Metalloenzymes play a vital role in metabolism, in particular, they are capable to functionalizing unactivated C-H bonds employing the “green” oxidant dioxygen. These processes are remarkable because these transformations proceed with high efficiency and stereo specificity and at ambient conditions. Understanding the catalytic mechanisms of enzymatic reactions at the atomic level will enhance our knowledge for further design and synthesis of novel low-molecular weight “green” catalysts. Quantum chemical approaches provide key contributions to this important field of investigation: (1) Calculation of the spectroscopic parameters of potential reaction intermediates and comparison with experimentally determined properties is vital for the structural identification of short lived species that are inaccessible to X-ray crystallography; (2) Optimization of the structures of transition states in order to “connect” these intermediates and to predict reaction rates and kinetic isotope effects; (3) Qualitative analysis of the electronic structures of the intermediates and transition states provides deep chemical insights into catalytic mechanisms, (4) The combined analysis triggers new ideas for conclusive experiments that probe the intricate details of the catalytic mechanisms.

Oxygen activation
The Fe^II- and a-ketoglutarate (aKG)-dependent dioxygenases constitute the largest class of mononuclear non-heme iron enzymes and catalyze a broad range of pivotal metabolic transformations. The most studied and prototypical member of aKG-dependent enzyme family is taurine (2-aminoethane-1-sulfonic acid)/aKG dioxygenase (TauD), which enables E. coli to use the aliphatic sulfonate taurine as a sulfur source during periods of sulfate starvation.
The proposed mechanism for Fe^II- and aKG-dependent dioxygenases (Figure 1) involves (1) addition of dioxygen to the square pyramidal quaternary enzyme:Fe(II):aKG:substrate complex to yield I, (2) attack of the uncoordinated O-atom in the O₂-moity on C2 of a KG to form the bicyclic intermediate II, (3) cleavage of the O-O bond and decarboxylation resulting in the Fe(IV)-oxo species III, (4) abstraction of an H-atom from the substrate to yield an Fe^III-hydroxide complex and a substrate radical, (5) hydroxylation via OH rebound, and (6) dissociation of the product.

Figure 1. Proposed consensus mechanism of the Fe^II- and KG-dependent dioxygenases.

Application of a combination of rapid kinetic and spectroscopic techniques to the reaction of TauD resulted in the detection of an intermediate termed J that is kinetically competent for C-H bond activation. Experimental findings suggested that it is best formulated as a high-valent Fe^IV species with an unusual HS (S = 2) configuration and that it features a very short iron ligand interaction (1.62 Å) that is typical for the Fe^IV-oxo group. Whereas the results provided definitive proof for the presence of the ferryl unit in J, they could not reveal other important structural features, such as the number, identity, and disposition of ligands in the Fe(IV) coordination sphere. To probe these important structural features, a series of models for J have been evaluated by density functional theory (DFT) calculations. Calculations of spectroscopic parameters (Mössbauer isomer shift, quadrupole splitting, and asymmetry parameter, ⁵⁷Fe hyperfine coupling tensor, and zero field splitting (ZFS) parameters, D and E/D) allowed us to differentiate various models. The calculated parameters of distorted octahedral models for J, in which one of the carboxylates serves as a monodentate ligand and the other as a bidentate ligand, and a trigonal bipyramidal model, in which both carboxylates serve as monodentate ligands, agree well with the experimental parameters, whereas the calculated parameters of a square pyramidal model, in which the oxo ligand is in the equatorial plane, are inconsistent with the data.

Table 1. Comparison of calculated spectroscopic parameters of different models for J with experiment.
The reaction mechanism of the oxygen activation by Fe^II- and aKG-dependent non-heme enzymes was investigated by DFT and high-level ab initio methods (Figure 2). The reaction may take place either on the septet or on the quintet surfaces with the same rate-limiting steps, whereas the triplet reaction channel is catalytically irrelevant. The rate-limiting step was calculated to be the nucleophilic attack of the distal O-atom of the O₂ adduct on the carbonyl group in aKG via a bicyclic transition state (^5,7TS1). Due to the opposite spin coupling between the HS ferric center and the aKG s-radical in ^5,7TS1 (Figure 3), the decay of ⁷TS1 leads to a ferric oxyl species, which undergoes a rapid intersystem crossing to form the experimentally observed ferryl intermediate. By contrast, a HS ferrous center ligated by a peroxosuccinate is obtained on the quintet surface following the rate-determining step. Thus, additional two single-electron transfer steps are required to afford the same Fe^IV-oxo species.

