Multiple mechanisms and multiple oxidants in P450-catalyzed hydroxylations

Cytochrome P450 enzymes catalyze a number of oxidations in nature including the difficult hydroxylations of unactivated positions in an alkyl group. The consensus view of the hydroxylation reaction 10 years ago was that a high valent iron-oxo species abstracts a hydrogen atom from the alkyl group to give a radical that subsequently displaces the hydroxy group from iron in a homolytic substitution reaction (hydrogen abstraction-oxygen rebound). More recent mechanistic studies, as summarized in this review, indicated that the cytochrome P450-catalyzed "hydroxylation reaction" is complex, involving multiple mechanisms and multiple oxidants. In addition to the iron-oxo species, another electrophilic oxidant apparently exists, either the hydroperoxo-iron intermediate that precedes iron-oxo or iron-complexed hydrogen peroxide formed by protonation of the hydroperoxo-iron species on the proximal oxygen. The other electrophilic oxidant appears to react by insertion of OH(+) into a C-H bond to give a protonated alcohol. Computational work has suggested that iron-oxo can react through multiple spin states, a low-spin ensemble that reacts by insertion of oxygen, and a high-spin ensemble that reacts by hydrogen atom abstraction to give a radical.     

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.     

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.     

One oxidant,many pathways: a theoretical perspective of monooxygenation mechanisms by cytochrome P450 enzymes

Density functional theoretical studies of monooxygenation reactivity of the high-valent oxoiron(IV) porphyrin cation-radical compound of cytochrome P450, the so-called Compound I, and of its precursor, the ferric(III)-hydroperoxide species, are described. The degeneracy of the spin states of Compound I, its electron deficiency, and dense orbital manifold lead to two-state and multi-state reactivity scenarios and may thereby create reactivity patterns as though belonging to two or more different oxidants. Most of the controversies in the experimental data are reconciled using Compound I as the sole competent oxidant. Theory finds ferric(III)-hydroperoxide to be a very sluggish oxidant, noncompetitive with Compound I. If and when Compound I is absent, P450 oxidation will logically proceed by another form, but this has to be more reactive than ferric(III)-hydroperoxide. Theoretical studies are conducted to pinpoint such an oxidant for P450.

Models and mechanisms of O-O bond activation by cytochrome P450. A critical assessment of the potential role of multiple active intermediates in oxidative catalysis.

Cytochrome P450 enzymes promote a number of oxidative biotransformations including the hydroxylation of unactivated hydrocarbons. Whereas the long-standing consensus view of the P450 mechanism implicates a high-valent iron-oxene species as the predominant oxidant in the radicalar hydrogen abstraction/oxygen rebound pathway, more recent studies on isotope partitioning, product rearrangements with 'radical clocks', and the impact of threonine mutagenesis in P450s on hydroxylation rates support the notion of the nucleophilic and/or electrophilic (hydro)peroxo-iron intermediate(s) to be operative in P450 catalysis in addition to the electrophilic oxenoid-iron entity; this may contribute to the remarkable versatility of P450s in substrate modification. Precedent to this mechanistic concept is given by studies with natural and synthetic P450 biomimics. While the concept of an alternative electrophilic oxidant necessitates C-H hydroxylation to be brought about by a cationic insertion process, recent calculations employing density functional theory favour a 'two-state reactivity' scenario, implicating the usual ferryl-dependent oxygen rebound pathway to proceed via two spin states (doublet and quartet); state crossing is thought to be associated with either an insertion or a radicalar mechanism. Hence, challenge to future strategies should be to fold the disparate and sometimes contradictory data into a harmonized overall picture.     

