Inorganic Biochemistry

Journal of Inorganic Biochemistry 100 (2006) 434447 www.elsevier.com/locate/jinorgbio

JOURNAL OF

Review article

High-valent iron in chemical and biological oxidations

Abstract Various aspects of the reactivity of iron(IV) in chemical and biological systems are reviewed. Accumulated evidence shows that the ferryl species [Fe(IV)@O]2+ can be formed under a variety of conditions including those related to the ferrous ionhydrogen peroxide system known as Fentons reagent. Early evidence that such a species could hydroxylate typical aliphatic CH bonds included regioselectivities and stereospecicities for cyclohexanol hydroxylation that could not be accounted for by a freely diusing hydroxyl radical. Iron(IV) porphyrin complexes are also found in the catalytic cycles of cytochrome P450 and chloroperoxidase. Model oxo-iron(IV) porphyrin complexes have shown reactivity similar to the proposed enzymatic intermediates. Mechanistic studies using mechanistically diagnostic substrates have implicated a radical rebound scenario for aliphatic hydroxylation by cytochrome P450. Likewise, several nonheme diiron hydroxylases, AlkB (X-hydroxylase), sMMO (soluble methane monooxygenase), XylM (xylene monooxygenase) and T4moH (toluene monooxygenase) all show clear indications of radical rearranged products indicating that the oxygen rebound pathway is a ubiquitous mechanism for hydrocarbon oxygenation by both heme and non-heme iron enzymes. 2006 Elsevier Inc. All rights reserved.
Keywords: Cytochrome P450; Chloroperoxidase; AlkB; sMMO; Oxygen rebound; Ferryl; Fenton; Mechanism

1. Introduction The oxidative transformations mediated by the ironcontaining oxygenases have attracted sustained attention for more than three decades [1]. The intense interest has derived from a most appealing combination of mechanistic complexity, unusual reactive intermediates, and potential synthetic and economic utility. Proteins such as cytochrome P450 [24], methane monoxygenase [5,6], AlkB [7,8] and TauD [9,10] are known to eect even the most difcult CH hydroxylations of saturated carbon centers in a wide variety of organic compounds. Aliphatic hydroxylation is of particular interest since the oxidative cleavage of such a strong bond is intrinsically dicult. Indeed, there are at present no general, direct, synthetic methods for specic replacement of an unactivated carbon-bound hydrogen with a hydroxyl group. Further, while there is an
*

emerging consensus regarding the mechanisms of oxygen activation and transfer for these systems, much is yet to be learned. These enzyme systems are known to activate molecular oxygen by the sequential, two-electron reduction of oxygen to the formal oxidation state of hydrogen peroxide. In all of the above systems the common mechanistic thread is the subsequent oxidation of iron(II) or iron(III) to the iron(IV) oxidation state. In this perspective I will summarize key insights and advances in the chemistry of model iron systems and relate them to our understanding of the iron-containing oxygenases. 2. Perspectives on the chemistry of Fentons reagent and ferryl intermediates Many of our notions of high-valent iron and its role in chemical and biological oxidation reactions stem from studies of Fentons reagent [1115], a mixture of hydrogen peroxide and ferrous salts. This extensive chemistry of ironhydrogen peroxide systems is a very useful resource

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for precedent and guidance in how to think about the enzymatic mechanisms. According to the generally held mechanism rst proposed by Haber and Weiss [16], the Fentons reagent oxidation of organic compounds proceeds via an initial one-electron reduction of hydrogen peroxide to yield free hydroxyl radicals and subsequent hydrogen abstraction. The formation of a ferryl ion intermediate (Fe(IV)@ O) in such non-enzymatic, ironperoxide systems was rst proposed by Bray and Gorin [17] and several other suggestions of similar species have appeared. Further, the possible competition of hydroxyl radical and ferryl ion pathways for the Fentons reagent mechanism have been discussed in terms of the equilibrium between Fe(III)+OH and Fe(IV)@O (Scheme 1). Two of the more important aspects of enzymatic hydroxylation that are striking in comparison to powerful chemical oxidants are oxygen transfer without equilibration with water and oxidation with net retention of conguration at the functionalized carbon. Notable precedents among organic oxidants for such an outcome are the CH insertion reactions of dioxiranes and peroxytriuoroacetic acid. Basic to the understanding of the iron-mediated processes is a catalog of the chemistry intrinsic to the higher oxidation state iron complexes such as Fe(IV)@O that has been implicated in these reactions. It was in this context that we initiated a program to examine the nature of Fentons reagent some years ago to look for parallels and insights that might be applicable to the chemistry of the iron-containing oxygenases. It is pertinent to review the salient features of those studies here. The oxidation of cyclohexanol by ferrous perchlorate and hydrogen peroxide in acetonitrile was found to aord a mixture of cyclohexanediols in addition to cyclohexanone [18]. The observed regioselectivities and stereoselectivities supported the formation of a ferryl ion-like intermediate. Curiously, cis-1,3-cyclohexanediol accounted for 72% of the diol produced when the reaction was run in acetonitrile-containing perchloric acid. Under these conditions, hydrogen removal at C-3 occurred stereoselectively cis to the hydroxyl group at C-1. A mechanism for this aliphatic hydroxylation that involved an initial hydroxyl-directed hydrogen abstraction was proposed. Evidence for the stepwise nature of the hydroxylation process was derived from analysis of the Fe2+/H2O2 oxidation of 7-hydroxynorbornane. Hydrogen abstraction from this substrate was found

