Mechanism of O2 Activation by Î±-Ketoglutarate Dependent

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Mechanism of O Activation by #-Ketoglutarate Dependent Oxygenases Revisited. A Quantum Chemical Study Anna Wójcik, Mariusz Radon, and Tomasz Borowski J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.5b12311 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 23, 2016

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The Journal of Physical Chemistry

Mechanism of O2 Activation by α-Ketoglutarate Dependent Oxygenases Revisited. A Quantum Chemical Study. Anna Wójcik∗2 , Mariusz Rado´ n3 , Tomasz Borowski∗1

1

Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,

ul. Niezapominajek 8, 30-239, Cracow, Poland. Telephone: +48 12 6395158 Fax: +48 12 4251923 E-mail: [email protected] 2

Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry,

Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Cracow, Poland E-mail: [email protected] 3

Department of Chemistry, Jagiellonian University,

ul. Ingardena 3, 30-060 Cracow, Poland

1 ACS Paragon Plus Environment


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Abstract Four mechanisms previously proposed for dioxygen activation catalyzed by α-keto acid dependent oxygenases (α-KAO) were studied with dispersion-corrected DFT methods employing B3LYP and TPSSh functionals in combination with triple-ζ basis set (cc-pVTZ). The aim of this study was to revisit mechanisms suggested in the last decade and resolve remaining issues related to dioxygen activation. The mechanism A, which runs on the quintet potential energy surface (PES) and includes formation of a Fe(III)-superoxide radical anion complex, subsequent oxidative decarboxylation and O-O bond cleavage, was found to be most likely. However, the mechanism B taking place on the septet PES involves a rate limiting barrier comparable to the one found for the mechanism A, and thus it cannot be excluded though two other mechanisms (C and D) were ruled out. The mechanism C is a minor variation of the mechanism A, whereas the mechanism D proceeds through formation of triplet Fe(IV)-alkyl peroxo bridged intermediate. The study covered also full optimization of relevant minimum energy crossing points (MECPs). The relative energy of critical intermediates was also studied with the CCSD(T) method in order to benchmark TPSSh and B3LYP functionals with respect to their credibility in predicting relative energies of septet and triplet spin states of the ternary enzyme-Fe-α-KG-O2 complex. Keywords: α-keto acid, dioxygenase, ferryl, density functional calculations, oxygen activation, coupled-cluster calculations


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1

Introduction

Oxygenases dependent on α-keto acids (α-KAO) represent a large family of biocatalyst involved in a broad range of biological processes, including regulation of gene expression,1–3 alkylated DNA repair,4, 5 cellular sensing of oxygen,6, 7 biosynthesis of collagen8 and antibiotics.9 α-KAO catalyze wide scope of chemical reactions on their specific substrates: including hydroxylation, desaturation, ring closure, epimerization, chlorination and demethylation.10–12 Based on the data obtained from crystal structures,13–19 it is known that these enzymes are characterized by a common double-stranded β-helix architecture motif called ’jellyrol’. α-KAO enzymes bind Fe(II) and require α-keto acid (usually α-keto glutarate) as a cosubstrate. In the resting state of α-KAO Fe(II) ion is bound in the catalytic center to the 2His-1Asp/1Glu facial triad motif and three solvent (H2 O) molecules. Steady-state kinetic studies showed that α-keto acid binds to Fe(II) prior to O2 and a specific substrate.13, 20–24

Figure 1: The key residues in the active site of taurine 2-oxoglutarate dioxygenase - a representative α-KAO, as revealed by the crystal structure PDB: 1OS7.



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Figure 2: Four mechanisms of O2 activation proposed in previous DFT studies.


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As revealed by results of spectroscopic and X-ray diffraction studies, α-keto glutarate (α-KG) is bound to ferrous center in the bidentate fashion replacing two water molecules; the geometry of the cofactor is best described as distorted octahedron.13–16, 18, 19, 25, 26 The carboxylic and keto groups of α-KG are bound to Fe(II) trans to histidine and glutamate or aspartate in the equatorial plane, whereas the vertical positions are occupied by water molecule and the second histidine (Fig. 1). Magnetic circular dichroism and resonance Raman spectroscopic studies on taurine 2-oxoglutarate dioxygenase (TauD) and clavaminic acid synthase (CAS) confirmed that the ferrous center is 6-coordinate at this stage of the catalytic cycle.13, 26–29 In the subsequent step, O2 and a specific substrate bind to the active site.13 Steady-state kinetic studies performed for prolyl hydroxylase indicate that oxygen binds prior to the specific substrate,13, 21, 22 whereas spectroscopic experiments devoted to TauD and CAS suggest that specific substrates bind to the enzyme before O2 .13, 26–30 Results of the latter studies showed that the process of substrate binding is most likely accompanied by displacement of the last water molecule from the Fe(II) first coordination shell, which yields a 5-coordinate ferrous center, i.e. the iron cofactor is prepared to bind O2 (Fig. 1).13 Dissociation of the water ligand upon binding of the specific substrate was confirmed by crystal structures solved for TauD and CAS in complexes with their specific substrates and α-KG (Fig. 1).14, 17 The vacant coordination site is located in the vertical position, trans to one of two histidines. The crystal structure solved for the CAS-αKG-NO complex (NO used as a surrogate of O2 )31 has provided indirect insight into the structure of α-KAO - molecular oxygen adduct; the NO ligand coordinates iron with its N-end and O atom is located above α-KG. In 1982 Hanauske-Abel and Gunzler, based on results obtained for prolyl-4-hydroxylase,32 suggested that the first few steps of the catalytic cycle include O2 activation, during which oxygen molecule reacts with Fe(II)-α-KG complex (in place of α-KG some enzymes, like hydroxymandelate synthase (HMS)33, 34 and 4-hydroxyphenylpyruvate dioxygenase (HPPD)33, 35 use other ketoacids). The oxidative decarboxylation of α-KG leads to formation of reactive Fe(IV)=O species (Fig. 2), that is responsible for the two-electron oxidation of the specific substrate.11, 36–39 In experimental studies on TauD, the Fe(IV)=O intermediate was trapped by rapid freezing and characterized with Raman and X-ray absorption spectroscopy.39–44 5 ACS Paragon Plus Environment


