The initial molecular structure of RibCbl was obtained from the high-resolution crystal structure of AdoCbl (Ouyang et al., 2004). The full structure of AdoCbl (Figure 1) consists of Co^III equatorially cooridinated via four nitrogen atoms to the corrin ring. The lower axial ligand, dimethylbenzimidazole (DBI) coordinates to the central Co atom via N_ax, with the other N atom bound to the side of the corrin plane by a nucleotide loop. In the actual AdoCbl structure, the upper Ado ligand is 5′-deoxy-5′-adenosine.

To reduce computational cost, calculations were performed using the simplified structural model of RibCbl, denoted Im-[Co^III(corrin)]-Rib⁺for consistency with previous studies (Jaworska et al., 2007; Lodowski et al., 2009, 2011). In the RibCbl model (see Figure 1), the Ado group was simplified to ribosyl (Rib) by omitting the purine ring, leaving only ribose in the upper corrin plane. The corrin ring was truncated by replacing the side chains with hydrogen atoms, and DBI with imidazole (Im). The resulting overall charge of the system was +1 due to removal of the PO⁻₄ nucleotide loop from below the plane. Previous studies have demonstrated that such simplified structural models accurately reproduce the electronic and spectroscopic properties of the full B₁₂ structure (Jaworska and Lodowski, 2003; Jensen and Ryde, 2003, 2009; Stich et al., 2003, 2004; Jaworska et al., 2005, 2007; Kuta et al., 2006, 2009; Liptak and Brunold, 2006; Kozlowski et al., 2007, 2012; Rovira and Kozlowski, 2007; Galezowski et al., 2008; Lodowski et al., 2009, 2011). More inclusive structural models of coenzyme B₁₂ will be employed in future studies utilizing multiple levels of theory.

Optimized RibCbl structures correspond to equilibrium geometries where Co-C_Rib = 2.02 Å and Co-N_Im = 2.21 Å in the gas phase, and Co-C_Rib = 2.02 Å and Co-N_Im = 2.19 Å in water (PCM). Optimized axial bond lengths are in reasonable agreement with X-ray data of Randaccio et al. (Ouyang et al., 2004) with corresponding experimental bond lengths of 2.04 Å for Co-C_Ado and 2.23 Å for Co-N_DBI, respectively. Molecular orbital (MO) energies and fragment contributions of RibCbl are collected in Table S5 for the gas phase, and Table S6 for water (PCM). The relevant MO diagram can be found in supporting information as Figure S2.

Frontier orbitals of RibCbl were determined by fragment analysis to be mainly corrin π, π^*, and metal d orbitals. In particular, HOMO, HOMO−2, −4, −6, and LUMO+1, +2, +3 show major contributions from d_Co and π^* while HOMO−3, −5, −8, −9, −10, and LUMO+3 have increased weights from the Rib group. On the other hand, LUMO and LUMO+4 are composed of nearly pure π orbitals of the corrin ring. Finally, HOMO-7 and LUMO+5 are both composed of excitations from the imidazole group. The change of environment from gas phase to water solution (Table S6) did not influence the character of the MOs significantly, differences being attributed to reversion of energies between the HOMO-5 and −7 in the gas, and HOMO-3 and −4 in the PCM model.

Simulated absorption (Abs) spectra generated from TD-DFT/BP86/TVZP vertical excitation energies with transition dipole moments, using corresponding optimized RibCbl geometries in the gas phase and in water, are shown in Figures 2, 3 respectively. Since hybrid B3LYP functional underestimates BDE as well as produce low-lying states as pure π_corrin → π^*_corrin excitations, we employed BP86 throughout this study which has been proved to produce correct BDE for both MeCbl and AdoCbl (Kuta et al., 2006; Kozlowski et al., 2007, 2012; Galezowski et al., 2008; Kornobis et al., 2011, 2013; Kumar et al., 2013). BP86 functional was also frequently applied in QM/MM calculations for the enzyme-active cofactors (Kumar et al., 2012; Kumar and Kozlowski, 2013). In both the gas phase and solvent-based calculations, forty excited states were calculated to cover a range appropriate for electronic states potentially involved in coenzyme B₁₂ photolysis. The lowest ten singlet states are listed in Table 1, and the lowest ten triplet states are listed in Table 2 for the gas phase. In addition, the corresponding solvent-based calculations are listed in Table 3 for singlet states and Table 4 for triplet states. Complete lists of the forty excited states for each system can be found in the supporting information (Tables S1–S4). Taking into account that light exposure used in experimental studies was no shorter than 400 nm to induce Co-C cleavage (Walker et al., 1998a,b; Shiang et al., 1999; Yoder et al., 2001; Cole et al., 2002; Sension et al., 2004, 2005a,b; Harris et al., 2007), in further discussion we will focus on the low-energy region.