to occur stereoselectively syn to the hydroxyl group and subsequent 2,6-hydride transfers resulted in a mixture of syn,exo and anti,exo diols. The regioselectivity observed for hydrogen abstraction from cyclohexanol and 7-hydroxynorbornane clearly indicated a directive role of the hydroxyl group. The most reasonable explanation for this behavior was metalalcohol complexation, which was conrmed by spectral changes observed upon addition of cyclohexanol to acetonitrile solutions of ferrous or ferric perchlorate. This sort of directive inuence might be expected if the reaction proceeded via the formation of a reactive ferry1 ion species [Fe(IV)@O] by two-electron oxidation of ferrous ion but would not be anticipated if the reaction was initiated by a freely diusing hydroxyl radical. Several arguments favored such a ferryl ion mediated process. Fe(II) and Fe(III) salts were known to be oxidized by hydrogen peroxide to give ferrate Fe(VI) salts in alkaline aqueous solution. Thus, intermediate Fe(IV) and Fe(V) states were implicated in these reactions as well. Likewise, treatment of ferrous ion with ozone aords transient a ferryl species in water. The observed regioselectivities for cyclohexanol and 7-hydroxynorbornane parallel the anticipated chemical availability of nearby hydrogen toward hydrogen abstraction by the iron-oxo intermediate through a cyclic transition state. Two stereoisomeric complexes representing the equatorial and axial conformers of the alcohol would be expected for the hydroxylation of cyclohexanol (Scheme 2). Only the cis-hydrogen at C-3 would be accessible to the bound

Scheme 1. Homolytic and heterolytic reactions of hydrogen peroxide with ferrous ion (Fe(II)).

Scheme 2. Directive eects in cyclohexanol hydroxylation by Fentons reagent in acetonitrile.

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ferry1 ion and accordingly, hydrogen abstraction occurred preferentially at that site. It was shown that the oxidation of cyclohexanol deuterated at C-3 gave 78% cis-hydrogen abstraction but 96% cis-1,3-diol formation. Accordingly, some radicals generated by trans-C-3 hydrogen abstraction, presumably from a non-directed component of this reaction, must have been oxidized to give the cis-diol. This result suggests that if hydrogen abstraction was the initial step in this process, 3-hydroxycyclohexyl radicals generated by an independent route would lead to stereoselective radical oxidation by ferric ion. As a test of this hypothesis, the reductive decarboxylation of cis- and trans-3-hydroxycyclohexaneperoxycarboxylic acid gave rise to the same pronounced preference for cis-diol formation, indicating that 3-hydroxycyclohexyl radicals generated by decarboxylation were hydroxylated stereoselectively by iron(III), as shown in Scheme 2. These ndings had important implications with regard to the mechanisms of biological hydroxylation. An early suggestion for the mechanism of enzymatic CH hydroxylation involved an oxene insertion process, largely to explain the net retention which was observed. This non-aqueous Fenton oxidation showed that net retention can also be the result of two successive stereoselective processes, hydrogen abstraction and radical oxidation. We also examined modications of Fentons reagent in which hydrogen peroxide had been replaced by peroxyacids [19]. Startling and revealing results were obtained for the hydroxylation of cyclohexanol by ferrous ionperoxyacid in non-aqueous systems. The results showed that about 90% of the product diols were cis- and trans-1,2cyclohexane diol and cis-1,3-cyclohexane diol. Critical to the understanding of the mechanism of this hydroxylation and any relationships with biological processes was the identication of the source of the new hydroxyl oxygen. Surprisingly, when the reaction was carried out in the presence of H2 18 O, the 18O/16O ratios for cis- and trans-1,2-

cyclohexanediols were found to be only 0.08 and 0.1, respectively. An 18O/16O ratio of 0.98 would have been expected if the hydroxyl oxygen were derived from water. Accordingly, it must have been the peroxy oxygen that had been inserted into the aliphatic CH bond (Scheme 3). Further, it is apparent that the reactive oxygen was capable of undergoing partial (ca 10%) exchange with water. This would be the expected result for the formation of a reactive iron-oxo intermediate that was coordinated with the hydroxyl group of the substrate and to water molecules in the medium. The hydroxylation of trans-2-deuteriocyclohexanol with ferrous ion/mCPBA (meta-chloroperoxybenzoic acid) again provided cis- and trans-1,2-cyclohexanediol with >90% retention of deuterium in the cis isomer while the trans isomer showed signicant loss of the label. An inescapable conclusion from these data was that cis-hydrogen abstraction had lead preferentially to cis-diol formation while transhydrogen abstraction aorded the trans-diol. Thus, what appeared supercially to be a nonselective process was, in fact, two predominantly stereospecic processes. The specic incorporation of the peroxy oxygen and the observed retention of conguration at C-2 put severe limitations on the range of considerable mechanisms for this Fenton-like process. Freely diusing hydroxyl radicals could be ruled out since there would be no way to explain the stereoselectivity or stereospecicity that had been observed. The results were completely consistent with the intermediacy of an oxo-iron species, formally a ferry1 ion (Fe(IV)@O) that we had proposed for these reactions (Scheme 3). This was the process we called oxygen rebound, since the oxygen trajectory had rebounded from iron to carbon with abstraction of the only three hydrogens chemically available to the coordinated iron-oxo complex. It was more problematical to nd an explanation for the retention of this stereochemical information. We had demonstrated the intermediacy of discrete carbon radicals and

Scheme 3. Stereoselectivity and oxygen exchange in CH hydroxylation by Fe(II)peroxyacid.

