Combined Spectroscopic and Computational Analysis of the

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Combined Spectroscopic and Computational Analysis of the Vibrational Properties of Vitamin B in its Co , Co , and Co Oxidation States 12

3+

2+

1+

Kiyoung Park, and Thomas C. Brunold J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp309392u • Publication Date (Web): 11 Mar 2013 Downloaded from http://pubs.acs.org on March 15, 2013

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The Journal of Physical Chemistry

Combined Spectroscopic and Computational Analysis of the Vibrational Properties of Vitamin B12 in its Co3+, Co2+, and Co1+ Oxidation States Kiyoung Park† and Thomas C. Brunold* Contribution from the Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 E-mail: [email protected] RECEIVED DATE (automatically inserted by publisher) †

Current address:

333 Campus Drive Stanford, CA 94305 *To whom correspondence should be addressed: 1101 University Ave. Madison, WI 53706 phone: (608) 265-9056 fax: (608) 262-6143 Running Title: Vibrational Properties of Vitamin B12 in its Co3+, Co2+, and Co1+ Oxidation States Key words: Density functional theory, time-dependent density functional theory, resonance Raman spectroscopy, excited-state distortions, normal mode descriptions

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Abstract While the geometric and electronic structures of vitamin B12 (cyanocobalamin, CNCbl) and its reduced derivatives Co2+cobalamin (Co2+Cbl) and Co1+cobalamin (Co1+Cbl-) are now reasonably well established, their vibrational properties, in particular their resonance Raman (rR) spectra, have remained quite poorly understood. The goal of this study was to establish definitive assignments of the corrin-based vibrational modes that dominate the rR spectra of vitamin B12 in its Co3+, Co2+, and Co1+ oxidation states. rR spectra were collected for all three species with laser excitation in resonance with the most intense corrin-based   * transitions. These experimental data were used to validate the computed vibrational frequencies, eigenvector compositions, and relative rR intensities of the normal modes of interest as obtained by density functional theory (DFT) calculations. Importantly, the computational methodology employed in this study successfully reproduces the experimental observation that the frequencies and rR excitation profiles of the corrin-based vibrational modes vary significantly as a function of the cobalt oxidation state. Our DFT results suggest that this variation reflects large differences in the degree of mixing between the occupied Co 3d orbitals and empty corrin * orbitals in CNCbl, Co2+Cbl, and Co1+Cbl. As a result, vibrations mainly involving stretching of conjugated C–C and C–N bonds oriented along one axis of the corrin ring may, in fact, couple to a perpendicularly polarized electronic transition. This unusual coupling between electronic transitions and vibrational motions of corrinoids greatly complicates an assignment of the corrin-based normal modes of vibrations on the basis of their rR excitation profiles.


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1. Introduction Corrinoids are naturally occurring coordination complexes consisting of a central cobalt ion and a tetrapyrrole ligand called the corrin ring.1 Their structural complexity was first revealed when the X-ray crystal structure of vitamin B12, the best known corrinoid species, was determined in 1956 (Figure 1).2,3 In vitamin B12, four nitrogen atoms, each belonging to one of the pyrrole rings of the corrin macrocycle, ligate the cobalt center (formally in the Co3+ oxidation state) equatorially, while a cyanide and a 5,6-dimethylbenzimidazole (DMB) group tethered to the macrocycle through a nucleotide loop coordinate to the cobalt center on the “upper” () and “lower” () faces, respectively, of the corrin ring. In the biologically active derivatives of vitamin B12, adenosylcobalamin (AdoCbl)4 and methylcobalamin (MeCbl),5 the cyanide ligand is replaced by an adenosyl moiety and a methyl group, respectively. Both prokaryotes and eukaryotes require cobalamin species as cofactors for numerous enzymes.6 AdoCbl is used to initiate the radical-induced rearrangement of substrates (e.g., by methylmalonyl-CoA mutase, MMCM; glutamate mutase, GM; diol dehydratase; and ethanolamine ammonia lyase)7-11 or the reduction of ribonucleotides (by ribonucleotide tri-phosphate reductase) via homolytic Co–C bond cleavage.12,13 Alternatively, MeCbl is used for the transfer of a methyl cation (e.g., by methionine synthase), whereby the Co–C bond is cleaved heterolytically.14 As in CNCbl, the formal oxidation state of the cobalt center in AdoCbl and MeCbl is +3; it is reduced to +2 and +1 upon homolytic and heterolytic Co–C bond cleavage, respectively.6,14,15 This change in the oxidation state of the cobalt center is accompanied by a decrease in coordination number from six in the native cofactors to five in Co2+Cbl, which only retains the DMB in the axial position, and to four in Co1+Cbl, which features an essentially square planar cobalt ion.16-18 The corrin ring is structurally related to the most ubiquitous naturally occurring tetrapyrrole ligand, the porphyrin ring. In fact, the de novo biosynthetic pathways of the corrin and porphyrin



