Π-Frontier Molecular Orbitals in S ¼ 2 Ferryl Species and Elucidation of Their Contributions to Reactivity

π-Frontier molecular orbitals in S ¼ 2 ferryl species and elucidation of their contributions to reactivity

Martin Srneca,1, Shaun D. Wonga,1, Jason Englandb, Lawrence Que, Jr.b,2, and Edward I. Solomona,2

aDepartment of Chemistry, Stanford University, Stanford, CA 94305; and bDepartment of Chemistry and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455

Contributed by Edward I. Solomon, July 30, 2012 (sent for review June 19, 2012)

S ¼ 2FeIV═O species are key intermediates in the catalysis of 1 (17) (Fig. 1A, Inset) and further correlated to density functional most nonheme iron enzymes. This article presents detailed spectro- theory (DFT) and multiconfigurational ab initio calculations to scopic and high-level computational studies on a structurally- elucidate the electronic structure and contribution of electronic defined S ¼ 2FeIV═O species that define its frontier molecular structure to reactivity. MCD spectroscopy provides a direct probe orbitals, which allow its high reactivity. Importantly, there are both of the reactive FMOs and, when applied to 1, reveals new S ¼ 2 π- and σ-channels for reaction, and both are highly reactive because π-channels at the transition state (TS). In the absence of a steric they develop dominant oxyl character at the transition state. These barrier (as would be the case for NHFe enzymes), these π-chan- π- and σ-channels have different orientation dependences defining nels would lead to new reactivity with interesting Fe spin-state how the same substrate can undergo different reactions (H-atom contributions. Furthermore, the MCD data reveal the presence abstraction vs. electrophilic aromatic attack) with FeIV═O sites in of extensive excited-state spin-orbit coupling (SOC) interactions different enzymes, and how different substrates can undergo dif- in the absorption spectrum—hitherto unobserved in other ferent reactions (hydroxylation vs. halogenation) with an FeIV═O FeIV═O species—that lead to interesting spectral consequences, species in the same enzyme. including a dip in the absorption (abs) band and different vibronic progressions within one excited state that reflect distorted poten- excited-state potential energy surfaces ∣ reaction coordinates ∣ magnetic tial energy surfaces (PES). circular dichroism ∣ density functional calculations ∣ multiconfigurational calculations Results MCD and Absorption Spectroscopy. Fig. 1 shows the UV-visible abs he key reactive intermediates in the catalytic cycles of many and VT MCD spectra of 1. The abs spectrum shows a weaker −1 −1 mononuclear nonheme iron (NHFe) enzymes are known to feature at 12;000 cm and an intense feature at 25;000 cm T −1 −1 be S ¼ 2 FeIV═O species capable of abstracting an H-atom with a shoulder at 19;000 cm . The abs band at 12;000 cm , from an inert C─H bond as strong as 106 kcal∕mol (1–5). These in fact, exhibits a dip in its intensity that corresponds to a sharp transient intermediates have been difficult to trap in appreciable positive feature in the MCD spectrum at the same energy (Fig. 1, concentrations amenable to spectroscopic studies, but recent vertical dashed line). These otherwise featureless abs bands developments hold promise for future geometric and electronic become much more well-resolved and feature-rich in the MCD structural characterization (4, 6). spectrum, which thus allows definitive band assignments for elec- The synthesis and structural characterization of stable biomi- tronic-structure elucidation. −1 metic model complexes have greatly aided in understanding the In the near-IR (NIR) 6;000–15;000 cm region of the MCD −1 geometric and electronic structures and reactivity of the FeIV═O spectra, three distinct bands centered around 11;500 cm can unit. A series of intermediate-spin S ¼ 1 FeIV═O model com- be identified: a positively signed Franck–Condon vibronic pro- plexes, supported by amine, pyridine, and deprotonated amido gression, a negatively signed vibronic progression, and a sharp ligands, has become available for structural, reactivity, and spec- positive feature (indicated by the dashed vertical line in Fig. 1) troscopic investigations (7–13). Recently, high-spin S ¼ 2 overlapping the positive progression. The temperature-depen- FeIV═O complexes have also become available, with two being dence behaviors of the two progressions indicate that they are structurally