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carbonium ions in the related ferrous ion/H2O2 mediated hydroxylation of cyclohexanol as discussed above. If such a stepwise process was also involved when mCPBA was the oxidant, hydrogen abstraction, electron transfer, and nucleophilic capture of the incipient carbonium ion must be rapid with respect to molecular rotation and ligand exchange at the iron center, which would equilibrate both the deuterium and oxygen labels. The compelling interpretation of these results was that C-2 hydrogen abstraction by an iron oxo species requires close approach of the iron to the intermediate carbon radical, allowing rapid, subsequent electron transfer and ligand delivery. The mechanistic interpretations of that early work were based almost exclusively on product distributions, stereoand regio-selectivities and isotopic labeling. While there were early indications that aqueous ferrous ion could be oxidized to Fe(IV) by ozone [20], it was not until 1992 that Loegager et al. showed that a discrete, short-lived intermediate, formulated as a ferryl species, was observed under these conditions [21]. The ferryl species, Fe(IV)@O, rather than an ironozonide complex was favored by fact that one equivalent of ozone aorded oxygen in addition to the ferryl. While this species decays in aqueous solution with a half-life of about 10 s, it has been possible to study the reaction kinetics with organic compounds and to spectroscopically characterize it.
Fe2 aq O3 ! FeIVO 2

O2

The aqueous ferryl species, [Fe(IV)O]2+, has been shown to be a reactive oxidant, exhibiting both single-electron hydrogen abstraction chemistry and two-electron oxidations of alcohols to ketones [22]. An interesting and significant correlation has been observed between the reactivity of [Fe(IV)O]2+ and that of a photo-excited uranyl complex, U*O2+, which is known to react with organic substrated via hydrogen abstraction (Fig. 1). Recent characterization of this aqueous [Fe(IV)O]2+ species by Mo ssbauer and EXAFS (extended X-ray absorption ne structure) spectroscopy has indicated a short ironoxygen bond distance characteristic of well characterized ferryl species [23,24] and that it has an

Fig. 1. Correlation of the reactivities of [Fe(IV)O]2+ and excited state U O2 2 toward organic substrates. Adapted from Ref. [22].

S = 1 triplet ground state [25]. Exchange of the Fe-oxo oxygen with coordinated water was shown to compete partially with oxygen atom transfer to a sulfoxide substrate. Interestingly, partial O-exchange described under the reaction conditions was similar to what we had found and described, and discussed above, for the CH hydroxylation of cyclohexanol by the proposed ferryl intermediate [19]. Another recent examination of Fentons reagent concluded that it is a ferryl species and not hydroxyl radical that is produced by aqueous Fe(II)/H2O2, on the basis of pH-dependant behavior [26]. The results were interpreted in terms of a prototropic equilibrium involving Fe@O2+ and FeOH3+, as has also been used to explain the reactivity of water-soluble oxoMn(V) porphyrins [27,28]. Thus, multiple forms of the ferryl oxidant may be contributing to the observed chemistry depending on the pH of the medium (Scheme 4). A recent study of the Fenton/ferryl system using combined DFT and CarParrinello molecular dynamics has led to the conclusion that formation of the ferryl species from a Fe(II)OOH precursor is intrinsically facile [29,30]. The reactivity of Fe(IV)@O with CH bonds follows the oxygen rebound scenario. We will now look at those results in some detail. The methane hydroxylation mechanism was found to proceed in two steps, hydrogen abstraction from methane by the Fe(IV)@O species to produce [Fe(III)OHCH3] followed by collapse of the carbon radical center onto the iron(III) hydroxyl oxygen. The energy prole for this process showed an initial, weak interaction between the methane substrate and the iron(IV) oxo complex (Fig. 2). The hydrogen atom transfer from methane CH to the ferryl intermediate was found to occur with only a small, 3.4 kcal barrier. Thus, this part of the reaction occurred on a at, high-energy plateau due to the simultaneous formation of the OH bond with the breaking of the CH bond. Signicantly, there was found to be a signicant stabilization of the methyl radical through interactions with the Fe(III)OH species. In spite of this interaction, there was still a 3 kcal/mol barrier to formation of the CO bond, even though this reaction was highly exothermic (46.6 kcal/ mol). The origin of the barrier to the oxygen rebound process was attributed to the atomic trajectory of the methyl carbon with respect to the iron-bound hydroxyl, changing from a nearly collinear CHO arrangement to a bent geometry before the CO bond can form. There is extensive literature devoted to various iterations of the GIF oxygenation system developed by Barton et al. [31,32]. In these complex mixtures, hydrocarbons could be oxidized by simple iron salts such as Fe-picolinates, in the presence of oxygen and a sacricial reducing agent such as zinc(0). Oxo-Fe(IV) and oxo-Fe(V) intermediates were proposed as the active species in these systems. A curious aspect of the selectivity of the GIF systems was the apparently higher reactivity of methylene groups (CH2) over the usually more reactive methine group (R3CH). This conundrum was ultimately shown by Stavropoulos to be due to selective trapping of tertiary carbon radicals in the

