Coordination chemistry of iron(III)-porphyrin-antibody complexes.

An artificial peroxidase-like hemoprotein has been obtained by associating a monoclonal antibody, 13G10, and its iron(III)-alpha,alpha,alpha,beta-meso-tetrakis(ortho-carboxyphenyl)porphyrin [Fe(ToCPP)] hapten. In this antibody, about two-thirds of the porphyrin moiety is inserted in the binding site, its ortho-COOH substituents being recognized by amino-acids of the protein, and a carboxylic acid side chain of the protein acts as a general acid base catalyst in the heterolytic cleavage of the O-O bond of H2O2, but no amino-acid residue is acting as an axial ligand of the iron.We here show that the iron of 13G10-Fe(ToCPP) is able to bind, like that of free Fe(ToCPP), two small ligands such as CN-, but only one imidazole ligand, in contrast to to the iron(III) of Fe(ToCPP) that binds two. This phenomenon is general for a series of monosubstituted imidazoles, the 2- and 4-alkyl-substituted imidazoles being the best ligands, in agreement with the hydrophobic character of the antibody binding site. Complexes of antibody 13G10 with less hindered iron(III)-tetraarylporphyrins bearing only one [Fe(MoCPP)] or two meso-[ortho-carboxyphenyl] substituents [Fe(DoCPP)] also bind only one imidazole. Finally, peroxidase activity studies show that imidazole inhibits the peroxidase activity of 13G10-Fe(ToCPP) whereas it increases that of 13G10-Fe(DoCPP). This could be interpreted by the binding of the imidazole ligand on the iron atom which probably occurs in the case of 13G10-Fe(ToCPP) on the less hindered face of the porphyrin, close to the catalytic COOH residue, whereas in the case of 13G10-Fe(DoCPP) it can occur on the other face of the porphyrin. The 13G10-Fe(DoCPP)-imidazole complex thus constitutes a nice artificial peroxidase-like hemoprotein, with the axial imidazole ligand of the iron mimicking the proximal histidine of peroxidases and a COOH side chain of the antibody acting as a general acid-base catalyst like the distal histidine of peroxidases does.     

Chemical properties of water-soluble porphyrins. 4. The reaction of a 'picket-fence-like' iron (III) complex with the superoxide oxygen couple

Solution properties of the iron-(III) 'picket-fence-like' porphyrin, Fe(III)-alpha,alpha,alpha, beta-tetra-ortho (N-methyl-isonicotinamidophenyl) porphyrin, (Fe(III)PFP) were investigated. These were acid/base properties of the aquo complex with pKa of 3.9 and its aggregation (formation of dimer with K = 1 X 10(-10) dm3 mol-1), complex formation with cyanide ions and 1-methyl imidazole (1-MeIm), spectral properties of the three iron complexes in their ferric and ferrous form and the one-electron reduction potential of these complexes. Knowing these properties, the reaction of the ferric complexes, aquo, dicyano and bis (1-MeIm), with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the iron (III) aquo complex which governs the catalytic efficiency of the metalloporphyrin upon the disproportionation of the superoxide radical was 7.6 X 10(7) dm3 mol-1 s-1, two orders of magnitude faster when compared to the reaction of each of the other complexes. The reduction by other radicals with all iron (III) complexes had similar second-order rate constants (10(9) to 10(10) dm3 mol-1 s-1). The reduction reaction in all cases produced Fe(II)PEP and no intermediate was found. The oxidation reaction of Fe(II)PEP by O2- was one order of magnitude faster when compared to the reduction of Fe(III)PFP by the same radical. Since the reactivity of O2- toward the three iron (III) porphyrin complexes follows their reduction potentials, it is suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The reactions of the Fe(II)PFP complexes with dioxygen were also studied. The aquo complex was found to be first order in O2 and second order in Fe(II)PFP, suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The intermediate formation was corroborated by evidence of the rapid CO binding reaction to the aquo complex of Fe(II)PFP. The two other complexes reacted very slowly with O2 as well as with CO.     