defined (14–20), but notably their reactivity does both (x, y)-polarized (analyzed in Scheme S1 and Fig. S1). This not surpass that of the most reactive S ¼ 1 complexes (15, 18, 21). pair of vibronic progressions forms a derivative-shaped pseudo-A S ¼ 1 FeIV═O complexes have been spectroscopically shown term (with its negative component at higher energy) that neces- 5 to possess one channel for reactivity—the β-dxz∕yz π -frontier sarily results from in-state spin-orbit splitting of a E excited state IV molecular orbital (FMO) (22)—and the S ¼ 2 species had been of the S ¼ 2 Fe ═O species in C3v symmetry. Importantly, computationally predicted to have an additional σ-FMO for as indicated by brackets in Fig. 1B, the vibronic spacing of the reactivity by stabilization of its α-dz 2 orbital through spin polar- negative progression is considerably larger than that of the posi- ization (i.e., exchange stabilization) (22–24). In the S ¼ 2 com- tive progression (ν ¼ 880 and 710 cm−1, respectively). IV −1 plex ðTMG3trenÞFe ═O(1), which is the subject of this study, In the 15;000–30;000 cm region of the MCD spectrum, there −1 it was shown that the accessibility of this α-dz 2 σ -FMO is are four bands under the envelope of the intense 25;000 cm restricted by an axial steric wall of methyl groups, giving a large absorption feature and its weaker low-energy shoulder. At steric contribution to its reaction barrier, rendering it only as 19;500 cm−1 and 23;500 cm−1, there are two z-polarized bands reactive as the S ¼ 1 complex ðN4PyÞFeIV═O (25). Importantly, ðN4PyÞFeIV═O also has a large steric barrier because the π-chan- ─ Author contributions: L.Q. and E.I.S. designed research; M.S., S.D.W., and J.E. performed nel requires an approach perpendicular to the Fe O bond for research; M.S., S.D.W., and E.I.S. analyzed data; and M.S., S.D.W., and E.I.S. wrote the orbital overlap, and this leads to the steric clash of the substrate paper. with the chelate ligand. In one case where the ligand completely The authors declare no conflict of interest. π S ¼ 1 σ excludes -approach, an complex can utilize a -channel 1M.S. and S.D.W. contributed equally to this work. S ¼ 2 for reactivity through a low-lying excited state (26). 2To whom correspondence may be addressed. E-mail: [email protected] or In this study, detailed variable-temperature (VT) magnetic [email protected]. circular dichroism (MCD) spectroscopic studies and analyses This article contains supporting information online at www.pnas.org/lookup/suppl/ are performed on the structurally defined S ¼ 2 model complex doi:10.1073/pnas.1212693109/-/DCSupplemental.

14326–14331 ∣ PNAS ∣ September 4, 2012 ∣ vol. 109 ∣ no. 36 www.pnas.org/cgi/doi/10.1073/pnas.1212693109 Downloaded by guest on October 2, 2021 Fig. 2. Spin-unrestricted MO energy-level diagram of 1 as determined by DFT calculations. Note the proximity in energy of the reactive α-dz 2 and β-dxz∕yz FMOs and the high-lying oxo π orbitals poised for oxo → Fe d CT.

for the lowest-energy 5E excited state (Fig. 1B). In the MCD Fig. 1. (A) UV-visible absorption spectrum and (Inset) structure of 1.(B)VT spectrum two low-energy z-polarized CT transitions are observed MCD spectrum of 1 and (Inset) VT MCD data of a higher-concentration 23;500 −1— sample in the 16;000–22;000 cm−1 region allowing resolution of the vibronic at 19,500 and cm the former is assigned in MCD and progression. VT MCD spectra taken at 2, 10, 20, 40, and 80 K. Arrows show Absorption Spectroscopy based on polarization and vibronic intensity behavior of bands with respect to increasing temperature. structure as the lowest-energy oxo π → Fe CT transition. The TD-DFTcalculations predict that the two lowest oxo π → Fe CT (from the temperature dependence of the MCD data; Scheme S1 transitions are z-polarized at 23,000 and 27;000 cm−1 (Fig. S3); and Fig. S1) that can be assigned to charge-transfer (CT) transi- thus, the two low-energy CT transitions in MCD are assigned as tions on the basis of their small MCD-to-absorption intensity oxo π → Fe CT transitions, the lowest in energy being the oxo (C0∕D0) ratios of 0.040 and 0.002, respectively. In contrast, the π → d −1 xz∕yz CT. In addition, the TD-DFT calculations predict that C0∕D0 ratio of the pseudo-A term at 11;500 cm (vide supra)is there are several (x, y)-polarized CT transitions originating from 5E 0.250, which indicates that this state corresponds to the lowest- the oxo group and equatorial nitrogens; two of