Functional models for catechol dioxygenases: iron(III) complexes of cis-facially coordinating linear 3N ligands

A series of 1:1 iron(III) complexes of simple and sterically hindered tridentate 3N donor ligands have been synthesized and studied as functional models for catechol dioxygenases. All of them are of the type [FeLCl3], where L is bis(pyrid-2-yl-methyl)amine (L1), N,N-bis(benzimidazol-2-ylmethyl)amine (L2), N-methyl-N'-(pyrid-2-ylmethyl)ethylenediamine (L3), N,N-dimethyl-N'-(pyrid-2-ylmethyl)-ethylenediamine (L4) and N-phenyl-N'-(pyrid-2-ylmethyl)ethylenediamine (L5). They have been characterised by spectral and electrochemical methods. The X-ray crystal structure of the complex [Fe(L4)Cl3] has been successfully determined. The complex crystallizes in the triclinic space group P1 with a = 7.250(6), b = 8.284(3), c = 12.409(4) angstroms, alpha = 80.84(3) degrees, beta = 86.76(6) degrees, gamma = 72.09(7) degrees and Z = 2. It possesses a distorted octahedral geometry in which the L4 ligand is cis-facially coordinated to iron(III) and the chloride ions occupy the remaining coordination sites. The systematic variation in the ligand donor atom type significantly influences the Lewis acidity of the iron(III) center and hence the binding interaction of the complexes with simple and substituted catechols. The spectroscopic and electrochemical properties of the catecholate complexes generated in situ have been investigated. All the complexes catalyze mainly the oxidative intradiol cleavage of 3,5-di-tert-butylcatechol (H2DBC) in the presence of dioxygen, which is unexpected of the cis-facial coordination of the ligands. The rate of intradiol catechol cleavage reaction depends upon the Lewis acidity of iron(III) center and steric demand and hydrogen-bonding functionalities of the ligands. Interestingly, the electron-sink property of N-phenyl substituent in [Fe(L5)Cl3] complex leads to enhancement in rate of cleavage. All these observations provide support to the substrate activation mechanism proposed for intradiol-cleaving enzymes.     

Some iron(III) complexes with polydentate Schiff base ligands

The iron(III) complexes of three Schiff base ligands are studied as their chloride or perchlorate salts and their electronic spectra, EPR spectra, and electrochemical behavior reported. Two of these ligands are formed from reaction between salicylaldehyde and 9 or 12-membered tri- or tetraazalkanes. EPR evidence indicates that one of the complexes, [1,12-bis(2-hydroxybenzylidene)-(1,4,9,12-tetraazadodec- 6-ene)iron(III)]perchlorate-1,5-water, is a spin-crossover species containing both high-spin and low-spin iron(III) in equilibrium. The third ligand comes from pyrrole-2-carboxaldehyde and a tetraazadodecane.     

The reactions of octaethylporphyrin and its iron (II) and iron (III) complexes with free radicals

The reactions of dilute solutions of octaethylporphyrin and its iron (II) and iron (III) complexes with methyl, 2-cyanopropyl, t-butoxy, and benzoyloxy radicals are described. The results are summarized: (i) The reactivity of the porphyrin and its high-spin iron (II) and iron (III) complexes toward alkyl and t-butoxy radicals stands in the order: Fe^II > Fe^III ? free porphyrin. For benzoyloxy radicals the order is Fe^II > Porp > Fe^III. (ii) The exclusive path of reaction of high-spin iron (II) porphyrin with radicals is the rapid reduction of the radical and generation of an iron (III) porphyrin. The dominant path of reaction of high-spin iron (III) porphyrin with alkyl and (presumably) t-butoxy radicals is a rapid axial inner sphere reduction of the porphyrin. An axial ligand of iron is transferred to the radical. (iv) The reaction of benzoyloxy radicals with high or low-spin iron (III) porphyrins occurs primarily at the meso position. With the low-spin dipyridyl complex in pyridine the attendant reduction to iron (II) can be observed spectrally. Methyl radicals also reduce this complex by adding to the meso position. (v) The reaction of a radical with either an iron (II) or an iron (III) porphyrin results in the generation of the other valence state of iron and consequently oxidation and reduction products emanating from both iron species are obtained. (vi) No evidence for an iron (IV) is intermediate is apparent. (vii) Iron (II) porphyrins in solvents that impart either spin state are easily oxidized by diacyl peroxides. The occurrence of both axial and peripheral redox reactions with the iron complexes supports an underlying premise of a recent theory of hemeprotein reactivity. The relevance of the work to bioelectron transfer and heme catabolism is noted.     