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Steady-state kinetic studies suggest that during turnover carbon dioxide is the first product released from the active site,22–24 however so far there is no experimental data for the reaction intermediates generated between O2 binding and formation of the Fe(IV)=O species. Therefore, insight into the mechanism of the oxygen activation by α-KAO were derived from computational studies, primarily employing DFT methods. However, different exchange-correlation functionals seem to favour different mechanisms for generation of ferryl species by α-KAO.33, 45–50 Based on the results obtained with DFT method four different mechanisms of oxygen activation were proposed (Fig. 2).50 In 2004, Borowski et al. performed DFT studies for CAS employing an active site model including Fe(II), first shell ligands and two residues: Tyr299, Arg297 from the second coordination shell.46 All geometry optimizations were performed with the B3LYP exchangecorrelation functional and double-ζ basis set (lacvp). The final energies combined electronic energies computed with triple-ζ basis set (lacv3p**), solvent and thermal corrections. The Gibbs free energy profiles obtained for different reaction pathways indicated that the most favourable mechanism of O2 activation proceeds through a quintet reaction channel, which is depicted as mechanism A in Fig. 2. In the mechanism A, formation of the 5 I1 intermediate (Fig. 2) is needed for subsequent decarboxylation and O-O bond heterolysis. In the next step, the superoxide radical anion coordinated to Fe(III) attacks α-keto carbon, which induces rate-limiting oxidative decarboxylation of α-KG and leads to Fe(II)-peracid intermediate in the ground quintet spin state (5 I2, Fig. 2). Subsequently, the O-O bond is heterolytically cleaved forming the experimentally observed quintet Fe(IV)=O intermediate.46, 50 In the same year (2004), Borowski et al. published results obtained with DFT/B3LYP method for O2 activation by HPPD, which does not require α-KG cosubstrate since the α-keto acid moiety is inbuilt in its specific substrate: 4-hydroxyphenylpyruvate (4-HPP).33 The cluster model included only the first shell metal ligands. Electronic energies were computed with the triple-ζ basis set (lacv3p**) and combined with the solvent corrections. Consistently with the results obtained for CAS,46 the main conclusions from this work was that quintet PES and formation of the quintet complex of high spin Fe(III) antiferromagnetically coupled with the superoxo radical anion is required for O2 activation according to the mechanism A (Fig. 2). However, formation of the septet O2 adduct and then con6 ACS Paragon Plus Environment

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certed decarboxylation and O-O bond cleavage followed by intersystem crossing had not been excluded as the barrier for the rate limiting process for septet was 8.2 kcal/mol higher than that for the quintet counterpart (without entropy contribution, which stabilizes loser septet TS) (mechanism B in Fig. 2). In a related work devoted to Fe[(hydrotris(3,5-diphenylpyrazol-1-yl)-(benzoylformate) biomimetic complex, which reproduces O2 activation catalyzed by α-KAO, it was found that O2 activation by this biomimetic complex occurs most efficiently through concerted decarboxylation and O-O heterolysis step on the septet PES leading to septet iron-oxo intermediate, which presumably decays to the lower lying quintet 5 I3 (mechanism B in Fig. 2).51 The computed enthalpy barrier of 10 kcal/mol is in reasonable agreement with the experimental data (6 kcal/mol). In 2006, Topol et al. published a report on the αKAO O2 activation for the active site models with different ions in the active site: Fe(II), Ni(II) and Co(II).47 The computational model was constructed based on the crystal structure of asparaginyl hydroxylase (PDB code: 1H2L) and consisted of: metal ion, side chains of His199, His279, Asp201, α-KG modelled by pyruvate and O2 . All stationary points were fully optimized with the DFT/B3LYP method employing modified LANL2DZdp basis set (diffuse functions only on oxygen atoms) and an appropriate pseudopotential on the metal. The relative electronic energies were computed with respect to the first minimum obtained on the quintet PES (5 I1, Fig. 2). Similar to previous works, reaction for the Fe-system was proposed to proceed mainly through the quintet reaction channel, but according to the mechanism C shown in Fig. 2. The 5 I1 → 5 I5 conversion (Fig. 2) was found as the rate limiting step with the transition state energy of 12.9 kcal/mol. Further decarboxylation of α-KG proceeds with a small barrier of 3.1 kcal/mol and leads to quintet Fe(II)-peracid intermediate (5 I2, Fig. 2), which is common for the the mechanisms A and C. Subsequent O-O bond cleavage yields ferryl species (5 I3, Fig. 2).47, 50 In 2007, de Visser published results for the TauD model obtained with the DFT/B3LYP method.48 As in the study by Topol et al., the model included Fe(II), side chains of His99, His255, Asp101, pyruvate replacing α-KG and O2 . All structures were fully optimized with the B3LYP/DFT method employing double-ζ basis set (lacvp). The electronic energies computed with the triple-ζ basis set (lacv3p+* for iron and 6-311G* on the other atoms) 7 ACS Paragon Plus Environment


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were combined with the Zero Point Energy correction (ZPE) and solvent correction computed with the dielectric constant of 5.7 and probe radius of 2.72 ˚ A. Similarly to the results obtained by Topol et al.,47 the mechanism C was found as the most efficient, formation of the bicyclic superoxo intermediate (5 I5) was found to be a rate limiting step and a small barrier (3.6 kcal/mol) was found for the decay of 5 I5 during the decarboxylation process. The mechanism D was suggested in 2011 by Diebold et al. In this work EPR, MCD and UV-vis absorption spectroscopic studies were combined with the DFT computations.45 The experimental part of the studies was devoted to HPPD-NO and HPPD-4-HPP-NO complexes, while computational studies were extended to encompass reactivity with O2 . The cluster model included first shell metal ligands and was constructed based on the crystal structure of CAS in complex with Fe(II), α-KG and NO (PDB code: 1GVG). All Stationary points were optimized employing a functional combining BP86 with 10% Hartree-Fock exchange. The basis set used was 6-311G* for: Fe, NO, O2 and αKG; and 6-31G* for the rest of atoms. Geometry optimizations were performed with constraints imposed on positions of the atoms corresponding to β carbons. Electronic energies were corrected by inclusion of the environment effects modelled by polarizable continuum with the dielectric constant of 4.0. For this particular and ad hoc proposed functional the Authors found best agreement between EPR data and the calculated electronic structure of NO-complexes, which was used as argument for applicability of this functional for study on the reaction mechanism. Based on the BP86 + 10% HF results, the O2 activation process was proposed to run according to the mechanism D (Fig. 2). In this reaction channel the triplet PES is involved. Moreover, the binding of oxygen was suggested to directly yield triplet Fe(IV)-alkyl peroxo bridged intermediate (3 I4, Fig. 2), which results from transfer of two electrons from iron to oxygen molecule. It was suggested that spin-crossover from triplet to the quintet PES initiates the following decarboxylation leading to quintet Fe(II)peracid species (5 I2, Fig. 2), common for the mechanisms A, C and D.45, 50 Intersystem crossing from the triplet to the quintet spin state is the rate-limiting step for the mechanism D, and it is supposed to take place when the bond between carboxyl and carbonyl carbons ˚ However, the mechanisms A-C were not tested with in α-KG is stretched to about 2.1 A. the proposed BP86 + 10% functional, and the critical crossing point between triplet and quintet PESs was located only approximately from two-dimensional relaxed scans. Hence, 8 ACS Paragon Plus Environment