these could z −1 energy ligand-field (LF) transition. From its -polarization, the correspond to the CT bands in MCD at 25,500 and 27;500 cm , 19;500 −1 negative band at cm can be assigned as the lowest-energy but because of the uncertainty in the polarizations of these two CHEMISTRY π → oxo Fe CT transition. This has a vibronic progression with a MCD bands, no definitive assignments can be made. 490 −1 spacing of cm and an intensity distribution indicating an These TD-DFTassignments of the transitions for 1 are consis- ─ excited-state distortion of the Fe O bond of approximately tent with multireference CASPT2 calculations (SI Materials and 0.21 Å (Ta b l e S 1 ). Finally, the two bands at 25;500 cm −1 and −1 Methods), showing the αðdxz∕yz → dz 2 Þ transition to be lowest in 27;500 cm have C0∕D0 ratios of 0.002 and 0.003, respectively, energy and the oxo πðpx;yÞ → dxz∕yz CT to be the lowest-energy and these can also be assigned as CT transitions. Their overlap CT (TableS2 and Fig. S4). For the 15;2000–30;2000 cm−1 energy precludes analysis of the VT MCD data in terms of polarization. −1 region, CASPT2 states are approximately 10;000 cm higher in energy than their corresponding bands in the MCD data; this DFT/Time-Dependent (TD)–DFT/Complete Active Space Second-Order is ascribed to the lack of inclusion of double-shell effects of the Perturbation Theory (CASPT2) Calculations. DFT calculations give 1 Fe 4d and oxo 3p orbitals (27). a geometry-optimized structure of in accord with the crystal 12;000 −1 structure (Fig. S2) and predict an S ¼ 2 ground state, in agree- In summary, the band at cm in the abs data exhibits an (x, y)-polarized pseudo-A term in the MCD spectrum, allowing ment with Mössbauer spectroscopy (15). This gives the (spin-un- 5A → 5E αðd → restricted) molecular orbital (MO) energy-level diagram shown in its assignment as the 1 transition involving the xz∕yz dz 2 Þ excitation. This transition is overlapped by a sharp peak in Fig. 2. As would be expected for a trigonal bipyramidal C3v ligand A field with a strong oxo axial ligand, the d manifold is split into MCD leading to a dip in abs intensity. The pseudo- term in two degenerate e levels, the dxy∕x 2−y 2 at lowest energy followed by MCD further shows different vibronic progression spacings for the dxz∕yz pair, and finally a nondegenerate a1 level (dz 2 ) that is its two components. The lowest-energy CT band in MCD at 4 19;500 −1 z πðp Þ → highest in energy. Thus, for a high-spin d FeIV species, the four cm is -polarized and assigned as the oxo x;y electrons are distributed unpaired into the two e sets (i.e., 4 α-d dxz∕yz CT transition from the TD-DFT and CASPT2 calculations. 5 orbitals are occupied as in Fig. 2, Left), resulting in a A1 ground state. Consequent to this electronic structure, in a spin-unrest- Analysis ricted formalism the α-dz 2 unoccupied orbital is spin polarized to Electronic Structure. Fano interference. The dip in the abs spectrum 11;820 −1 lower energy, comparable to that of the β-dxz∕yz π -FMOs, a and the sharp positive peak in MCD, both observed at cm phenomenon that was identified in previous studies (22–24). (Fig. 3), originate from a Fano-type interference/antiresonance Compared to FeIV═O S ¼ 1 species, which possess only a π-path- (28–30) that occurs when a sharp transition from a spin-forbidden way for reactivity, this shows the availability of an additional α-dz 2 state overlaps and weakly interacts with a broad vibronic continuum σ-FMO σ-pathway for reactivity in an S ¼ 2 FeIV═O species. associated with an allowed state (i.e., an intense band that only TD-DFTcalculations predict that the lowest-energy LF excited changes slowly in the vicinity of the sharp peak). This Fano inter- −1 5 5 state at 18;100 cm , originating from the A1 → E transition, ference arises from SOC between the spin-forbidden and spin- involves excitation from the degenerate α-dxz∕yz to the α-dz 2 level allowed states, and the fact that it is a peak in MCD but a dip in −1 (Fig. S3). This is overpredicted in energy by around 6;500 cm abs is caused by the different contributions of the Ms sublevels to as compared with the MCD experimental value of 11;500 cm−1 the Fano intensity for MCD versus abs (SI Analysis).

Srnec et al. PNAS ∣ September 4, 2012 ∣ vol. 109 ∣ no. 36 ∣ 14327 Downloaded by guest on October 2, 2021 lower in energy than j − 1; −1i by 2λ (þ200–300 cm−1, from a fit of the pseudo-A term with two overlapping bands; SI Analysis).