Complexation of iron(III) and iron(II) by citrate. Implications for iron speciation in blood plasma

Estimates of the concentrations and identity of the predominant complexes of iron with the low-molecular-mass ligands in vivo are important to improve current understanding of the metabolism of this trace element. These estimates require a knowledge of the stability of the iron-citrate complexes. Previous studies on the equilibrium properties of the Fe(III)-citrate and Fe(II)-citrate are in disagreement. Accordingly, in this work, glass electrode potentiometric titrations have been used to re-determine the formation constants of both the Fe(III)- and Fe(II)-citrate systems at 25 degrees C in 1.00 M (Na)Cl and the reliability of these constants has been evaluated by comparing the measured and predicted redox potentials of the ternary Fe(III)-Fe(II)-citrate system. The formation constants obtained in this way were used in computer simulation models of the low-molecular-mass iron fraction in blood plasma. Redox equilibria of iron are thus included in large models of blood plasma for the first time. The results of these calculations show the predominance of Fe(II)-carbonate complexes and a significant amount of aquated Fe(II) in human blood plasma.     

Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III)

Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition.     

Siderophore-Mediated microbial iron(III) uptake: An exercise in chiral recognition

Molecular recognition by microbial receptors for siderophores [natural iron(III) carriers] is examined with synthetic iron(III) carriers as structural probes. The iron(III) carriers have been designed to reproduce the two essential features of the natural siderophores: the capability to form octahedral iron(III) binding cavities and to fit specific membrane receptors. Specifically, analogs of tripodal ferrichrome and linear ferrioxamines have been prepared and examined. The ferrichrome analogs rely on C₃-symmetric binders that are assembled from triscarboxylates as anchors, amino acids as bridges, and terminal hydroxamate groups as binding sites. The ferrioxamine analogs are based on linear assemblies of three identical monomers, each derived from a chiral amino acid. The deliberate use of animo acid residues as variable building blocks enables us to systematically modify the molecules' envelopes and the preferred absolute configuration of the iron(III) complexes until optimal performance is reached. Examination of the synthetic analogs in Pseudomonas putida demonstrates that the domains around the iron(III) center and their chiral sense dictate the extent of recognition by the membrane receptors. It is also shown that the synthetic siderophore analogs may be designed to either exert a broader, or a more narrow range of microbial activity than the natural siderophores. The implications of these findings are discussed in relation to the possible design of species-specific antimicrobial agents. © 1993 Wiley-Liss, Inc.     

Interaction between iron(II) and hydroxamic acids: oxidation of iron(II) to iron(III) by desferrioxamine B under anaerobic conditions

Interaction between iron(II) and acetohydroxamic acid (Aha), alpha-alaninehydroxamic acid (alpha-Alaha), beta-alaninehydroxamic acid (beta-Alaha), hexanedioic acid bis(3-hydroxycarbamoyl-methyl)amide (Dha) or desferrioxamine B (DFB) under anaerobic conditions was studied by pH-metric and UV-Visible spectrophotometric methods. The stability constants of complexes formed with Aha, alpha-Alaha, beta-Alaha and Dha were calculated and turned out to be much lower than those of the corresponding iron(II) complexes. Stability constants of the iron(II)-hydroxamate complexes are compared with those of other divalent 3d-block metal ions and the Irving-Williams series of stabilities was found to be observed. Above pH 4, in the reactions between iron(II) and desferrioxamine B, the oxidation of the metal ion to iron(III) by the ligand was found. The overall reaction that resulted in the formation of the tris-hydroxamato complex [Fe(HDFB)]+ and monoamide derivative of DFB at pH 6 is: 2Fe2+ + 3H4DFB+ = 2[Fe(HDFB)]+ + H3DFB-monoamide+ + H2O + 4H+. Based on these results, the conclusion is that desferrioxamine B can uptake iron in iron(III) form under anaerobic conditions.     

Synthesis of rhodium(III), iridium(III) and osmium(III) complexes with tris(2-aminoethanethiolato)cobalt(III)

Polynuclear sulfur bridged complexes where the neutral complex tris(2-aminoethanethiolato)cobalt(III) acts as a tridentate ligand to rhodium(III), iridium(III) and osmium(III) have been prepared. These complexes have been characterized by electronic spectroscopy, vibrational spectroscopy and nuclear magnetic resonance spectroscopy. Along with the previously prepared complexes of iron(III), ruthenium(III) and cobalt(III), these complexes form two series of complexes with the group 8 and group 9 elements from all three transition series.     

