Insights into the P-to-Q conversion in the catalyticcycle of methane monooxygenase from a syntheticmodel systemGenqiang Xuea, Adam T. Fiedlera, Marlene Martinhob, Eckard Munckb,1, and Lawrence Que, Jr.a,1

aDepartment of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, 207 Pleasant Street SE, Minneapolis, MN 55455; and bDepartmentof Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213

Edited by Edward I. Solomon, Stanford University, Stanford, CA, and approved October 29, 2008 (received for review August 29, 2008)

For the catalytic cycle of soluble methane monooxygenase(sMMO), it has been proposed that cleavage of the O–O bond in the(�-peroxo)diiron(III) intermediate P gives rise to the diiron(IV)intermediate Q with an Fe2(�–O)2 diamond core, which oxidizesmethane to methanol. As a model for this conversion, (�–oxo)-diiron(III) complex 1 ([FeIII

2(�–O)(�–O2H3)(L)2]3�, L � tris(3,5-di-methyl-4-methoxypyridyl-2-methyl)amine) has been treated con-secutively with one eq of H2O2 and one eq of HClO4 to form 3([FeIV

2(�–O)2(L)2]4�). In the course of this reaction a new species, 2,can be observed before the protonation step; 2 gives rise to acationic peak cluster by ESI-MS at m/z 1,399, corresponding to the{[Fe2O3L2H](OTf)2}� ion in which 1 oxygen atom derives from 1 andthe other two originate from H2O2. Mossbauer studies of 2 revealthe presence of two distinct, exchange coupled iron(IV) centers,and EXAFS fits indicate a short Fe–O bond at 1.66 Å and an Fe–Fedistance of 3.32 Å. Taken together, the spectroscopic data point toan HO-FeIV-O-FeIV � O core for 2. Protonation of 2 results in the lossof H2O and the formation of 3. Isotope labeling experiments showthat the [FeIV

2(�–O)2] core of 3 can incorporate both oxygen atomsfrom H2O2. The reactions described here serve as the only biomi-metic precedent for the conversion of intermediates P to Q in thesMMO reaction cycle and shed light on how a peroxodiiron(III) unitcan transform into an [FeIV

2(�–O)2] core.

diiron(IV) � iron-oxo � Mossbauer spectroscopy � nonheme �oxygen activation

The biological conversion of methane to methanol is carriedout by methane monooxygenases (MMO) found in meth-

anotrophic bacteria (1). The soluble form of this enzyme(sMMO) has been well characterized by crystallographic, spec-troscopic, and kinetic methods (2–5). It has a nonheme diironactive site that reacts with O2 to generate in sequence twointermediates, a (�-peroxo)diiron(III) species called P (orHperoxo) (6–8) and then a diiron(IV) species called Q that isresponsible for oxidizing CH4 (6, 7, 9). Extended X-ray absorp-tion fine structure (EXAFS) analysis of Q revealed a short Fe–Fedistance of 2.5 Å, leading us to propose an [FeIV

2(�–O)2]diamond core structure (10).

To date, the mechanistic details of the P-to-Q conversion arenot understood. Two limiting scenarios can be considered on thebasis of bioinorganic precedents, and these are shown in Scheme1. The first scenario (Scheme 1 Upper) is based on recentquantum mechanics/molecular mechanics (QM/MM) studiesthat favor an [FeIII

2(�–�2:�2–O2)] structure for P and propose aconcerted isomerization of the [FeIII

2(�–�2:�2–O2)] core to an[FeIV

2(�–O)2] diamond core (5, 11–13), as established for anumber of dicopper-peroxo complexes (14, 15). The alternativescenario (Scheme 1 Lower) starts with a (�-1,2-peroxo)-diiron(III) structure for P based on the similarity of its spectro-scopic properties to those of the better characterized peroxointermediate of the D84E R2 protein of the ribonucleotidereductase from Escherichia coli (16). The P-to-Q conversion isthen postulated to involve protonation of the bound peroxide,

isomerization to a (�-1,1-hydroperoxo) moiety, and heterolyticcleavage of the O–O bond in a cytochrome P450-like mechanism(3, 16). Consistent with this alternative are kinetics experimentson the sMMO from Methylosinus trichosporium Ob3b that haverevealed a pH dependence in the conversion of P to Q (17).These two scenarios make different predictions concerning thefate of the oxygen atoms derived from O2, with one or both ofthe oxygen atoms of O2 becoming incorporated into the Fe2O2core of Q. To distinguish between the scenarios requires vibra-tional information on the Fe2O2 cores of P and Q. However,attempted resonance Raman experiments have thus far not beensuccessful, so there is no experimental basis upon which to makea choice between the two mechanisms in Scheme 1.