5 3 3 Interactions of the E state with spin-forbidden E and A1 states. The fact that (in Fig. 1B) both the RCP and LCP progressions in the MCD spectrum of the 5E state have different vibronic spacings, with the RCP negative progression having a larger vibronic splitting than the ground-state value (820 cm−1) (25), implies strong SOC interaction between one component of the 5E state and other nearby spin-forbidden excited states (31). Also, the presence of a Fano dip in the abs spectrum (Fig. 1A) indicates a weak SOC to the second component of the 5E. As presented in Fig. 4B, starting at a and a 0, two candidates are identified to be close in energy to the 5E state—a 3E state (Fig. 4B, a)at −1 3 0 −1 −30 cm and a A1 state (Fig. 4B, a )atþ1;210 cm , relative to the 5E—from nonrelativistic CASPT2 calculations at the ground-state equilibrium geometry. In-state SOC is introduced (Fig. 4B, a → b and a 0 → b 0) with CASPT2/spin-orbit complete Fig. 3. (A) NIR abs spectrum showing a dip in the continuum background active space state interaction (SO–CASSI) calculations, splitting 5 −1 profile; (B) NIR MCD spectrum at 2 K (bold solid line) fit with three separate the E state into five doublets, each pair separated by 49 cm . vibronic Franck–Condon progressions and one sharp peak, where S is the Next, out-of-state SOC is turned on (Fig. 4B, b → c and b 0 → c 0), Huang–Rhys parameter (see MCD and Absorption Spectroscopy), ΔQ is the showing that the 3E state interacts strongly with one sublevel (in- Fe─O bond distortion, and E00 is the zero-phonon energy; Fano analyses 3 dicated in green) and the A1 state interacts weakly with another of the abs dip and sharp positive MCD feature indicated by the arrow provided in Fig. S5. sublevel (indicated in red). Finally, out-of-state SOC among the lowest-energy states (Fig. S6A) is included (Fig. 4B, c, c 0 → d)to 5 MCD 5E pseudo-A term. Aspresentedabove,inadditiontothe assess the overall SOC perturbations to the E state. −1 3 3 sharp positive feature at 11;820 cm , the NIR MCD spectrum Evaluation of the PES interactions of the E and A1 states 5 exhibits a pseudo-A term with vibronically resolved progressions, with the E state along the Fe─O coordinate (Fig. 5 C and D) resulting from in-state SOC of the LF 5Eðxz; yzÞ state. Consider- shows that the strongest interactions occur within 0.05 Å of the ing the C3v double group and the SOC operator λLzSz [appro- ground-state equilibrium geometry. This reveals two significant priate for an (x, y)-polarized transition] acting on a set of insights into the electronic structure, which can be correlated with eff jML ¼1;MS ¼2; 1; 0i wave functions (where λ is the the NIR MCD spectra: (i) among the 10 sublevels (from five 5 5E j1; −1i many-electron state-specific SOC constant) the E state is split doublets) originating from in-state SOC, only the sub- into five twofold degenerate sublevels separated in energy by λ level, which results in LCP, positive MCD intensity (in Fig. 4B), is 3A (Fig. 4A). For an (x, y)-polarized transition, MCD intensity coupled to the spin-forbidden 1 state, with a SOC matrix element 160 −1 ≤1% requires an applied magnetic field H parallel to the z axis of of approximately cm leading to mutual admixture of j − 1; −1i 5E the molecule, which lowers the symmetry to C3 and splits the the two states; and (ii)the sublevel of the state, which doublets. For an applied field of 7 T, the positive axial zero-field 5 −1 splitting of the A1 ground state (D ¼þ5 cm ) (15) results in the Ms ¼ −1 component being the lowest-energy MCD-active 5 sublevel for the A1 ground state (Fig. 4A). Because transitions 5 require ΔMs ¼ 0, the two sublevels from the E excited state that are MCD-active are j1; −1i and j − 1; −1i, resulting in left circularly polarized (LCP,positive) and right circularly polarized (RCP, negative) MCD C-term intensities, respectively, with j1; −1i lying

5 5 Fig. 5. (A) PES of the A1 ground state and LF E and CT excited states along 5 5 Fig. 4. (A) The E excited-state in-state spin-orbit splitting and A1 ground- the Fe─O bond coordinate, evaluating (B) the evolution of wave function state zero-field splitting without and with magnetic field along z [for an characters. Note that the CT state is composed of two dominant configura- eff (x, y)-polarized transition]. Sublevels labeled in terms of (ML , MS). LCP tions. Energies of CASPT2 PESs shifted to match experimental values (for 5 5 and RCP transitions in MCD (numbered according to Fig. 3) indicated by unshifted CASPT2 PES, see Fig. S7). (C) PES of the A1,LF E, and CT excited vertical arrows. (B) Energy diagram connecting nonrelativistic states [(B, a) states, and one triplet state (3E) interacting with 5E. Only strong SOC 3 5 0 3 5 3 5 5 E and E;(B, a ) A1 and E)] with corresponding spin-orbit states (B, d between E and E in the vicinity of the A1 PES minimum is considered; and Fig. S6A) through in-state spin-orbit perturbed states (B, b and b 0) and otherwise, PES are calculated with nonrelativistic CASPT2 approach. PES 3 5 0 3 5 two-state spin-orbit perturbed states [(B, c) E∕ E;(B, c ) A1∕ E). MCD- shifted to match experimental values (for unshifted PES, see Fig. S7). (D) SOC- active states indicated in red (LCP) and green (RCP). driven strong CI mixing between 3E and 5E PES.