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these aspects call for further testing before definite conclusions can be drawn. In 2012, Ye et al. studied O2 activation in α-KAO employing the cluster model based on the crystal structure of TauD (PDB code: 1GQW).49 The model included the first shell metal ligands: two histidines modelled by imidazoles, aspartate reduced to acetate, α-ketoglutarate replaced by propionate and O2 , and it was fully optimized with the use of the B3LYP functional in combination with triple-ζ basis set (TZVP) on Fe, N, O and SVP on the rest of atoms (RIJDX approximation).49 Additionally, for even smaller models of the active site, CASSCF and NEVPT2 ab initio computations were performed to compare the relative energies of O2 adducts with different multiplicities and CCSD(T) calculations were used to assess the energy of transition state for α-KG decarboxylation on the septet PES. Electronic energies were combined with the thermal corrections, zero-point energies and entropy terms obtained from calculated Hessians. The Authors of this study concluded that the O2 activation reaction may proceed either on the quintet (mechanism A) or the septet (mechanism B) PES (Fig. 2), whereas the mechanism D was found to be catalytically irrelevant due to high relative energy of the triplet bicyclic intermediate. Summarizing the results of the previous DFT studies, the mechanisms A, B and C were suggested based on B3LYP computations.33, 46, 49 On the other hand, BP86 + 10% HF results were apparently supportive for the mechanism D, yet neither other mechanisms were tested with this functional, nor proper minimum energy crossing point (MECP) was optimized and shown to connect the right minima.45, 50 In addition, applicability of the BP86 + 10% HF functional to study PESs is an open question. Therefore, in this work we have undertaken additional computational studies with the purpose to clarify remaining issues pertaining to dioxygen activation by α-KAO. Hence, two dispersion-corrected DFT methods, namely B3LYP-D3 and TPSSh-D3 (the latter contains 10% of HF exchange) were used in combination with reliable all-electron triple-ζ basis set cc-pVTZ to revisit the mechanisms that have been suggested in the last decade. Several MECPs were fully optimized and characterised by following IRC to the nearby minima on the two crossing surfaces. In addition, benchmark calculations of the relative spin-state energetics were carried out with the CCSD(T) method. The most important result of this study is that TPSSh, like BP86 + 10% HF, overstabilizes the triplet Fe(IV)-peroxo species and that the energy landscape including MECPs is 9 ACS Paragon Plus Environment


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shaped in such a way, that, irrespective of the functional used, the decarboxylation reaction proceeds through classical transition state and not MECP.

2

Models and Methods

An active site cluster model employed in the present study was constructed based on the crystal structure of taurine 2-oxoglutarate dioxygenase in complex with Fe(II) and α-keto glutarate (PDB code: 1OS7), where first shell metal ligands are arranged in unsaturated octahedral geometry with five-coordinated ferrous ion (Fig. 1). Our cluster model includes first shell metal ligands: two histidines (His255, His99), aspartate (Asp101), α-KG and the O2 molecule added in the originally vacant site trans to His255. Histidines were modelled by imidazoles, aspartate by acetate and α-KG was replaced by pyruvate (Fig. 3). Quantummechanical studies were performed with Gaussian 09 program.52 All calculations were carried out without constraints imposed on the atomic coordinates and reaction paths for three multiplicities were considered, i.e. septet, quintet and triplet. B3LYP and TPSSh hybrid exchange-correlation functionals with D3 Grimme’s dispersion correction52, 53 (for B3LYP with Becke-Johnson damping54 ) were used in combination with triple-ζ basis set (cc-pVTZ) for optimization of all intermediates, transition states and MECPs proposed to be involved in the mechanisms: A, B, C and D. Intermediates and transition states are labelled as

m

Xn, where X stands for either I (intermediate) or T (transition state)

and m stands for multiplicity. Electronic energies of intermediates and transition states were combined with the thermal correction to Gibbs free energy or enthalpy and solvent correction. The latter correction - describing effects due to the surrounding of the active site were modelled with macroscopic continuum PCM model with the dielectric constant of ˚ Minimum Energy Crossing Points (MECPs) were optimized 4.0 and probe radius of 1.4 A. with the use of a meta-program provided courtesy of J. Harvey.55 The energy barrier connected with the surface crossing was computed from electronic energies of optimized MECP and two electronic intermediates that a given MECP connects and combined with solvation corrections (no thermal corrections included). In addition, a series of constrained MECP optimizations was carried out at the B3LYP-D2/cc-pVDZ level with the C1-C2


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distance fixed (Fig. 3). For all stationary points optimized with the B3LYP-D3/cc-pVTZ method, the significance of the relativistic effects was assessed with the B3LYP-D3/cc-pVTZ-DK DouglasKroll-Hess second order scalar relativistic approximation implemented in Gaussian 09 program.52, 56–59 Comparison of DFT and coupled-cluster (CC) calculations was performed for simplified mimics of 7 I1’ and 3 I4. Structures of these mimics were constructed from the structure of the two intermediates optimized at the TPSSh/cc-pVTZ level by replacing both histidines with ammonia molecules and the Asp101 moiety with formate. The capping H atoms were A, the N–H distance of 1.008 ˚ A) added at standard positions (the C–H distance of 1.000 ˚ and not optimized any further. The CC calculations were carried out using Molpro60 at the RCCSD(T) level based on an ROHF reference.61, 62 Comparison with UCCSD(T) is provided in Supporting Information. Explicitly correlated RCCSD(T)-F12 calculations were also performed using the RCCSD-F12a approximation by Werner et al. scalled contribution to correlation energy due to the triples, (T*).63 Two types of basis sets were used in the CC calculations. The smaller bsI was composed of cc-pwCVTZ for Fe and cc-pVDZ for all other atoms. The larger bsII was composed of cc-pwCVTZ for Fe, cc-pVTZ for O2 moiety and all atoms in the first coordination sphere of Fe, and cc-pVDZ for all other atoms. In the relativistic calculations with bsI the appropriate DK-recontractions of the atomic basis sets (cc-pwCVTZ-DK, cc-pVDZ-DK)64, 65 were used. As in the recent work of Harvey et al.,66 the following auxiliary basis sets were used in the CCSD(T)-F12 calculations: aug-cc-pVnZ/MP2FIT67 as the density-fitting basis set; def2-TZVPP/JKFIT68 (for Fe) or aug-cc-pVnZ/JKFIT69 (for other elements) as the density fitting basis set for computation of the exchange and Fock operators; and def2-TZVPP/JKFIT (for Fe) or aug-cc-pVnZ/OPTRI70 (for other elements) as the resolution-of-identity basis set (in each case n being the same as for the orbital basis set of a given atom, i.e., either T or D).

3

Results and discussion

Four previously suggested mechanisms for dioxygen activation by α-KAO denoted as A, B, C and D (Fig. 2) were considered and potential energy surfaces for three spin states:



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triplet, quintet and septet were explored.33, 45–50 In all energy diagrams we present relative Gibbs free energies with respect to separately optimized geometries of reactants, i.e. O2 and the enzyme-Fe(II) complex.