	Exptl.	Distorted octahedron	Trigonal bipyramid	Square pyramid
d	0.30	0.27	0.27	0.17
?EQ	–0.90	–0.65	–0.82	–0.77

Figure 2. The calculated potential energy profile for the oxygen activation by the Fe^II- and aKG dependent dioxygenases with the B3LYP density functional (I, II, III indicate the proposed intermediates in Figure 1).

Figure 3. Schematic spin-allowed electron transfer pathways for the decay of ^7,5TS1.

The biological role of aKG played in the oxygen activation reaction is dual. The aKG LUMO (C=O p*) serves as an electron acceptor for the nucleophilic attack of the superoxide monoanion. This process lowers the reduction potential of the O₂ motif, and hence speeds up the second electron reduction of O₂. On the other hand, the nucleophilic attack on the carbonyl group decreases the oxidation potential of aKG, which makes aKG as a potential electron donor at the active site of enzymes. The KG HOMO (C1-C2 s) provides the second and third electrons for the further reduction of the superoxide.
According to the computed mechanism (Figure 2), the intermediates that can be trapped experimentally other than the ferryl species is the initial O₂ adduct, which is a {FeO₂}⁸ species. However, this intermediate of the aKG-dependent non-heme iron enzymes have not been trapped and characterized; thus, the corresponding NO–adduct, which is TauD-{FeNO}⁸ species, has been prepared recently by low-temperature g-irradiation of TauD-{FeNO}⁷. The combined experimental and theoretical spectroscopic studies demonstrated that the TauD-{FeNO}⁸ species is best described as having a triplet ground state, arising from antiferromagnetic coupling between a HS ferrous center (S_Fe = 2) and a triplet NO^- (S_NO = 1). This electronic structure description is identical to that calculated for the triplet O₂ adduct. By contrast, distinct electronic structures were found between the septet and quintet TauD-{FeNO}⁸ species and the corresponding O₂ analogues.
Table 2. Comparison of the predicted electronic structures of {FeNO}⁸ with their O₂ derivatives.

C-H bond activation
Ferryl intermediates function as C-H cleaving agent in the catalytic cycles of a wide range of heme and nonheme oxygen activating iron enzymes. The reaction coordinate for alkane oxidation by Fe^IV-oxo species is calculated to be lengthening of the Fe-oxo and the cleaving C-H bonds. The detailed analysis of the electronic structure changes along the reaction coordinate suggests that the real C-H bond cleaving agent is an oxyl-ferric species that is generated by lengthening of the Fe-oxo bond in the ferryl reactant en route to H-atom abstraction transition state. This can be traced back to the rather covalent nature of Fe-oxo interaction. Importantly, the oxyl formation enhances the ability of the (FeO)²⁺ to react with the bonding C-H s-orbital since the O-based p-orbital more efficiently overlaps with the C-H s-orbital of the substrate, compared to the Fe 3d-based orbital which has limited O-p character.

	S = 1	S = 2	S = 3
{FeNO}8	HS-FeII-3O2 (AF)	HS-FeIII-2O2– (AF)	HS-FeIII-2O2– (F)
{FeO2}8	HS-FeII-3NO– (AF)	HS-FeII-1NO–	HS-FeII-3NO– (F)

Figure 4. Schematic MO diagrams for the quintet Fe^IVoxo species (left) and the evolution of its electronic structure as a function of the Fe-oxo bond length (right).