Hydroperoxoferric heme intermediate as a second electrophilic oxidant in cytochrome P450-catalyzed reactions

Experimental evidence supporting the catalytic activity of the peroxoferric and hydroperoxoferric cytochrome P450 intermediates as alternative oxidants to the compound I (ferryl) state in the oxygenation of organic substrates is reviewed. The peroxoferric P450 state is proposed to function as a nucleophile in the lyase step of the P450-aromatase reaction. Several systems are reviewed in which the hydroperoxoferric P450 intermediate likely functions as a second electrophilic oxidant, the two-oxidants model. These include alkene epoxidation, sulfoxidation, and hydroxylation of methyl groups on cyclopropane rings. The key use of the P450 mutants from different sources in which the conserved threonine in the distal substrate binding pocket is replaced with alanine, in order to minimize the formation of the compound I intermediate and unmask the reactivity of the hydroperoxoferric state, is emphasized. These data are discussed in the context of the two-states model, which proposes that the compound I P450 intermediate has both high- and low-spin states with different reactivities. A complicated reaction profile emerges for the wide range of P450 reactions involving up to three reactive intermediates, of which the most reactive, the compound I P450 state, has two spin states with different reactivities.     

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and M?ssbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.     

Ipso-substitution by cytochrome P450 with conversion of p-hydroxybenzene derivatives to hydroquinone: evidence for hydroperoxo-iron as the active oxygen species.

Evidence for multiple functional active oxidants in cytochrome P450-catalyzed reactions was previously obtained in this laboratory with mutants in which proton delivery was perturbed by replacement of the highly conserved threonine residue in the active site by alanine, thus apparently interfering with the conversion of the peroxo-iron to the hydroperoxo-iron and the latter to the oxenoid-iron species. These enzymes have now been employed to examine the reaction in which cytochrome P450 in liver microsomes is known to effect ipso-substitution, the elimination of p-substituents in phenols to yield hydroquinone. As shown with purified NH(2)-truncated cytochromes in a reconstituted enzyme system, the reaction exhibits an absolute requirement for cytochrome P450 and NADPH-cytochrome P450 reductase. Under optimal conditions truncated cytochrome P450 2E1 is active with 10 of the p-substituted phenols examined. Of particular interest, the corresponding cytochrome with threonine-303 replaced by alanine is from 1.5- to 50-fold higher in activity with the p-chloro, -bromo, -nitro, -cyano, -hydroxymethyl, -formyl, and -acetyl derivatives, and the reaction with the p-benzoyl, -methyl, and -t-butyl compounds is catalyzed by the mutant enzyme only. The results implicate the hydroperoxo-iron species as an electrophilic active oxidant in cytochrome P450-catalyzed aromatic ipso-substitution.     

Freeze-quenched iron-oxo intermediates in cytochromes P450

Since the discovery of cytochromes P450 and their assignment to heme proteins a reactive iron-oxo intermediate as the hydroxylating species has been discussed. It is believed that the electronic structure of this intermediate corresponds to an iron(IV)-porphyrin-pi-cation radical system (Compound I). To trap this intermediate the reaction of P450 with oxidants (shunt pathway) has been used. The common approaches are stopped-flow experiments with UV-visible spectroscopic detection or rapid-mixing/freeze-quench studies with EPR and M?ssbauer spectroscopic characterization of the trapped intermediate. Surprisingly, the two approaches seem to give conflicting results. While the stopped-flow data indicate the formation of a porphyrin-pi-cation radical, no such species is seen by EPR spectroscopy, although the M?ssbauer data indicate iron(IV) for P450cam (CYP101) and P450BMP (CYP102). Instead, radicals on tyrosine and tryptophan residues are observed. These findings are reviewed and discussed with respect to intramolecular electron transfer from aromatic amino acids to a presumably transiently formed porphyrin-pi-cation radical.     

Thoughts on thiolate tethering. Tribute and thanks to a teacher

A unique feature of P450 enzymes is in the presence of a thiolate ligand heme but its exact function in catalysis is a matter of debate. For P450 dependent monooxygenases the "active oxygen" complex seems to exist only as a transition state in which the thiolate ligand provides electron density in order to prevent pi-backbonding of the oxygen to the iron (-S-Fe-O(z.rad;)). The corresponding ground state (Compound I) would be a ferryl species (Fe(IV)z.dbnd6;O) with an electron hole either at the porphyrin or at the sulfur. Apart from this role we postulate that a second function is related to the electronic structure of Compound II as an electron acceptor and this property is shared among monooxygenases, thromboxane synthase, prostacyclin synthase, allene oxide synthase, P450(NOR(-)) and chloroperoxidase. As a common step in all P450 enzymes an extremely rapid electron uptake by Compound II allows that the primary substrate radicals are oxidized to cations which immediately combine with a neighbouring nucleophile. Thus "electron transfer" may substitute for "oxygen rebound" as the final step leading to product formation. The same principle also applies methane monooxygenases in which the role of the thiyl sulfur is replaced by a ferryl-oxyl entity.     

