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.     

Valence-tautomerism in high-valent iron and manganese porphyrins

Iron and manganese hemes are "high-valent" when the valence state of the metal exceeds III. Redox chemistry of the high valent metal complexes involves redistribution of holes and electrons over the metal ion and the porphyrin and axial ligands, defined as valence tautomerism. Thus, catalytic pathways of heme-containing biomolecules such as peroxidases, catalases and cytochromes P450 involve valence tautomerism, as do pathways of biomimetic oxygen transfer catalysis by manganese porphyrins, robust catalysts with potential commercial value. Determinants of the site of electron abstraction are key to understanding valence tautomerism. In model systems, metal-centered oxidation is supported by hard anionic axial ligands that are also strongly pi-donating, such as oxo, aryl, bix-methoxy and bis-fluoro groups. Manganese(IV) is more stable than iron(IV) and metal-centered one-electron oxidations occur with weaker pi-donating axial ligands such as bisazido, -isocyanato, -hypochlorito and bis chloro groups. Virtually all known high-valent iron porphyrin complexes oxidized by two-electrons above the ferric state are coordinated by the strongly pi-donating oxo or nitrido ligands. In all well-characterized oxo complexes, iron is in the ferryl state and the second oxidizing equivalent resides on the porphyrin. Complexes with iron(V) have not been definitively characterized. One-electron oxidation of oxomanganese(IV) porphyrin complexes gives the oxomanganese(IV) porphyrin pi-cation redicals. In aqueous solution, oxidation of Mn(III) complexes of tetra cationic N-methylpyridiniumylporphyrin isomers by monooxygen donors yields a transient oxomanganese(V) species.     

Remarkable axial thiolate ligand effect on the oxidation of hydrocarbons by active intermediate of iron porphyrin and cytochrome P450

In order to examine the reactivity of active intermediate derived form iron porphyrins, competitive oxidations of alkane and alkene were carried out. It has been proposed that the first step of alkane hydroxylation is H atom abstraction and that of alkene is one-electron transfer. Therefore, it is expected that alkene-alkane competitive oxidation can be used as a probe for discrimination of differences in chemical properties among active species. Cytochrome P450 and SR complex, which is a stable thiolate-ligated iron porphyrin, mediated the oxidation of alkane much more preferentially than iron porphyrin coordinated by imidazole or chloride. These results indicate that thiolate coordination alters the reactivity of the two-electron-oxidized intermediate in a manner that is much more favorable to alkane hydroxylation than the case of chloride or imidazole coordination.     

Delocalization over the heme and the axial ligands of one of the two oxidizing equivalents stored above the ferric state in the peroxidase and catalase Compound-I intermediates: indirect participation of the proximal axial ligand of iron in the oxidation reactions catalyzed by heme-based peroxidases and catalases?

 A comparison of the exchange interactions arising in the peroxidase and catalase Compound I intermediates and their iron(IV)-oxo porphyrin π-cation radical models, both of which are two oxidizing equivalents above the ferric state, suggests that in the models the oxidizing equivalent is localized on the porphyrin ring, while in the proteins it is partly delocalized onto the proximal ligand. Thus, the proximal axial ligand of iron participates indirectly in the oxidation reactions catalyzed by the enzymes. Possible roles of the axial ligand in the catalytic mechanism of these heme-based enzymes are discussed. Received and accepted: 7 May 1996     

One electron oxidation of benzyltrialkylsilanes catalysed by lignin peroxidase: comparison with the oxidation induced by chemical oxidants

Lignin peroxidase catalyses the H(2)O(2)-induced oxidation of 4-methoxybenzyltrimethylsilane by an electron transfer mechanism. The intermediate radical cation undergoes preferentially C(alpha)[bond]H deprotonation to give 4-methoxybenzaldehyde whereas C(alpha)[bond]Si bond cleavage is a minor fragmentation pathway and leads to 4-methoxybenzyl alcohol. Similar results are obtained in the oxidation catalysed by the water soluble model compound 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrinatoiron(III) pentachloride. Instead, in the oxidation promoted by the genuine one-electron transfer oxidant potassium dodecatungstocobalt(III)ate C(alpha)[bond]Si bond cleavage is the exclusive fragmentation process of the intermediate radical cation. It is suggested that in the enzymatic and biomimetic oxidations of 4-methoxybenzyltrimethylsilane the deprotonation of the intermediate radical cation is promoted by the reduced form [PorFe(IV)[double bond]O] of the active oxidant, which is an iron-oxo porphyrin radical cation.     