Fe^IV-oxo complexes can exst in either quintet or triplet ground states and the quintet species is predicted to be more reactive towards C-H bond activation than its corresponding triplet partner. For the quintet reaction, the Fe-d_z2 based s-antibonding molecular orbital (MO) serves as the final electron accepting orbital, whereas the energy of the corresponding MO in the triplet reactant is too high to be an electron acceptor. Thus, the lower energy p-antibonding MOs that mainly consist of the Fe-d_xz/yz and the O-p_x/y fragment orbitals act as electron acceptors on the triplet surface. As a consequence, a s-pathway is often suggested to be operative on the quintet surface, while the triplet reaction proceeds via a p-channel. The differential reactivity stems from the fact that the two spin states have different requirements for the optimal angle at which the substrate should approach the (FeO)²⁺ core because distinct electron accepting orbitals are employed on the two surfaces. The quintet s-pathway requires an essentially linear attack; by contrast the triplet p-channel would favor an ideal attack angle near 90°. However, the latter is not possible due to steric crowding; thus, the attenuated orbital interaction and the unavoidably increased Pauli repulsion result in the lower reactivity of the triplet ferryl complexes. Further studies demonstrated that the reaction of alkane hydroxylation by ferryl model compounds can take place through both the s- and the p-pathways on the quintet and triplet surfaces with barrier heights displaying the order ⁵s > ⁵p » ³p > ³s (Figure 5). In line with the above analysis, the quintet p-channel encounters a comparable barrier to that calculated on the triplet surface due to the enhanced Pauli repulsion and the reduced orbital overlap relative to the quintet s-pathway.



Figure 5. Viable eletron transfer pathways and potential energy surfaces for ethane hydroxylation by [Fe^IV(O)(NH₃)₅]²⁺ system.

Ye S., W. Kaim, B. Sarkar, B. Schwederski, F. Lissner, T. Schleid, C. Duboc-Toia and J. Fiedler:
First crystal structure determination and high-frequency EPR study of an organoarsanecopper radical complex.
Inorg. Chem. Commun. 6, 1196-1200 (2003)

Ye S., W. Kaim, M. Albrecht, F. Lissner and T. Schleid:
Complexes of the imine/thioether mixed-donor ligand 8-methylthioquinoline with d6-configurated transition metal centers: synthesis, structures and comparison with complexes of 1-methyl-2-(methylthiomethyl)-1H-benzimidazole.
Inorg. Chim. Acta 357, 3325-3330 (2004)

Ye S., W. Kaim, M. Niemeyer and N. S. Hosmane:
Stereospecific Intramolecular S→Pt Transmethylation from 8-Methylthioquinoline to Coordinated PtMe₂: Formation of syn-[fac-Pt^IVMe₃(μ-8-quinolinethiolato)]₂ with Stacked Quinoline Rings.
Organometallics 24, 794-796 (2005)

Ye S., B. Sarkar, M. Niemeyer and W. Kaim:
Mixed-ligand copper complexes with 8-methylthioquinoline and triphenylphosphane or the o-semiquinone/catecholate redox system.
European Journal of Inorganic Chemistry 4735-4738 (2005)

Ye S., B. Sarkar, C. Duboc, J. Fiedler and W. Kaim:
The redox series [M(bpy)₂(Q)]ⁿ⁺, M = Ru or Os, Q = 3,5-di-tert-butyl-N-phenyl-1,2-benzoquinonemonoimine. Isolation and a complete X and W band EPR study of the semiquinone states (n = 1).
Inorg. Chem. 44, 2843-2847 (2005)

Ye S., B. Sarkar, F. Lissner, T. Schleid, J. van Slageren, J. Fiedler and W. Kaim:
Three-spin system with a twist: A bis (semiquinonato)copper complex with a nonplanar configuration at the copper(II) center.
Angew. Chem. Int. Ed. 44, 2103-2106 (2005)

Ye S.
Combined Quantum Chemical and Spectroscopic Studies on Transition Metal Complexes with Coordinating Radicals.
Chemtracts (Special Volume on Computational Inorganic Chemistry), 19, 77-86 (2006).