On the identity and reactivity patterns of the "second oxidant" of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.     

On the identity and reactivity patterns of the “second oxidant” of the T252A mutant of cytochrome P450cam in the oxidation of 5-methylenenylcamphor

Density functional calculations show that in the absence of Compound I, the primary oxidant species of P450, the precursor species, Compound 0 (FeOOH), can effect double bond activation of 5-methylenylcamphor (1). The mechanism is initiated by homolytic cleavage of the O–O bond and formation of an OH radical bound to the Compound II species by hydrogen bonding interactions. Subsequently, the so-formed OH radical can either activate the double bond of 1 or attack the meso position of the heme en route to heme degradation. The calculations show that double bond activation is preferred over attack on the heme. Past the double bond activation, the intermediate can either lead to epoxidation or to a glycol formation. The glycol formation is predicted to be preferred, although in the P450_cam pocket the competition may be closer. Therefore, in the absence of Compound I, Compound 0 will be capable of epoxidizing double bonds. Previous studies [E. Derat, D. Kumar, H. Hirao, S. Shaik, J. Am. Chem. Soc. 128 (2006) 473–484] showed that in the case of a substrate that can undergo only C–H activation, the bound OH prefers heme degradation over hydrogen abstraction. Since the epoxidation barrier for Compound I is much smaller than that of Compound 0 (12.8 vs. 18.9 kcal/mol), when Compound I is present in the cycle, Compound 0 will be silent. As such, our mechanism explains lucidly why T252A P450_cam can epoxidize olefins like 5-methylenylcamphor but is ineffective in camphor hydroxylation [S. Jin, T.M. Makris, T. A. Bryson, S.G. Sligar, J.H. Dawson, J. Am. Chem. Soc. 125 (2003) 3406–3407]. Our calculations show that the glycol formation is a marker reaction of Compound 0 with 5-methylenylcamphor. If this product can be found in T252A P450_cam or in similar mutants of other P450 isozymes, this will constitute a more definitive proof for the action of Cpd 0 in P450 enzymes.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Unusual Cytochrome P450 Enzymes and Reactions

Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO³⁺), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function.     

The oxo/peroxo debate: a nonheme iron perspective

The oxygen activation mechanisms proposed for nonheme iron systems generally follow the heme paradigm in invoking the involvement of iron-peroxo and iron-oxo species in their catalytic cycles. However, the nonheme ligand environments allow for end-on and side-on dioxygen coordination and impart greater flexibility in the modes of dioxygen activation. The currently available evidence for nonheme iron-peroxo and iron-oxo intermediates is summarized and discussed in light of the ongoing discussion on the nature of the oxidant(s) in heme enzymes.     

How does the push/pull effect of the axial ligand influence the catalytic properties of Compound I of catalase and cytochrome P450?

