Frontier Molecular Orbital Contributions to Chlorination versus Hydroxylation Selectivity in the Non-Heme Iron Halogenase SyrB2

The ability of an Fe^IVO intermediate in SyrB2 to perform chlorination versus hydroxylation was computationally evaluated for different substrates that had been studied experimentally. The π-trajectory for H atom abstraction (Fe^IVO oriented perpendicular to the C–H bond of substrate) was found to lead to the <i>S</i> = 2 five-coordinate HO–Fe^III–Cl complex with the C^• of the substrate, π-oriented relative to both the Cl^– and the OH^– ligands. From this ferric intermediate, hydroxylation is thermodynamically favored, but chlorination is intrinsically more reactive due to the energy splitting between two key redox-active dπ* frontier molecular orbitals (FMOs). The splitting is determined by the differential ligand field effect of Cl^– versus OH^– on the Fe center. This makes chlorination effectively competitive with hydroxylation. Chlorination versus hydroxylation selectivity is then determined by the orientation of the substrate with respect to the HO–Fe–Cl plane that controls either the Cl^– or the OH^– to rebound depending on the relative π-overlap with the substrate C radical. The differential contribution of the two FMOs to chlorination versus hydroxylation selectivity in SyrB2 is related to a reaction mechanism that involves two asynchronous transfers: electron transfer from the substrate radical to the iron center followed by late ligand (Cl^– or OH^–) transfer to the substrate

Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S­(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (<i>H</i>_DA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S­(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates <i>H</i>_DA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites

Molecular Origin of Rapid versus Slow Intramolecular Electron Transfer in the Catalytic Cycle of the Multicopper Oxidases

Kinetic measurements on single-turnover processes in laccase established fast type-1 Cu to trinuclear Cu cluster (TNC) intramolecular electron transfer (IET) in the reduction of the native intermediate (NI), the fully oxidized form of the enzyme formed immediately after O–O bond cleavage in the mechanism of O₂ reduction. Alternatively, slow IET kinetics was observed in the reduction of the resting enzyme, which involves proton-coupled electron transfer on the basis of isotope measurements. The >10³ difference between the IET rates for these two processes confirms that the NI, rather than the resting enzyme that has been defined by crystallography, is the fully oxidized form of the TNC in catalytic turnover. Computational modeling showed that reduction of NI is fast because of the larger driving force associated with a more favorable proton affinity of its μ₃-oxo moiety generated by reductive cleavage of the O–O bond. This defines a unifying mechanism in which reductive cleavage of the O–O bond is coupled to rapid IET in the multicopper oxidases

The Role of Chloride in the Mechanism of O₂ Activation at the Mononuclear Nonheme Fe(II) Center of the Halogenase HctB

Mononuclear nonheme Fe­(II) (MNH) and α-ketoglutarate (α-KG) dependent halogenases activate O₂ to perform oxidative halogenations of activated and nonactivated carbon centers. While the mechanism of halide incorporation into a substrate has been investigated, the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure. Here, we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl-halogenase HctB. Stopped-flow kinetic studies show that the oxidative transformation of the Fe­(II)-α-KG-enzyme complex is >200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate. By contrast, the presence of substrate, which generally brings about O₂ activation at enzymatic MNH centers, only has an ∼10-fold effect in the absence of chloride. Circular dichroism (CD) and magnetic CD (MCD) studies demonstrate that chloride binding triggers changes in the metal center ligation: chloride binding induces the proper binding of the substrate as shown by variable-temperature, variable-field (VTVH) MCD studies of non-α-KG-containing forms and the conversion from six-coordinate (6C) to 5C/6C mixtures when α-KG is bound. In the presence of substrate, a site with square pyramidal five-coordinate (5C) geometry is observed, which is required for O₂ activation at enzymatic MNH centers. In the absence of substrate an unusual trigonal bipyramidal site is formed, which accounts for the observed slow, uncoupled reactivity. Molecular dynamics simulations suggest that the binding of chloride to the metal center of HctB leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme–substrate complex. Results are discussed in relation to other MNH dependent halogenases

Comparison of High-Spin and Low-Spin Nonheme Fe^III–OOH Complexes in O–O Bond Homolysis and H‑Atom Abstraction Reactivities

The geometric and electronic structures and reactivity of an <i>S</i> = 5/2 (HS) mononuclear nonheme (TMC)­Fe^III–OOH complex are studied by spectroscopies, calculations, and kinetics and compared with the results of previous studies of <i>S</i> = 1/2 (LS) Fe^III–OOH complexes to understand parallels and differences in mechanisms of O–O bond homolysis and electrophilic H-atom abstraction reactions. The homolysis reaction of the HS [(TMC)­Fe^III–OOH]²⁺ complex is found to involve axial ligand coordination and a crossing to the LS surface for O–O bond homolysis. Both HS and LS Fe^III–OOH complexes are found to perform direct H-atom abstraction reactions but with very different reaction coordinates. For the LS Fe^III–OOH, the transition state is late in O–O and early in C–H coordinates. However, for the HS Fe^III–OOH, the transition state is early in O–O and further along in the C–H coordinate. In addition, there is a significant amount of electron transfer from the substrate to the HS Fe^III–OOH at transition state, but that does not occur in the LS transition state. Thus, in contrast to the behavior of LS Fe^III–OOH, the H-atom abstraction reactivity of HS Fe^III–OOH is found to be highly dependent on both the ionization potential and the C–H bond strength of the substrate. LS Fe^III–OOH is found to be more effective in H-atom abstraction for strong C–H bonds, while the higher reduction potential of HS Fe^III–OOH allows it to be active in electrophilic reactions without the requirement of O–O bond cleavage. This is relevant to the Rieske dioxygenases, which are proposed to use a HS Fe^III–OOH to catalyze <i>cis</i>-dihydroxylation of a wide range of aromatic compounds