In our efforts to generate synthetic precedents for proposedintermediates in the catalytic cycle of sMMO, we have reportedthe only synthetic complex to have the [FeIV

2(�–O)2] diamondcore structure postulated for Q (complex 3 in Scheme 2) (18).This was accomplished by electrochemical oxidation of itsiron(III)iron(IV) precursor, which had been shown by crystal-lography to have an [Fe2(�–O)2] diamond core (19). In thisarticle, we report that this synthetic [FeIV

2(�–O)2] complex canalso be formed in high yield by treatment of a (�–oxo)-diiron(III) complex (1, Scheme 2) with stoichiometric H2O2followed by addition of 1 eq of HClO4. Furthermore, we havealso been able to trap the precursor of 3 before the protonationstep and characterize it as a (�–oxo)diiron(IV) species withinequivalent iron sites (2b). These results provide a valuableexperimental model for the conversion of P to Q in the sMMOcatalytic cycle and show how a nonheme diiron center canpromote the cleavage of the peroxo O–O bond.

Author contributions: G.X., E.M., and L.Q. designed research; G.X., A.T.F., and M.M.performed research; G.X. contributed new reagents/analytic tools; G.X., A.T.F., M.M., andE.M. analyzed data; and G.X., A.T.F., M.M., E.M., and L.Q. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence may be addressed. E-mail: larryque@umn.edu or emunck@cmu.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808512105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

Scheme 1. Possible mechanisms for the conversion of P to Q. PT, protontransfer.

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Results and DiscussionWe have shown previously that the reaction of the (�–oxo)diiron(III) complex [FeIII

2(�–O)(OH)(H2O)(TPA)2](ClO4)3(TPA � tris(2-pyridylmethyl)amine) with excess H2O2 in MeCN at�40 °C afforded a bright green chromophore that was character-ized to arise from a valence delocalized FeIIIFeIV complex with an[Fe2(�–O)2] core structure (19, 20). That study suggested thepossibility that high-valent [Fe2(�–O)2] diamond core structuresmay be involved in the catalytic cycles of oxygen activating diironenzymes such as MMO. Recently, we demonstrated that amember of this family of FeIIIFeIV complexes could be oxidizedby bulk electrolysis to the corresponding [FeIV

2(�–O)2] complex(18). The lifetime of this diiron(IV) complex was extended by theintroduction of electron donating methyl groups at C-3 and C-5 anda methoxyl group at C-4 of the pyridine rings of the TPA ligand,thereby allowing its formation in �85% yield and its spectroscopiccharacterization. When [FeIII

2(�–O)(OH)(H2O)(L)2]3� (1 inScheme 2), was reacted with stoichiometric D2O2 in MeCN at�40 °C, a new species 2 formed that has a broad absorptionfeature with �max � 705 nm (� � 2.5 mM�1�cm�1) (Fig. 1, dashedline). Upon addition of 1 eq of HClO4, species 1 converted to[FeIV

2(�–O)2(L)2]4� (3). The conversion yield of 1 to 3 was�80%, as determined by UV-vis (Fig. 1) and Mossbauer spec-troscopies. These results demonstrate that the stoichiometriccombination of a (�–oxo)diiron(III) complex and hydrogenperoxide can produce a species with an [FeIV

2(�–O)2] core inhigh yield. If we make the reasonable assumption that it mustproceed through a diiron(III)-peroxo adduct, this reaction servesas a synthetic precedent for the P-to-Q conversion in the sMMOcatalytic cycle.