Air-stable,heme-like water-soluble iron(II) porphyrin: in situ preparation and characterization

Preparation of the water-soluble, kinetically labile, high-spin iron(II) tetrakis(4-sulfonatophenyl)porphyrin, Fe(II)TPPS⁴⁻, has been realized in neutral or weakly acidic solutions containing acetate buffer. The buffer played a double role in these systems: it was used for both adjusting pH and, via formation of an acetato complex, trapping trace amounts of iron(III) ions, which would convert the iron(II) porphyrins to the corresponding iron(III) species. Fe(II)TPPS⁴⁻ proved to be stable in these solutions even after saturation with air or oxygen. In the absence of acetate ions, however, iron(II) ions play a catalytic role in the formation of iron(III) porphyrins. While the kinetically inert iron(III) porphyrin, Fe(III)TPPS³⁻, is a regular one with no emission and photoredox properties, the corresponding iron(II) porphyrin displays photoinduced features which are typical of sitting-atop complexes (redshifted Soret absorption and blueshifted emission and Q absorption bands, photoinduced porphyrin ligand-to-metal charge transfer, LMCT, reaction). In the photolysis of Fe(II)TPPS⁴⁻ the LMCT process is followed by detachment of the reduced metal center and an irreversible ring-opening of the porphyrin ligand, resulting in the degradation of the complex. Possible oxygen-binding ability of Fe(II)TPPS⁴⁻ (as a heme model) has been studied as well. Density functional theory calculations revealed that in solutions with high acetate concentration there is very little chance for iron(II) porpyrin to bind and release O₂, deviating from heme in a hydrophobic microenvironment in hemoglobin. In the presence of an iron(III)-trapping additive that is much less strongly coordinated to the iron(II) center than the acetate ion, Fe(II)TPPS⁴⁻ may function as a heme model.     

Iron normal mode dynamics in (nitrosyl)iron(II)tetraphenylporphyrin from X-ray nuclear resonance data

The complete iron atom vibrational spectrum has been obtained by refinement of normal mode calculations to nuclear inelastic x-ray absorption data from (nitrosyl)iron(II)tetraphenylporphyrin, FeTPP(NO), a useful model for heme dynamics in myoglobin and other heme proteins. Nuclear resonance vibrational spectroscopy (NRVS) provides a direct measurement of the frequency and iron amplitude for all normal modes involving significant displacement of (57)Fe. The NRVS measurements on isotopically enriched single crystals permit determination of heme in-plane and out-of-plane modes. Excellent agreement between the calculated and experimental values of frequency and iron amplitude for each mode is achieved by a force-field refinement. Significantly, we find that the presence of the phenyl groups and the NO ligand leads to substantial mixing of the porphyrin core modes. This first picture of the entire iron vibrational density of states for a porphyrin compound provides an improved model for the role of iron atom dynamics in the biological functioning of heme proteins.     

Reduction of Fe(III) porphyrin hydroxides by heterocyclic aromatic amines

The reduction of iron(III) porphyrin hydroxides by the heterocyclic aromatic amines, pyridine, 1-methylimidazole and derivatives, occurs in toluene to give the bisamine iron(II) porphyrin complexes. The reaction has not been fully characterized but is found to proceed through a different mechanism from that reported for the similar reductions by 1° and 2° amines in the absence of hydroxide ion. Preliminary data indicate that the first step in the reduction is formation of the bisamine Fe(III) porphyrin complex from the hydroxide. Nucleophilic attack by hydroxide ion on the aromatic ring of an axially ligated pyridine or methylimidazole of the Fe(III) complex followed by homolytic cleavage of the FeN bond is proposed.     

The nature of the high-valent complexes in the catalytic cycles of hemoproteins

We report geometry optimization results on heme compound I (ferryl-oxo + porphyrin cation radical), compound II (ferryl-oxo) and ferric-hydroxo species with thiolate or imidazole axial ligands. We also examine protonated forms of compound I and compound II species, prompted by recent reports that, in at least two different hemoproteins, compound II may in fact contain a hydroxo rather than an oxo ligand. We propose that the stable compound I and compound II species of hemoproteins (e.g., peroxidases and myoglobin) most likely contain a hydroxo rather than the oxo ligand traditionally assumed, whereas the extremely transient compound I species of monooxygenase hemoproteins (P450) would contain an oxo atom. We show evidence impacting the previously accepted notion in hemoprotein computational chemistry that non-covalent interactions and medium polarization effects are essential in properly describing the electronic structure of heme-thiolate high-valent complexes. On a different note, we find that the charge density on the iron remains essentially the same throughout the catalytic cycles of heme-containing oxygenases and peroxidases, despite clear changes in bond lengths and spin densities suggestive of various iron oxidation states. The iron thus appears to simply relay the electron flux between the porphyrin and the axial dioxygen/superoxo/peroxo/oxo/hydroxo ligands.     