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macrocycles share a common tetrapyrrole precursor, termed uroporphyrinogen III.19,20 From there, however, the two pathways diverge, with a unique step in the corrin biosynthesis being the ring contraction between the pyrrole rings A and D (Figure 1). Consequently, the corrin ring has a direct link between the C1 and C19 atoms, rather than a methine group as found in the porphyrin ring at this position.19,20 As a result, the corrin ring displays a larger degree of conformational freedom than the porphyrin ring.21 This increased flexibility of the corrin ring, which is well documented by the numerous X-ray crystal structures of naturally occurring corrinoids and related model complexes,17,22 prompted researcher to speculate that AdoCbl binding to an apoenzyme is coupled to an upward flexing of the corrin ring so as to trigger homolytic cleavage of the cofactor’s Co–C bond.23-25 An excellent probe of conformational changes of the corrin ring upon corrinoid binding to enzyme active sites is provided by resonance Raman (rR) spectroscopy. Due to the presence of intense absorption bands associated with corrin * transitions in the visible spectral region,26-29 the vibrations of corrinoids can be selectively enhanced. This property was exploited to investigate the interaction of AdoCbl with apo-MMCM and apo-GM using rR spectroscopy.30-32 While the Co– C bond strength was found to be virtually unaffected by the binding of AdoCbl to apo-MMCM despite the DMBhistidine (His) axial ligand switch that accompanies this process, all vibrational features in the high-frequency region (~ 1490 – 1620 cm1) of the protein-bound cofactor spectrum displayed noticeable up-shifts.31 From a comparison to trends established previously for metalloporphyrins, these up-shifts were interpreted as indicating a flattening of the corrin ring upon AdoCbl binding to apo-MMCM; however, no attempts were made to assign these high-frequency features to specific vibrational modes. As expected, the energies and relative intensities of the high-frequency features in the rR spectra of Co3+corrinoids show little dependence on the identities of the axial ligands.33-37


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Invariably, these features are strongly enhanced for laser excitation in the UV/Vis spectral region where intense corrin * transitions occur, because in the corresponding excited states the cofactor is primarily distorted along the conjugated C–C and C–N bonds of the corrin ring. However, the development of specific assignments of the corrin-based vibrational modes is a relatively daunting task, given the large size and low symmetry of the corrin ring.38 Four major bands associated with corrin-based vibrational modes are observed in the ~1490  1620 cm1 spectral region of rR spectra of vitamin B12 and other Co3+corrinoids. A band at ~1500 cm1 dominates rR spectra obtained with laser excitation at 514.5 nm, which is close in energy to the socalled -band absorption feature that arises from a corrin * transition polarized along the C5C15 axis (the corrin long axis LA in Figure 1). Alternatively, a band at ~1550 cm1 is the most strongly enhanced vibrational feature upon near-UV laser excitation into the so-called -band absorption feature, which is due to a corrin * transition polarized along the CoC10 axis (the corrin short axis SA in Figure 1). Based on these rR enhancement patterns, the ~1500 and 1550 cm1 features have been assigned as vibrational modes primarily involving stretching motions of conjugated C–C and C–N bonds oriented along the LA (the LA-stretching motion) and the SA (the SA-stretching motion), respectively.33-36,39 These assignments are consistent with the following two observations: (i) the frequencies of C=C stretching modes in polyenes decrease as the chain length increases, so the LA-stretching mode should have a lower frequency than the SA-stretching mode,40,41 and (ii) upon H/D exchange at C10 under acidic conditions, only the band at ~1550 cm1 shows a significant down-shift.39 Yet, a density functional theory (DFT)-assisted normal mode analysis for MeCbl revealed that these assignments may not actually be correct.38 First, DFT predicted that the normal mode formally involving the LA-stretching motion has a higher frequency than the SA-stretching mode.



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Second, in the computed off-resonance Raman spectrum, the feature associated with the LAstretching mode at higher frequency is considerably more intense than that due to the SA-stretching mode at lower frequency. Also, in the case of Co2+corrinoid species, no obvious connection was found to exist between the polarizations of the electronic transitions and the rR enhancement patterns of the corrin-based vibrational modes.29 For example, in the rR spectrum of Co2+Cbl obtained with laser excitation in resonance with the “-band-type” corrin * transition, two corrin-based vibrational modes are strongly resonance enhanced, rather than a single mode as observed in the Co3+Cbl spectra.28,29 Only in the case of Co1+Cbl did DFT frequency calculations provide results in good agreement with the experimentally observed frequencies, excitation profiles, and isotope shifts upon H/D exchange at C10 of the corrin-based vibrational modes.27 The goal of this study was to establish conclusive assignments of the relevant corrin-based vibrational modes of vitamin B12 in all three Co oxidation states. The approach we have used involved calculating the vibrational frequencies and relative rR intensities of the normal modes of interest within the framework of DFT, and to validate the computational results on the basis of experimental rR spectra obtained for vitamin B12 in its Co3+, Co2+, and Co1+ oxidation states. Importantly, the computational methodology employed successfully reproduces the experimental observation that the frequencies and rR excitation profiles of the corrin-based vibrational modes vary significantly as a function of the cobalt oxidation state. Collectively, the results obtained in this study greatly enhance the current understanding of the vibrational spectra and normal-mode compositions of corrinoids and provide an excellent basis for interpreting rR spectra obtained for enzyme-bound B12 cofactors.


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2. Experimental and Computational Details 2.1. Sample preparation and spectroscopy. All chemicals including cyanocobalamin (CNCbl), the chloride salt of aquacobalamin ([H2OCbl]Cl), potassium formate (HCOOK), deuterium chloride (DCl), deuterium oxide (D2O), 2-(N-morpholino)ethanesulfonic acid (MES), zinc dust, and ammonium chloride (NH4Cl) were purchased from Sigma and used as obtained. The samples of base-on and base-off CNCbl were prepared in pH 6 MES buffer and 0.5 M HCl solution, respectively.42 The solution of Co2+Cbl was obtained anaerobically by degassing an aqueous solution of H2OCbl+ under vacuum and subsequent reduction with HCOOK.29 The sample of Co1+Cbl was also derived from H2OCbl+, but in this case by anaerobic reduction with Zn dust in an NH4Cl-saturated aqueous solution.43 H/D isotopic labeling at C10 was accomplished by incubating CNCbl in a 0.5 M DCl/D2O solution overnight.44 Since under acidic conditions the DMB group is protonated, the product from this reaction was base-off CNCbl(d-10). To prepare base-on CNCbl(d-10), a portion of the acidic solution was neutralized with 12 M NaOH/D2O solution and the pH was adjusted to 6 with MES buffer prepared in D2O. The same procedure was used to prepare a solution of H2OCbl+(d-10), which was subsequently reduced to Co2+Cbl(d-10) or Co1+Cbl(d-10) as described above. All rR spectra were obtained on frozen solution samples contained in NMR tubes that were placed in a finger dewar filled with liquid N2. A Coherent I-305 Ar+ ion laser was used as the excitation source, keeping the power at the sample in the 10–50 mW range to avoid photodecomposition. The ~135 backscattered light was dispersed by an Acton Research triple monochromator equipped with 1200 and 2400 groves/mm gratings and analyzed with a Princeton Instruments Spec X:100BR deep depletion, back-thinned CCD camera. The ice peak at 228 cm1 was used as an internal standard.