Formation of Complex 2. The broad absorption feature of 2 isnearly identical to that of a short-lived species with �max � 705nm (� � 1.8 mM�1�cm�1) observed in a stopped-flow study of thereaction of [FeIII

2(�–O)(OH)(H2O)(TPA)2]3� with H2O2 in

MeCN at �40 °C (21). Based on the kinetic analysis, a diiro-n(III)-peroxo species was proposed, but no other spectroscopiccharacterization of this intermediate could be performed due toits short lifetime and further reaction with the excess hydrogenperoxide in the solution (21). Introduction of the 3 electrondonating substituents on the pyridine rings and substitution of Hwith D on the benzylic carbon atoms of the TPA ligand (Scheme2) significantly increased the lifetime of this intermediate (t1�2

�4h at �40 °C), allowing it to be characterized by a variety ofspectroscopic methods. The use of D2O2 in place of H2O2 alsoenhanced the yield of 2 by �10% by slowing further reaction of2 with H2O2 to form [FeIIIFeIV(�–O)2(L)2]3� (4), the one-electron-reduced derivative of 3 (see below). Indeed, titration of1 with substoichiometric D2O2 resulted in the linear increase ofthe 705 nm absorption band (Fig. 2), with its intensity plateauingat �1 eq of D2O2 added (Fig. 2 Inset), indicating the stoichio-metric reaction of 1 with D2O2 to yield 2.

Electrospray Ionization Mass Spectrometry (ESI-MS) Characterizationof 2. The ESI-MS spectrum of 2 (generated with H2O2) revealsa prominent mono-cation peak cluster (Fig. 3A) with m/z � 1,399and an isotope distribution pattern corresponding to an[(Fe2O3L2H)(OTf)2]� formulation (diiron(III) precursor withtrif luoromethanesulfonate (OTf) rather than ClO4

� counter

Scheme 2. Reaction of 1 with H2O2 to form diiron(IV) complexes. Unlike forconversion of 2b to 3, the fate of the 3 oxygen atoms cannot be determinedfor the conversion of 2b to 4, due to rapid exchange of the latter with H2O.

Fig. 1. UV-vis spectra showing the conversion of 2b (dashed line) to 3 (boldsolid line) upon addition of 1 eq (WRT 1, 0.5 mM) of HClO4. (Inset) Plot ofabsorbance at 705 nm (circles, decay of 2b) and 875 nm (squares, formation of3) vs. the amount of HClO4 added. Complex 3 is completely formed (bold solidline, 80% yield as estimated from absorbance at 875 nm with � � 2,200M�1�s�1) when 0.8 eq of HClO4 is added.

Fig. 2. UV-vis spectroscopic change upon titration of D2O2 into 0.7 mMcomplex 1 (dotted line) solution in MeCN at �40 °C. The bold solid linerepresents the spectrum of 2b with 1 eq of D2O2 added. (Inset) Plot ofabsorption at 705 nm vs. an equivalent amount of D2O2 added.

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anions used for all ESI-MS studies reported here to avoidcomplications arising from the Cl isotope distribution patternand simplify calculations for the fraction of 18O-incorporation).None of the 3 O-atoms in 2 exchanged with H2

18O (up to 200 eq)at �40 °C. However, the m/z value increased by 1 unit uponexchange with D2O (Fig. 3B), by 2 units when 18O-labeled 1 wasused to generate the sample (Fig. 3C) and by 4 units when H2

18O2was used (Fig. 3D). These results demonstrate that 2 has 3 oxygenatoms, 1 originally from 1 and 2 from H2O2. Moreover, nomonolabeled 2 was detected when a 1:1 mixture of H2O2 andH2

18O2 was used in the reaction with 1, showing that 2 O-atomsoriginate exclusively from 1 molecule of H2O2. Based on theseresults, we propose that the reaction of 1 with H2O2 results in thelikely displacement of the hydroxide and water ligands of 1 byH2O2 with the loss of 2 molecules of H2O and the coordinationof HOO� to the diiron center, corresponding to our initialformulation of 2 as 2a (Scheme 2).

Mossbauer Characterization of 2. We have studied with Mossbauerspectroscopy several preparations of 2 in MeCN and MeCN/PrCN mixtures, prepared at �40 °C by addition of either H2O2or D2O2 to 1 and determined that 2 can be formed in 65–80%yield. We present here data for a sample we have studied mostthoroughly, namely one prepared in 1:1 MeCN/PrCN with 0.8 eqof H2O2. For this sample, we have analyzed Mossbauer spectrarecorded between 1.5 K and 140 K in applied magnetic fields upto 8.0 T; we will report the rather involved analysis of the appliedfield spectra elsewhere. Fig. 4A shows a zero field Mossbauerspectrum of 2 recorded at 4.2 K. The sample contains 3 species,namely intermediate 2 (�65% of total Fe), diiron(III) species(�30%), and FeIIIFeIV complex 4 (�5% according to EPR andUV-vis analysis). The solid line in Fig. 4A represents thecombined contributions of 1 and 4 whose spectra are well knownto us from parallel studies of pure samples. (Because 1 wastreated with 0.8 eq of H2O2, the sample is expected to containat least 20% unreacted 1, plus perhaps diiron(III) decay prod-ucts; for lack of resolution we have modeled all diiron(III)species by 1.) The spectrum of Fig. 4B, obtained by subtractingthe diiron(III) species and 4 from the raw data, corresponds tothat of intermediate 2 and exhibits two doublets of equalintensity with �EQ(A) � 1.96 (3) mm�s�1