A tetrahydrobiopterin radical forms and then becomes reduced during Nomega-hydroxyarginine oxidation by nitric-oxide synthase

Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from l-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H4B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-l-arginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy). Significant biopterin radical (>0.5 per heme) formed during reactions catalyzed by iNOSoxy that contained either H4B or 5-methyl-H4B. Biopterin radical formation was kinetically linked to conversion of a heme-dioxy intermediate to a heme-NO product complex. The biopterin radical then decayed within a 200-300-ms time period just prior to dissociation of NO from a ferric heme-NO product complex. Measures of final biopterin redox status showed that biopterin radical decay occurred via an enzymatic one-electron reduction process that regenerated H4B (or 5MeH4B). These results provide evidence of a dual redox function for biopterin during the NOHA oxidation reaction. The data suggest that H4B first provides an electron to a heme-dioxy intermediate, and then the H4B radical receives an electron from a downstream reaction intermediate to regenerate H4B. The first one-electron transition enables formation of the heme-based oxidant that reacts with NOHA, while the second one-electron transition is linked to formation of a ferric heme-NO product complex that can release NO from the enzyme. These redox roles are novel and expand our understanding of biopterin function in biology.     

The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

The dehaloperoxidase paradox

The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.     

The dehaloperoxidase paradox

The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.     

The kinetics of flavine oxidation-reduction. II. Metal ion interactions.

The oxidation-reduction reactions of tetraacetylriboflavine in the presence of various metal ions in dimethylformamide have been investigated using the stopped-flow technique under anaerobic conditions. Dismutation kinetics in the presence of redox-inactive dissociated divalent metal ions such as Cd2+, Zn2+, and Fe2+ are typically triphasic. Metal ions act primarily upon an intermediate flavine dimer formed by fast association of flavoquinone and flavohydroquinone, resulting in a parallel formation and neutral and chelated radicals. A competition between metal ions and proton donors, e.g. the neutral flavohydroquinone (FredH3), is observed at the level of this intermediate complex. Small spectral changes occur secondarily as an ill-defined intermediate phase which could correspond to the reorganization of the solvation of radical chelate. The neutral radical is finally chelated at a much slower rate, the yield of total radical formation remaining almost unchanged during this kinetic phase. The oxidation of flavohydroquinone by ferric ions, either dissociated or strongly coordinated within a porphyrin, is complete and proceeds through biphasic kinetics. The first phase (Fred leads to F) is much faster than the second one (F leads to Fox). Dismutation resulting from the transient accumulation of neutral flavosemiquinone competes with the direct oxidation with ferric ions for the completion of the second oxidation step. The relative rate of dismutation is essentially limited by acidic-basic reactions in the absence of an excess of ferrous ion. The kinetic analysis of the direct oxidation reactions favors an outer-sphere mechanism for the electron transfer to the ferric ion, either free or strongly coordinated. The formation of a ferrous radical chelate can result from the dismutation reactions only when the amount of ferric ion initially present is not sufficient for complete oxidation.     

Reaction of Mycobacterium tuberculosis truncated hemoglobin O with hydrogen peroxide: evidence for peroxidatic activity and formation of protein-based radicals

In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H(2)O(2) to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme pi-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr(55) and Tyr(115), which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H(2)O(2) and the one-electron donor 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs.     

Kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations with organic reductants