Since the studied RibCbl model is a truncated form of full coenzyme B₁₂, no direct comparison to the experimental spectrum can be made. However, the predicted spectra can be correlated to that of AdoCbl with good agreement (Shiang et al., 1999). Several calculated electronic transitions can be assigned to experimental spectral bands. Specifically, the S₂ at 2.36 eV and the S₃ at 2.37 eV agree well with the experimental transition at 2.35 eV (528 nm), the S₆ at 2.87 eV agrees with the experimental transition at 2.90 eV (428 nm), and the S₁₀ state at 3.10 eV can be assigned to absorption observed at 3.07 eV (404 nm). The same trend applies to PCM geometries (Table 3). Namely, the S₂, S₃, S₆, and S₉ can all be correlated with experimental absorption bands for AdoCbl observed at 2.35 eV (528 nm), 2.90 eV (428 nm), and 3.07 eV (404 nm), respectively. Furthermore, the simulated RibCbl Abs spectra are also in reasonable agreement with the simulated AdoCbl spectra published by Andruniow et al. (2009) further supporting the ability of the truncated AdoCbl model, (i.e., RibCbl) to accurately reproduce the experimental spectroscopic features of AdoCbl.

The absorption spectrum of RibCbl calculated in the gas phase at the BP86/TZVP level is shown in Figure 2, and assignments for the lowest forty singlet states are collected in Table S1. Based on these results it can be deduced that the α/β bands are located in the low energy region from 2.00 to 2.80 eV, covering first five calculated singlet transitions, the D/E band ranges from 2.80 to 3.30 eV and is composed of multiple excitations, and the high-energy region, or so-called γ band, has significant intensity covering several transitions from 3.30 to 4.40 eV. Based on the nature of the calculated electronic excited states (Table 1), the α/β bands involve primarily d_Co/π → π^* contributions, the D/E bands result from various electronic transitions with more contributions from the Rib group, and the same tendency is observed in the γ band, where oscillator strength increases significantly. It should be noted that ribose increases its role in photoelectronic transitions in the high energy region, i.e., the D/E and γ bands, respectively.

Table 1 summarizes the ten lowest calculated excited singlet states of RibCbl in the gas phase. As shown in Table 1, the S₁ excitation present at 2.24 eV, carries only a small oscillator strength (0.008). The major contribution from the S₁ state is an excitation from HOMO-1 to LUMO, in which π + d_xz/d_z² → π^* is dominant. The MO diagram (Figure S2), shows that S₁ excitation also involves a transition from a σ orbital of axial bonding to the π^* of the corrin ring, and is identified as SBLCT (Lodowski et al., 2009). The second noticeable contribution from the S₁ state is the transition from HOMO-2 to LUMO, identified as π + d_xz → π^*. Based on the MO diagram it is apparent that LUMO is a pure corrin π^* orbital, to which all the excitations in the α/β band generally take place. The next two states, the S₂ and S₃, are adjacent closely in energy. The most significant components of the S₂ and S₃ states are HOMO → LUMO and HOMO-2 → LUMO, where HOMO is a mixture of π and d_yz and HOMO-2 refers to π and d_xz. Therefore, the d to π^* transition is dominant in this region, and should be described as MLCT transition (Cole et al., 2002; Jaworska et al., 2007; Lodowski et al., 2009, 2011).

The S₄ state has even less oscillator strength intensity compared to that of S₁, in which both HOMO-3 and HOMO-4 contribute almost evenly to excitation at 2.58 eV. HOMO-3 derives from ribose, according to MO diagram, while HOMO-4 is composed from the Co d_{x² −y²} orbital. Coincidently, the S₅ involves the same set of orbitals which contribute to the S₄ state. The only difference, considering their specific characters, is that the S₅ state mixes a minor contribution from the HOMO-1→ LUMO transition. However, the larger oscillator strength of the S₅ state makes it the strongest component of the α/β band.