, �(A) � 0.00 (1) mm�s�1,

and �EQ(B) � 0.92 (2) mm�s�1, �(B) � �0.03 (1) mm�s�1. (Theseparameters correspond to a nested assignment of the 4 absorp-tion lines, which is also the only assignment consistent with ouranalysis of the applied field spectra to be presented elsewhere.)The isomer shifts of both doublets are typical of S � 1 FeIV

complexes (22), and we thus assign these two doublets toinequivalent subsites of a diiron(IV) complex, 2b. Throughoutthe H2O2 titrations doublets A and B were observed to haveequal intensity, as they must if they represent subsites of adinuclear complex. Spectra recorded in strong applied fields, tobe presented elsewhere, unambiguously show that 2b is a ferro-magnetically coupled FeIVFeIV complex with local SA � SB � 1sites. Interestingly, the �EQ and � values of site B are essentiallyidentical to those obtained for the mononuclear oxoiron(IV)complex [FeIV(O)(L)(NCCH3)]2�, �EQ � 0.95 (2) mm�s�1 and� � 0.01 (2) mm�s�1 (Fig. 4C and Fig. S1). This resemblance,which extends to the magnetic hyperfine parameters extractedfrom the analysis of applied field spectra, and our previousdemonstration that the Mossbauer parameters of closely related

Fig. 5. Fe K-edge EXAFS data [k3��(k) (Inset)] and the corresponding Fouriertransform of 2 obtained by fluorescence detection at 10 K. The sample wasobtained from the reaction of 5 mM 1 with 0.8 eq of D2O2 in CH3CN at �40 °C.Back-transformation range: 0.35 to 3.40 Å; Fourier-transformed range: k �2.1 � 14.9 Å�1; experimental data (solid line); and best fit (dotted line). Fitting(fit 5, Table 1): 1 O/N at 1.65 Å (�2, 0.0048), 1 O/N at 1.80 Å (0.0038), 4 N/O at1.98 Å (0.0054), 6 C at 2.89 Å (0.0079), and 1 Fe at 3.32 Å (0.0040).

Fig. 3. ESI-MS of 2 from reaction of 1-OTf with H2O2. The black columnsrepresent the experimental isotope profile and the shaded columns representthe theoretical one with formula of {[Fe2(O)3(H)(L)2](OTf)2}�. The differentimages show the ESI-MS of 2 with no isotope labeling (A), in the presence of50 eq of D2O (B), from reaction of 18O-labeled 1 with H2O2 (C), and fromreaction of 1 with H2

18O2 (D).

Fig. 4. Mossbauer characterization of complex 2. Shown are 4.2 K Mossbauerspectraof samplesof1treatedwith0.8eqofH2O2 in1:1MeCN/PrCN(dashed line)(A) and [FeIV(O)(L)(NCCH3)]2� generated by reacting [FeII(L)(MeCN)2](OTf)2 inMeCN with 1 eq of peracetic acid (C). The solid line in A is the sum of thecontributions of diiron(III) species (30%) and 4 (5%). The spectrum in B, repre-senting 2, was obtained by subtracting the two contaminants from the spectrumof A. The 2 FeIV doublets are indicated by the brackets. The spectrum shown in Cwas obtained from a sample that contained a diiron(III) decay product (48%),which we have subtracted from the raw data.

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[FeIV(O)(TPA)(X)]n� complexes are not very sensitive to thenature of the X ligand (23) lead to the conclusion that site B of2b has a terminal oxo ligand. In contrast, the large �EQ for siteA of 2b suggests an iron(IV) center without a terminal oxo ligandlike that of [FeIV(OH)(OOtBu)(BPMCN)]2� (�EQ � 1.76 (2)mm�s�1 and � � 0.10 (2) mm�s�1, BPMCN � N,N�-bis(2-pyridylmethyl)-N,N�-dimethyltrans-1,2-diaminocyclohexane)(22, 24). These observations lead us to conclude that only site Bhas a terminal oxo ligand.