Iron(IV)-oxo porphyrin radical cations are observed intermediates in peroxidase and catalase enzymes, where they are known as Compound I species, and the putative oxidizing species in cytochrome P450 enzymes. In this work, we report kinetic studies of reactions of iron(IV)-oxo porphyrin radical cations that can be compared to reactions of other metal-oxo species. The iron(IV)-oxo radical cations studied were those produced from 5,10,15,20-tetramesitylporphryinato-iron(III) perchlorate (1), 5,10,15,20-tetramesitylporphryinato-iron(III) chloride (2), both in CH(3)CN solvent, and that from 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(III) perchlorate (3) in CH(2)Cl(2) solvent. The substrates studied were alkenes and activated hydrocarbons diphenylmethane and ethylbenzene. For a given organic reductant, various iron(IV)-oxo porphyrin radical cations react in a relatively narrow kinetic range; typically the second-order rate constants vary by less than 1 order of magnitude for the oxidants studied here and the related oxidant 5,10,15,20-tetrakis(pentafluorophenyl)porphyrinato-iron(IV)-oxo porphyrin radical cation in CH(3)CN solvent. Charge transfer in the transition states for epoxidation reactions of substituted styrenes by oxidants 1 and 2, rho(+) values of -1.9 and -0.9, respectively, mirrors results found previously for related species. Competition kinetic reactions with a catalytic amount of porphyrin iron(III) species and a terminal oxidant give relative rate constants for oxidations of competing substrates that are somewhat smaller than the ratios of absolute rate constants. Water in CH(3)CN solutions has an apparent modest stabilizing effect on oxidant 1 as indicated in slightly reduced rate constants for oxidation reactions. The iron(IV)-oxo porphyrin radical cations are orders of magnitude less reactive than porphyrin-manganese(V)-oxo cations and a corrole-iron(V)-oxo species. The small environment effects found here suggest that high energy demanding hydrocarbon oxidation reactions catalyzed by cytochrome P450 enzymes might require highly reactive iron(V)-oxo transients as oxidants instead of the more stable, isomeric iron(IV)-oxo porphyrin radical cations.     

Methane monooxygenase catalyzed oxygenation of 1,1-dimethylcyclopropane. Evidence for radical and carbocationic intermediates

Methane monooxygenase catalyzes the oxygenation of 1,1-dimethylcyclopropane in the presence of O2 and NADH to (1-methylcyclopropyl)methanol (81%), 3-methyl-3-buten-1-ol (6%), and 1-methyl-cyclobutanol (13%). Oxygenation by 18O2 using the purified enzyme proceeds with incorporation of 18O into the products. Inasmuch as methane monooxygenase catalyzes the insertion of O from O2 into a carbon-hydrogen bond of alkanes, (1-methylcyclopropyl)methanol appears to be a conventional oxygenation product. 3-Methyl-3-buten-1-ol is a rearrangement product that can be rationalized on the basis that enzymatic oxygenation of 1,1-dimethylcyclopropane proceeds via the (1-methylcyclopropyl)carbinyl radical, which is expected to undergo rearrangement with ring opening to the homoallylic 3-methyl-3-buten-1-yl radical in competition with conventional oxygenation. Oxygenation of the latter radical gives 3-methyl-3-buten-1-ol. 1-Methylcyclobutanol is a ring-expansion product, whose formation is best explained on the basis that the 1-methylcyclobutyl tertiary carbocation is an oxygenation intermediate. This cation would result from rearrangements of carbocations derived by one-electron oxidation of either radical intermediate. The fact that both 3-methyl-3-buten-1-ol and 1-methylcyclobutanol are produced suggests that the oxygenation mechanism involves both radical and carbocationic intermediates. Radicals and carbocations can both be intermediates if they are connected by an electron-transfer step. A reasonable reaction sequence is one in which the cofactor (mu-oxo)diiron reacts with O2 and two electrons to generate a hydrogen atom abstracting species and an oxidizing agent. The hydrogen-abstracting species might be the enzymic radical or another species generated by the iron complex and O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction

The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate. Thus, ascorbate is not required for O(2) activation or product formation. Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.     

A new thioether-ligated iron porphyrin as a model of a protonated form of P450 active site

Thioether-ligated iron porphyrin (complex 1) was synthesized as a model of the protonated form of P450 to explore the possible involvement of the protonated form in the catalytic cycle, and ether-ligated iron porphyrin (complex 2) was also synthesized for comparison. The thioether and ether ligands enhanced heterolytic O-O bond cleavage of peroxy acid-iron porphyrin complex even in highly hydrophobic media without the assistance of acid or base, using mCPPAA as an oxidant. Competitive oxidation of cyclooctane/cyclooctene catalyzed by iron porphyrins showed that complexes 1 and 2 are less effective than heme thiolate (P450 and a synthetic heme thiolate (SR complex)) in oxidizing alkane. The possibility that thiol-ligated heme, which is a protonated form of heme thiolate, is not involved in the active intermediate structure of P450 is indicated by this result. This is the first report concerning the oxidizing ability of a thioether-ligated iron porphyrin.     