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Low-temperature electronic absorption (Abs) spectra were collected on samples containing 60% (v/v) glycerol (to ensure glass formation upon freezing) using a Jasco J-715 spectropolarimeter in conjunction with an Oxford Instruments SM-4000 8T magnetocryostat. By measuring Abs spectra at reduced temperatures (4.5 K for CNCbl and Co2+Cbl, 250 K for Co1+Cbl), significantly better spectral resolution was achieved than at room temperature. 2.2. Computational models and frequency calculations. The initial geometries of the computational models for CNCbl5 and Co2+Cbl45 were taken from the corresponding X-ray crystal structures. Because attempts to characterize Co1+Cbl by X-ray crystallography have not yet been successful, the input coordinates for the Co1+Cbl model were derived from the Co2+Cbl structure by simply removing the axial DMB ligand. In all calculations described below, the CNCbl and Co1+Cbl models were treated using the spin-restricted formalism, while the Co2+Cbl model was treated using the spin-unrestricted formalism. As in previous computational studies of corrinoid species,27-29 the peripheral side chains were replaced with H atoms at a distance of 1.1 Å from the adjacent C atoms. However, because some corrin-based vibrational modes of interest involve significant methine-bridge movement, the methyl groups on C5 and C15 were preserved in all of our models. To prevent excessive flattening of the corrin ring in the CNCbl and Co2+Cbl models during the geometry optimizations,28,29 the methyl group on the B5 carbon of the DMB group was maintained, while the one attached at the B6 position was replaced with a H atom. The geometries of these truncated models were then optimized via DFT energy minimizations using the ADF 2008.01 program46 with the Vosko-Wilk-Nusair (VWN) local density approximation and the Perdew-Burke-Ernzerhof (PBE) gradient-corrected exchange and correlation functional,47,48 an integration constant of 5.0, and the TZP (core double- and valence triple-, polarized) Slater-type orbital basis set with a frozen core through 1s (C, N, O) or 2p (Co).49-53 Cartesian coordinates for all three optimized models are provided in the Supporting Information, Tables S1 – S3. To calculate ACS Paragon Plus Environment

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harmonic frequencies, the two-point numerical differentiation approach with ±0.01 Å Cartesian coordinate displacements of the atoms was employed, where the absence of negative frequencies confirmed that the geometry optimizations had converged to true minima on the corresponding energy hypersurfaces. Vector representations of the computed normal-mode descriptions were generated using Jmol.54 2.3. Assessment of the relative rR intensities of corrin-based vibrational modes.55 For the closed-shell systems CNCbl and Co1+Cbl, rR intensity calculations were performed in ADF on the geometry-optimized models by using the block keyword “Vibron” and subkeyword “Resraman”,56-59 which invoked the computation of excited state energy gradients (hereafter, this approach is referred to as Method 1). Two additional methods were employed to estimate the origin shifts () of the electronic excited-state potentials relative to the ground-state potential along totally symmetric corrin-based vibrational modes (collectively called s modes). In each case, TDDFT calculations were performed first using the ORCA 2.6 program developed by Dr. Frank Neese to identify the dominant single-electron excitations contributing to the major Abs bands.60,61 In these calculations, the PBE exchange correlation functional47 was employed and the resolution of the identity (RI) approximation was chosen to speed up the calculation of the Coulomb terms.62-68 A valence double- and polarized Gaussian-type orbital basis set in conjunction with the DeMon-J Coulomb fitting basis set were used for all atoms except Co, for which Ahlrichs valence triple- polarized basis set was used.69,70 At least 60 excited states were calculated using the Tamm-Dancoff approximation within an orbital energy window of  3 hartrees.71 To facilitate the interpretation of the computational results, isosurface plots of the molecular orbitals (MOs) and electron density difference maps (EDDMs) were generated with the gOpenMol program developed by Laaksonen, using isodensity values of 0.03 and 0.001 au, respectively.72-74



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On the basis of these TDDFT calculations, the corrin 7-based MO was chosen as the donor MO and the corrin 8*- and corrin 9*-based MOs were chosen as the acceptor MOs for the corrin * transitions associated with the -band and the -band, respectively. These excited electronic configurations were specified using the block keyword “Occupations” in subsequent ADF calculations with the same functionals and basis sets as described above for the ground state geometry optimizations.46 While for Co2+Cbl the self-consistent field procedure failed to convergence during excited state geometry optimizations, optimized geometries in the electronic excited states of interest could be obtained for the CNCbl and Co1+Cbl models using this approach. The changes in internal coordinates between the optimized geometries in the electronic ground and excited states for CNCbl and Co1+Cbl were then projected onto the s mode eigenvectors to estimate the relative rR intensities of these modes (Method 2). Since neither of the abovementioned methods could be applied to the Co2+Cbl model, the potential energy curves for the relevant electronic excited states of this species were determined by systematically distorting the model along the s modes of interest and performing excited-state single-point DFT calculations using the ADF program (Method 3).