X-Ray Absorption Spectroscopy (XAS) Characterization of 2. XAScharacterization was carried out on a sample prepared from 5mM 1 and 0.8 eq of D2O2 in MeCN. The 4.2 K Mossbauerspectrum of this sample, which was not enriched in 57Fe (2.2%57Fe) exhibited 2 and a diiron(III) species in a molar ratio of�6:1. In agreement, an 57Fe-enriched (20%) sample preparedunder identical conditions revealed that �70–75% of the iron ofthis sample belonged to 2 and �12–15% to diiron(III) species,with the remainder to a heterogeneous distribution of mononu-clear high-spin Fe3� species.* This sample exhibited an FeK-edge energy of 7,128.8 eV, slightly blue-shifted relative to the7,130.1 eV value found for its proton-derived product [FeIV

2(�–O)2(L)2]4� (3) (18) but still consistent with an iron(IV) oxidationstate (Table S1). Both 2 and 3 exhibit preedge features with adominant 1s33d transition near 7,113.6 eV and multiple weakerfeatures to higher energy (Table S1), with those of 2 approxi-mately 2-fold more intense than those of 3 (Fig. S2). Thedominant preedge transition observed for the sample of 2 has anarea of 24.1 units [no correction made for the contributions ofthe remaining fractions, which are expected to have smallerpreedge areas*]. Such an intense transition is comparable withthose previously reported for mononuclear oxoiron(IV) species(25) and supports the presence of a terminal oxo group in 2, assuggested by the Mossbauer analysis above.

The Fourier transform (FT, r� space) of the Fe K-edge EXAFSdata of 2 exhibits prominent features at r� � 1.6, 2.4 and 2.9 Å(Fig. 5), where r� is the actual metal-scatterer distance (r) aftera phase-shift correction of �0.4 Å. As shown in EXAFS Fit 1 inTable 1, the features at r� � 1.6 and 2.4 correspond mainly to Nand C scatterers arising from the TPA ligand at �2 and 2.9 Å,respectively, as found previously for other S � 1 FeIV(TPA)complexes (23, 25). Further improvements to the fit are obtainedby the introduction of an O scatterer at 1.69 Å in Fit 2, which canbe assigned to a terminal oxo atom (22, 25), and an Fe scattererat 3.32 Å in Fit 3. In Fit 4, the inclusion of 2 scatterers in thenearest shell yields significant improvement in fit quality, al-though the resulting Debye–Waller factor (�2 � 0.0124) isunacceptably large. This problem is resolved in fit 5 by splittingthe first shell into an O scatterer at 1.80 Å with concomitant shiftof the other O scatterer to 1.66 Å, thereby accounting for theremaining O atom(s) present in the ESI-MS-based formulationof 2. The observation that the Fe–Fe distance of 2 is 0.6 Å longerrelative to the 2.72 Å distance associated with the Fe2(�–O)2core of 3 (18) indicates that the 2 Fe atoms of 2 are bridged bya single oxo atom. This oxo bridge can then be assigned as theO scatterer at 1.80 Å (26).

The metrical information provided by XAS, together with theESI-MS and Mossbauer data, allows us to assign 2 as 2b of

Scheme 2, the diiron(IV) isomer of 2. This structure accounts forvarious constraints imposed by the accumulated data namely: (i)the ESI-MS formulation of the complex as [Fe2(L)2O3H]3�, (ii)the Mossbauer requirement for 2 inequivalent Fe(IV) centersthat are coupled to each other, and (iii) EXAFS evidence for aterminal oxo ligand. Structure 2b also rationalizes the distinctMossbauer quadrupole splittings observed for the 2 iron(IV)centers, with the smaller splitting similar to that of [FeIV-

(O)(L)(NCMe)]2� (Fig. 4C) and associated with the Fe � O halfof the complex and the larger splitting similar to that of[FeIV(OH)(OOtBu)(BPMCN)]2� (�EQ � 1.76 (2) mm�s�1) (24)and associated with the Fe–OH half of the complex.