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Scheme 4. Ferryl ion prototropy in Fentons reagent. Adapted from Ref. [26].

in which oxo- or hydroxo-iron(III) are shown to be reactive toward CH bonds. A useful paradigm for understanding this kind of reactivity to appreciate that an FeOH bond is being formed as the CH bond is broken during aliphatic hydroxylation. Thus, as in typical organic radical chemistry, it is the strength of the new FeOH bond that ultimately dominates the kinetics and thermodynamics of these reactions. Mayer has insightfully discussed these reactions in terms of the Marcus-like EvansPolyani relationship [36]. Accordingly, if we can measure or estimate the redox potential of FenO, and the pKa of Fen 1OH, the strength of the FeOH bond can be determined. The driving force for a particular CH hydrogen atom abstraction by FenO can then be estimated to be the dierence between the bond dissociation energies of the scissile CH bond and that of the newly formed FeOH bond. This formalism nicely explains the reactivity of oxo- and hydroxo-iron(III) complexes, which by this analysis have typical Fe(II)OH BDEs in the range of 80 kcal/mol (Scheme 5). Stack has suggested that this mode of reactivity can explain the homolytic CH bond cleavage mediated by lipoxygenases [37,38]. Indeed, a methoxy-Fe(III) complex has been shown capable of CH bond cleavage. Likewise, Borovik has shown that even a fully deprotonated Fe(III)-oxo group can be formed when the oxygen is suitably stabilized by hydrogen bonds [3941]. This species was also able to cleave relatively reactive CH bonds by homolytic hydrogen abstraction (Scheme 6).

Fig. 2. Oxygen rebound mechanism for methane hydroxylation by (aq)Fe(IV)@O. Adapted from Ref. [30].

system by the co-solvent pyridine [33]. These products had not been accounted for in the earlier Barton work. Further, it was shown by Minisci [34] and by Ingold [35] that the GIF systems displayed typical radical behavior. Accordingly, the GIF systems now also seem to come within the bounds of typical ferryl ion behavior. It is of interest to compare the reactivity of the ferryl species under discussion here to the relatively few cases

Scheme 5. Fe(II)OH bond dissociation energy determined from Fe(OH2) pKas and redox potentials. Adapted from Ref. [37].

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Scheme 6. Preparation of an oxo-Fe(III) complex. Adapted from Ref. [41].

In leaving this discussion of the reactivity of ferryl species, we note the major recent advances in the isolation and full characterization of authentic, small-molecule non-heme complexes with the Fe(IV)@O formulation. The chemistry of these compounds has been extensively reviewed both in this volume by Que and elsewhere [23,24,42]. Likewise, a reactive ferryl intermediate has been characterized recently by Krebs in the non-heme iron, aketoglutarate dependent hydroxylase TauD [10,4345]. It is this ferryl intermediate that is responsible for aliphatic hydroxylation of the substrate taurine (Scheme 7). 3. Models and mechanisms of oxidative catalysis by heme iron and cytochrome P450 The reactions catalyzed by the heme-containing monooxygenase cytochrome P450 continue to be a central focus of attention [24]. The drivers behind this world-wide eort

are the fundamental desire to understand the details of biological oxygen activation and transfer and, as well, to advance the opportunity oered by this chemistry for the development of new, selective catalysts that are beginning to be of considerable economic value. In this section we will highlight several important advances of the 1980s when the rst iron(IV) porphyrins were isolated and characterized and the mechanisms of their remarkable oxygenation reactions were rst investigated. The heme-containing metalloenzymes cytochrome P450 [4] and chloroperoxidase (CPO) [4649], and their relatives catalyze a host of crucial biological oxidation reactions. Highly specic P450s are involved in the selective oxygenations of steroid and prostaglandin biosynthesis. Certain fungal chloroperoxidases and bacterial P450s have been genetically engineered for large-scale biotransformations [5053]. The active sites of these protein families, known in detail from a number of X-ray crystal structures [46,5456], are remarkably similar. Both have an ironprotoporphyrin IX center coordinated to a cysteine thiolate. Both are oxidoreductases that activate molecular oxygen (O2), in the case of P450, or hydrogen peroxide in the case of CPO, at the iron center and incorporate one of the oxygen atoms into a wide variety of biological substrates. Both proteins are proposed to initiate their chemistry through the oxidation of a resting iron(III) state (1) to a reactive oxoiron(IV)porphyrin cation radical intermediate (2) (Fig. 3). For CPO that reactive ferryl-porphyrin cation radical species has been spectroscopically characterized. Further the one-electron reduced Fe(IV) intermediate (CPO compound II) has been shown to be protonated on the ferryl oxygen, based on the observation of a long FeO distance determined by EXAFS spectroscopy [49]. Studies using synthetic metalloporphyrins as models for cytochrome P450 have aorded important insights into the nature of the enzymatic processes [57,58]. Indeed, each of the proposed intermediates shown in Scheme 8 has been independently identied by model studies using synthetic analogs, especially meso-tetraaryl porphyrins [59,60].