3. Results and Analysis 3.1. Electronic absorption spectroscopy. The Abs spectra of vitamin B12 (CNCbl) in its Co3+, Co2+, and Co1+ oxidation states are shown in Figure 2. As expected on the basis of the large changes in color of vitamin B12 solutions in response to Co ion reduction, from red for CNCbl to orange for Co2+Cbl and green for Co1+Cbl, the Abs spectra of these three species are markedly different with regards to the number and positions of the dominant features. Because the Abs spectra of CNCbl and other Co3+corrinoids closely resemble that of the metal-free corrin


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macrocycle, with the main features being the - and -bands in the visible and near-UV spectral regions, respectively (Figure 2a), it was suggested early on that these Abs spectra are dominated by corrin * transitions (note, however, that Co3+Cbl species possessing strongly -donating upper axial ligands, such as alkylcobalamins, exhibit considerably different Abs spectra).26 Consistent with this proposal, previous TDDFT studies of Co3+corrinoids revealed that the frontier MOs participating in the most intense transition are indeed corrin-based.28,75 In general, the highestenergy occupied MO (HOMO) and the lowest-energy unoccupied MO (LUMO) of the metal-free corrin macrocycle, termed the corrin 7 and 8* orbitals, respectively, also serve as the HOMO and LUMO of Co3+corrinoids. The transition responsible for the -band is essentially a HOMOLUMO electronic excitation, while the -band transition primarily involves HOMOLUMO+1(corrin 9*) electronic excitation, though it also contains a sizeable contribution from the HOMO-1(corrin 6)LUMO excitation. As a result, the transition dipole moments of these corrin * transitions are oriented perpendicular to one other; i.e., the -band transition is LA-polarized, whereas the -band transition is SA-polarized (see Figure 1 for the corrin LA and SA orientations).28,75 Because of the decreased effective nuclear charge of the cobalt center in the reduced vitamin B12 derivatives, the filled Co 3d-based MOs of Co2+Cbl and Cb1+Cbl- are raised in energy, shifting between the corrin *orbitals.27-29 Consequently, the HOMOs of these species have primarily Co 3d rather than corrin orbital character. Nevertheless, the dominant features in the Abs spectra of Co2+Cbl and Co1+Cbl can still be attributed to corrin * transitions. Previous TDDFT studies of these species have revealed that the main Abs band in the visible spectral region (peaking at 476.0 nm for Co2+Cbl and 386.5 nm for Co1+Cbl) primarily involves an electronic excitation from the corrin 7-based MO to the corrin 8*-based MO, analogous to the -band



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transition of Co3+corrinoids, though in the case of Co1+

this band also contains sizable

contributions from metal-to-ligand charge transfer (MLCT) excitations.27-29 Alternatively, the dominant near-UV feature was shown to arise from a transition between the corrin 7-based and 9*-based MOs, which corresponds to the -band transition of the oxidized cofactor.27,29 3.2. rR spectroscopy. The relevant regions of the rR spectra obtained for CNCbl, Co2+Cbl, and Co1+Cbl are shown in Figure 3. Although these spectra were collected over a much wider range (150 – 2400 cm-1), only features in the frequency range characteristic of conjugated C=C and C=N bond stretching motions (shown in Figure 3) are enhanced, in agreement with the results obtained in previous rR studies of corrinoids.27-29,33,36,37,76 This finding supports our assignments of the major UV/Vis Abs features exhibited by these species as corrin * transitions, since corrinbased vibrational modes are expected to couple most strongly to these electronic transitions.77 In the rR spectrum of CNCbl obtained with laser excitation at 514.5 nm so as to enhance the vibrational modes coupling to the -band transition, the peak at 1505 cm1 shows by far the largest enhancement, though three additional weak peaks can be discerned in the higher-frequency region (Figure 3a, solid black). Alternatively, when the laser is tuned into the -band (excitation at 363.8 nm), the rR spectrum of CNCbl is dominated by a peak at 1550 cm1 that is roughly twice as intense as that at 1505 cm1 (Figure 3a, dashed gray). Note that on the basis of a recent computational study it was suggested that the first excited state of CNCbl has both corrin * and charge transfer character, with the latter prevailing in solution phase.78,79 However, the lack of any noticeable enhancement of the Co-C(N) and Co-N(DMB) stretching modes in our rR spectrum presented in Figure 3, as well as a detailed analysis of the CNCbl absorption spectrum within the framework of time-dependent Heller theory carried out earlier,28 do not support this hypothesis. Also, we interpret the striking similarities between the Abs spectra of CNCbl and the metal-free


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corrin macrocycle80 as an indication that the Co 3d orbitals do not contribute in any significant way to the donor and acceptor orbitals associated with the dominant electronic transitions of CNCbl. Interestingly, two peaks at 1482 and 1587 cm1 are similarly enhanced in the rR spectrum of Co2+Cbl obtained with 476.5 nm laser excitation (Figure 3b), even though the electronic transition associated with the 476-nm Abs feature of Co2+Cbl involves the same corrin-based donor and acceptor MOs as the -band transition of CNCbl. Moreover, as reported previously,36 no specific enhancement of corrin-based vibrational modes is observed in the rR spectrum of Co2+Cbl obtained upon near-UV laser excitation (data not shown). This variation in the rR enhancement patterns of the corrin-based vibrational modes as a function of the oxidation state of the cobalt ion also extends to Co1+Cbl. For 514.5 nm laser excitation, the dominant peak in the rR spectrum of Co1+Cbl is observed at 1503 cm1 (Figure 3c, solid black), while laser excitation at 454.5 nm gives rise to predominant enhancement of two peaks at 1487 and 1567 cm1 (Figure 3c, solid gray). As the laser is tuned into the dominant Abs band at 386.5 nm (excitation at 363.8 nm), which arises from the same corrin-based electronic transition as the -band of CNCbl, the peak at 1487 cm1 is no longer visible, and the peaks at 1503 and 1567 cm1 now show the largest rR enhancements. To identify the rR peaks associated with vibrational modes involving motion of conjugated C–C and C–N bonds oriented roughly along the SA of the corrin ring (Figure 1), the hydrogen at the C10 position of the corrin was exchanged with a deuterium atom as described in Section 2.1.44 Due to the rapid exchange of this hydrogen at high pH, the isotope shifts for the oxidized cofactor were determined by using base-off CNCbl at low pH and base-on CNCbl at pH 6. As summarized in Table 1, the rR peaks of these species at ~1500 and ~1610 cm1 are insensitive to H/D exchange at C10, while the feature at ~1550 cm1, which shows the largest enhancement for laser excitation into the -band (Figure 3a), is down-shifted by 2 and 4 cm in the rR spectra of base-on and base-