14328 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1212693109 Srnec et al. Downloaded by guest on October 2, 2021 results in RCP, negative MCD intensity, is coupled to the j − 1; −1i Reactivity. Model complex 1 has been shown to perform electro- sublevel of the 3E state, with a SOC matrix element of approxi- philic H-atom abstraction from exogenous substrates, including mately 220 cm −1 giving rise to considerable (approximately 50%) 1,4-cyclohexadiene (CHD) and THF, as well as undergo self- mixing of both states caused by their energy proximity. decay via H-atom abstraction from a methyl group on the These theoretical findings directly correlate to the experimen- TMG3tren ligand (15, 17). For reactivity with exogenous sub- 3 tal MCD data. First, the A1 state can be assigned as the sharp strates, because of ligand sterics a σ-attack is the only possible Fano interference. Second, the MCD pseudo-A term with differ- orientation to access the oxo moiety, whereas self-decay necessarily ent progressions within the formally-same transition can be attrib- proceeds via a π-attack because of the approximately 90° Fe─O─C uted to strong SOC between the 3E state and the RCP component angle presented by the closest target methyl group (Fig. 6, Center). of the 5E (leading to a close-to-equal mixing of both states). This This self-decay reactivity is ideal for probing the efficacy of the IV strong SOC leads to two RCP (negative) vibronic progressions, β-dxz∕yz π -FMOs in an S ¼ 2 Fe ═O species for comparison whose PES are shaped by strong SOC-driven configuration inter- with its α-dz 2 σ -FMO for exogenous-substrate reactivity. 5 σ π action (CI) in the vicinity of the A1 ground-state geometry (see The -channel H-atom abstraction from THF and -channel Fig. 5 C and D). Alternatively, the LCP (positive) component of self-decay reactivities were evaluated computationally, and the the 5E is unperturbed by the 3E and only weakly interacts with the DFT-calculated reaction coordinate energy profiles are shown 3A in Fig. 5. For the THF σ-attack pathway, the Gibbs energy 1 (Fig. 4B). Reasonable agreement between this model and ex- ‡ perimental data is obtained from a fit of the pseudo-A term with barrier, ΔG , is calculated to be 16.8 kcal∕mol, in reasonable one positive and two negative vibronic progressions (labeled “2,” agreement with the experimental value of 18.9 kcal∕mol (17). “1,” and “3,” respectively, in Fig. 3B; see also Table S1): (i) The At the TS, an electron has been partially transferred (39%) from ─ α d 2 relative energy positions of the three vibronic progressions from the C H bond of THF into the - z FMO, a description agreeing 1 the MCD Franck–Condon fits match well with the corresponding with a previous study on the reaction of with CHD (25) as well as calculated CASPT2 vertical-transition energies for the three the electronic structure obtained from a CASPT2 calculation on states responsible for the progressions; (ii) excited-state distor- the TS structure (Fig. S9). Further along the reaction coordinate, S ¼ 5∕2 III─ tions relative to that of the ground state (ΔQ ─ s), as estimated an Fe OH product is formed, consistent with the Fe O σ from the CASPT2 PES in Fig. 5C, reproduce the change in Mössbauer spectroscopic results (17). Note that the -attack pro- 1 S ¼ 5∕2 III─ the Fe─O bond length in the excited state, relative to the ground duct of with CHD is also an Fe OH (17, 25). π state obtained from the MCD Franck–Condon analyses (Fig. 3B For the self-decay -pathway, the DFT-calculated and experi- ΔG‡s 19 4 ∕ and Table S1); and (iii) the frequencies of the excited-state Fe─O mental (both . kcal mol) are in agreement. In addition, ΔH ‡ was determined from temperature-dependent kinetic stretch, derived from the curvatures of the CASPT2 PES, are in 16 7 ∕ accord with the experimental values (Table S1). studies (17) to be . kcal mol (at 0 °C), also corresponding well