3.1

O2 binding

The first step of O2 activation in α-KAO is binding of the triplet O2 molecule to the five coordinated Fe(II) center, which may lead to triplet, quintet or septet O2 adduct intermediate. The optimized B3LYP-D3 geometries of these intermediates: 3 I1, 5 I1, 7 I1 and 7 I1’ are presented in Fig. 3 and Fig. 4. The relative Gibbs free energies of the triplet, quintet and two septet O2 adducts are shown on the energy diagram in Fig. 5. Relative Gibbs free energies of O2 adducts differ significantly, 7 I1’, 5 I1 and 3 I1 are less stable then 7

I1 by 8.1, 9.5 and 0.5 kcal/mol, respectively. To a large extent, this is a consequence of a

(structurally) tighter binding of O2 to Fe in the case of 5 I1 and 7 I1’ compared with 3 I1 and 7 ˚ 3.29 ˚ I1; the Fe-O1 distances being 3.42 A, A for 3 I1 and 7 I1 but only 2.11 ˚ A, 2.21 ˚ A for 5

I1 and 7 I1’, respectively (see Fig. 5 and Table 1). Thus, the Fe-O2 fragment has a much

larger vibrational entropy in the case of 7 I1 and 3 I1 than in the case of 5 I1 and 7 I1’. Considering enthalpies (see Fig. S 1), 3 I1 and 7 I1 intermediates are by 1.3 kcal/mol more stable then 5 I1, which is in good agreement with results obtained by Ye et al.,49 who had obtained relative enthalpies equal to 2.6 kcal/mol, 3.1 kcal/mol and 3.9 kcal/mol for triplet, septet and quintet O2 adducts, respectively. For the septet spin state two O2 adducts have been found, which are characterized by nearly the same relative enthalpies (7 I1’ is 0.5 kcal/mol less stable than 7 I1). They might be described as Fe(II) in quintet spin state in complex with triplet O2 (7 I1) and Fe(III)7 O•− 2 ( I1’) and these complexes differ significantly in the length of the Fe-O1 bond: 3.29 ˚ A in 7 I1 and 2.21 ˚ A in 7 I1’ which also manifests in entropy difference, i.e. their Gibbs free

energies differ by 8.1 kcal/mol (Fig. 5). Hence, the O2 moiety is very loosely bound to the Fe center in 7 I1. The structure of the septet O2 adduct analogous to the present 7 I1’, with the Fe-O1 distance of 2.32 ˚ A, was previously found by Ye et al.49 Its electronic structure has been described as a resonance of two structures: one characterized by ferromagnetic coupling between high spin ferrous center (SF e =2) and triplet oxygen molecule (SO2 =1) and second including high spin Fe(III) ion ferromagnetically coupled with superoxide radical 12 ACS Paragon Plus Environment

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49 O•− 2 (SO2•− =1/2).

Figure 3: Structures of 3 I1, 5 I1 and of 7 I1 optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames.

Figure 4: Structure of 7 I1’ optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frame. In this work, the electronic structure of 3 I1 is best described as Fe(II) high spin center (SF e =2) antiferromagnetically coupled with triplet O2 , which is only loosely bound to the Fe(II) center. The O1-Fe distance is equal to 3.42 ˚ A. The structure of 5 I1 stays in a very good agreement with the previously published results. The atomic spin populations of 5 I1 indicate that high spin Fe(III) is antiferromag13 ACS Paragon Plus Environment


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Figure 5: Reaction energy profiles obtained with the B3LYP-D3/cc-pVTZ and TPSShD3/cc-pVTZ methods: in green for triplet, in red for quintet, while in blue for septet spin state (in parenthesis, in black - Gibbs free energies obtained with the B3LYP-D3/cc-pVTZDK method with the Douglas-Kroll-Hess approximation). netically coupled with the superoxo radical ligand, which is bound to ferric center by a coordination bond of 2.11 ˚ A length, see Fig. 3 (2.11 ˚ A in previous studies49 ). Inclusion of Douglas-Kroll-Hess corrections gave a decrease in the relative Gibbs free energies of 5 I1 and 7 I1’ by: 2.5 kcal/mol and 2.2 kcal/mol, respectively, whereas virtually no effect was observed for 3 I1 and 7 I1 (the DKH corrected ∆G values are given in parentheses in Fig. 5), i.e. the relativistic effects stabilize the quintet and septet Fe(III)-O•− 2 complexes. The relative Gibbs free energies computed for O2 adducts with the use of the TPSSh-D3 functional (Fig. 5, S 1 and Table 1) revealed that 3 I1 lies only 2.3 kcal/mol below 7 I1’, while 5 I1 is by 0.5 kcal/mol less stable then 7 I1’. Comparison of the relative enthalpies of O2 adducts obtained with TPSSh-D3 and B3LYP-D3 functionals (Fig. S 1) reveals that in contrast to the B3LYP-D3 results, according to the TPSSh-D3 method, the 5 I1 species (-7.1 kcal/mol) is more stable then 3 I1 (-2.1 kcal/mol) and has similar enthalpy to 7 I1’ (-7.4 14 ACS Paragon Plus Environment

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Table 1: Important interatomic distances (in ˚ A) for triplet, quintet and septet O2 adducts optimized with B3LYP-D3 and TPSSh-D3 functionals. 3

Bond Fe-O1 Fe-Asp101 Fe-His99 Fe-His255 Fe-O3 Fe-O4 O2-C1 C1-C2 O1-O2

B3LYP-D3 3.42 1.98 2.15 2.12 2.60 1.96 3.34 1.55 1.20

I1 TPSSh-D3 3.55 1.96 2.14 2.10 2.54 1.95 3.36 1.55 1.21

5

B3LYP-D3 2.11 1.89 2.15 2.15 2.14 1.96 2.15 1.54 1.29

I1 TPSSh-D3 2.14 1.88 2.15 2.13 2.08 1.95 1.98 1.54 1.29

7

B3LYP-D3 3.29 1.97 2.15 2.12 2.57 1.96 3.24 1.55 1.21

I1 TPSSh-D3 -

7

B3LYP-D3 2.21 1.89 2.14 2.14 2.13 1.96 2.18 1.54 1.28

I1’ TPSSh-D3 2.23 1.89 2.14 2.12 2.06 1.95 1.93 1.54 1.29

kcal/mol). The geometries of the oxygen adducts are similar to those obtained with the B3LYP-D3 functional. Interestingly enough, using the TPSSh-D3 functional in the septet spin state only 7 I1’ gave a stable energy minimum, with Fe-O1 distance of 2.23 ˚ A, whreas 7 I1 could not be obtained. The most important interatomic distances for the considered O2 adducts are reported in Table 1.