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off CNCbl(d-10), respectively. Additionally, deuteration at C10 causes the weak rR peak at ~1580 cm to down-shift by 6 cm in the case of base-off CNCbl, but has no effect on the corresponding feature of base-on CNCbl. Intriguingly, the dominant peaks at 1482 cm1 and 1587 cm in the rR spectrum of Co2+Cbl obtained with laser excitation in resonance with the “-band-type” transition (Figure 3b) down-shift by 6 and 2 cm-1, respectively, in response to H/D exchange at C10, which implies that modes involving significant SA-stretching motion couple to an LA-polarized electronic transition. Additional isotopic shifts are displayed by the weak features at 1529 cm (–3 cm) and 1555 cm (–9 cm-1); however, these features show negligible rR enhancement, indicating that the corresponding modes are minimally coupled to the “-band-type” transition of Co2+Cbl. Finally, in the case of Co1+Cbl, the peak at 1487 cm undergoes the largest down-shift (by 4 cm) upon deuteration at C10, while the other resonance-enhanced features shift by a mere 1 – 2 cm. Based on their energies and large isotope shifts, the 1487-cm mode of Co1+Cbl– and the 1482-cm1 mode of Co2+Cbl appear to be similar in nature; however, only the latter shows a large enhancement upon laser excitation in resonance with the “-band-type” transition (cf. Figures 3b and 3c). 3.3. Geometry optimizations and frequency calculations. To aid in the interpretation of the experimental data presented above, computational studies were performed on vitamin B12 models in each of the three cobalt oxidation states. In previous DFT studies of corrinoids, the replacement of the peripheral amide groups with hydrogen atoms was found to be acceptable for the prediction of spectroscopic properties.27-29 Therefore, the same truncation scheme was employed in this study, except that the methyl groups at C5 and C15 were retained, because they strongly influence the frequencies of vibrational modes involving motion of the methine bridges.27


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Additionally, the methyl group at the B5 position of the DMB ligand was included to preserve the steric bulk below the corrin macrocycle.28,29 Although this precaution did not prevent the corrin ring from adopting a slightly flatter conformation during the DFT energy minimizations, the optimized models properly reproduce the experimental trend that the corrin ring becomes less folded upon reduction of the cobalt center (Figure 4 and Table 2). Analytical frequency calculations for each of these geometry-optimized models predict three corrin-based vibrational modes in the 1400–2000 cm1 range that can be classified as totally symmetric in the parent C2v point group. All of these modes, which are referred to as s(#1~3), primarily involve a combination of SA-stretching motion along the N22–C9 and N23–C11 bonds, LA-stretching motion along the N21–C4 and N24–C16 bonds, and methine-stretching motion along the C5–C6 and C15–C14 bonds (Figure 5). With the exception of s(#2), the eigenvector representation of any given mode varies only slightly as a function of the oxidation state of the cobalt ion and, thus, the number of axial ligands. In each case, the s(#1) mode involves in-phase coupling of SA-stretching and methine-stretching motions. The s(#2) modes of CNCbl and Co2+Cbl are characterized by an in-phase coupling of LA-stretching and methine-stretching motions, with an out-of-phase contribution from SA-stretching motion. In the case of Co1+Cbl, the composition of s(#2) is qualitatively similar, except that the methine-stretching motion contributing to this mode involves the C5–C4 and C15–C16 bonds rather than the C5–C6 and C15– C14 bonds. Lastly, the s(#3) mode involves out-of-phase coupling of methine-stretching and LAstretching motions. The calculated frequencies for the s(#1~3) modes are listed in Table 1. Since the totally symmetric vibrational modes are expected to show the largest rR enhancement, each of the DFTpredicted s modes should correspond to one of the dominant rR peaks observed experimentally.



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Indeed, the calculated frequencies for the s modes agree well with the positions of the three most strongly resonance-enhanced features in the experimental rR spectra (Table 1). For each of the corrinoid models considered (Figure 4), the DFT frequency calculations also predict one corrinbased anti-symmetric stretching mode, as, with a frequency between those of s(#2) and s(#3). Consistent with this prediction, a peak is observed in the experimental rR spectra between the features associated with the s(#2) and s(#3) modes whose intensity varies only slightly as a function of laser excitation wavelength (Figure 3). Our experimental data indicate that H/D exchange at C10 causes the rR peaks attributed to the s(#2) and as modes of CNCbl to down-shift, while those assigned to the s(#1) and s(#3) modes remain unaffected (Table 1). Computationally, however, the isotope shift for the s(#1) mode of CNCbl is predicted to be similar to those of the s(#2) and as modes. To examine if this discrepancy between the experimental and computed isotope shifts results from the deletion of the corrin side chain in the computational model, an additional frequency calculation was performed for a hypothetical model whose hydrogen atoms were assigned masses equivalent to those of the entire side chains in the actual cofactor. Due to the minor contributions from the scissoring and wagging motions of CH2 groups on the saturated outer ring of the corrin to the s and as modes, this increase in the masses of the peripheral hydrogen atoms resulted in a relatively uniform downshift of the frequencies predicted for these modes in the hypothetical model (Table 1). However, the calculated shifts of the s(#2), s(#3), and as modes for deuteration at C10 of the hypothetical model remained very similar to those predicted for the original computational model. In fact, the computed shift for the s(#1) mode became even larger for the hypothetical model, indicating that the truncation of peripheral side chains has a substantial, and somewhat unpredictable, effect on the computed isotope shift for the s(#1) mode. For this reason and because of the good overall