Heat-resistant oxygen-carrying hemoproteins consist of recombinant xylanases and synthetic iron(II) porphyrin

Synthetic iron(II) porphyrin (FeP) is equivalently incorporated into recombinant Thermotoga maritima xylanase B (TMX; family F/10 of glycoside hydrolase), producing a heat-resistant artificial hemoprotein (TMX-FeP) that can bind and release oxygen (O(2)) in aqueous medium (pH 7.3, 25 degrees C) in the same manner as hemoglobin and myoglobin. The oxygenated species was sufficiently stable; the half-lifetime against the ferric state (tau(1/2)) was 5 h. This O(2)-carrying hemoprotein showed a high degree of thermal stability over a wide range of temperatures up to 90 degrees C (tau(1/2) = 5 min at 90 degrees C and 9 min at 75 degrees C). Dictyoglomus thermophilum xylanase B (DTX; family G/11) also incorporates FeP, and DTX-FeP showed identical O(2)-binding parameters and thermostability. TMX-FeP is capable of catalyzing the beta-1,4-d-xylan hydrolysis reaction. Its larger K(m) value compared to that of TMX itself suggested competitive FeP binding to the active site of the host enzyme.     

Characterization of the reduction steps of Fe(III) porphyrins

Polarographic studies have shown that Fe(III) porphyrins undergo successively three one-electron reduction steps in dimethylformamide. The first involves the Fe(III)/Fe(II) redox couple. The second step proceeds to a second reduction of the metal ion and is attributed to the Fe(II)/Fe(I)_couple. This new reduction state of iron porphyrins has been characterized by ESR spectra and by absorption spectra in various solvents. This compound is not axially liganded by strong nucleophilic bases but is sensitive to solvation, the lone electron being localised in the d_z2 orbital. The third reduction step is assumed to involve a reduction of the porphyrin π-electron system.All these results have been confirmed by chemical reductions in tetrahydrofuran.     

Models for ferrous cytochrome b5: Sign inversions in the magnetic circular dichroism spectra of bis-imidazole ferrous porphyrin systems

The magnetic circular dichroism (MCD) spectrum of bis-imidazole ferrous tetraphenylporphyrin in the Soret region is nearly the mirror image of the spectrum of ferrous cytochrome b₅, a bis-imidazole (histidine)-ligated hemoprotein. Based on previous MCD studies of model and protein heme systems, a sign inversion in the spectra of two heme chromophores having essentially the same coordination structure is unexpected. To investigate whether the nature of the porphyrin itself could account for the observed spectral discrepancy, two additional model complexes, bis-imidazole ferrous protoporphyrin IX dimethylester and bis-imidazole ferrous octaethylporphyrin, whose peripheral porphyrin substituent patterns more closely match that of the protein- bound porphyrin, have been prepared and their MCD spectra measured. In these cases, the band pattern of the ferrous protein in the Soret region is successfully reproduced. It therefore appears that the anomalous MCD spectrum of the tetraphenylporphyrin complex can be attributed to the nature and positioning of the peripheral substituents on the porphyrin ring. Although iron tetraphenylporphyrin complexes are frequently used as models for protoporphyrin- containing hemoproteins, one should be aware that such differences in the peripheral porphyrin substituents may significantly affect the spectral properties of the model complex.     

First success of catalytic epoxidation of olefins by an electron-rich iron(III) porphyrin complex and H2O2: imidazole effect on the activation of H2O2 by iron porphyrin complexes in aprotic solvent

An electron-rich iron(III) porphyrin complex (meso-tetramesitylporphinato)iron(III) chloride [Fe(TMP)Cl], was found to catalyze the epoxidation of olefins by aqueous 30% H₂O₂ when the reaction was carried out in the presence of 5-chloro-1-methylimidazole (5-Cl-1-MeIm) in aprotic solvent. Epoxides were the predominant products with trace amounts of allylic oxidation products, indicating that Fenton-type oxidation reactions were not involved in the olefin epoxidation reactions. cis-Stilbene was stereospecifically oxidized to cis-stilbene oxide without giving isomerized trans-stilbene oxide product, demonstrating that neither hydroperoxy radical (HOO·) nor oxoiron(IV) porphyrin [(TMP)Fe^IV=O] was responsible for the olefin epoxidations. We also found that the reactivities of other iron(III) porphyrin complexes such as (meso-tetrakis(2,6-dichlorophenyl)porphinato)iron(III) chloride [Fe(TDCPP)Cl], (meso-tetrakis(2,6-difluorophenyl)porphinato)iron(III) chloride [Fe(TDFPP)Cl], and (meso-tetrakis(pentafluorophenyl)porphinato)iron(III) chloride [Fe(TPFPP)Cl] were significantly affected by the presence of the imidazole in the epoxidation of olefins by H₂O₂. These iron porphyrin complexes did not yield cyclohexene oxide in the epoxidation of cyclohexene by H₂O₂ in the absence of 5-Cl-1-MeIm in aprotic solvent; however, addition of 5-Cl-1-MeIm to the reaction solutions gave high yields of cyclohexene oxide with the formation of trace amounts of allylic oxidation products. We proposed, on the basis of the results of mechanistic studies, that the role of the imidazole is to decelerate the O–O bond cleavage of an iron(III) hydroperoxide porphyrin (or H₂O₂–iron(III) porphyrin adduct) and that the intermediate transfers its oxygen to olefins prior to the O–O bond cleavage.     