Scheme 7. Proposed mechanism for aliphatic hydroxylation by TauD. Adapted from Ref. [43].

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Fig. 3. Oxidation of Fe(III)heme by hydrogen peroxide, the peroxide shunt pathway.

The rst report of a simple iron porphyrin system that eected stereospecic olen epoxidation and alkane hydroxylation was reported in 1979 [61]. This system introduced the use of iodosylbenzene as an oxygen transfer agent to mimic the chemistry of cytochrome P450. The choice of this reagent was informed in part by the chemistry of typical alkyl hydroperoxides, ROOH, which show extensive and varied modes of metal-mediated free radical chain reactions. To suppress such reactions, an oxo-donor reagent was needed that could not easily propagate the chain. Our idea was that iodosyl benzene, which is a polymeric solid and did not contain a weak OH, could be a useful reagent in this regard. Another important discovery at this time came from the reports by Balch, et al. that an iron(II) porphyrin would oxidize readily in air to aord a reactive oxo-iron(IV) porphyrin, analogous to peroxidase compound II. The process was considered to proceed through a l-peroxo iron(III) dimer, Fe(III)O OFe(III) (Scheme 9). At about the same time we discovered that the oxidation of Fe(III) porphyrins could produce either a red oxo-iron(IV) species, shown to be the same that Balch had produced aerobically, or a green oxoiron(IV) porphyrin cation radical species. The oxoiron(IV) porphyrin cation radical complexes were shown to exchange their oxo oxygen with water and to be highly reactive as oxygen atom transfer agents toward olens and hydrocarbons, i.e., reacting with olens to aord epoxides and with alkanes to give alcohols (Fig. 4) [6264]. Another type of iron(IV) porphyrin isolated at that time was a dimethoxy derivative [65]. These compounds have been extensively studied and well characterized by various spectroscopic techniques, including visible spectroscopy, NMR, EPR (electron paramagnetic resonance), Mo ssbauer and EXAFS [6573].

Scheme 8. Concensus mechanism for the cytochrome P450 reaction cycle.

The substrate oxygenations catalyzed by the cytochrome P450 family of enzymes are sometimes accompanied by a variety of molecular rearrangements. How these rearrangements inform the discussion about the mechanisms of oxygen transfer from the oxidized heme intermediates to the substrate has been the source of lively debate. A hydrogen abstraction scenario is consistent with the stereochemical, regiochemical and allylic scrambling results observed in the oxidation of norbornane, camphor and cyclohexene by cytochrome P450. The hydroxylation of a saturated methylene (CH2) in norbornane was accompanied by a signicant amount of epimerization at the carbon center [74] and the hydroxylation of camphor by P450cam [75] gave exo-alcohol with retention of the exo-deuterium label. The hydroxylation of selectively deuterated cyclohexene proceeded with substantial allylic scrambling [76]. Signicantly, similar results were obtained for a fully reconstituted oxygen/NADPH/P450 system and peroxide shunt pathways that used alkyl hydroperoxides or iodosyl bezene as oxo-transfer agents. Thus, one concludes that the epimerization and allylic scrambling processes are intrinsic properties of the oxygen transfer event from an oxoiron complex. The hydroperoxy iron(III) complex in Scheme 8 has also been suggested to eect substrate oxygenations based on observed changes in product ratios and loss of hydrogen peroxide (uncoupling) upon P450 active site mutations [7779]. However, there is no experimental evidence that such a porphyrinFe(III)OOH would be suciently electrophilic at the distal oxygen to react with a CH bond. DFT calculations indicate that such a species would be nucleophilic but not electrophilic [80]. Bach has recently described an alternative DFT reaction pathway for an iron(III) hydroperoxide that involves OO bond homolysis to produce a reactive species formulated as a ferryl-associated hydroxyl radical [(por)Fe(IV)@OHO] [81]. An important experimental advance has been the development of cryo-spectroscopic studies by Homan et al. that have allowed the stepwise interrogation of intermediates along the hydroxylation pathway [82]. While no ferryl intermediate was observed, the data indicated that the product alcohol was formed with its oxygen atom coordinated to the heme iron (Scheme 10). This arrangement has important mechanistic implications since, if a ferryl species were the immediate precursor of the product complex, then coordination of the product hydroxyl oxygen would be a necessary consequence. Evidence for short-lived substrate radicals has derived from studies of the mechanistically diagnostic probe molecule norcarane by cytochrome P450 [83,84]. The successful application of this substrate to the analysis of enzymatic hydroxylation mechanisms stems from the fact that the cyclopropyl carbinyl radicals and cations for this substrate rearrange via dierent pathways. Typical radical reactions such as tin hydride dehalogenations or chlorinations with alkyl hypochlorites have shown a 50:1 kinetic preference for opening of the exocyclic cyclopropyl bond to aord

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Scheme 9. Aerobic oxidation of an iron(II) porphyrin.