with the DFT-calculated value of 19.2 kcal∕mol. An internal π → Fe d reaction coordinate scan proceeding from the TS results in a pro- PES of oxo xz∕yz CT state. In analogy to the vibronically re- duct with an S ¼ 3∕2 FeIII─OH ferromagnetically coupled to the solved NIR MCD features, the excited-state parameters obtained – ligand radical. However, this is a local minimum, and relaxation from Franck Condon analysis of the CT progression (Fig. 1B, of the wavefunction of this product and reoptimization of the Inset), along with theoretical values derived from the correspond- structure leads to the energetically favored S ¼ 5∕2 FeIII─OH þ CHEMISTRY ing CASPT2 PES (Fig. 5C), are provided in Table S1. The calcu- π → d π ligand radical product comparable in endergonicity to that of the lated oxo xz∕yz ( ) CT transition has a lower excited-state THF σ-pathway reaction (Fig. 6A). Thus, DFT evaluation of the frequency and a larger excited-state distortion than any of the π- and σ-attack pathways of 1 shows that the reaction barriers three LF 5E progressions as observed experimentally, reflecting ─ are close in energy, in concord with experimental kinetics data. a weak Fe O bond in this excited state. The barrier ΔE‡ of the π-pathway (self-decay) and σ-pathway for an exogenous substrate with comparable C─H bond strength FeIII Fe─O Evolution of -oxyl character along coordinate. The spectro- (CH3NH2; see Fig. S10) can be decomposed into steric and elec- scopically calibrated electronic-structure model with the two low- tronic components (25), showing that deformation of TMG3tren 5 est excited states, E (dxz∕yz → dz 2 ) and CT (oxo π → dxz∕yz), can in the π-pathway has a much larger steric contribution (13.4 vs. be extended to examine the evolution of the wavefunction along 4.4 kcal∕mol) to its ΔE‡, resulting in similar electronic barriers the Fe─O stretching coordinate that is relevant to reaching the TS in reactivity (see Reactivity). Using the multireference CASPT2 approach, the wavefunction character is inspected in terms of the dominant electronic configurations. As presented in Fig. 5 A and B, elongation along the Fe─O coordinate to bond lengths close to the TS at approximately 1.75 Å (Reactivity) leads to a large change in the ground- and ex- 5 cited-state wavefunctions. The A1 ground-state wavefunction III develops Fe ðS ¼ 5∕2Þ—oxylðpz; σÞ character (at the Fe─O dis- 5 tance of 1.88 Å there is approximately 45% oxyl character in A1; see Fig. 5 A and B and Fig. S8), whereas the 5E and oxo π CT III III states develop Fe ðS ¼ 5∕2Þ—oxylðpx; πÞ and Fe ðS ¼ 3∕2Þ —oxylðpx; πÞ character, respectively. For the first excited state, the oxyl character dominates starting from an Fe─O distance Fig. 6. Two possible reaction channels for 1 (Center): σ-attack for exogenous of 1.72 Å (Fig. 5 A and B and Fig. S8). Moreover, both CASPT2 substrate (THF) and π-attack for self-decay of closest endogenous methyl excited-state PES cross the ground-state PES (at 1.9 and 2.0 Å, group. The next-closest methyl group (circled) is a second potential target respectively), which indicates accessibility of the two π-oxyl chan- for self-decay (Fig. S10). The σ-attack (A) and π-attack (B) energy profiles, in ¼ nels in addition to the σ-oxyl channel for the S ¼ 2 FeIV═O kcal/mol. Solvated energies (Polarized Continuum Model, solvent CH3CN) shown in the order ΔE ‡∕ΔH‡∕ΔG‡ for TS and ΔE°∕ΔH°∕ΔG° for products. species. Thus, the π- as well as the σ-channels can play important ‡ Experimental ΔG values calculated from second-order rate constants k2 roles in reactivity, and, importantly, all three states have domi- using the Eyring equation. Transition-state lowest unoccupied MOs involved nant oxyl character at these crossing points, indicating their effec- in H-atom transfer shown. Note that DFT-calculated bond strengths of target tiveness in electrophilic attack. C─H bonds are 94 (THF) and 97 (-CH3) kcal∕mol.

