Mechanistic Insights for Formation of an Organometallic Co–C Bond in the Methyl Transfer Reaction Catalyzed by Methionine Synthase

Methionine synthase (MetH) catalyzes the transfer of a methyl group from methyltetrahydrofolate (CH₃–H₄Folate) to the cob­(I)­alamin intermediate to form an organometallic Co–C bond, a reaction similar to that of CH₃–H₄Folate:corrinoid/iron–sulfur protein (CFeSP) methyltransferase (MeTr). How precisely it is formed remains elusive because the displacement of a methyl group from the tertiary amine is not a facile reaction. To understand the electronic structure and mechanistic details of the MetH–cob­(I)­alamin:CH₃–H₄Folate reaction complex, we applied quantum mechanics/molecular mechanics (QM/MM) computations. The hybrid QM/MM calculations reveal the traditionally assumed S_N2 mechanism for formation the CH₃–cob­(III)­alamin resting state where the activation energy barrier for the S_N2 reaction was found to be ∼8–9 kcal/mol, which is comparable with respect to the determined experimental rate constant. However, the possibility of an electron transfer (ET) based radical mechanism consistent with the close-lying diradical states observed from triplet and open-shell singlet states has also been suggested as an alternative, where first an electron transfer from His-on cob­(I)­alamin to the pterin ring of the protonated CH₃–H₄Folate takes place, forming the Co^II(d⁷)–pterin radical (π*)¹ diradical state, followed by a methyl radical transfer. Although the predicted energy barrier for the ET-mediated radical reaction is comparable to that of the S_N2 pathway, the major advantage of ET is that a methyl radical can be transferred at a longer distance, which does not require the close proximity of two binding modules of MetH as does the S_N2 type. In addition, based on the energy barrier of the transition state (TS) in both the protonated (∼8–9 kcal/mol) and the unprotonated N5 (39 kcal/mol) species of the CH₃–H₄Folate, it can be inferred that the protonation event must takes place either prior to or during the methyl transfer reaction in a ternary complex. The results of the present study including mechanistic insights can have implications to a broad class of corrinoid–methyltransferases, which utilize a CH₃–H₄Folate substrate or its related analogues as methyl donor

Charge Separation Propensity of the Coenzyme B₁₂–Tyrosine Complex in Adenosylcobalamin-Dependent Methylmalonyl–CoA Mutase Enzyme

We report the electrophilic Fukui function analysis based on density functional reactivity theory (DFRT) to demonstrate the feasibility of the proton-coupled electron transfer (PCET) mechanism. To characterize the charge propensity of an electron-transfer site other than the proton-acceptor site of the coenzyme B₁₂–tyrosine complex, several structural models (ranging from minimal to actual enzyme scaffolds) have been employed at DFT and QM/MM computations. It is shown, based on the methylmalonyl-CoA mutase (MCM) enzyme that substrate binding plays a significant role in displacing the phenoxyl proton of the tyrosine (Y89), which initiates the electron transfer from Y89 to coenzyme B₁₂. PCET-based enzymatic reaction implies that one electron-reduced form of the AdoCbl cofactor induces the cleavage of the Co–C bond, as an alternative to its neutral analogue, which can assist in understanding the origin of the observed trillion-fold rate enhancement in MCM enzyme

Cob(I)alamin: Insight Into the Nature of Electronically Excited States Elucidated via Quantum Chemical Computations and Analysis of Absorption, CD and MCD Data

The nature of electronically excited states of the super-reduced form of vitamin B₁₂ (i.e., cob­(I)­alamin or B_12s), a ubiquitous B₁₂ intermediate, was investigated by performing quantum-chemical calculations within the time-dependent density functional theory (TD-DFT) framework and by establishing their correspondence to experimental data. Using response theory, the electronic absorption (Abs), circular dichroism (CD) and magnetic CD (MCD) spectra of cob­(I)­alamin were simulated and directly compared with experiment. Several issues have been taken into considerations while performing the TD-DFT calculations, such as strong dependence on the applied exchange-correlation (XC) functional or structural simplification imposed on the cob­(I)­alamin. In addition, the low-lying transitions were also validated by performing CASSCF/MC-XQDPT2 calculations. By comparing computational results with existing experimental data a new level of understanding of electronic excitations has been established at the molecular level. The present study extends and confirms conclusions reached for other cobalamins. In particular, the better performance of the BP86 functional, rather than hybrid-type, was observed in terms of the excitations associated with both Co d and corrin π localized transitions. In addition, the lowest energy band was associated with multiple metal-to-ligand charge transfer excitations as opposed to the commonly assumed view of a single π → π* transition followed by vibrational progression. Finally, the use of the full cob­(I)­alamin structure, instead of simplified molecular models, shed new light on the spectral analyses of cobalamin systems and revealed new challenges of this approach related to long-range charge transfer excitations involving side chains