Electronic transitions in the D/E band are more complex than those in the α/β band, and the oscillator strengths are noticeably smaller. This region can be represented by electronic transitions from the S₆ to S₁₄ with three strong excitations occurring at 2.87 (S₆), 3.10 (S₁₀), and 3.21 eV (S₁₂) (Figure 2). The S₆ and S₁₀ have d_Co/π → π^* character while the S₁₂ shows a d_Co/π → σ^*_Co−C nature. The other small intensity transitions in this region are of similar character.

More than twenty singlet states can be associated with γ band ranging from 3.31 (S₁₅) to 4.23 eV (S₄₀) (Table S1). Among them, the S₂₅ and S₂₇ have the largest transition dipole moment and are composed of n_Rib/σ_Rib + d_xz/d_z² + π →d_xy −n + π^* transitions with a minor d_Co/ π → π^* contribution. The S₂₉ is dominated by d_Co/π → π^* transitions, while the S₃₂ has d_Co/π → π^*_Im character. Near the end of the simulated spectrum, another noticeable transition of n_Rib/σ_Rib + d_xz/d_z² + π → σ^*(d_z²) + n type is found at 4.14 eV corresponding to the S₃₇ state (Table S1).

Since triplet excitations have zero transition dipole moments, we will not take them into account for spectral analysis. The first three triplet states are energetically below the S₁ (2.24 eV), where the T₁ occurs at 1.72 eV from an electronic transition of HOMO→LUMO, while T₂ and T₃ are transitions at 1.94 and 2.15 eV, originating from HOMO-1→LUMO and HOMO-2→LUMO, respectively. All three electronic transitions can be described as d_Co/π → π^* type (see Table 2 and Table S2).

The Abs spectrum of RibCbl calculated in water solution using the same level of theory was plotted in Figure 3. The energy of the γ band remains unchanged in comparison to gas phase calculation. However, the α/β band is narrowed due to the blue shift of the S₅ to the D/E region. The S₁, S₂, and S₃ are assigned to d_Co/π → π^* transition. A significant contribution to S₁ state is composed of a π + d_xz/d_z² → π^* excitation and, like in the gas phase, is characterized as SBLCT. The S₄ has pure d_{x² −y²} → π^* character. The D/E band covers states S₅–S₁₃. The states S₅ and S₇ are identified as d/π → π^*/d−n excitations with a small admixture of long range CT (LRCT) type transitions (n_Rib/σ_Rib → π^*) (Andruniow et al., 2009). The S₆ state at 2.90 eV is a mixture of a dominant LRCT, and minor d/π → π^*/d−n character. In contrast, the S₇ state is mainly composed of a d_Co/π → π^* transition, with minor contributions from SBLCT. The other intense singlet transitions in this region are assigned to the d_Co/π → π^* transition, which is consistent with singlet excited states observed in the gas phase calculation. The complete assignment of electronic transitions is presented in Table S3. In water solvated system, the T₁–T₄ states are below the S₁ state (Table 4), and all possess contributions from the d_Co/π → π^* transition.

In order to simulate the photodissociation of AdoCbl, the Co-C_Rib bond in RibCbl was elongated to mimic the light induced bond scission. The length of the Co-C_Rib bond was systematically stretched with the increment of 0.05 Å in a range from 1.90 to 2.65 Å. For each constrained distance, the ground state geometry was optimized at the BP86/TZVP level, followed by TD-DFT calculations for both singlet and triplet states. Figure 4 depicts the potential energy curves of a manifold of excited states computed as a function of Co-C_Rib bond length.