Srnec et al. PNAS ∣ September 4, 2012 ∣ vol. 109 ∣ no. 36 ∣ 14329 Downloaded by guest on October 2, 2021 between the π- and σ-pathways (9.8 vs. 9.5 kcal∕mol, respectively) for similar C─H bond strengths. Finally, the product in both cases is an S ¼ 5∕2 FeIII─OH with similar free energies of reaction. Because both pathways have similar reaction barriers and driving forces, both the α-dz 2 σ -FMO and the β-dxz∕yz π - FMO are equally-viable pathways in 1 for H-atom abstraction reactivity, which is in contrast to a recent computational study (32). Interestingly, there are two possible electronic descriptions of the TS of the π-pathway, raising implications for π-reactivity. The DFTcalculations describe the TS as transferring 57% of a β-spin electron from the C─H bond into the Fe─O π-FMO, giving FeIIIðS ¼ 3∕2Þ character (Scheme S2, Left), whereas in CASPT2 Scheme 1. (Left) MCD spectroscopy describes the electronic structure of calculations the dominant (35–40%) configuration contributing the FeIV═O unit at its equilibrium geometry, and (Middle) computational extension to the TS Fe─O bond length leads to (Right)twoπ-FeIII-oxyl and to the TS involves an αðoxo π → dz 2 Þ excitation, resulting in an one σ-FeIII-oxyl descriptions activated for electrophilic attack. FeIIIðS ¼ 5∕2Þ center and an oxyl radical ready to accept an α electron from the C─H bond (Fig. S9B and Scheme S2, Right). the π-FMO to give FeIIIðS ¼ 3∕2Þ─O•− character, whereas (ii) Importantly, these two electronic structure descriptions in- CASPT2 calculations show a dominant contribution from an III •− volved in π reactivity directly correlate to the first two excited states Fe ðS ¼ 5∕2Þ─O ðpx; πÞ configuration. These two states are observed in MCD spectroscopy and corresponding CASPT2 PESs observed directly in the MCD data and their evolution into oxyl- (Fig. 5 A and C). The 5E LF excited state evolves into an FeIIIðS ¼ dominant character is observed in the PES calculated by CASPT2 5∕2Þþπ-oxyl configuration (at long Fe─O distances), whereas the in Scheme 1. The importance of these two π-channels in the re- III IV first oxo π → dxz∕yz CT excited state corresponds to an Fe ðS ¼ activity of S ¼ 2 Fe ═O NHFe enzyme intermediates and the 3∕2Þþπ-oxyl configuration (Fig. 5 A and B), both highly activated associated spin-state differences on Fe along the reaction coor- to accept an electron from C─H into the oxyl radical. Thus, in dinate are the subjects of current study. addition to the σ-channel considered earlier (Fig. 6A, Center), The availability and viability of both σ- and π-pathways to MCD spectroscopy has defined two possible π-channels for the NHFe enzyme FeIV═O intermediates, which both have similar reactivity of S ¼ 2 FeIV═O intermediates. electronic reaction barriers but different substrate orientation requirements for electrophilic reaction, can dictate both the spe- Discussion cificity of their initial reaction and the path of their subsequent The S ¼ 2 FeIV═O biomimetic model complex 1 is capable of reactivity. In (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) H-atom abstraction through a σ-attack by the Fe─O moiety on and (4-hydroxy)mandelate synthase (HmaS), the same substrate, an exogenous substrate. Underpinning this reactivity is the spin depending on its orientation, undergoes electrophilic aromatic polarization of the unoccupied α-dz 2 MO, which lowers it in en- attack via the σ-pathway, or H-atom abstraction via the π-path- ergy and makes it accessible as an FMO for electrophilic attack. way (24). The presence of both pathways allows substrate orien- For 1, however, the methyl groups of the ligand present a steric tation by the protein to control reactivity; in the halogenase barrier hindering axial access to the FeIV═O unit, as discussed SyrB2, either Cl• transfer (native) or OH• rebound (nonnative) above and emphasized in previous studies on 1 (15, 25). In the reactivity can take place after the initial rate-determining H-atom present study, this σ-attack has been shown to proceed via an abstraction step (33), depending on the carbon-chain length of FeIIIðS ¼ 5∕2Þ–oxyl state, where elongation of the Fe─O bond the substrate and thus the orientation of the C─H bond relative transfers an α electron from the oxo pz σ orbital to the Fe dz 2 , to the Fe═O bond. This also implies that the enzyme can mod- leaving a low-lying oxo pz σ unoccupied orbital available to accept ulate its σ vs. π reactivity through the active site ligand environ- an α electron from the C─H bond of the substrate. ment; for example, differences in axial