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agreement between the experimental and DFT computed vibrational data, no further modifications of our CNCbl model were considered. For Co2+Cbl, the agreement between the experimental and computed frequencies and H/D isotope shifts for the s and as modes is excellent, with no difference exceeding 5 cm-1 (Table 1). Importantly, the computation nicely reproduces the experimental observation that the s(#1) and as modes show considerably larger down-shifts in response to H/D exchange at C10 than the other two modes. In the case of Co1+Cbl, the DFT computed vibrational data also agree well with our experimental results (Table 1). As predicted previously,27 maintaining the methyl substituents on the C5 and C15 methine bridges of Co1+Cblleads to a decrease in the computed frequency of the s(#3) mode (by 23 cm-1 for the model used in our calculations), thereby yielding considerably better agreement with the experimental value (Table 1). Note, however, that while the experimental s(#1) frequency is larger for Co1+Cbl than for Co2+Cbl, our computations predict the opposite trend and overestimate the isotope shift of the as mode for Co1+Cbl. Yet, considering the complex geometric and electronic structures of vitamin B12 and its reduced forms, these minor discrepancies are well within the acceptable range for DFT frequency calculations. 3.4. TDDFT calculations. Although the computed eigenvector representations of the three corrin-based s modes show very little variation between CNCbl, Co2+Cbl, and Co1+Cbl (Figure 5), the experimental rR enhancement patterns for these modes vary considerably as a function of the cobalt oxidation state (Figure 3). To explore the origin of this variation and to corroborate the DFT frequency calculation-based assignments of the rR spectra presented above, the relative rR intensities of the s modes at various laser excitation wavelengths were evaluated within the framework of DFT calculations. First, the donor and acceptor MOs involved in the dominant electronic transitions were identified by carrying out TDDFT calculations. As noted in previous



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computational studies, the agreement between the TDDFT-predicted and experimental Abs spectra is quite remarkable (Figure 6).27-29, 75 In the TDDFT-computed Abs spectrum for CNCbl, all of the major features observed experimentally, in particular the -band in the visible region and the -band in the near-UV region, are reproduced very well, though their energies are consistently overestimated. The one-electron excitations producing the dominant contributions to the - and - bands are listed in Table 3, and a subset of the corresponding donor and acceptor MOs are shown in Figure 7. Consistent with the rR data analysis presented above, our TDDFT results indicate that the - and -bands arise from corrin * transitions. Specifically, the transition responsible for the -band is an essentially pure MO #143 (HOMO)  MO #144 (LUMO) one-electron excitation, while the -band transition contains contributions from several one-electron excitations mostly involving MOs with predominant corrin * character. As expected on the basis of the transformation behavior of the MOs involved in these transitions with respect to reflection about the pseudo-mirror plane of the corrin ring (Figure 7), TDDFT predicts the -band and -band transitions of CNCbl to be LA-polarized and SApolarized, respectively (Figure 6 and Table 3). The TDDFT-computed Abs spectrum for Co2+Cbl correctly reproduces the presence of two major bands in the experimental Abs spectrum below ~35,000 cm-1, as well as the fact that the dominant Abs band in the visible spectral region is blue-shifted from that of CNCbl (Figure 6). Because Co2+Cbl is a paramagnetic S = ½ species, calculations on the corresponding model were carried out using the spin-unrestricted formalism, allowing the spin-up and spin-down electrons to localize in spatially and energetically distinct MOs. The one-electron excitations producing the largest contributions to the dominant Abs bands of Co2+Cbl below 35,000 cm-1 are listed in Table 3, where the labels “a” and “b” are used to distinguish between MOs carrying spin-up and spin-down


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electrons, respectively. Inspection of Table 3 and Figure 7 reveals that the corrin * transitions associated with the - and -bands of CNCbl also serve as the principal contributors to the major Abs bands of Co2+Cbl in the visible (State 20) and near-UV (State 70) spectral regions (note that MOs #134b, #137b, and #139b are nearly identical in composition to MOs #134a, #138a, and #139a, respectively, the latter of which are shown in Figure 7). On the basis of these qualitative considerations alone, the same set of corrin-based normal modes of vibration should dominate rR spectra of Co2+Cbl obtained with 476.5 nm laser excitation and of CNCbl obtained with 514.5 nm laser excitation. In the case of Co1+Cbl, the TDDFT-computed Abs spectrum mimics the overall shape of the experimental spectrum extremely well, including the presence of a dominant band in the nearUV spectral region and a series of weaker features at lower energy, though it generally overestimates the transition energies and intensities (Figure 6c). The transition associated with the most intense band in the simulated Abs spectrum of Co1+Cbl(State 12) is LA-polarized and contains contributions from the corrin 78* (MO#99 and MLCT one-electron excitations. The same LA-polarized corrin 78* excitation also contributes to the transition to State 8, which corresponds to the experimental Abs band with a peak position closest to the 514.5 nm laser excitation wavelength used in our rR experiments. Alternatively, the transition to State 11, which includes a significant contribution from the SA-polarized “-band-type” electronic excitation from the corrin 7-based MO to the corrin 9*-based MO, can be assigned to the experimental Abs band peaking closest to the 454.5 nm laser excitation wavelength. 3.5. Relative rR intensities of s modes. The TDDFT-based descriptions of the relevant electronic transitions for the three corrinoid species investigated show that in each case the LApolarized electronic excitation from the corrin 7-based MO to the corrin 8*-based MO and the