Reaction between iron(III) and pyrazoic acid

In the reaction of [Fe(H₂O)₆]³⁺ with pyrazoic acid, reduction of iron(III) to iron(II) is observed. When an excess of iron is present, the reaction involves a transfer of four electrons per mole of acid. At room temperature the redox reaction, which is dependent on hydrogen ion, iron(III) and pyrazoic acid concentrations, is rather slow and is the rate-determining step. The kinetic study was carried out at 50.0 ± 0.1 °C. The redox reaction is followed by a fast reaction of the iron(II) with an excess of ligand, resulting in the production of well-known complexes, where the acid acts as a chelating ligand through the nitrogen and oxygen atoms.     

Reaction of iron(III)-polyaminocarboxylate complexes with hydrogen peroxide: Correlation between ligand structure and reactivity

Reactions of iron(III) complexes with five polyaminocarboxylates and hydrogen peroxide in an alkaline solution were investigated. Iron(III) complexes of which the ring including two nitrogen and iron atoms is five-membered formed a well-known stable side-on peroxo adduct. On the other hand, iron(III) complexes which have a six-membered ring formed a short-lived side-on peroxo adduct and then changed to iron(II) complex and superoxide. Electrochemical measurements showed that the redox potentials of the iron complexes having a six-membered ring are higher than those of the complexes having a five-membered ring. These results indicate that the chelate size is an important factor for tuning the redox potential of the iron center and for the reactivity toward hydrogen peroxide.     

Investigation of the reactions of iron(III) halobisdithiocarbamates with halogens: a novel series of fluxional homobinuclear iron(III) complexes with two different coordination spheres around the magnetic centers

In addition to the variety of products formed during the reaction of iron(III) halobisdithiocarbamates with halogens, some novel fluxional homobinuclear iron(III) complexes with two different coordination spheres around the magnetic centers have also been synthesized and studied. The formation of these products depends on the nature of both the molecular halogen and the reagent complex, as well as on the reaction conditions. The new compounds have been characterized chemically and by means of spectroscopic methods and magnetic susceptibility measurements. The volatility characteristics and thermogravimetric analysis data for the complexes were also studied. Finally, a general mechanism accounting for the variety of products formed in the reactions of iron(III) halobisdithiocarbamates with molecular halogens is proposed.     

A specific transporter for iron(III)-phytosiderophore in barley roots

Iron acquisition of graminaceous plants is characterized by the synthesis and secretion of the iron-chelating phytosiderophore, mugineic acid (MA), and by a specific uptake system for iron(III)-phytosiderophore complexes. We identified a gene specifically encoding an iron-phytosiderophore transporter (HvYS1) in barley, which is the most tolerant species to iron deficiency among graminaceous plants. HvYS1 was predicted to encode a polypeptide of 678 amino acids and to have 72.7% identity with ZmYS1, a first protein identified as an iron(III)-phytosiderophore transporter in maize. Real-time RT-PCR analysis showed that the HvYS1 gene was mainly expressed in the roots, and its expression was enhanced under iron deficiency. In situ hybridization analysis of iron-deficient barley roots revealed that the mRNA of HvYS1 was localized in epidermal root cells. Furthermore, immunohistological staining with anti-HvYS1 polyclonal antibody showed the same localization as the mRNA. HvYS1 functionally complemented yeast strains defective in iron uptake on media containing iron(III)-MA, but not iron-nicotianamine (NA). Expression of HvYS1 in Xenopus oocytes showed strict specificity for both metals and ligands: HvYS1 transports only iron(III) chelated with phytosiderophore. The localization and substrate specificity of HvYS1 is different from those of ZmYS1, indicating that HvYS1 is a specific transporter for iron(III)-phytosiderophore involved in primary iron acquisition from soil in barley roots.     

Potentiometric study of vitamin D3 complexes with manganese(II), iron(II), iron(III) and zinc(II) in water-ethanol medium.

Vitamin D3 (LH) complexes with manganese(II), iron(II), iron(III) and zinc(II) were identified in water-ethanol medium (30/70). Their stability constants were determined at 298 K and at a constant ionic strength of 0.100 M using potentiometric methods. The computerisation of the experimental data showed the presence of ML (M = metal, L = deprotonated vitamin D3) and ML2 species in all cases; in addition, the ML3 iron(III) complex was detected. The calculated overall stability constants beta for MnIIL, FeIIL, FeIIIL and ZnIIL are, respectively, in logarithms, 12.4, 16.5, 28.5 and 16.5. Under the experimental conditions, the only protonated species MLH detected was with iron(III).     

