molecular modeling of the proximal cavity in the active site of H93G Mb indicates that the cavity is of sufficient size to accommodate benzoate and phenolate in conformations that allow their oxygen atoms to come within binding distance of the heme iron. In addition, benzoate may occupy the cavity in an orientation which allows one carboxylate oxygen atom to ligate to the heme iron while the other carboxylate oxygen is within hydrogen bonding distance of serine 92. The ferric phenolate and benzoate complexes have been prepared and characterized by UV-visible and MCD spectroscopies. The benzoate adduct shows characteristics of a six-coordinate high-spin complex. To our knowledge, this is the first known example of a six-coordinate high-spin heme complex with an anionic oxygen donor proximal ligand. The benzoate ligand is displaced at alkaline pH and upon reaction with hydrogen peroxide. The phenolate adduct of H93G Mb is a five-coordinate high-spin complex whose UV-visible and MCD spectra are distinct from those of the histidine 93 to tyrosine (H93Y Mb) mutant of sperm whale myoglobin. The phenolate adduct is stable at alkaline pH and exhibits a reduced reactivity with hydrogen peroxide relative to that of both native ferric myoglobin, and the exogenous ligand-free derivative of ferric H93G Mb. These observations indicate that the identity of the proximal oxygen donor ligand has an important influence on both the heme iron coordination number and the reactivity of the complex with hydrogen peroxide.     

Spectroscopic characterization and ligand-binding properties of chlorite dismutase from the chlorate respiring bacterial strain GR-1.

Chlorite dismutase (EC 1.13.11.49), an enzyme capable of reducing chlorite to chloride while producing molecular oxygen, has been characterized using EPR and optical spectroscopy. The EPR spectrum of GR-1 chlorite dismutase shows two different high-spin ferric heme species, which we have designated 'narrow' (gx,y,z = 6.24, 5.42, 2.00) and 'broad' (gz,y,x = 6.70, 5.02, 2.00). Spectroscopic evidence is presented for a proximal histidine co-ordinating the heme iron center of the enzyme. The UV/visible spectrum of the ferrous enzyme and EPR spectra of the ferric hydroxide and imidazole adducts are characteristic of a heme protein with an axial histidine co-ordinating the iron. Furthermore, the substrate analogs nitrite and hydrogen peroxide have been found to bind to ferric chlorite dismutase. EPR spectroscopy of the hydrogen peroxide adduct shows the loss of both high-spin and low-spin ferric signals and the appearance of a sharp radical signal. The NO adduct of the ferrous enzyme exhibits a low-spin EPR signal typical of a five-co-ordinate heme iron nitrosyl adduct. It seems that the bond between the proximal histidine and the iron is weak and can be broken upon binding of NO. The midpoint potential, Em(Fe3+/2+) = -23 mV, of chlorite dismutase is higher than for most heme enzymes. The spectroscopic features and redox properties of chlorite dismutase are more similar to the gas-sensing hemoproteins, such as guanylate cyclase and the globins, than to the heme enzymes.     

Mechanisms of catalase activity of heme peroxidases

In the absence of exogenous electron donors monofunctional heme peroxidases can slowly degrade hydrogen peroxide following a mechanism different from monofunctional catalases. This pseudo-catalase cycle involves several redox intermediates including Compounds I, II and III, hydrogen peroxide reduction and oxidation reactions as well as release of both dioxygen and superoxide. The rate of decay of oxyferrous complex determines the rate-limiting step and the enzymes’ resistance to inactivation. Homologous bifunctional catalase-peroxidases (KatGs) are unique in having both a peroxidase and high hydrogen dismutation activity without inhibition reactions. It is demonstrated that KatGs follow a similar reaction pathway as monofunctional peroxidases, but use a unique post-translational distal modification (Met⁺-Tyr-Trp adduct) in close vicinity to the heme as radical site that enhances turnover of oxyferrous heme and avoids release of superoxide. Similarities and differences between monofunctional peroxidases and bifunctional KatGs are discussed and mechanisms of pseudo-catalase activity are proposed.     