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SA-polarized electronic excitation from the corrin 7-based MO to the corrin 9*-based MO are key contributors to the major visible/near-UV Abs bands that give rise to predominant enhancement of the corrin-based vibrations (Figures 6 and 7, Table 3). Despite this similarity, the rR spectra obtained in resonance with any given corrin * transition show dramatic differences in terms of the enhancement pattern of the corrin-based vibrational modes as a function of the cobalt oxidation state. To explore the origin of this puzzling observation, three different DFT-based computational approaches were employed for predicting the relative rR intensities of the s modes at laser excitation wavelengths roughly matching the peak positions of the dominant Abs features. For the closed-shell systems CNCbl and Co1+Cbl, the magnitude of the excited state energy gradient along a given normal mode Q at the ground state equilibrium geometry, (Exs/Q)eq, could be computed directly using the ADF software package. The relative rR intensities were then estimated from I  [(Exs/Q)eq]2, which assumes identical harmonic potentials for the ground and excited states (Method 1).55,81 Alternatively, the dimensionless displacements of the excited state potential energy surfaces (PESs) from the ground state PES along the s modes, Q[s(#1~3)], were estimated by performing excited-state geometry optimizations (Method 2). Although convergence was not achieved in some cases, we were able to obtain the optimized geometries of CNCbl and Co1+Cbl in the electronic excited states corresponding to the LA-polarized and SA-polarized corrin * transitions of interest. The excited-state distortions were then expressed in terms of internal coordinate changes and projected onto the s modes of the corrinoids in their electronic ground states, and the rR intensities were estimated from I  [Q]2 (only the -conjugated portion of the macrocycle was considered in this analysis, while the minute scissoring and wagging motions of the sp3 hybridized CH2 groups were ignored). Finally, to estimate the Q[s(#1~3)] values for Co2+Cbl, the PESs for the relevant electronic excited states were computed by distorting the computational


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model from its optimized ground-state geometry in fixed increments along the three s modes and performing excited-state single-point DFT calculations (Method 3). For comparison, the same approach was also applied to the CNCbl and Co1+Cbl models. While the peak associated with the s (#1) mode dominates the experimental rR spectrum of CNCbl obtained with laser excitation into the -band (Figure 3a), the computational Method 1 (i.e., the ADF-implemented rR intensity calculation) predicts the s (#2) mode to be similarly enhanced as the s (#1) mode for excitation in resonance with this transition (Table 4). Alternatively, both Methods 2 and 3 correctly predict that the PES of the excited state associated with the -band transition (modeled as the MO#143MO#144 one-electron excitation) is displaced more substantially along the s (#1) mode than the remaining s modes. Therefore, the calculated relative rR intensity of the s (#1) mode using Methods 2 and 3 is greater than those of the other two s modes, though it is still somewhat underestimated (Table 4). Turning to the -band transition in the near-UV region, all three methods predict the s (#2) mode to be preferentially enhanced for laser excitation in resonance with this transition (represented by the MO#143MO#145 excitation). This prediction is qualitatively consistent with the experimental rR spectrum obtained with 364-nm excitation (Figure 3a), though the relative intensity of the s (#1) mode is largely underestimated by all three methods (Table 4). As described above, the rR spectra of Co2+Cbl and CNCbl obtained with laser excitation in resonance with the “-band-type” corrin * transition differ in two important ways (cf. Figures 3a and 3b). First, in addition to the s (#1) mode that dominates the CNCbl spectrum, the s (#3) mode also shows significant enhancement in the rR spectrum of Co2+Cbl. Second, while H/D exchange at C10 has no effect on the s (#1) mode of CNCbl, as expected for a mode coupling to a LA-polarized corrin * transition, it causes the s (#1) mode of Co2+Cbl to down-shift by 6 cm-1



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(Table 1), implying the involvement of significant SA-stretching motion in this mode. Consistent with these findings, our rR intensity calculations for the Co2+Cbl model using Method 3 predict the PES of the excited state corresponding to the “-band-type” transition to be displaced along several coordinates, including the s (#1) mode with a computed down-shift of 5 cm upon deuteration at C10 (Table 4). The computed intensities for the s (#2) and s (#3) modes using Method 3 are, however, somewhat over- and underestimated, respectively. Because we were unable to excite directly into the dominant Abs band of Co1+Cbl at 386.5 nm, the experimental rR intensities reported in Table 4 do not precisely reflect the enhancement of the s modes for laser excitation in resonance with the “-band-type” transition. However, from a comparison of the rR spectra of Co1+Cbl obtained with laser excitation at 406.7 and 363.8 nm,27 it can be inferred that tuning the laser from 363.8 nm to 386.5 nm would cause the relative rR intensities of the s (#2) and s (#3) modes to decrease and increase, respectively. Consistent with this assumption, all three methods predict the s (#3) mode to couple most strongly to the LApolarized “-band-type” transition (represented by the MO#99MO#103 excitation), though in each case the relative intensity of the s (#2) mode is presumably underestimated. Likewise, all three computational methods concur with the experimental finding that the s (#1) mode of Co1+Cbl, which involves significant SA-stretching motion based on its 4-cm-1 down-shift upon H/D exchange at C10 (Table 1), is most strongly coupled to the SA-polarized “-band-type” transition (modeled as the MO#99MO#104 one-electron excitation).