Co²⁺/Co⁺ Redox Tuning in Methyltransferases Induced by a Conformational Change at the Axial Ligand

Density functional theory and quantum mechanics/molecular mechanics computations predict cob­(I)­alamin (Co⁺Cbx), a universal B₁₂ intermediate state, to be a pentacoordinated square pyramidal complex, which is different from the most widely accepted viewpoint of its tetracoordinated square planar geometry. The square pyramidality of Co⁺Cbx is inspired by the fact that a Co⁺ ion, which has a dominant d⁸ electronic configuration, forms a distinctive Co⁺--H interaction because of the availability of appropriately oriented filled d orbitals. This uniquely H-bonded Co⁺Cbx may have catalytic relevance in the context of thermodynamically uphill Co²⁺/Co⁺ reduction that constitutes an essential component in a large variety of methyltransferases

Electronic Structure of One-Electron-Oxidized Form of the Methylcobalamin Cofactor: Spin Density Distribution and Pseudo-Jahn–Teller Effect

The electronic and structural properties of the one-electron-oxidized form of methylcobalamin (MeCbl) cofactor have been investigated using density functional theory (DFT) and CASSCF/MC-XQDPT2 calculations. We applied two types of functionals (hybrid and GGA) which produced quite different results in terms of spin density profiles: the B3LYP description was consistent with Co­(III) and the π-cation corrin radical while the BP86 result was more in line with the Co­(IV) oxidation state. A closer inspection of both outcomes indicates that the oxidized species have a mixed π-cation corrin radical and Co­(III)/Co­(IV) character. This mixed character was further supported by high-level <i>ab initio</i> CASSCF/MC-XQDPT2 calculations, which reveal the strong mixing of the electronic states due to nondynamical correlation effects. The near degeneracy, which takes place between the ground and first excited state, was consistent with the presence of a pseudo-Jahn–Teller (pJT) effect in the oxidized form of MeCbl. In addition, the DFT-based investigation of the structurally related porphyrin complexes gives a description consistent with corrin-based analogues and reveals that the corrin species have more Co­(IV) character. The most important finding of the present study, regardless of the type of functional used, was the significant lowering of dissociation energy (∼35%), which might be due to the partial depopulation of the Co–C σ orbital upon removal of an electron

Mechanism of Co–C Bond Photolysis in Methylcobalamin: Influence of Axial Base

A mechanism of Co–C bond photolysis in the base-off form of the methylcobalamin cofactor (MeCbl) and the influence of its axial base on Co–C bond photodissociation has been investigated by time-dependent density functional theory (TD-DFT). At low pH, the MeCbl cofactor adopts the base-off form in which the axial nitrogenous ligand is replaced by a water molecule. Ultrafast excited-state dynamics and photolysis studies have revealed that a new channel for rapid nonradiative decay in base-off MeCbl is opened, which competes with bond dissociation. To explain these experimental findings, the corresponding potential energy surface of the S₁ state was constructed as a function of Co–C and Co–O bond distances, and the manifold of low-lying triplets was plotted as a function of Co–C bond length. In contrast to the base-on form of MeCbl in which two possible photodissociation pathways were identified on the basis of whether the Co–C bond (path A) or axial Co–N bond (path B) elongates first, only path B is active in base-off MeCbl. Specifically, path A is inactive because the energy barrier associated with direct dissociation of the methyl ligand is higher than the barrier of intersection between two different electronic states: a metal-to-ligand charge transfer state (MLCT), and a ligand field state (LF) along the Co–O coordinate of the S₁ PES. Path B initially involves displacement of the water molecule, followed by the formation of an LF-type intermediate, which possesses a very shallow energy minimum with respect to the Co–C coordinate. This LF-type intermediate on path B may result in either S₁/S₀ internal conversion or singlet radical pair generation. In addition, intersystem crossing (ISC) resulting in generation of a triplet radical pair is also feasible