Two-Electron Reduction versus One-Electron Oxidation of the Type 3 Pair in the Multicopper Oxidases

Multicopper oxidases (MCOs) utilize an electron shuttling Type 1 Cu (T1) site in conjunction with a mononuclear Type 2 (T2) and a binuclear Type 3 (T3) site, arranged in a trinuclear copper cluster (TNC), to reduce O₂ to H₂O. Reduction of O₂ occurs with limited overpotential indicating that all the coppers in the active site can be reduced via high-potential electron donors. Two forms of the resting enzyme have been observed in MCOs: the alternative resting form (AR), where only one of the three TNC Cu’s is oxidized, and the resting oxidized form (RO), where all three TNC Cu’s are oxidized. In contrast to the AR form, we show that in the RO form of a high-potential MCO, the binuclear T3 Cu­(II) site can be reduced via the 700 mV T1 Cu. Systematic spectroscopic evaluation reveals that this proceeds by a two-electron process, where delivery of the first electron, forming a high energy, metastable half reduced T3 state, is followed by the rapid delivery of a second energetically favorable electron to fully reduce the T3 site. Alternatively, when this fully reduced binuclear T3 site is oxidized via the T1 Cu, a different thermodynamically favored half oxidized T3 form, i.e., the AR site, is generated. This behavior is evaluated by DFT calculations, which reveal that the protein backbone plays a significant role in controlling the environment of the active site coppers. This allows for the formation of the metastable, half reduced state and thus the complete reductive activation of the enzyme for catalysis

Correlation of the Electronic and Geometric Structures in Mononuclear Copper(II) Superoxide Complexes

The geometry of mononuclear copper­(II) superoxide complexes has been shown to determine their ground state where side-on bonding leads to a singlet ground state and end-on complexes have triplet ground states. In an apparent contrast to this trend, the recently synthesized (HIPT₃tren)­Cu^IIO₂^•– (<b>1</b>) was proposed to have an end-on geometry and a singlet ground state. However, reexamination of <b>1</b> with resonance Raman, magnetic circular dichroism, and ²H NMR spectroscopies indicate that <b>1</b> is, in fact, an end-on superoxide species with a triplet ground state that results from the single Cu^IIO₂^•– bonding interaction being weaker than the spin-pairing energy

Substrate and Metal Control of Barrier Heights for Oxo Transfer to Mo and W Bis-dithiolene Sites

Reaction coordinates for oxo transfer from the substrates Me₃NO, Me₂SO, and Me₃PO to the biologically relevant Mo­(IV) bis-dithiolene complex [Mo­(OMe)­(mdt)₂]⁻ where mdt = 1,2-dimethyl-ethene-1,2-dithiolate­(2-), and from Me₂SO to the analogous W­(IV) complex, have been calculated using density functional theory. In each case, the reaction first proceeds through a transition state (TS1) to an intermediate with substrate weakly bound, followed by a second transition state (TS2) around which breaking of the substrate X–O bond begins. By analyzing the energetic contributions to each barrier, it is shown that the nature of the substrate and metal determines which transition state controls the rate-determining step of the reaction

Substrate and Metal Control of Barrier Heights for Oxo Transfer to Mo and W Bis-dithiolene Sites

Reaction coordinates for oxo transfer from the substrates Me₃NO, Me₂SO, and Me₃PO to the biologically relevant Mo­(IV) bis-dithiolene complex [Mo­(OMe)­(mdt)₂]⁻ where mdt = 1,2-dimethyl-ethene-1,2-dithiolate­(2-), and from Me₂SO to the analogous W­(IV) complex, have been calculated using density functional theory. In each case, the reaction first proceeds through a transition state (TS1) to an intermediate with substrate weakly bound, followed by a second transition state (TS2) around which breaking of the substrate X–O bond begins. By analyzing the energetic contributions to each barrier, it is shown that the nature of the substrate and metal determines which transition state controls the rate-determining step of the reaction

Second-Sphere Effects on Methane Hydroxylation in Cu-Zeolites

Two [Cu₂O]²⁺ cores have been identified as the active sites of low temperature methane hydroxylation in the zeolite Cu-MOR. These cores have similar geometric and electronic structures, yet different reactivity with CH₄: one reacts with a much lower activation enthalpy. In the present study, we couple experimental reactivity and spectroscopy studies to DFT calculations to arrive at structural models of the Cu-MOR active sites. We find that the more reactive core is located in a constricted region of the zeolite lattice. This leads to close van der Waals contact between the substrate and the zeolite lattice in the vicinity of the active site. The resulting enthalpy of substrate adsorption drives the subsequent H atom abstraction stepa manifestation of the “nest” effect seen in hydrocarbon cracking on acid zeolites. This defines a mechanism to tune the reactivity of metal active sites in microporous materials