the cyclohexenylmethyl radical and corresponding products derived from that radical. This radical ring-opening rate has been measured to be 2 108 s1. The behavior of norcarane-derived cations has been studied by examining products of the solvolysis of the dinitrobenzoate ester of 2-norcaranol in aqueous acetone. This reaction was reported to aord mostly 2-norcaranols and 10% 3-cyclohepten-1-ol as the only rearranged product. Similar results have been reported for the corresponding mesylates [84]. Under more forcing conditions (HClO4/HOAc) that allow for repeated reionizations, 2-norcaranol aorded 3-cyclohepten-1-yl acetate and 3-acetoxymethylcyclohexene in a ratio of 96:4. Further, we have shown that solvolysis of the dinitrobenzoate ester of 3-hydroxy-methylcyclohexanol in aqueous acetone gave the same mixture of products as for the 2-norcaranol ester with 90% 2-norcaranols, 9% cycloheptenol, and <1% hydroxymethylcyclohexene [83]. Thus, while it has been suggested that norcarane my behave anomalously as a probe substrate [84], all of the available experimental evidence indicates that hydroxymethyl cyclohexene is produced in signicant amounts only

Fig. 4. Substrate (S) oxygenation by a family of oxoiron(IV) tetramesitylporphyrin cation radical species, Fe(IV)(O)(TMP)+(X).

Scheme 10. Hydrogen abstraction, oxygen rebound pathway for aliphatic hydroxylation by cytochrome P450.

via radical pathways while the only signicant cation rearrangement product for this probe is cycloheptenol. Accordingly, norcarane is a revealing mechanistic probe for enzymatic and model CH hydroxylations. Among the products found with P450 oxidations of norcarane was ca 0.5% of the radical rearrangement product hydroxymethylcyclohexene (Fig. 5). The cation-derived product cycloheptenol was observed in amounts similar to the radical alcohol product in some cases but not in others. The extent of observed rearrangement with a panel of P450 enzymes leads to a radical lifetime in the picosecond regime, certainly long enough to be considered an intermediate. We have suggested that the cationic rearranged products that sometimes appear during P450 turnover derive either from a reionization of the initially formed alcohol or from an electron-transfer oxidation of the incipient carbon radical that competes in some cases with oxygen rebound. An important precedent for this kind of behavior derives from the inner-sphere vs outer-sphere paradigm for carbon radical oxidation that has been extensively studied by Rollick and Kochi [85]. In this very informative work, the rate of electron transfer from an alkyl radical to an iron(III)phenanthroline oxidant to form a carbocation and iron(II) was timed by competition with a homolytic bromine atom transfer from bromotrichloromethane. Signicantly, carbocation formation via electron-transfer oxidation of even primary alkyl radicals was found to compete with innersphere atom transfer processes. Moreover, the extent of carbocation formation was a very sensitive function of the oxidation potentials of both the iron(III) oxidant and the alkyl radical while the inner-sphere processes were most sensitive to steric eects. In the case of the stepwise hydroxylation process mediated by cytochrome P450, oxygen rebound to form the product alcohol is the inner-sphere process while competing electron-transfer processes would occur to the extent that the rebound rate, taken to be about 1010 s1 competes with the electron-transfer rate. Thus, the electron-transfer oxidation of an incipient carbon radical at the active site in the P450 rebound process could well be oxidized by an intermediate oxoiron(IV) species to explain the products that appear to arise from cationic rearrangement processes. Recently it has been shown that further oxidation of the desaturation product norcarene can lead to a variety of secondary metabolites [86], the presence of which can make the product analysis more dicult. Fig. 6 shows data for

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Fig. 5. Norcarane as a molecular probe of radical intermediates during CH hydroxylation by cytochrome P450.

the oxidation of norcarane by cytochrome P450 2B4 showing limited over-oxidation and a 0.5% yield of the radical product hydroxymethyl cyclohexene in this case [87]. Ortiz de Montellano has recently described informative results for the hydroxylation of a-thujone and b-thujone by P450cam and P450BM3 [8890]. These isomeric substrates were hydroxylated at C-4 to aord both rearranged and unrearranged hydroxylation products. Radical intermediates rather than cationic intermediates were indicated by the nature of the products obtained. The carbon radical initially produced by hydrogen abstraction at C-4 was shown to undergo both inversion of stereochemistry and cyclopropyl ring-opening, acting as a two-zone radical clock (Scheme 11). Some desaturation was also reported. The possibility of a cationic ring-opening mechanism was discounted due top the lack of any phenol products in the mixture. From the extent of ring-opened products obtained, which were of a similar degree discussed above for norcarane, the radical rebound rates in these systems were found to be between 0.2 1010 and 2.8 1010 s1. Parallel reactivity of multiple oxidants (Fe(III)OOH and ferryl) [78,81,91,92] and multiple spin states of a single metal-oxo intermediate [9396] are two alternative mechanisms that have been advanced to explain the fact that the extent of rearrangement seen for particularly fast radical probes is less than expected based on rearrangement rates determined for these probes. It has proved to be very dicult to devise experiments to distinguish between these hypotheses. One very interesting set of results that does address this point was an examination of kinetic isotope eects for the N-demethylation of aromatic amines reported by Jones and co-workers [97]. Here, the aerobic

Scheme 11. Products deriving from radical rearrangements during the P450-mediated hydroxylation of a- and b-thujone. Adapted from Ref. [90].