Synthesis, characterization, and antitumor activity of iron(II) and iron(III) complexes of alpha-N-heterocyclic carboxaldehyde thiosemicarbazones

Complexes of iron(II) and iron(III) with 1-formylisoquinoline thiosemicarbazone (1-iqtsc-H), 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone (4-Me-5-NH2-1-iqtsc-H) and 4-(m-aminophenyl)-2-formylpyridine thiosemicarbazone (4-m-NH2ph-2-pytsc-H) were synthesized and characterized by elemental analysis, conductance measurements, magnetic susceptibilities (from room temperature down to liquid N2 temperature), and M?ssbauer, electronic, and infrared spectral studies. On the basis of these studies, a highly distorted, high-spin, five-coordinate structure for Fe(HL)SO4 (HL = 1-iqtsc-H, 4-Me-5-NH2-1-iqtsc-H or 4-m-NH2ph-2-pytsc-H) and a distorted, low-spin, octahedral structure for Fe(HL)Cl2 are suggested. The EPR spectra of iron(III) complexes show that all have dxy low-spin ground state. All these complexes have been screened for their antitumor activity against the P 388 lymphocytic leukemia test system in mice and have been found to possess significant activity at the dosages employed.     

Formation of stable and metastable porphyrin- and corrole-iron(IV) complexes and isomerizations to iron(III) macrocycle radical cations

Oxidations of three porphyrin-iron(III) complexes (1) with ferric perchlorate, Fe(ClO₄)₃, in acetonitrile solutions at −40 °C gave metastable porphyrin-iron(IV) diperchlorate complexes (2) that isomerized to known iron(III) diperchlorate porphyrin radical cations (3) when the solutions were warmed to room temperature. The 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetramesitylporphyrin (TMP), and 2,3,7,8,12,13,17,18-octaethylporphyrin (OEP) systems were studied by UV-visible spectroscopy. Low temperature NMR spectroscopy and effective magnetic moment measurements were possible with the TPP and TMP iron(IV) complexes. Reactions of two corrole systems, 5,10,15-tris(pentafluorophenyl)corrole (TPFC) and 5,15-bis(pentafluorophenyl)-10-p-methoxyphenylcorrole (BPFMC), also were studied. The corrole-iron(IV) chlorides reacted with silver salts to give corrole-iron(IV) complexes. The corrole-iron(IV) nitrate complexes were stable at room temperature. (TPFC)-iron(IV) toslyate, (TPFC)-iron(IV) chlorate, and (BPFMC)-iron(IV) chlorate were metastable and rearranged to their electronic isomers iron(III) corrole radical cations at room temperature. (TPFC)-iron(III) perchlorate corrole radical cation was the only product observed from reaction of the corrole-iron(IV) chloride with silver perchlorate. For the metastable iron(IV) species, the rates of isomerizations to the iron(III) macrocycle radical cation electronic isomers in dilute acetonitrile solutions were relatively insensitive to electron demands of the macrocyclic ligand but reflected the binding strength of the ligand to iron. Kinetic studies at varying temperatures and concentrations indicated that the mechanisms of the isomerization reactions are complex, involving mixed order reactivity.     

Electron spin echo envelope modulation evidence for carbonate binding to iron(III) and copper(II) transferrin and lactoferrin

Iron binding to transferrin and lactoferrin requires a synergistic anion, which is carbonate in vivo. The anion is thought to play a key role in iron binding and release. To understand better the iron-carbonate interaction, experiments were performed with iron(III) and copper(II) complexes of human milk lactoferrin and serum transferrin with carbon-13-labeled carbonate. Modulation frequencies were present in the Fourier transforms of two-pulse and three-pulse electron spin echo envelope modulation data for the Fe(III) and Cu(II) complexes, consistent with binding of carbonate to both metals. The metal-13C interaction was similar for the lactoferrin and transferrin complexes. Spin coupling to the nitrogen of a coordinated histidine imidazole was observed for both metals. Both the metal-nitrogen and the metal-carbon spin coupling constants were about a factor of 5 smaller for the iron complexes than for the copper complexes, which indicated substantial similarity in the metal-carbonate and metal-imidazole binding for the two metals.     

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.     

Monitoring of iron(III) removal from biological sources using a fluorescent siderophore.