Cyclopropylglyoxylate as a mechanistic probe of thiamine pyrophosphate dependent pyruvate-metabolizing enzymes

Four pyruvate-decarboxylating enzymes with thiamine pyrophosphate (TPP) cofactors catalyze the decarboxylation of the cyclopropyl substrate analog cyclopropylglyoxylate. Pyruvate: ferredoxin oxidoreductase, an archaebacterial enzyme which catalyzes oxidation of the hydroxyethyl-TPP (HETPP) intermediate by two one-electron transfers to an iron-sulfur center, generates the coenzyme A thioester of cyclopropylcarboxylic acid. A long-lived free radical, HETPP is thought to be an intermediate in the pyruvate to acetyl-CoA conversion; however, cleavage of the cyclopropyl ring was not detected. Pyruvate decarboxylase, pyruvate oxidase, and pyruvate dehydrogenase also generate the corresponding cyclopropyl products. The applicability of cyclopropyl substrate analogs as indicators of free-radical enzyme mechanisms is discussed in light of these results.     

Intra- and intermolecular oxidation of oxymyoglobin and oxyhemoglobin induced by hydroxyl and carbonate radicals

The mechanism of the reactions of myoglobin and hemoglobin with *OH and CO3*- in the presence of oxygen was studied using pulse and gamma-radiolysis. Unlike *NO2, which adds to the porphyrin iron, *OH and CO3*- form globin radicals. These secondary radicals oxidize the Fe(II) center through both intra- and intermolecular processes. The intermolecular pathway was further demonstrated when BSA radicals derived from *OH or CO3*- oxidized oxyhemoglobin and oxymyoglobin to their respective ferric states. The oxidation yields obtained by pulse radiolysis were lower compared to gamma-radiolysis, where the contribution of radical-radical reactions is negligible. Full oxidation yields by *OH-derived globin radicals could be achieved only at relatively high concentrations of the heme protein mainly via an intermolecular pathway. It is suggested that CO3*- reaction with the protein yields Tyr and/or Trp-derived phenoxyl radicals, which solely oxidize the porphyrin iron under gamma-radiolysis conditions. The *OH particularly adds to aromatic residues, which can undergo elimination of H2O forming the phenoxyl radical, and/or react rapidly with O2 yielding peroxyl radicals. The peroxyl radical can oxidize a neighboring porphyrin iron and/or give rise to superoxide, which neither oxidize nor reduce the porphyrin iron. The potential physiological implications of this chemistry are that hemoglobin and myoglobin, being present at relatively high concentrations, can detoxify highly oxidizing radicals yielding the respective ferric states, which are not toxic.     

The monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450

This review examines the monooxygenase, peroxidase, and peroxygenase properties of cytochrome P450 (P450)1 enzymes and their mechanisms of action in archaeal, bacterial, and mammalian systems. In the P450 catalytic cycle, a transient iron oxo monooxygenating species is generated that reacts with substrate to produce a monooxygenated product. We describe results of early investigations that endeavored to trap and detect this elusive monooxygenating species, as well as results of experiments that attempted to generate and characterize this active oxidant spectroscopically after reacting ferric P450 enzymes with peroxy compounds (e.g. peroxides, peracids) or single oxygen atom donors (e.g. periodate, iodosobenzene). Surrogate oxidants were able to promote P450-catalyzed monooxygenations in a manner similar to that of O2/NAD(P)H, suggesting involvement of a common transitory monooxygenating species in the two pathways. This common P450 oxidant was characterized as a porphyrin radical iron(IV) oxo complex and assigned a Compound I structure (Por+FeIV=O) exhibiting a formal FeV oxidation state. Other reactive oxidants, such as the ferric oxenoid complex (PorFeIII=O), ferryloxy radical species (PorFeIV-O·), and perferryloxo entity (PorFeV=O), were also proposed to function as P450 monooxygenating species. We also discuss the possible involvement of the ferriperoxo (PorFeIII-OO-) and ferrihydroperoxo (PorFeIII-OOH) species as alternative oxidants in P450-mediated monooxygenation reactions.