3.2

Mechanism A

The mechanism A is suggested to proceed only through the quintet reaction channel (Fig. 2). The first step of this pathway is decarboxylation of the Fe(III)-superoxide radical complex (5 I1), which is the rate limiting process of the mechanism A connected with Gibbs free energy accumulated barrier of 21.3 kcal/mol (B3LYP-D3); with the inclusion of the relativistic effects the barrier drops to 18.8 kcal/mol (see Fig. 5). The enthalpy barrier equals to 5.3 kcal/mol (Fig. S 1). The geometry of the transition state 5 T1 is presented in Fig. 6. Table 2: Important interatomic distances (in ˚ A) for 5 T1, 5 I2, 5 T2 and 5 I3. Bond Fe-O1 Fe-Asp101 Fe-His99 Fe-His255 Fe-O3 Fe-O4 O2-C1 C1-C2 O1-O2

5 T1 B3LYP-D3 TPSSh-D3 2.05 2.05 1.89 1.80 2.15 2.17 2.15 2.15 2.04 2.04 2.07 2.05 1.46 1.48 1.71 1.67 1.36 1.36

5

B3LYP-D3 1.95 1.98 2.16 2.13 2.38 3.73 1.31 3.69 1.44

I2 TPSSh-D3 1.92 1.96 2.13 2.11 2.33 3.74 1.31 3.78 1.47

5 T2 B3LYP-D3 TPSSh-D3 1.80 1.81 1.96 1.95 2.13 2.12 2.12 2.12 2.25 2.25 3.69 3.75 1.28 1.29 4.26 3.87 1.70 1.65


5

B3LYP-D3 1.62 2.07 2.08 2.12 1.93 3.92 1.22 4.00 3.21

I3 TPSSh-D3 1.63 2.06 2.14 2.09 1.92 3.98 1.22 4.03 3.15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6: Structures of the transition state for decarboxylation on the quintet PES (5 T1) and product of this reaction (5 I2) optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames.

Figure 7: Structures of the transition state for O-O bond cleavage (5 T2) and Fe(IV)=O complex - product of this reaction (5 I3) optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames. In 5 T1 the C1-C2 bond is elongated from 1.54 ˚ A (in 5 I1) to 1.71 ˚ A and the C1-O2 distance equals to 1.46 ˚ A (in the reactant 5 I1: 2.15 ˚ A), which agrees well with the corre16 ACS Paragon Plus Environment

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sponding distances obtained in the previous studies: 1.72-1.74 ˚ A for C1-C2 and 1.46-1.51 ˚ A for C1-O2.33, 46, 49 The experimentally determined Gibbs free energy barriers for the rate limiting decarboxylation step amount to 15.2 kcal/mol (rate constant of 42±9 s−1 ) for the enzyme-Fe(II)αKG-taurine-O2 complex30 and 18.8 kcal/mol for a biomimetic model system.71 For the model system a large contribution to the experimental barrier comes from the entropy term (-T∆S = 12.8 kcal/mol, determined experimentally), while the enthalpy of activation equals to 6.0 kcal/mol, which is in a good agreement with the present result: ∆H‡ = 5.3 kcal/mol. However, when the entropic effects are included, which in the present study amounts to 17.0 kcal/mol, the Gibbs free energy barrier of 21.3 kcal/mol (B3LYP-D3) is higher then the experimental value, which might be a consequence of inadequacy of small cluster model for estimation of free energy effects connected with binding of O2 to the enzyme. Moreover, the estimates of entropic effects accompanying the results of quantum chemical calculations are based on rather crude assumptions (e.g., harmonic oscillations, rigid rotations, ideal gas approximation). Similarly to the results obtained for the B3LYP-D3 functional, TPSSh-D3 gave the mechanism A as the most favourable (Fig. 5), yet the Gibbs free energy barrier is only 11.4 kcal/mol. The most important interatomic distances for critical points defining the mechanism A are reported in Table 2.

3.3

Mechanism B

The mechanism B starts with the septet O2 adduct (Fig. 3, 4), and as found previously, might proceed via decarboxylation coupled with O1-O2 bond cleavage, which yields the septet Fe(III)-O•− intermediate (7 I3, Fig. 2).33, 51

7

I3 is supposed to decay to 5 I3 via a

minimum energy crossing point (7−5 M4, see Fig. 2). In the present studies a two step reaction was found, where first step includes attack of O2 on the carbonyl carbon and C1-C2 bond cleavage and the second step is O1-O2 bond cleavage. The decarboxylation process running through 7 T1 is the rate limiting step with the Gibbs free energy barrier of 21.6 kcal/mol (19.1 kcal/mol with the relativistic effects, Fig. 5). The C1-C2 and C1-O2 distances equal to 2.46 and 1.39 ˚ A, respectively, and hence 7

T1 can be classified as a late transition state. The product (7 I2) is an unstable peracid 17 ACS Paragon Plus Environment


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Figure 8: Structures of the transition state for decarboxylation on the septet PES (7 T1) and product of this reaction (7 I2) optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames.

Figure 9: Structures of the transition state for O-O bond cleavage (7 T2), Fe(III)-O• complex - product of this reaction (7 I3) and minimum energy crossing point (7−5 M4) for septet (7 I3) and quintet (5 I3) potential energy surfaces optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames. intermediate (Fig. 2 and 8), featuring an unpaired electron on carbon C1. The next step is O1-O2 bond cleavage which is connected with a tiny barrier of 0.5 kcal/mol (0.5 kcal/mol when relativistic effects are included). The critical O1-O2 distance elongates from 1.44 ˚ A in 7 I2 to 1.51 ˚ A in 7 T2 (Fig. 8 and 9). Taking into account the fact that 7 I2 lies in a very 18 ACS Paragon Plus Environment

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Table 3: Important interatomic distances (in ˚ A) for 7 T1, 7 I2, 7 T2, 7 I3 and 7


T1 B3LYP-D3 TPSSh-D3 1.89 1.88 1.91 1.91 2.10 2.10 2.09 2.09 1.99 1.98 3.71 3.81 1.39 1.40 2.46 2.57 1.42 1.43

7

I2 B3LYP-D3 TPSSh-D3 1.90 1.88 1.92 1.92 2.11 2.11 2.09 2.09 1.98 1.97 3.65 3.88 1.40 1.41 3.82 3.19 1.44 1.44

7

T2 B3LYP-D3 TPSSh-D3 1.89 1.89 1.92 1.92 2.11 2.11 2.09 2.09 2.00 1.99 3.65 3.72 1.36 1.36 3.70 3.72 1.51 1.51

7

I3 B3LYP-D3 TPSSh-D3 1.92 1.92 1.91 1.91 2.22 2.21 2.13 2.12 2.35 2.28 4.08 4.11 1.28 1.28 3.41 3.45 2.82 2.80

7−5

M4

7−5

M4 B3LYP-D3 TPSSh-D3 1.86 1.91 1.91 2.03 2.21 2.12 2.12 2.22 2.35 1.91 4.05 4.10 1.28 1.23 6.03 3.48 2.79 2.81

shallow minimum it seems fairly safe to conclude that the two steps: decarboxylation and O1-O2 bond cleavage are effectively coupled but asynchronous on the septet PES, which is in line with the results of the previous studies.33, 51 The final step of the mechanism B is the intersystem crossing proceeding through the minimum energy crossing point (Fig. 9). The energy cost of reaching