Effects of citrinin on iron-redox cycle

The ability of the mycotoxin citrinin to act as an inhibitor of iron-induced lipoperoxidation of biological membranes prompted us to determine whether it could act as an iron chelating agent, interfering with iron redox reactions or acting as a free radical scavenger. The addition of Fe3+ to citrinin rapidly produced a chromogen, indicating the formation of citrinin-Fe3+ complexes. An EPR study confirms that citrinin acts as a ligand of Fe3+, the complexation depending on the [Fe3+]:[citrinin] ratios. Effects of citrinin on the iron redox cycle were evaluated by oxygen consumption or the o-phenanthroline test. No effect on EDTA-Fe2+-->EDTA-Fe3+ oxidation was observed in the presence of citrinin, but the mycotoxin inhibited, in a dose-dependent manner, the oxidation of Fe2+ to Fe3+ by hydrogen peroxide. Reducing agents such as ascorbic acid and DTT reduced the Fe3+-citrinin complex, but DTT did not cause reduction of Fe3+-EDTA, indicating that the redox potentials of Fe3+-citrinin and Fe3+-EDTA are not the same. The Fe2+ formed from the reduction of Fe3+-citrinin by reducing agents was not rapidly reoxidized to Fe3+ by atmospheric oxygen. Citrinin has no radical scavenger ability as demonstrated by the absence of DPPH reduction. However, a reaction between citrinin and hydrogen peroxide was observed by UV spectrum changes of citrinin after incubation with hydrogen peroxide. It was also observed that citrinin did not induce direct or reductive mobilization of iron from ferritin. These results indicate that the protective effect on iron-induced lipid peroxidation by citrinin occurs due to the formation of a redox inactive Fe3+-citrinin complex, as well as from the reaction of citrinin and hydrogen peroxide.     

Chemistry for an essential biological process: the reduction of ferric iron

In biological systems, the predominant form of iron is the trivalent Fe(III) form, which is potentially not readily bioavailable because of its hydrolysis and polymerization to insoluble forms. It is also the easiest of the two predominant forms of iron to chelate selectively. In a short overview of iron chemistry, we point out some of the pitfalls using standard redox potentials, comment on the interaction of ferric complexes with hydrogen peroxide to give hydroxyl radicals and address the release of iron from ferrisiderophores. In biological systems there are two classes of ferric reductases, the soluble flavin reductases found in prokaryotes, and the membrane-bound cytochrome b-like reductases found in eukaryotes. Finally the role of dissimilatory ferric reduction in microbial respiration and biomineralization is discussed.     

The secondary coordination sphere and axial ligand effects on oxygen reduction reaction by iron porphyrins: a DFT computational study

Oxygen reduction reaction (ORR) catalyzed by a bio-inspired iron porphyrin bearing a hanging carboxylic acid group over the porphyrin ring, and a tethered axial imidazole ligand was studied by DFT calculations. BP86 free energy calculations of the redox potentials and pK _a’s of reaction components involved in the proton coupled electron transfer (PCET) reactions of the ferric-hydroxo and -superoxo complexes were performed based on Born–Haber thermodynamic cycle in conjunction with a continuum solvation model. The comparison was made with iron porphyrins that lack either in the hanging acid group or axial ligand, suggesting that H-bond interaction between the carboxylic acid and iron-bound hydroxo, aquo, superoxo, and peroxo ligands (de)stabilizes the Fe–O bonding, resulting in the increase in the reduction potential of the ferric complexes. The axial ligand interaction with the imidazole raises the affinity of the iron-bound superoxo and peroxo ligands for proton. In addition, a low-spin end-on ferric-hydroperoxo intermediate, a key precursor for O–O cleavage, can be stabilized in the presence of axial ligation. Thus, selective and efficient ORR of iron porphyrin can be achieved with the aid of the secondary coordination sphere and axial ligand interactions.