31P chemical shifts as structural probes for heme environments. 31P-NMR study of the binding of trimethyl phosphine to various hemoglobins and myoglobins

A 31P-NMR study of trimethyl phosphine bound to the heme iron(II) atom in natural myoglobins, hemoglobins and synthetic porphyrin iron(II) shows that the iron-bound trimethylphosphine 31P chemical shifts are sensitive to the presence of globin:separated NMR signals can be observed for 31PMe3 bound to the hemes of the alpha and beta chains. On the basis of previous hemoprotein studies, the markedly high field resonance observed with one of the two PMe3-rabbit subunits is consistent with a specific role of the distal histidine (E7) in rabbit alpha-subunit.     

Hemozymes peroxidase activity of artificial hemoproteins constructed from the Streptomyces lividans xylanase A and iron(III)-carboxy-substituted porphyrins

To develop artificial hemoproteins that could lead to new selective oxidation biocatalysts, a strategy based on the insertion of various iron-porphyrin cofactors into Xylanase A (Xln10A) was chosen. This protein has a globally positive charge and a wide enough active site to accommodate metalloporphyrins that possess negatively charged substituents such as microperoxidase 8 (MP8), iron(III)-tetra-alpha4-ortho-carboxyphenylporphyrin (Fe(ToCPP)), and iron(III)-tetra-para-carboxyphenylporphyrin (Fe(TpCPP)). Coordination chemistry of the iron atom and molecular modeling studies showed that only Fe(TpCPP) was able to insert deeply into Xln10A, with a KD value of about 0.5 microM. Accordingly, Fe(TpCPP)-Xln10A bound only one imidazole molecule, whereas Fe(TpCPP) free in solution was able to bind two, and the UV-visible spectrum of the Fe(TpCPP)-Xln10A-imidazole complex suggested the binding of an amino acid of the protein on the iron atom, trans to the imidazole. Fe(TpCPP)-Xln10A was found to have peroxidase activity, as it was able to catalyze the oxidation of typical peroxidase cosubstrates such as guaiacol and o-dianisidine by H2O2. With these two cosubstrates, the KM value measured with the Fe(TpCPP)-Xln10A complex was higher than those values observed with free Fe(TpCPP), probably because of the steric hindrance and the increased hydrophobicity caused by the protein around the iron atom of the porphyrin. The peroxidase activity was inhibited by imidazole, and a study of the pH dependence of the oxidation of o-dianisidine suggested that an amino acid with a pKA of around 7.5 was participating in the catalysis. Finally, a very interesting protective effect against oxidative degradation of the porphyrin was provided by the protein.     

Change in spectral properties of nitrosohemoglobin induced by UV light

The effect of UV radiation on the spectral characteristics of human nitrosohemoglobin in a wide dose range (151-4530 J/m2) was studied. It was shown that the irradiation of hemoprotein solutions with low doses of UV light (151-453 J/m2) led to local conformational rearrangements of the iron porphyrin moiety of the molecule. The apoprotein exhibited a high photostability under the conditions of the experiment. High doses of UV radiation (1359-4530 J/m2) induced the accumulation of methemoglobin in the protein sample. A scheme of the photoinduced formation of methemoglobin was elaborated.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

The coordination of imidazole and substituted pyridines by the hemeoctapeptide N-acetyl-ferromicroperoxidase-8 (FeIINAcMP8)