P450-mediated process was compared with a peroxide shunt pathway using a set of related amine N-oxides. The central result was that for P450cam and P4502E1, variations in the intramolecular kinetic isotope eect for the hydrogen abstraction that initiated N-demethylation showed a very nearly identical pattern of isotope shifts upon variation of a para substituent on the aromatic ring. For multiple oxidants, such as Fe(III)OOH and ferryl, acting in parallel to produce such results would require an extraordinary coincidence of substitution eects on both pathways. The results were much more easily accommodated by assuming a single ferryl intermediate that may be reacting from multiple spin states as suggested by Shaik [93,94,98]. 4. Metalloporphyrins as detectors and decomposition catalysts of peroxynitrite The rapid reaction rates of peroxides with metalloporphyrins and the detection of observable oxometalloporphyrin species, especially in the reactions of manganese porphyrins with various oxidants (m-CPBA, NaOCl, KHSO5) [99], has also inspired the use of these porphyrins as detectors for a transient biological oxidant, peroxynitrite (ONOO). ONOO, a strong one- and two-electron oxidant, has been implicated in a number of pathological

Fig. 6. GCMS ion chromatogram for the norcarane-derived product alcohols from a cytochrome P450 2B4 oxidation. The inset shows the very characteristic spectrum of the radical rearrangement product hydroxymethyl cyclohexene, labeled R at 6.64 min. The small peak at 6.57 min is a secondary oxidation product. Sample kindly supplied by M. Newcomb (see Ref. [84]).

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conditions [100]. Structurally, ONOO is the conjugate base of peroxynitrous acid and, like other similar peroxides, it rapidly oxidizes manganese porphyrins [57,99, 101]. Thus, its reactions with manganese porphyrins to generate oxomanganese intermediates allowed the sensitive detection of this transient molecule under cell-like conditions [57,99]. Using manganese porphyrins as ONOO detectors, it has been shown that ONOO can quickly diuse across biological membranes and react with its biological targets [99,102]. The fast oxidation of manganese(III) porphyrins to oxomanganese(IV) species by ONOO satised the prerequisite for the porphyrins to be ecient ONOO decomposition catalysts [99]. Indeed, manganese porphyrins, redox-coupled with biological antioxidants (such as ascorbate, glutathione, Trolox), rapidly reduce ONOO and thus prevent the oxidation and nitration of biological substrates by this toxic oxidant [101,103]. Amphiphilic analogs of the watersoluble metalloporphyrins have been developed that are suitable for liposomal delivery [104]. Further, these amphiphilic metalloporphyrins in sterically stabilized liposomes are highly active in decomposing ONOO [104], and thus are potential therapeutic agents for the treatment of ONOO-related diseases. Iron(III) porphyrins are also rapidly oxidized by ONOO, and Stern et al. have reported that water-soluble iron porphyrins have the ability to

decompose ONOO by isomerizing it to nitrate and these compounds, moreover, have shown signicant biological responses in animals [105]. The mechanism of action of the iron porphyrins is signicantly dierent than the manganese case. Signicantly, both Fe(III) and oxoFe(IV) states are catalytically active toward ONOO (Scheme 12) [106,107]. Compounds of this sort have been shown to be highly potent in animal models of inammation related pathologies such as diabetes and colitis [108]. 5. Molecular probes of the mechanism of the diiron hydroxylases The non-heme diiron hydroxylases [5,7,109111], such as the x-hydroxylase from Pseudomonas putida (AlkB), toluene monooxygenase (T4moH), soluble methane monooxygenase [6] (sMMO) and xylene monooxygenase (XylM) have been studied with a variety of structural, spectroscopic and mechanistic probes. The consensus mechanisms for the heme and diiron hydroxylases are remarkably similar and analogous to the cytochrome P450 cycle described above. The process is initiated by a two-electron reduction of dioxygen via the di-ferrous form of the enzyme to aord a peroxo-Fe(III)Fe(III) state. For sMMO this peroxo-form is known to be oxidized to a reactive species, compound Q, that has been characterized as a bis-l-oxo-iron(IV)

Scheme 12. Mechanism of peroxynitrite decomposition by FeTMPS.

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intermediate. It is this strongly oxidizing intermediate that is responsible for substrate oxygenation. We have examined the reactions of the diagnostic substrate norcarane with AlkB [8,112], sMMO [84,113], T4moH [114] and XylM [115]. As discussed above, this simple substrate allows a dierentiation of radical and cationic intermediates that may be formed during hydroxylation since dierent ring-scission rearrangements predominate for these two potential reaction pathways, as discussed above. All four diiron hydroxylase systems have shown the radical rearrangement product, hydroxymethylcyclohexene in signicant amounts (Scheme 13). For the histidine-rich hydroxylase, AlkB, the results were particularly striking since fully 15% of the product was indicative of the radical rearrangement pathway, putting the lifetime of the intermediate radical at about 1 ns [112]. Interestingly, it was found that longer apparent radical lifetimes, up to 19 ns, were observed when the substrate was maintained at low concentrations [8]. Notably, this eect was observed in whole cell oxidations both with wild-type P. oleoverans and with an Escherichia coli clone into which the AlkB gene had been inserted. No such eect was observed with T4moH expressed in E. coli. While the reasons for this variability in the radical lifetime for AlkB are not yet clear, this protein is known to have a substrate entrance channel that could be lled either with water or