Density functional theory (DFT) calculations on the chemoselective epoxidation versus hydroxylation reactions of propene by oxoiron porphyrin models mimicking the active sites of catalase, cytochrome P450 (P450) and horseradish peroxidase Compound I (CpdI) are presented. The catalase reactions are concerted and proceed via two-state reactivity patterns on competing doublet and quartet spin state surfaces, but the lowest barrier is the one leading to epoxide products on the doublet spin surface. The results are compared with earlier DFT studies of models of cytochrome P450, horseradish peroxide (HRP), taurine/alpha-ketoglutarate dioxygenase and some synthetic oxoiron catalysts. The catalase barriers are midway in between those obtained for HRP and P450 models, so that tyrosinate ligated heme systems should be able to catalyze C-H hydroxylation and C=C epoxidation reactions. We show that for heme systems the barrier height of epoxidation linearly correlates with the electron affinity of Compound I as expected from the electron transfer mechanism of the rate determining step. Our studies show that the axial ligand does not influence the chemoselectivity of a reaction but that it does regulate the barrier heights and rate constants. Finally, we estimated the effect of the axial ligand on the oxoiron group and derived that it contributes from a field effect due to the charge of the ligand and a quantum mechanical effect as a result of orbital mixing. In catalase, the major component is the field effect, while the quantum mechanical effect is negligible. This is in contrast to P450 CpdI, where both effects are of similar order of magnitude.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Manganese substituted Compound I of cytochrome P450 biomimetics: a comparative reactivity study of Mn(V)-oxo versus Mn(IV)-oxo species

Manganese-oxo porphyrins have been well studied as biomimetic models of cytochromes P450 and are known to be able to catalyze substrate hydroxylation reactions. Recent experimental studies [J.Y. Lee, Y.-M. Lee, H. Kotani, W. Nam, S. Fukuzumi, Chem. Commun. (2009) 704] showed that Mn(V)-oxo porphyrins react rapidly with 10-methyl-9,10-dihydroacridine (AcrH(2)) via a proton-coupled-electron-transfer followed by an electron transfer. In this work, we present a computational study on the reactivity patterns of Mn(V)-oxo and Mn(IV)-oxo with respect to AcrH(2). This study shows that although both oxidants are capable of hydroxylating AcrH(2), the Mn(V)-oxo species is the more active oxidant. We have generalized these observations with thermodynamic cycles that explain the reaction mechanisms and electron transfer processes. For the Mn(V)-oxo mechanism the reactions proceed with a fast spin state crossing from the ground state singlet to the triplet spin state prior to a hydrogen atom transfer followed by another electron transfer. The present results are fully consistent with previous studies on iron-oxo porphyrins and manganese-oxo porphyrins and shows that the interplay of low lying singlet and triplet spin state surfaces influences the reaction mechanisms and kinetics.     

Thianthrene 5-oxide as a probe of the electrophilicity of hemoprotein oxidizing species.

Thianthrene 5-oxide (T-5-O), which is oxidized to the 5,10- and 5,5-dioxides, respectively, by electrophilic and nucleophilic agents, has been used to determine the electronic properties of hemoprotein oxidizing species. Cytochrome P450 oxidizes T-5-O to the 5,10- rather than the 5,5-dioxide but oxidizes the 5,5-dioxide rapidly and the 5,10-dioxide slowly to the 5,5,10-trioxide. Chloroperoxidase oxidizes T-5-O to the 5,10-dioxide but very poorly oxidizes it further to the 5,5,10-trioxide. It does, however, readily oxidize the 5,5-dioxide to the trioxide. The oxidizing species of cytochrome P450 and chloroperoxidase are thus comparably electrophilic, but the former is more powerful. T-5-O is not detectably oxidized by horseradish peroxidase/H2O2 but is oxidized exclusively to the 5,5-dioxide when the peroxide is replaced by dihydroxyfumaric acid (DHFA). The oxygen incorporated into the 5,5-dioxide in this reaction derives from molecular oxygen. This is consistent with the involvement of a DHFA-derived co-oxidizing species. Oxidation of T-5-O by human hemoglobin and H2O2 yields the 5,5- and 5,10-dioxides and the 5,5,10-trioxide. The oxygen in these products derives primarily (greater than 80%) from H2O2. Hemoglobin and H2O2 thus form both a P450-like electrophilic oxidant (5,10-dioxide) and a peroxide-derived nucleophilic oxidant (5,5-dioxide). A large difference in the cis:trans ratios of the 5,10-dioxides produced from T-5-O by cytochrome P450 (1.3:1) and chloroperoxidase (2.5:1) vs hemoglobin (0.1:1) suggests that the hemoglobin active site severely constrains the geometry of the electrophilic oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)