The N-terminus acetylated ferric hemeoctapeptide from cytochrome c, N-acetylmicroperoxidase-8 (Fe(III)-NAcMP8) can be reduced by dithionite in aqueous solution to produce Fe(II)-NAcMP8. The UV-Vis spectrum has a broad Soret band and relatively poorly defined Q bands which is consistent with a mixture of a five-coordinate high spin species with His as the axial ligand and a six-coordinate, predominantly high spin species with His/H(2)O as axial ligands. There are two spectroscopically observable pK(a)s at 8.7+/-0.1 and 10.9+/-0.2 which are attributed to ionization of a heme propionic acid group and coordinated H(2)O, respectively; a pK(a) > or = 14 is due to ionization of the proximal His ligand. Equilibrium constants were determined by UV-Vis spectrophotometry at 25.0+/-0.2 degrees C and 0.5 M ionic strength (NaClO(4)) for the coordination of imidazole and a number of substituted pyridines, and complement available data for the ferric hemepeptide, allowing a comparison to be made of the affinity of an iron porphyrin with Fe in the +2 and +3 oxidation states towards these ligands. Imidazole is coordinated more strongly by the ferric porphyrin (log K=4.08) than by the ferrous porphyrin (log K=3.40). The equilibrium constants for coordination of pyridines by the ferric and ferrous porphyrins increase and decrease, respectively, with increasing ligand basicity. Values determined by cyclic voltammetry show the same dependence on the identity of the ligand. In the ferric porphyrin, the stability of the complex increases with the basicity of the ligand and hence its ability to donate electron density onto the metal. In the case of the more electron rich ferrous porphyrin, greater stability occurs with pyridine ligands that have an electron withdrawing group and hence can accept electron density from the metal. This is consistent with the midpoint reduction potentials E(1/2) of the pyridine complexes determined by cyclic voltammetry; E(1/2) is linearly dependent on, and becomes more negative with an increase in, ligand basicity. Log K for coordination of pyridines by the ferrous hemepeptide correlates well with the energy of the ligand frontier orbital with pi symmetry, suggesting that pi-bonding effects are significant in determining the strength of binding of pyridines by a ferrous porphyrin.     

EPR and ENDOR studies of Fe(II) hemoproteins reduced and oxidized at 77 K

γ-irradiation of frozen solutions of Fe(II) hemoproteins at 77 K generates both electron paramagnetic resonance (EPR) active singly reduced and oxidized heme centers trapped in the conformation of the Fe(II) precursors. The reduction products of pentacoordinate (S = 2) Fe(II) globins, peroxidases and cytochrome P450cam show EPR and electron–nuclear double resonance (ENDOR) spectra characteristic of (3d ⁷) Fe(I) species. In addition, cryoreduced Fe(II) α-chains of hemoglobin and myoglobin exhibit an S = 3/2 spin state produced by antiferromagnetic coupling between a porphyrin anion radical and pentacoordinate (S = 2) Fe(II). The spectra of cryoreduced forms of Fe(II) hemoglobin α-chains and deoxymyoglobin reveal that the Fe(II) precursors adopt multiple conformational substates. Reduction of hexacoordinate Fe(II) cytochrome c and cytochrome b ₅ as well as carboxy complexes of deoxyglobins produces only Fe(II) porphyrin π-anion radical species. The low-valent hemoprotein intermediates produced by cryoreduction convert to the Fe(II) states at T > 200 K. Cryogenerated Fe(III) cytochrome c and cytochrome b ₅ have spectra similar to these for the resting Fe(III) states, whereas the spectra of the products of cryooxidation of pentacoordinate Fe(II) globins and peroxidases are different. Cryooxidation of CO–Fe(II) globins generates Fe(III) hemes with quantum-mechanically admixed S = 3/2, 5/2 ground states. The trapped Fe(III) species relax to the equilibrium ferric states upon annealing at T > 190 K. Both cryooxidized and reduced centers provide very sensitive EPR/ENDOR structure probes of the EPR-silent Fe(II) state. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chloroperoxidase compound I: Electron paramagnetic resonance and M?ssbauer studies