ligation could modulate Additionally, 1 self-decays by attacking one of its own ligand the α-dz 2 σ -FMO for electrophilic attack. Thus, it is now impor- methyl groups, and this takes place necessarily via a side-on π- tant to apply this combined spectroscopic and computational ─ ─ IV attack because of the bent Fe O Cmethyl angle (approximately approach to Fe ═O enzyme intermediates to evaluate their 90°) imposed by the covalent linkage of the methyl substrate selectivity in reaction coordinates. to the ligand. Spectroscopic data on 1 show a near featureless Both the abs and MCD spectra exhibit a sharp feature at absorption profile, providing minimal insight, but become rich in 11;820 cm−1; in abs it is a dip, whereas in MCD it is a peak. Fano information content upon going to VT MCD spectroscopy. From analysis of the spectroscopic data was correlated to advanced VT MCD data, two excited states have been identified, both cap- quantum-chemical methods to reveal the shared origin of these able of providing additional π-channels for reactivity in an S ¼ 2 features, which is a weak spin-orbit interaction between the broad IV 5 5 3 Fe ═O species (Scheme 1). In the case of the first E LF excited spin-allowed Eðxz; yzÞ and the nearby spin-forbidden A1 state state where a dxz∕yz electron is excited into the dz 2 orbital, elon- (Scheme 2A). In addition, the Franck–Condon analyses corre- gation of the Fe─O bond along this 5E PES allows it to mix with lated with the same level of calculations demonstrated that the 5 the ground-state A1 PES (because of the Cs distortion associated with the substrate interaction) while transferring an α electron from the oxo px π orbital into the dxz orbital, creating an FeIIIðS ¼ 5∕2Þ─O•− species with an oxo π-hole (Scheme 1, Right). Alternatively, the first oxo π → dxz∕yz (π ) CT transition results in an FeIIIðS ¼ 3∕2Þ─O•− species that decreases in energy with elongation of the Fe─O bond to the TS and can similarly accept an electron into its oxo π-hole. These provide two viable channels for π-reactivity, supported by experimental spectroscopic and kinetic data. π Similarly, computational evaluation of the self-decay reac- Scheme 2. (A) Weak CI between one component of the 5E state with the 1 3 tion coordinate of revealed that the TS can be described by A1 results in a sharp Fano antiresonance feature, whereas (B) strong CI mix- two possible electronic configurations: (i) DFTcalculations show ing of the 5E with the 3E (represented by dotted lines) results in one narrow a β electron being partially transferred from the substrate to and one broad PES (solid lines) with different vibrational-level spacings.

14330 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1212693109 Srnec et al. Downloaded by guest on October 2, 2021 MCD pseudo-A term with variable vibronic spacing is a result of the evolution of these states along the Fe─O reaction coordinate the in-state spin-orbit–perturbed 5Eðxz; yzÞ state having strong for catalysis. SOC with another nearby spin-forbidden 3E state; this strong CI distorts the interacting PES (Scheme 2B). Together, these give Materials and Methods rise to one positive and two negative vibronic progressions and Samples of 1 were prepared as described previously (15, 17), but in butyro- nitrile. UV-visible abs data were recorded on an HP8453A and a Cary 500 one sharp positive peak in the NIR MCD spectrum. It is essential spectrophotometer. MCD was performed on Jasco J-730 and J-810 spectro- to note that these important effects of excited-state CI (a dip and polarimeters equipped with Oxford Instruments SM-4000 superconducting a distortion of the PES) could only be identified and quantified magnets. DFT calculations were performed with Gaussian 03/09 and multi- through their differential effects on the MCD relative to the abs configurational/multireference calculations with MOLCAS 7.4. spectra because of their different selection rules, which result in different coupling interactions. ACKNOWLEDGMENTS. M.S. thanks the Rulíšek group at the Institute of Organ- These experimental observations and theoretical findings pro- ic Chemistry and Biochemistry, Prague, for use of their computational S ¼ 2 resources. Funding for research was provided by the National Institutes of vide fundamental insight into the electronic structure of Health (Grants GM 40392 to E.I.S. and GM33162 to L.Q.) and the National FeIV═O species, the interactions among its excited states, and Science Foundation (Grant CHE1058248 to L.Q.).

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