The S₁–S₅ singlet states, which belong to α/β band, have non-repulsive character, and the gap between the S₁ and higher states gets larger when the bond length increases. States denoted as S₂ and S₃ are parallel at short Co-C_Rib distance but cross at ~2.50 Å. On the other hand the S₄ and S₅ keep similar energy but visibly separate from the first group of transitions (S₁–S₃). However, at ~2.30 Å these two states show avoiding crossing, and the S₄ approaches the S₂ at long length bond region. The excited states higher in energy (S₆–S₁₀), display multiple crossings at even short Co-C distances. According to the TD-DFT data, low-lying singlet states do not participate in photodissociation because there is no ¹(σ_Co−C → σ^*_Co−C) dominant. Most likely such a transition requires an energetically high excitation and none of singlet states with predominant ¹(σ_Co−C → σ^*_Co−C) character are in a reasonable transition region. Additionally, it is expected that such states would not dissociate to the cob(II)alamin and ribosyl radicals, but rather to ionic fragments. However, the low-lying triplet states appear to have σ_Co−C → σ^*_Co−C character in the achievable energy region. By connecting the energy points of σ_Co−C-featured triplet states at different dissociation distances, the ³(σ_Co−C → σ^*_Co−C) state with repulsive character can be located (black curve in Figure 4). At 2.35 Å, this state becomes the lowest level in terms of energy, and displays an avoided crossing with T₁ state, that typically accounts for d_Co/π → π^* excitation. Together with an increasing Co-C_Rib distance, the energy of the triplet state drops dramatically above a Co-C bond distance of 2.40 Å, indicating that dissociation may not be properly described with a single-determinant wavefunction approximation at long Co-C_Rib distances (Kumar et al., 2011a,b). It is also reasonable to postulate that there is a conical intersection existing between the two states. Although the current study has limitations on the longer bond distances, it is expected that the photolysis happens at much shorter bond lengths, where ³(σ_Co−C → σ^*_Co−C) excitation is described properly. Further multi-configuration wave functions would be required to obtain a more detailed picture.

Since the photoexcitation of B₁₂ derivatives involves primarily the structural changes of the axial ligand involved in N-Co-C bonding, this investigation was extended to the PESs computed as a function of both Co-C_Rib and Co-N_Im distances. The S₁ and S₂ PESs were constructed from the equilibrium structure of the RibCbl (S₀ state), and systematically stretching the two axial bonds with an increment of 0.05 Å. Geometries at each grid point were optimized by BP86/TZVP, and the vertical excitations were calculated by TD-DFT at the BP86/TZVP level of theory (Figures 5, 6).

In gas phase PES contours (Figure 5), the S₀ shows an equilibrium geometry with the lowest energy at 2.02 Å for Co-C_Rib and 2.21 Å for Co-N_Im, while for the S₁ PES, the minimum occurs at the longer Co-C_Rib bond (~2.17 Å) but shorter Co-N_Im (~2.00 Å) distance. According to pump-probe time resolved experiments, an intermediate state of AdoCbl is populated through photoelectronic excitation, leading to a partial mixture of adenosyl radical and cob(II)alamin. It is therefore reasonable to postulate that such an intermediate state can be identified as the S₁, which according to TD-DFT calculations has the Co-C_Rib bond elongated and Co-N_Im shortened due to an electron density shift from the metal to the equatorial bonds of the corrin ring. The PES plot of the S₂ is nearly parallel to the S₀ surface. Estimates based on PES projections (Figure 5) predict the energy minimum of the S₂ surface is located at Co-C_Rib ≈ 2.04 Å, and Co-N_Im ≈ 2.23 Å, ± 0.02 Å, compared to the equilibrium geometry. This indicates no significant change of axial bond distances occur from the S₀ to S₂ transition. If this is true, the energy difference between the S₀ and S₂ states are mainly attributed the geometry changes of the corrin ring. At this point, the distortion of the corrin ring, mainly due to π→π^* electronic transitions, increases the potential energy of the system. Since the geometry change in the S₁ state involves Co-C_Rib bond stretching, it is likely that the S₁ state is the most involved in the photolytic cleavage of Co-C, and thus the formation of the radical pair generated when the Co-C bond is stretched.

The PES of RibCbl in water retains nearly all of the general features of the gas phase PES (Figure 6). However, although the equilibrium geometry of RibCbl in water has similar axial bond lengths to those obtained in the gas phase calculation (with the exception of a slightly reduced Co-N bond distance of 0.02 Å), there remain subtle differences in energies between the S₀ and S₁, and S₀ and S₂ states. The energy gaps between S₀ and S₁, as well as S₀ and S₂, are larger for the solvated geometry than those of the gas phase. The vertical energy gaps between the minimum point of solvated ground state and the lowest two excited states are ~2.30 eV (S₀–S₁), and ~2.40 eV (S₀–S₂) respectively, while the vertical energy gaps in the gas phase calculation are ~2.20 eV (S₀–S₁) and ~2.35 eV (S₀–S₂). It can be reasoned that the larger vertical energy gaps from the ground state to the first two excited states in the PCM calculation, that the polar effect of solvent tends to increase the energies required for RibCbl excitation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.