The green primary compound of chloroperoxidase was prepared by freeze-quenching the enzyme after rapid mixing with a 5-fold excess of peracetic acid. The electron paramagnetic resonance (EPR) spectra of these preparations consisted of at least three distinct signals that could be assigned to native enzyme, a free radical, and the green compound I as reported earlier. The absorption spectrum of compound I was obtained through subtraction of EPR signals measured under passage conditions. The signal is well approximated by an effective spin Seff = 1/2 model with g = 1.64, 1.73, 2.00 and a highly anisotropic line width. M?ssbauer difference spectra of compound I samples minus native enzyme showed well-resolved magnetic splitting at 4.2 K, an isomer shift delta Fe = 0.15 mm/s, and quadrupole splitting delta EQ = 1.02 mm/s. All data are consistent with the model of an exchange-coupled spin S = 1 ferryl iron and a spin S' = 1/2 porphyrin radical. As a result of the large zero field splitting, D, of the ferryl iron and of intermediate antiferromagnetic exchange, S.J.S'.J approximately 1.02 D, the system consists of three Kramers doublets that are widely separated in energy. The model relates the EPR and M?ssbauer spectra of the ground doublet to the intrinsic parameters of the ferryl iron, D/k = 52 K, E/D congruent to 0.035, and A perpendicular (gn beta n) = 20 T, and the porphyrin radical.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intramolecular photoredox reactions in iron(III) cytochrome c and its azide derivative

The irradiation of deaerated solutions of horse heart cytochrome c causes the reduction of Fe(III) to Fe(II). The dependence of the photoreaction quantum yield on pH shows that the photoreactive species is a form of cytochrome c which contains methionine-80 and histidine-18 as heme ligands. The primary photochemical event consists of an electron transfer from the sulphur of methionine- 80 to iron. The re-oxidation of the photochemically obtained Fe(II) protein gives a Fe(III) cytochrome which exhibits a typical low-spin absorption spectrum, lacking the 695-nm band and indicating that a strong field ligand, other than methionine-80, coordinates to the sixth binding site of the heme iron. Spectrophotometric titration of the photochemically modified Fe(III) cytochrome shows that histidine- 18 remains bound in the fifth position.The substitution of methionine-80 with the more oxidizable azide ligand increases the efficiency of the intramolecular electron transfer. Azide radicals, detected by spin-trapping ESR technique, are formed in the primary act. Visible-UV spectral data indicate that histidine-18 and methionine-80 occupy the fifth and sixth position, respectively, in the photoreaction product. All the results obtained correlate well with those previously obtained in investigations concerning the photoredox behavior of iron porphyrin complexes.     

Iron-porphyrin catalysis of the oxidative dealkylation of para-nitro-anisole and 7-ethoxycoumarin by cumylhydroperoxide: a possible model for the corresponding cytochrome P 450-dependent reactions

A biphasic system containing an iron porphyrin, Fe (TPP) (C1)¹ or [Fe(TPP)]₂O, efficiently catalyzes the cumyl-or tertiobutyl-hydroperoxide-supported dealkylation of p-nitroanisole and 7-ethoxycoumarin to the corresponding phenol and formaldehyde. Stoichiometric amounts of iron porphyrin and hydroperoxide give a quantitative reaction. Catalytic amounts of iron porphyrin give reaction rates and yields which are proportional to substrate concentration. With increasing hydroperoxide concentrations, the rates level offto limit values and the yield rapidly decreases. The maximum rates obtained approach those of the reactions mediated by cytochrome P 450-dependent monooxygenases.     

The ferroxidase activity of yeast frataxin

Frataxin is required for maintenance of normal mitochondrial iron levels and respiration. The mature form of yeast frataxin (mYfh1p) assembles stepwise into a multimer of 840 kDa (alpha(48)) that accumulates iron in a water-soluble form. Here, two distinct iron oxidation reactions are shown to take place during the initial assembly step (alpha --> alpha(3)). A ferroxidase reaction with a stoichiometry of 2 Fe(II)/O(2) is detected at Fe(II)/mYfh1p ratios of < or = 0.5. Ferroxidation is progressively overcome by autoxidation at Fe(II)/mYfh1p ratios of >0.5. Gel filtration analysis indicates that an oligomer of mYfh1p, alpha(3), is responsible for both reactions. The observed 2 Fe(II)/O(2) stoichiometry implies production of H(2)O(2) during the ferroxidase reaction. However, only a fraction of the expected total H(2)O(2) is detected in solution. Oxidative degradation of mYfh1p during the ferroxidase reaction suggests that most H(2)O(2) reacts with the protein. Accordingly, the addition of mYfh1p to a mixture of Fe(II) and H(2)O(2) results in significant attenuation of Fenton chemistry. Multimer assembly is fully inhibited under anaerobic conditions, indicating that mYfh1p is activated by Fe(II) in the presence of O(2). This combination induces oligomerization and mYfh1p-catalyzed Fe(II) oxidation, starting a process that ultimately leads to the sequestration of as many as 50 Fe(II)/subunit inside the multimer.