DFT geometry optimizations were performed to assess theviability of the model proposed above for 2b, and key metricparameters for the resulting structure are shown in Fig. 6 (seeTable S2 for list of atom coordinates). Importantly, the Fe-ligandbond distances provided by DFT match reasonably with theexperimental values determined by EXAFS (Table 1, fit 5). Inaddition, DFT predicts a hydrogen bond from the terminalhydroxide to the terminal oxo with r(Ohydroxo���Ooxo) � 2.46 Å,similar to that observed between the terminal hydroxo and aqualigands of the diiron(III) precursor (20). This interaction givesthe (�–oxo)diiron(IV) core additional structural rigidity that isreflected in the intensity of the r� � 2.9 Å peak in Fig. 5 and theconsequently small Debye–Waller factor (0.004) associated withthe Fe scatterer. The FEFF program (27, 28) was then used tocalculate EXAFS spectra (Fig. S3) for a truncated version of theDFT-derived structure. Differences in ligand environments sur-rounding the 2 iron centers made it necessary to first computeEXAFS spectra for each Fe site individually and then sum theresulting data to obtain the composite diiron spectrum shown inFig. S3. The FEFF-derived Fourier-transform nicely reproducesthe r�-positions and intensities all of the major features in theexperimental data, thus providing independent verification thatour putative structure of 2b is quite accurate.

Conversion of 2b to 3. Addition of 1 eq of HClO4 instantly converts2b to the [FeIV

2(�–O)2] complex 3, as indicated by the appear-ance of its characteristic bands at 485 nm and 875 nm (Fig. 1).The 4.2 K Mossbauer spectrum of the sample shows a quadru-pole doublet with � � �0.04 mm�s�1, �EQ � 2.0 mm�s�1,identical with that of 3 reported (18). Our best attempt in theconversion of 2b to 3, as estimated by its absorbance at 875 nmand Mossbauer analysis, gave rise to an 80% yield of 3 withrespect to 1, which corresponded to that of 2b. Furthermore, thepresence of the 3 isosbestic points at 637, 806 and 910 nm in thecourse of the conversion suggests that 3 derives directly from 2b.

The use of 18O-labeled 2b to study its conversion to 3 providesinsights into which of the 3 oxygen atoms present in 2b areincorporated into the [FeIV

2(�–O)2] core of 3. The ESI-MSspectrum of 3 derived from the protonation of singly 18O-labeled2b (obtained from labeled 1) shows that half the moleculesincorporate one 18O label (Fig. S4A), while that of 3 generated

*These iron(III) contaminants are unlikely to alter the major conclusions of the XAS analysispresented here. The FeAO scatterer at 1.66 Å must arise from 2, since Fe(III) complexes donot possess such short Fe–O bonds. While the Fe–Fe distance of 3.32 Å determined for 2resembles those found for some diiron(III) complexes, it is unlikely that a �15% contam-inant could give rise to such an intense feature assigned to the Fe scatterer in the Fouriertransformed EXAFS spectrum of 2b. Indeed, the data is well-fit with N � 1.0 for the Fescatterer. Finally, it is significant that the experimental EXAFS data was adequatelysimulated using a geometry optimized model of 2b, suggesting that contributions fromimpurities to the overall form of the EXAFS spectrum are minor.

Fig. 6. The computational model of 2b obtained from DFT geometry opti-mization. Bond lengths are in Ångstroms.

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from protonation of doubly 18O-labeled 2b (generated fromH2

18O2) shows that approximately half of the product (52%) isdoubly labeled and the other half (48%) is singly labeled (Fig.S4B). The presence of equal amounts of singly and doubly18O-labeled 3 in the latter preparation was also confirmed byresonance Raman spectroscopy (Fig. S4C). Thus, 1.5 oxygenatoms from H2O2 and 0.5 of the oxo bridge of 1 becomeincorporated into 3, which implies that label scrambling verylikely occurs between only 2 of the 3 oxygen atoms of 2b in thesubsequent loss of water to form 3. Given that the terminal oxoshould be the least basic oxygen atom on 2b, protonation wouldvery likely occur at either the oxo bridge or the terminalhydroxide to initiate the label scrambling (29). Most signifi-cantly, the above labeling experiments demonstrate that it ispossible to react a diiron(III) complex with H2O2 and incorpo-rate both its oxygen atoms into an [FeIV

2(�–O)2] core, like thatof 3.