multiple substrate molecules depending upon the substrate concentration. If this is so, the radical rebound rate must be sensitive to the nature of other small molecules present at the active site, causing a kind of solvent eect. Similar nanosecond radical timings have been obtained recently using norcarane as a radical clock for the related diiron hydroxylase XylM [115], which also has a histidine-rich active site. A signicant aspect of this work was that while some of the studies were performed on with isolated, puried proteins, the AlkB and XylM studies relied on comparisons of results for diagnostic substrate oxidations by whole, wild-type cells and E. coli clones into which the AlkB and XylM genes had been introduced. These are membrane-bound proteins that have been dicult to isolate and purify. None-the-less it has been possible to obtain mechanistically useful information is this whole-cell milieu. Sequence analysis has indicated that AlkB spans the phosphilipid membrane with six parallel peptide helices (Fig. 7) [7,8,52]. The diiron active site is suggested to be near the membrane interface and the substrate binding pocket is arranged to accommodate a medium sized hydrocarbon. The examination of isolated toluene monooxygenase (T4moH), and several active site mutants with diagnostic substrates was particularly informative [114]. First, norcarane was an excellent substrate for this enzyme, giving turn-over rates and coupling eciencies nearly as large as

Scheme 13. Hydroxylation of norcarane by non-heme diiron proteins with diagnostic substrate radical rearrangement.

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Fig. 7. Proposed membrane-spanning arrangement and active site position in AlkB.

the similarly sized natural substrate toluene. Second, it was possible in this case to dissect the mechanism into two divergent pathways, one leading to radical-type products and the other involving cationic rearrangements. For norcarane the ratio of unrearranged 2-norcaranyl alcohols to hydroxymethyl cyclohexene, the radical rearranged alcohol, was about 20:1, indicating a radical lifetime of 260 ps. No cycloheptenol, the product of cationic rearrangement, was observed. For diethyl cyclopropane, however, the story was more complex since products of both radical and cationic rearrangement pathways were observed. Here the amount of radical rearranged alcohol, 3-ethyl-3-penten-1-ol, was 1.8%, consistent with the slower radical rearrangement rate of this cyclopropylcarbinyl probe. However, there was now a signicant amount of the 1-ethyl-2-methylcyclobutanol (8.5%) that is indicative of a cation rearrangement for this substrate. When this reaction was run under 18O2, however, the radical alcohol product and the unrearranged product were shown to have similarly high ratios of labeled oxygen incorporation. By contrast, the mass spectrum of the cyclobutanol product indicated that 99% of the hydroxyl oxygen had derived from water in the medium. These results are consistent with a scenario in which an intermediate substrate radical is formed by initial hydrogen abstraction by the active oxygen species, presumably similar to compound Q. Indeed, for the reaction of sMMO with norcarane compound Q was detected and determined kinetically to be the reactive species [113]. The lifetime of this radical, in the hundreds of picoseconds, is long enough for multiple rotations and molecular rearrangement of the radical intermediate near the active site but too short for diusion out of the active site. Accordingly, the rearranged radical builds up over time and both the rearranged radical and the unrearranged radical rebound to an iron-bound oxygen that has derived

Scheme 14. Oxygen isotopic labeling allows dissection of radical and cationic pathways in diethylcyclopropane hydroxylation by T4moH. Unrearranged and radical rearranged products derive their hydroxyl oxygen from O2 whereas the cation rearranged product derives its oxygen from water.

from molecular oxygen (Scheme 14). By contrast, the cationic rearrangement pathways must diverge from this radical rebound mechanism since it is the water pool that traps the cations and not the iron-bound, O2-derived oxygens. This is the rst time that such a mechanistic dissection of substrate hydroxylation by a metalloenzyme has been detected. Just how this mechanistic divergence takes place is under continued investigation. 6. Conclusion I have summarized here some of the important experimental advances that have led the study of the iron-containing oxygenases forward and the parallel development of our concepts about how these fascinating proteins work. Indeed, it was the lack of chemical precedents and any mechanistic underpinnings that led us to initiate these studies three decades ago. Today the eld is burgeoning as the

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reader can see in the many other contributions in this special issue of the Journal of Inorganic Biochemistry and, as well, in the huge body of eort cited in those papers. The vitality of the eld has been driven by the wonderfully diverse family of oxidation reactions mediated by these iron enzymes and the realization that reactive, high-valent iron is essential to their function. Direct observation of many of the proposed intermediates, the synthesis and characterization of model compounds that display many aspects of this remarkable enzymatic reactivity and the application of ever more sophisticated physical and computational methods have shown that the ferryl intermediate, which was once an intuitive idea about how nature might activate molecular oxygen, now has a rich and rapidly developing chemistry. Acknowledgements Special thanks are due to all the members of my research group and my collaborators who participated in the projects described here from our laboratory and contributed so many ideas, inspirations and insights. Their names are indicated in the referenced papers. We are grateful to the National Science Foundation, NSF CHE-0316301 and CHE-0221978 through the Environmental Molecular Sciences Institute CEBIC at Princeton University, and the National Institutes of Health, NIH GM 036298, for support of this research. References
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