7−5

7−5

M4

M4 is only 0.8 kcal/mol, 0.4 kcal/mol with the

relativistic effects, using the B3LYP-D3 functional (Fig. 2). The reaction energy profile and geometries of stationary points computed with the TPSSh-D3 functional are similar to those obtained with the B3LYP-D3 method (see Fig. 2 and Table 3). Despite, that the results obtained with both methods unanimously indicate that the mechanism A is more probable than the mechanism B, the relative Gibbs free energies of the rate limiting steps of these channels (5 T1 and 7 T1) differ only by 0.3 kcal/mol and 4.00 kcal/mol for B3LYP-D3 and TPSSh-D3 functionals, respectively (Fig. 5). Since this difference is smaller than the expected uncertainty of the applied methodology, the mechanism B cannot be excluded.

3.4

Mechanism C

The mechanism C is a slight variation of the mechanism A, where the nucleophilic attack of the superoxide on the carbonyl carbon and decarboxylation are decoupled from each other. The 5 I5 intermediate is supposed to intervene between these two steps.47, 48 In the present work, 5 I5 has been found and characterized as an intermediate spin Fe(III) complex with α-KG directly bound to the superoxide radical anion (Fig. 10 and Table 4). ˚ and 1.62 ˚ The Fe-O1 and O2-C1 distances equal to 2.47 A A, respectively. The variant of this intermediate containing high spin Fe(III) has also been sought. However, it turned out that if the ferric ion is in the high spin state, superoxide spontaneously detaches from α-KG 19 ACS Paragon Plus Environment


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Figure 10: Structure of 5 I5 optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frame. Table 4: Important interatomic distances (in ˚ A) for 5 I5. 5


B3LYP-D3 2.47 1.87 2.01 2.15 1.89 1.88 1.62 1.54 1.30

I5 TPSSh-D3 2.42 1.86 2.01 2.16 1.88 1.88 1.61 1.54 1.31

and binds to iron ion. Importantly, 5 I5 is by 4.0 kcal/mol less stable (3.6 kcal/mol with the relativistic corrections, Fig. 5) than transition state connected with the rate limiting step of the mechanism A (5 T1) and 3.7 kcal/mol (3.3 kcal/mol with relativistic effects included) compared to 7 T1 from the mechanism B. Also without entropic contributions the mechanisms A and B are preferred over the mechanism C (Fig. S 1). We have also tried to obtain a septet version of 5 I5, yet such species is unstable on the septet PES and all attempts of its optimization ended up in 7 I1’. Hence, the mechanism C is considered to be less probable than the mechanisms A and B. Results obtained with the use of TPSSh-D3 functional gave the Gibbs free energy of 5 I5 by 2.9 kcal/mol higher than transition state connected with decarboxylation on the quintet


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PES, i.e. 5 T1, however, 1.1 kcal/mol below the decisive transition state of the mechanism B, i.e. 7 T1 (Fig. 5). Most important interatomic distances for 5 I5 are reported in Table 4.

3.5

Mechanism D

In the mechanism D (Fig. 2), the first O2 binding step occurs on the triplet PES, whereas decarboxylation is accomplished via intersystem crossing, which leads to the quintet Fe(II)peracid intermediate 5 I2. It was suggested that O2 binding yields triplet Fe(IV) - alkyl peroxo bridged intermediate (3 I4, Fig. 11).45 In the present work, 3 I4 was optimized with the use of B3LYP-D3 and TPSSh-D3 functionals and the resulting structures and their stabilities are significantly different (see Fig. 5). With B3LYP-D3 this triplet intermediate lies 24.3 kcal/mol (20.3 with relativistic effects) above the parent triplet intermediate 3 I1, while with TPSSh-D3 3 I4 is by 0.2 kcal/mol less stable than 3 I1. Thus, the difference exceeds 20 kcal/mol meaning that this aspect of the energy profile is described qualitatively different by the two functionals (B3LYP, TPSSh) and hence it remains somewhat uncertain. Exclusion of the entropic effects leads to 3 I4 being by 11.5 kcal/mol (7.5 kcal/mol with relativistic effects) less stable than 3 I1 with B3LYP-D3 and by 13.0 kcal/mol more stable than 3 I1 with TPSSh-D3 (Fig. S 1). Mechanistic significance of 3 I4 was proposed based on studies performed with the BP86 + 10% HF functional, which has the same amount of HF exchange as TPSSh. The issue of relative stability of 3 I4 with respect to the entrance complex 7 I1 was tackled with the use of coupled-cluster calculations, whose results are described in section 3.6. In short, the CCSD(T) energies indicate that TPSSh substantially overstabilizes the 3 I4 species, which with this post-HF method is predicted to be less stable than the septet entrance complex 7

I1’. According to the mechanism D, once 3 I4 is formed it undergoes an intersystem crossing

to the quintet spin state, which is supposed to be coupled with decarboxylation yielding the Fe(II)-peracid intermediate 5 I2. However, unconstrained optimization of minimum energy crossing point (MECP) in the vicinity of 3 I4 yields a MECP (3−5 M1) that connects, as unambiguously demonstrated by following IRC on both PESs, 3 I4 with 5 I1, i.e. surpassing 21 ACS Paragon Plus Environment


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Figure 11: Structures of the triplet Fe(IV) - alkyl peroxo bridged intermediate (3 I4), transition state for decarboxylation on the triplet PES (3 T1) and product of this reaction (3 I2) optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames.

Figure 12: Structures of the minimum energy crossing points joining triplet and quintet potential energy surfaces: 3−5 M1 and 3−5 M2 optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frames. this MECP is not coupled with decarboxylation (see Fig. 5). With B3LYP-D3 reaching 3−5

M1 from 3 I4 costs 1.9 kcal/mol (5.1 kcal/mol with the inclusion of the relativistic effects,

Fig. 5, Table 6) and hence this process is predicted to be faster than decarboxylation on the triplet PES, which requires an additional energy of 2.5 kcal/mol (5.6 kcal/mol on the enthalpy profile, Fig. S 1) and 4.6 kcal/mol on the triplet electronic energy surface including only solvent corrections. 22 ACS Paragon Plus Environment

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Table 5: Important interatomic distances (in ˚ A) for 3 I4, 3 T1, 3 I2, 3 T2 and 3 I3. 3