Kirchner B., F. Wennmohs, S. Ye and F. Neese:
Theoretical bioinorganic chemistry: the electronic structure makes a difference.
Curr. Opin. Chem. Biol. 11, 134-141 (2007)

Sinnecker S., N. Svensen, E. W. Barr, S. Ye, J. M. Bollinger, Jr., F. Neese and C. Krebs:
Spectroscopic and computational evaluation of the structure of the high-spin Fe(IV)-oxo intermediates in taurine: alpha-Ketoglutarate dioxygenase from Escherichia coli and its His99Ala ligand variant.
J. Am. Chem. Soc. 129, 6168-6179 (2007)

Ye S., T. Tuttle, E. Bill, L. Simkhovich, Z. Gross, W. Thiel and F. Neese:
The Electronic Structure of Iron Corroles: A Combined Experimental and Quantum Chemical Study.
Chem. Eur. J. 14, 10839-10851 (2008)

Roemelt M., S. Ye and F. Neese:
Calibration of Modern Density Functional Theory Methods for the Prediction of ⁵⁷Fe Mössbauer Isomer Shifts: Meta-GGA and Double-Hybrid Functionals.
Inorg. Chem. 48, 784-785 (2009)

Sundararajan M., D. Ganyushin, S. Ye and F. Neese:
Multireference ab initio studies of zero-field splitting and magnetic circular dichroism spectra of tetrahedral Co(II) complexes.
Dalton Trans. 6021-6036 (2009)

Ye S. and F. Neese:
Quantum chemical studies of C-H activation reactions by high-valent nonheme iron centers.
Curr. Opin. Chem. Biol. 13, 89-98 (2009)

Geng C.-Y., S. Ye and F. Neese:
Analysis of Reaction Channels for Alkane Hydroxylation by Nonheme Iron(IV)-Oxo Complexes.
Angew. Chem. Int. Ed. 49, 5717-5720 (2010)

Neese F., W. Ames, G. Christian, M. Kampa, D. G. Liakos, D. A. Pantazis, M. Roemelt, P. Surawatanawong and S. Ye:
Dealing with Complexity in Open-Shell Transition Metal Chemistry from a Theoretical Perspective: Reaction Pathways, Bonding, Spectroscopy.
Advances in Inorganic Chemistry:
Theoretical and Computational Inorganic Chemistry, Vol 62. R. H. J. Van Eldik. 62, 301-349 (2010).

Ye S. and F. Neese:
The Unusual Electronic Structure of Dinitrosyl Iron Complexes.
J. Am. Chem. Soc. 132, 3646-3647 (2010)

Ye S. and F. Neese:
Accurate Modeling of Spin-State Energetics in Spin-Crossover Systems with Modern Density Functional Theory.
Inorg. Chem. 49, 772-774 (2010)

Ye S., F. Neese, A. Ozarowski, D. Smirnov, J. Krzystek, J. Telser, J.-H. Liao, C.-H. Hung, W.-C. Chu, Y.-F. Tsai, R.-C. Wang, K.-Y. Chen and H.-F. Hsu:
Family of V(III)-Tristhiolato Complexes Relevant to Functional Models of Vanadium Nitrogenase: Synthesis and Electronic Structure Investigations by Means of High-Frequency and -Field Electron Paramagnetic Resonance Coupled to Quantum Chemical Computations.
Inorg. Chem. 49, 977-988 (2010)

Ye S., J. C. Price, E. W. Barr, M. T. Green, J. M. Bollinger, Jr., C. Krebs and F. Neese:
Cryoreduction of the NO-Adduct of Taurine:alpha-Ketoglutarate Dioxygenase (TauD) Yields an Elusive {FeNO}⁸ Species.
J. Am. Chem. Soc. 132, 4739-4751 (2010)

Neese F., D. G. Liakos and S. Ye:
Correlated wavefunction methods in bioinorganic chemistry.
J. Biol. Inorg. Chem. 16, 821-829 (2011)

Ye S. and F. Neese:
Nonheme oxo-iron(IV) intermediates form an oxyl radical upon approaching the C-H bond activation transition state.
Proc. Natl. Acad. Sci. U.S.A. 108, 1228-1233 (2011)