Conversion of 2b to 4. Complex 2b also reacts with H-atom donorsto form 4, the one-electron-reduced derivative of 3, [FeII-

IFeIV(�–O)2(L)2]3�. This transformation is indicated by theappearance of its intense chromophore at 620 nm, its charac-teristic S � 3/2 EPR signal and a Mossbauer quadrupole doubletwith parameters � � 0.11 mm�s�1 and �EQ � 0.44 mm�s�1 (18,20). With 2,4,6-tri-tert-butylphenol (TTBP) as donor, 1 eq issufficient to reduce 2b rapidly and quantitatively to 4. Thespontaneous formation of 4 (as monitored by a rise in theabsorption at 620 nm) is concurrent with formation of 2,4,6-tri-tert-butylphenoxyl radical (from absorption at 400 nm) (Fig. S5).UV-vis and EPR analyses show stoichiometric formation of both4 and the phenoxyl radical, providing evidence that the conver-sion of 2b to 4 involves abstraction of 1 hydrogen atom.

H2O2 can also serve as the H-atom donor. Indeed, addition ofexcess H2O2 to 2b converts it to 4 in quantitative yield. Theconversion of 2b to 4 exhibits pseudofirst-order behavior, andthe kobs dependence on H2O2 concentration gives a second orderrate constant of 5.9 M�1�s�1 at �40 °C (Fig. S6). These resultsconfirm earlier postulates that H2O2 could serve as a reductantin the formation of FeIIIFeIV complexes analogous to 4 (21, 30).

Summary Remarks. We have demonstrated in this work that H2O2reacts stoichiometrically with (�–oxo)diiron(III) complex 1 at�40 °C to afford an unsymmetric diiron(IV) intermediate 2b.Subsequent treatment with 1 eq of strong acid converts 2b to 3,which has been shown to have an [FeIV

2(�–O)2] diamond core

(18). We postulate in Scheme 2 that a peroxodiiron(III) adductlike 2a must initially form upon reaction of 1 with H2O2, but thatthis adduct is too fleeting to be observed under the conditionsof the experiment. On the assumption that Scheme 2 is reason-able, this series of reactions serves as a synthetic precedent forthe conversion of MMO intermediate P to Q.

Scheme 1 presents two limiting scenarios posited for theP-to-Q conversion that implicate different fates for the O2-derived atoms into the [FeIV

2(�–O)2] core of Q. Owing to thelack of vibrational information on P or Q, there is no experi-mental basis for choosing between the two alternatives. With thediscovery of the reactions described in this article and our abilityto monitor these transformations by resonance Raman and/orelectrospray mass spectrometry, we can determine the fate of theoxygen atoms derived from the H2O2 used to generate 3 in ourmodel system, gaining insights not currently available from theenzymatic system. The observation that 2 of the 3 oxygen atomsin 2b originate from 1 molecule of H2O2 strongly suggests thecoordination of H2O2, most likely in �-1,2 bridging mode, to thediiron(III) center, as postulated for 2a. This obligatory, but todate unobserved, peroxo intermediate then undergoes O–Obond cleavage to hydrooxidize the diiron(III) center to thediiron(IV) state and convert the peroxo group to the terminaloxo and hydroxide ligands of 2b (Scheme 2). This conversionmay be facilitated by the proton that remains in 2a, because the[FeIII

2(�–O)(�–1,2-O2)] core appears to be quite stable asjudged by the number of complexes characterized with suchcores (30–33). Indeed, the relatively stable complex [FeIII

2(�–O)(�–1,2-O2)(L�)2] (L� � tris(6-methyl-2-pyridylmethyl)amine)converts to a high-valent iron(III)iron(IV) complex upon addi-tion of strong acid (30), which to date is the only example of ahigh-valent diiron complex directly generated from an observedperoxo intermediate.

Upon treatment with strong acid, H218O2-labeled 2b converts

to 3 with the [FeIV2(�–O)2] core consisting of 16O18O and 18O18O

isotopomers in a 1:1 ratio. The observation of the latter isoto-pomer shows that there is a mechanism by which the oxo bridgeof 1 is lost as H2O and both atoms of H2O2 become incorporatedinto the [FeIV

2(�–O)2] core. This unprecedented experimentalresult lends credence to recent DFT calculations that favor thedicopper-like pathway for the conversion of intermediate P to Q(Scheme 1 Upper) (5, 11–13). An alternative that retains bothperoxo oxygen atoms in the diamond core of Q derives frombroken symmetry DFT calculations; it assigns P to have acis-(�-1,2 peroxo)diiron(III) unit and posits its rearrangement to

Scheme 3. Proposed mechanism for P-to-Q conversion. PT, proton transfer.