B3LYP-D3 1.88 1.88 2.00 1.98 1.87 1.92 1.53 1.56 1.34

I4 TPSSh-D3 1.80 1.87 2.00 2.00 1.84 1.88 1.47 1.55 1.41

3 T1 B3LYP-D3 TPSSh-D3 1.84 1.82 1.89 1.88 2.01 1.99 2.02 2.01 1.99 1.96 1.94 1.93 1.31 1.31 2.69 2.64 1.46 1.47

3

B3LYP-D3 1.89 1.96 2.28 2.01 1.98 3.49 1.30 3.86 1.47

I2 TPSSh-D3 1.84 1.96 2.27 1.99 1.95 3.59 1.30 4.04 1.50

3 T2 B3LYP-D3 TPSSh-D3 1.80 1.96 2.27 2.00 1.96 3.59 1.29 3.98 1.55

Table 6: Important interatomic distances (in ˚ A) for 3−5


M1 B3LYP-D3 TPSSh-D3 2.00 2.07 1.87 1.89 2.05 2.09 2.04 2.09 1.98 1.99 1.94 1.94 2.08 1.88 1.54 1.54 1.29 1.30

3−5

M2 B3LYP-D3 TPSSh-D3 1.87 1.96 2.31 2.01 1.98 3.47 1.30 3.95 1.47 -

3−5

M1,

3−5

3

B3LYP-D3 -

M2 and

I3 TPSSh-D3 1.59 1.91 1.91 2.11 1.93 3.11 1.23 3.65 3.22

3−5

M3.

3−5

M3 B3LYP-D3 TPSSh-D3 1.71 1.94 2.15 2.10 2.03 3.93 1.23 3.87 2.25

For TPSSh-D3 the barrier of the intersystem crossing via 3−5 M1 is 13.3 kcal/mol, which is higher than the barrier obtained for decarboxylation on the triplet PES, amounting to 9.6 kcal/mol (3 T1, Fig. 5). Thus the TPSSh-D3 results suggest that MECP might take place after decarboxylation process. Without entropic effects there is no difference between enthalpies of 3−5

3−5

M1 and 3 T1 (Fig. S 1) and with the exclusion of all thermal corrections

M1 lies 1.3 kcal/mol below 3 T1. In the vicinity of the minimum for Fe(II)-peracid species (I2) another MECP (3−5 M2)

was found with B3LYP-D3, and it connects 3 I2 with 5 I2, i.e. no change in bonding takes place during this intersystem crossing. Due to instability of 3 I2 with the TPSSh-D3 functional (see Fig. 5), for this level of approximation attempts to optimize it ended up with

3−5

3−5

M2 most likely does not exist (all

M3), see Fig. 5. The critical distances for these

MECPs are reported in Table 6. In an attempt to directly verify the hypothesis of Diebold et al., the approximate MECP that connects 3 I4 with 5 I2, i.e. where intersystem crossing elicits decarboxylation, was sought. To this end, during the MECP optimization the C1-C2 distance had to be fixed at the length found for 5 T1, while all other coordinates were fully optimized. However, this particular ”MECP” (3−5 M5) lies higher than

3−5

M1 and

3−5

M2, as well as 5 T1, irrespec-



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tive of the functional used and thermal correction added to 5 T1 (Fig. 5 and Fig. S 1). Hence, from the architecture of the PESs it follows that the decarboxylation step is not connected with an intersystem crossing, which is beneficial for the efficiency of the process, as the probability of surface hopping at MECP is always lower than one. The optimized triplet-quintet ”MECP” (3−5 M5) is presented in Fig. 13. 3−5 M5, compared with 5 T1, is characterized by a slightly shorter Fe-O1 distance (1.95 ˚ A vs 2.05 ˚ A), shorter coordination bonds between Fe and His99 (2.06 ˚ A vs 2.15 ˚ A), His255 (2.05 ˚ A vs 2.15 ˚ A), O3 (1.93 ˚ A vs 2.04 ˚ A) and O4 (2.00 ˚ A vs 2.07 ˚ A). The important interatomic distances for

3−5

M5 are

reported in Table 7. Additional calculations performed for triplet-quintet spin crossover, which justify the procedure used to optimize 3−5 M5, are presented in the following section.

Figure 13: Structure of the 3−5 M5 optimized with the B3LYP-D3/cc-pVTZ method. Relevant spin populations are reported in frame.

Table 7: Important interatomic distances for distance [˚ A] / method Fe-O1 Fe-Asp101 Fe-His99 Fe-His255 Fe-O3 Fe-O4 O2-C1 C1-C2 O1-O2

5

M5 B3LYP-D3 TPSSh-D3 1.95 1.97 1.89 1.88 2.06 2.08 2.05 2.10 1.93 1.97 2.00 2.01 1.44 1.47 1.71 1.67 1.37 1.37


3−5

M5.

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Figure 14: Energy profiles obtained from relaxed energy scans: in blue - MECP optimized with constraints imposed on the C1-C2 distance, in green and red energies for triplet and quintet states, respectively, optimized with the C1-C2 bond fixed at a given length. For three MECP geometries IRC (intrinsic reaction coordinate) calculations were performed for triplet and quintet multiplicities, the directions in which the IRC calculations evolved are denoted by arrows: green for triplet and red for quintet. Insight into the triplet → quintet intersystem crossing In order to search for a possible region of PES, where intersystem crossing could be coupled to decarboxylation, an array of MECP optimizations for a set of C1-C2 fixed distances was performed (Fig. 14). To reduce computational burden, the scan was performed with the use of double-ζ basis set (cc-pVDZ). The results are presented in Fig. 14, where the relative energies are calculated with respect to the electronic energy of fully optimized 5 T1 with the use of cc-pVDZ basis set. To find triplet-quintet MECP, which is coupled to decarboxylation and lead directly from triplet bicyclic peroxo structure (3 I4) to quintet Fe(II)-peracid species (5 I2), we chose the C1-C2 distance window spanning from 1.53 to 1.93 ˚ A (1.72 ˚ A for 5 T1 optimized with cc-pVDZ and 1.71 ˚ A for 5 T1 optimized with cc-pVTZ). In order to verify conclusions from these 1-dimensional scans, we have also performed intrinsic reaction coordinate calculations 25 ACS Paragon Plus Environment


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Table 8: Relative Energy for Small Mimics of 7 I1’ and 3 I4 from CCSD(T) and DFT Calculations. E(3 I4) − E(7 I1’) (kcal/mol) CCSD(T) / bsIa DKH CCSD(T) / bsIa NR CCSD(T)-F12b / bsIIa NR CCSD(T) best estimatec B3LYP/cc-pVTZ-DK DKH TPSSh/cc-pVTZ-DK DKH

6.7 8.8 4.2 2.1 3.6 −12.3

a

See Models and Methods section for definition of basis set bsI and bsII. b Approximation F12a. c CCSD(T)-F12 / bsII NR + relativistic correction. The correction is estimated as the difference between the DKH and NR results at the CCSD(T) / bsI level.

starting from these ”MECPs” optimized with constraints imposed on the C1-C2 distance. The directions in which the IRC downward trajectories went are marked in Fig. 5 and they are in agreement with the shapes of energy profiles obtained for triplet and quintet PESs (see Fig. 14). Hence, ”MECP” with C1-C2 distance of 1.63 ˚ A connects 3 I4 with 5 I1, whereas ”MECPs” with C1-C2 of 1.73 and 1.83 ˚ A connect 3 I4 with 5 I2. However, the first ”MECP” found that is coupled with decarboxylation (for C1-C2 of 1.73 ˚ A) is located in the transition state region for quintet decarboxylation (5 T1) and due to the lower stability of triplet species such ”MECP” does not offer any advantage over the quintet reaction pathway. Notably, even if the order of triplet and quintet PESs were reversed, such ”MECP” would also most likely lie in the same (5 T1) region, and hence the mechanism D still would not offer any advantage, at least not for the critical decarboxylation step.