We present here the physicochemical and biochemical properties of NBD-DFO, the 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of the siderophore, desferrioxamine B (DFO) (Lytton et al., Mol. Pharmacol. 40, 584, 1991). Modification of DFO at its terminal amine renders it more lipophilic, imparts to it fluorescent properties, and is conservative of the high-affinity iron(III) binding capacity. NBD-DFO partitions readily from aqueous solution into n-octanol (Pcoeff = 5) and displays solvent-induced shifts in absorption and fluorescence spectra. The relative quantum yield of the probe's fluorescence increases over a 10-fold range with decreasing dielectric constant of the solvent. Fluorescence is quenched upon binding of iron(III) to the probe. We demonstrate here the application of NBD-DFO for the specific detection and monitoring of iron (III) in solutions and iron(III) mobilization from cells. Interactions between fluorescent siderophore and the ferriproteins ferritin and transferrin were monitored under physiological conditions. Iron removal from ferritin was evident by the demonstrable quenching of NBD-DFO fluorescence by scavenged iron(III). Quantitation of iron sequestered from cells by NBD-DFO or from other siderophore-iron(III) complexes was accomplished by dissociation of NBD-DFO-Fe complex by acidification and addition of excess ethylenediamin-etetraacetic acid. The sensitivity of the method and the iron specificity indicate its potential for monitoring chelatable iron under conditions of iron-mediated cell damage, iron overload, and diseases of iron imbalance such as malaria.     

The displacement of iron(III) from its complexes with the anticancer drugs piroxantrone and losoxantrone by the hydrolyzed form of the cardioprotective agent dexrazoxane

Piroxantrone and losoxantrone are new DNA topoisomerase II-targeting anthrapyrazole antitumor agents that display cardiotoxicity both clinically and in animal models. A study was undertaken to see whether dexrazoxane or its hydrolysis product ADR-925 could remove iron(III) from its complexes with piroxantrone or losoxantrone. Their cardiotoxicity may result from the formation of iron(III) complexes of losoxantrone and piroxantrone. Subsequent reductive activation of their iron(III) complexes likely results in oxygen-free radical-mediated cardiotoxicity. Dexrazoxane is in clinical use as a doxorubicin cardioprotective agent. Dexrazoxane presumably acts through its hydrolyzed metal ion binding form ADR-925 by removing iron(III) from its complex with doxorubicin, or by scavenging free iron(III), thus preventing oxygen-free radical-based oxidative damage to the heart tissue. ADR-925 was able to remove iron(III) from its complexes with piroxantrone and losoxantrone, though not as efficiently or as quickly as it could from its complexes with doxorubicin and other anthracyclines. This study provides a basis for utilizing dexrazoxane for the clinical prevention of anthrapyrazole cardiotoxicity.     

4-Nitrocatechol as a colorimetric probe for non-heme iron dioxygenases.

4-Nitrocatechol is examined as an active site probe for non-heme iron dioxygenases and found to be of value, particularly with those containing iron in the Fe(II) oxidation state. 4-Nitrocatechol is astrong competitive inhibitor of substrate oxygenation by protocatechuate 3,4-dioxygenase, forming a reversible complex with this enzyme, and by pyrocatechase. The number of binding sites per enzyme molecule titrated spectrophotometrically with 4-nitrocatechol agrees with results from previous studies with either the principal substrate or other analogues, as expected of an effective probe. Despite these facts and the observation that both enzymes cleave the same substrates at the same carbon-carbon bond, the optical and electron paramagnetic resonance (EPR) spectra of their 4-nitrocatechol complexes are remarkably different. The 4-nitocatechol-protocatechuate 3,4-dioxygenase optical spectra resemble that of the 4-nitrocatecholate ion shifted 20 to 30 nm to longer wavelength. Concomitant with this change the EPR signal centered at g equal 4.28 shows increased rhombicity (g values at 4.74, 4.28, and 3.74).In contrast, the spectrum of the 4-nitrocatechol-pyrocatechase complex has a maximum at the same wavelength as that of a 1:1 solution of free Fe(II) and 4-nitrocatechol in the absence of enzyme after titration of the catecholic protons with base and the g equal 4.28 EPR signal is not resolved at liquid N-2 temperature. These changes are interpreted as resulting in part from a pronounced change in the ligand fields about the irons at the active sites which in the case of protocatechuate 3,4-dioxygenase leads to enzyme inactivation. The results also are the first indication that substrate analogues change their ionization form upon complexation with Fe (III) dioxygenases. The interaction of the probe with metapyrocatechase, an Fe(III) containing dioxygenase, and with several additional oxygenases and hydroperoxidases is also briefly examined. The probe is not specific for any particular class of non-heme iron dioxygenases.