Table 1. EXAFS fitting results for 2

Fe–O Fe–O Fe–N Fe–C Fe–Fe

Fit n r, Å �2 � 103 n r, Å �2 � 103 n r, Å �2 � 103 n r, Å �2 � 103 n r, Å �2 � 103 GOF

1 6 1.97 15.4 6 2.94 7.8 1212 1 1.69 6.2 5 1.97 8.8 6 2.92 7.7 673 1 1.69 5.9 5 1.96 8.9 6 2.90 8.0 1 3.32 4.0 204 2 1.72 12.4 4 1.97 6.1 6 2.89 8.1 1 3.32 4.1 145 1 1.66 4.8 1 1.80 3.8 4 1.98 5.4 6 2.89 7.9 1 3.32 4.0 12

Back-transformation range: 0.35 to 3.40 Å; Fourier-transformed range: k � 2.1 � 14.9 Å�1 (resolution 0.12 Å). �2 in units of Å2. he first two shells were fitusing a scatterer with oxygen parameters, whereas the third employed nitrogen parameters, based on what we expect the donor atoms to be at such distances.However, backscatterers differing by Z � 1 cannot be distinguished by EXAFS. GOF, goodness of fit.

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a trans-�-1,2 coordination mode before O–O bond cleavage (34).However, unlike the DFT results, our model system points to akey role for a proton in promoting O–O bond cleavage, implyinginvolvement of some aspects of the P450-like pathway (Scheme1 Lower) (3, 16). Our experimental results in fact raise thepossibility of a hybrid mechanism (Scheme 3) that invokesproton-facilitated O–O bond lysis to form 2 distinct FeIV sitesanalogous to those in 2b before [FeIV

2(�–O)2] core assembly togenerate Q.

Experimental ProceduresMaterials. 2-Chloromethyl-4-methoxy-3,5-dimethylpyridine hydrochloride,acetonitrile anhydrous were purchased from Aldrich and used directly. H2

18O(97.2% 18O), H2

18O2 (� 90% 18O) and D2O2 (98% d) were purchased from ICONIsotopes.

The tris(4-methoxy-3,5-dimethyl-2-pyridylmethyl)amine ligand was syn-thesized following published procedures (18). To obtain the deuterated li-gand (L), 1.5 g (3.2 mmol) of the undeuterated ligand and 0.46 g of (19 mmol)NaH were suspended in 30 mL of acetonitrile-d3, and the mixture was stirredat 50 °C under Ar for 24 h. A total of 20 mL of D2O was then introduced toquench the reaction. Upon removal of acetonitrile under vacuum, the residuewas extracted with CH2Cl2 and the organic layer was washed with 100 mL of

H2O. A yellow oil was obtained when CH2Cl2 was removed. The oily productwas washed with H2O to give rise to a white powder. Yield: 1.3 g (83%). NMRand ESI-MS analyses showed that �98% of the protons on the methylenegroups were deuterated.

[FeIII2(O)(OH)(H2O)(L)2](ClO4)3 (complex 1-ClO4) was synthesized following

a published procedure (18). Preparations of [FeIII2(O)(OH)(H2O)(L)2](OTf)3 (1-

OTf) and complex 2 are described in SI Text.

Physical Methods. UV-vis, NMR, Mossbauer, XAS, and resonance Raman ex-periments were carried out following published methods (18), with detailsgiven in SI Text. Information about ESI-MS measurements, the geometryoptimization of 2b and the EXAFS spectral simulation is also described in SIText.

ACKNOWLEDGMENTS. We thank Professor Thomas Brunold (University ofWisconsin-Madison, Madison, WI) for graciously providing access to his com-puter cluster. This work was supported by National Institutes of Health GrantsGM38767 (to L.Q.) and EB-001475 (to E.M.) and Postdoctoral FellowshipFGM079839 (to A.T.F.). XAS data were collected on beamline 9–3 at theStanford Synchrotron Radiation Laboratory, a national user facility operatedby Stanford University on behalf of the U.S. Department of Energy, Office ofBasic Energy Sciences. The SSRL Structural Molecular Biology Program issupported by the Department of Energy, Office of Biological and Environ-mental Research, and by the National Institutes of Health, National Center forResearch Resources, and Biomedical Technology Program.

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