3.6

Benchmark CCSD(T) calculations

To resolve the issue of relative stability of 7 I1’ and 3 I4 (for which we found in the preceding section that B3LYP-D3 and TPSSh-D3 methods provide qualitatively different results) we performed comparative CCSD(T) and DFT calculations for small mimics of these intermediates. The results of benchmark calculations are summarized in Table 8. In order to minimize basis set error in CCSD(T) calculations, we applied the explicitly correlated (F12)


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approach.72, 73 However, since the CCSD(T)-F12 method is presently implemented only for non-relativistic (NR) Hamiltonian, a correction for scalar relativistic effects was estimated from ordinary CCSD(T) calculations using either NR or Douglas-Kroll-Hess (DKH) Hamiltonian and a smaller basis set. (As shown in Table 8, the relativistic correction amount to ∼ 2 kcal/mol in favour of the triplet state, which is consistent with the results of DFT calculations for full model presented above.) The resulting “best estimate” of the 7 I1’ – 3

I4 splitting from the CCSD(T) calculations is thus 2.1 kcal/mol, with the septet lying

below the triplet state. This is not far from the B3LYP splitting 3.6 kcal/mol obtained for the same small models (cf Table 8). In contrast, at the TPSSh level the same splitting is −12.3 kcal/mol, with the triplet lying below the septet state. A comparable splitting is observed with the BP86+10% HF functional. These results are contradictory to the CCSD(T) benchmark and point to a conclusion that, in this case, the TPSSh functional noticeably overstabilize the triplet PES as compared with the septet PES (at least for this initial step of dioxygen activation). We note that although 5 I1 has not been studied in this benchmark (to avoid issues with spin contamination in CCSD(T) calculations) the above results are also relevant for relative stability of the quintet and triplet species. This is because the 5 I1 and 7 I1’ species are predicted to be nearly degenerate using both B3LYP-D3 and TPSSh-D3 levels of theory (cf Fig. 5), and this is easily explained by the electronic structure of these species (antiferromagnetic / ferromagnetic coupling in 5 I1 / 7 I1’; see above). Other functionals behave consistently with the typical trends established in the literature: i.e., nonhybrid BP86 point to a very strong overstabilization of the low-spin state, whereas OLYP and B3LYP* provide the energetics in between TPSSh and B3LYP. We also note that the present finding should not be viewed as an argument against the usage of TPSSh in general. For instance, TPSSh was previously found to provide reliable spin gaps for a number of spin-crossover complexes.74 It is also known that functionals containing a small amount of exact exchange (like 10% in TPSSh) or even no exact exchange (non-hybrid functionals) may be necessary to reproduce the spin density distributions and related spectroscopic properties for nitrosyliron complexes45, 75 where the metal–ligand bond is very covalent. However, the correct choice of functional may be system- and propertydependent.76 In the particular case studied above the relative stability of the septet and triplet intermediates seems to be more correctly described by B3LYP (20% exact exchange) 27 ACS Paragon Plus Environment


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than by TPSSh (10% exact exchange). Hence, the present benchmark argues against the plausability of the mechanism D because it suggests that the bicyclic intermediate 3 I4 is already too unstable to make the triplet pathway efficient; this is in qualitative agreement with the B3LYP-D3 reaction profile (cf Fig. 5). However, we note that even according to the TPSSh-D3 energy profile, where 3

I4 is strongly favoured (and possibly overstabilized), the high energy barrier to surpass on

the triplet PES makes the mechanism D ineffective compared with A and B (see previous section).

4

Conclusions

In this study four different mechanisms of O2 activation by α-KAO enzymes proposed in the literature by different authors have been comparatively studied using a single, consistent methodology, employing two different dispersion-corrected DFT methods (B3LYP-D3, TPSSh-D3). Taking together the results obtained in this study, it is the mechanism A, which was found to be most likely responsible for oxidative decarboxylation of α-KG in the catalytic cycle of α-KAO. However, the mechanism B with the rate limiting step lying only 0.3 kcal/mol higher than 5 T1 on Gibbs free energy profile cannot be excluded. The mechanism A is a two steps process characterised by rate limiting decarboxylation occurring with the Gibbs free energy barrier of 21.3 kcal/mol and subsequent O1-O2 bond cleavage (∆G‡ = 4.0 kcal/mol) leading to experimentally observed Fe(IV)=O intermediate (Fig. 5). The mechanism B consists of three steps: rate limiting decarboxylation (∆G‡ = 21.6 kcal/mol), O1-O2 bond cleavage (∆G‡ = 0.5 kcal/mol) and final intersystem crossing leading to Fe(IV)=O quintet intermediate (0.8 kcal/mol). Although a large difference between the two functionals used was found for the relative stability of quintet and septet vs triplet states of the Fe-α-KG-O2 adduct, the CCSD(T) benchmark calculations support the B3LYP-D3 spin-state energetics. Moreover, in regard to the feasibility of the mechanism D, it was found that previously proposed triplet-quintet crossing point that would couple intersystem crossing to decarboxylation is not a rigorous MECP and it lies above the quintet transition state for decarboxylation (5 T1). Therefore,


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the mechanism involving such a crossing is unlike.



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Acknowledgements This research was supported in part by PL-Grid Infrastructure. Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. This research project was supported by grant No UMO-2014/14/E/NZ1/00053 from the National Science Center, Poland. MR acknowledges the deparmental funds of Jagiellonian University and scholarship for outstanding young scientists from the Ministry of Science and Higher Education, Poland. Supporting Information Available: Enthalpy profiles obtained with the B3LYP-D3/ccpVTZ and TPSSh-D3/cc-pVTZ methods. Figures presenting the geometries fully optimized with the TPSSh-D3/cc-pVTZ method. Table including diagnostics of multireference character from CCSD(T) calculations and comparison between RCCSD(T) (the Default) and UCCSD(T) energetics. Coordinates of stationary points obtained with the B3LYP-D3/ccpVTZ and TPSSh-D3/cc-pVTZ methods.

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Mechanism of O2 Activation by Î±-Ketoglutarate Dependent

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