Mechanism of Co–C Bond Photolysis in the Base-On Form of Methylcobalamin

A mechanism of Co–C bond photodissociation in the base-on form of the methylcobalamin cofactor (MeCbl) has been investigated employing time-dependent density functional theory (TD-DFT), in which the key step involves singlet radical pair generation from the first electronically excited state (S₁). The corresponding potential energy surface of the S₁ state was constructed as a function of Co–C and Co–N_axial bond distances, and two possible photodissociation pathways were identified on the basis of energetic grounds. These pathways are distinguished by whether the Co–C bond (path A) or Co–N_axial bond (path B) elongates first. Although the final intermediate of both pathways is the same (namely a ligand field (LF) state responsible for Co–C dissociation), the reaction coordinates associated with paths A and B are different. The photolysis of MeCbl is wavelength-dependent, and present TD-DFT analysis indicates that excitation in the visible α/β band (520 nm) can be associated with path A, whereas excitation in the near-UV region (400 nm) is associated with path B. The possibility of intersystem crossing, and internal conversion to the ground state along path B are also discussed. The mechanism proposed in this study reconciles existing experimental data with previous theoretical calculations addressing the possible involvement of a repulsive triplet state

Beyond Metal-Hydrides: Non-Transition-Metal and Metal-Free Ligand-Centered Electrocatalytic Hydrogen Evolution and Hydrogen Oxidation

A new pathway for homogeneous electrocatalytic H₂ evolution and H₂ oxidation has been developed using a redox active thiosemicarbazone and its zinc complex as seminal metal-free and transition-metal-free examples. Diacetyl-bis­(<i>N</i>-4-methyl-3-thiosemicarbazone) and zinc diacetyl-bis­(<i>N</i>-4-methyl-3-thiosemicarbazide) display the highest reported TOFs of any homogeneous ligand-centered H₂ evolution catalyst, 1320 and 1170 s^–1, respectively, while the zinc complex also displays one of the highest reported TOF values for H₂ oxidation, 72 s^–1, of any homogeneous catalyst. Catalysis proceeds via ligand-centered proton-transfer and electron-transfer events while avoiding traditional metal-hydride intermediates. The unique mechanism is consistent with electrochemical results and is further supported by density functional theory. The results identify a new direction for the design of electrocatalysts for H₂ evolution and H₂ oxidation that are not reliant on metal-hydride intermediates

Mode Recognition in UV Resonance Raman Spectra of Imidazole: Histidine Monitoring in Proteins

The imidazole side-chains of histidine residues perform key roles in proteins, and spectroscopic markers are of great interest. The imidazole Raman spectrum is subject to resonance enhancement at UV wavelengths, and a number of UVRR markers of structure have been investigated. We report a systematic experimental and computational study of imidazole UVRR spectra, which elucidates the band pattern, and the effects of protonation and deprotonation, of H/D exchange, of metal complexation, and of addition of a methyl substituent, modeling histidine itself. A consistent assignment scheme is proposed, which permits tracking of the bands through these chemical variations. The intensities are dominated by normal mode contributions from stretching of the strongest ring bonds, C₂N and C₄C₅, consistent with enhancement via resonance with a dominant imidazole π–π* transition

Translation of Ligand-Centered Hydrogen Evolution Reaction Activity and Mechanism of a Rhenium-Thiolate from Solution to Modified Electrodes: A Combined Experimental and Density Functional Theory Study

The homogeneous, nonaqueous catalytic activity of the rhenium-thiolate complex ReL₃ (L = diphenylphosphinobenzenethiolate) for the hydrogen evolution reaction (HER) has been transferred from nonaqueous homogeneous to aqueous heterogeneous conditions by immobilization on a glassy carbon electrode surface. A series of modified electrodes based on ReL₃ and its oxidized precursor [ReL₃]­[PF₆] were fabricated by drop-cast methods, yielding catalytically active species with HER overpotentials for a current density of 10 mA/cm², ranging from 357 to 919 mV. The overpotential correlates with film resistance as measured by electrochemical impedance spectroscopy and film morphology as determined by scanning and transmission electron microscopy. The lowest overpotential was for films based on the ionic [ReL₃]­[PF₆] precursor with the inclusion of carbon black. Stability measurements indicate a 2 to 3 h conditioning period in which the overpotential increases, after which no change in activity is observed within 24 h or upon reimmersion in fresh aqueous, acidic solution. Electronic spectroscopy results are consistent with ReL₃ as the active species on the electrode surface; however, the presence of an undetected quantity of catalytically active degradation species cannot be excluded. The HER mechanism was evaluated by Tafel slope analysis, which is consistent with a novel Volmer–Heyrovsky–Tafel-like mechanism that parallels the proposed homogeneous HER pathway. Proposed mechanisms involving traditional metal-hydride processes vs ligand-centered reactivity were examined by density functional theory, including identification and characterization of relevant transition states. The ligand-centered path is energetically favored with protonation of cis-sulfur sites culminating in homolytic S–H bond cleavage with H₂ evolution via H atom coupling