4. Discussion From the experimental and computational results obtained in this study, several interesting trends can be noted with regards to variations in the frequencies and relative rR intensities of the


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corrin-based vibrational modes of corrinoids as a function of the Co oxidation state. First, the frequency of a given mode tends to decrease as the effective nuclear charge of the cobalt ion decreases from CNCbl to Co2+Cbl and Co1+Cbl (Figure 3 and Table 1). This trend can be understood in terms of the different electronic structures of the three corrinoid species investigated. The orbital plots in Figure 7 show that the two lowest-energy, corrin *-based unoccupied MOs possess variable amounts of Co 3dxz and 3dyz orbital character, signifying varying degrees of mixing between the filled Co 3dxz and 3dyz orbitals and the empty corrin * frontier orbitals. The extent of this Co 3dcorrin * “backbonding” is controlled by the energy gap between interacting orbitals; hence, it increases from CNCbl (where the filled Co 3d orbitals are too low in energy to interact appreciably with the corrin * orbitals) to Co2+Cbl and Co1+Cbl. The partial population of the corrin * orbitals with decreasing Co oxidation state leads to a weakening of the conjugated C– C and C–N bonds of the corrin ring and, thus, a decrease in the frequencies of the corrin-based s modes. Second, in rR spectra obtained upon laser excitation in resonance with the “-band-type” transition, the ratio of the relative intensities of the s (#1) and s (#3) modes shows a marked decrease from CNCbl to Co2+Cbl and Co1+Cbl (Figure 3 and Table 1). The s (#1) mode primarily involves in-phase coupling of SA-stretching and methine-stretching motions, while the s(#3) mode mainly entails out-of-phase coupling of LA-stretching and methine-stretching motions of the corrin ring (Figure 5). In the case of CNCbl, thes (#1) mode shows by far the greatest rR enhancement of all corrin-based vibrational modes for laser excitation into the -band (Figure 3), indicating that in the excited state associated with the LA-polarized -band transition, the corrin ring is, in fact, primarily distorted along its SA. Alternatively, for Co2+Cbl, the s (#1) and s (#3) modes are similarly enhanced in the rR spectrum obtained with laser excitation in resonance with the



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analogous LA-polarized corrin * transition, and in the case of Co1+Cbl, it is the LA-polarized s (#3) mode that most strongly couples to this transition. Lastly, upon excitation in resonance with a given corrin * transition, the relative rR enhancement of the s (#2) mode, which involves both LA- and SA-stretching motions, changes considerably as a function of the Co oxidation state. In the case of CNCbl, this mode is predominantly enhanced upon laser excitation into the -band, while for Co1+Cbl, it displays the largest enhancement for excitation in resonance with the “-band-type” transition. No significant enhancement of this mode is observed in rR spectra of Co2+Cbl obtained with laser excitation across the entire visible/near-UV spectral region. Even without an explicit knowledge of the origin of these peculiar trends, which will be explored further below, it can be concluded that the corrin-based vibrational modes of corrinoids should not be assigned solely on the basis of their rR excitation profiles. Vibrations mainly involving stretching of conjugated C–C and C–N bonds oriented along one axis of the corrin ring may in fact couple to a perpendicularly electronic transition; e.g., the LA-polarized -band transition of CNCbl gives rise to predominant enhancement of the SA-polarized s (#1) mode, as described above. Another experimental technique that has been used frequently for distinguishing between SA- and LA-polarized corrin ring vibrations is to carry out H/D exchange at C10, which should primarily affect the frequencies of SA-polarized modes. However, as is evident from the data summarized in Table 1, isotope shifts for the SA-polarized s (#1) mode of corrinoids vary substantially as a function of the Co oxidation state. While H/D exchange at C10 leads to an obvious downshift of the s (#1) mode for Co2+Cbl and Co1+Cbl, it does not appear to have any noticeable effect in the case of CNCbl, though this may be due, at least in part, to fast D/H backexchange at high pH. Our DFT frequency calculations qualitatively reproduce these experimental


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findings, predicting the smallest, albeit still quite significant, isotope shift for the s (#1) mode of CNCbl. Interestingly, by assigning the hydrogen atoms in our CNCbl model masses equivalent to those of the entire side chains in the actual cofactor, the computed isotope shift for the s(#1) mode became even larger (Table 3), indicating that caution must be exercised when interpreting computed isotope shifts for truncated corrinoid models. Despite the fact that our computations using Methods 2 and 3 do not explicitly account for contributions from MLCT excitations to the “-band-type” and “-band-type” transitions, which are expected to become increasingly important from CNCbl to Co2+Cbl and Co1+Cbl,82 they reproduce quite nicely the experimental finding that the relative rR intensities of the corrin-based s modes vary considerably as a function of the Co oxidation state. A key electronic structural difference between CNCbl, Co2+Cbl, and Co1+Cbl is the degree of mixing between the Co 3d and corrin frontier orbitals, which modulates the compositions of the corrin /*-based MOs and, thus, the rR enhancement patterns of the corrin-based s modes. Because the Co 3dyz and corrin 8* orbitals are anti-symmetric with respect to reflection about the pseudo-mirror plane oriented along the corrin SA, these orbitals can mix. The degree of this mixing, or “backbonding”, is inversely proportional to the energy gap between interacting orbitals. Hence, the amount of Co 3dyz orbital character in the corrin 8*-based MO increases from 3.3% in CNCbl (MO #144) to 18.3% in Co1+Cbl (MO #103 in Figure 7). Similarly, the Co 3dxz and corrin 9* orbitals, both of which are symmetric with respect to reflection about the pseudo-mirror plane of the corrin ring, display an increasing degree of mixing with decreasing Co oxidation state, with the Co 3dxz orbital contribution to the corrin 9*-based MO increasing from 6.0 % in CNCbl (sum of contributions to MOs #145 and #146) to 16.5% in Co1+Cbl (MO#104 in Figure 7).



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Because in the corrin 8*- and 9*-based MOs the Co ion and N atoms of the corrin ring engage in -antibonding interactions (Figure 7), the way by which the corrin ring distorts upon population of these two MOs in electronic excited states will depend on the extent of Co 3dcorrin * “backbonding” and, thus, the Co oxidation state. For example, when an electron is promoted to the corrin 8*-based MO of Co1+Cbl (MO #103, Figure 7), a corrin ring distortion along the SApolarized s(#1) mode, involving a lengthening of the N22–C9 and N23–C11 bonds, would increase the Co–N -antibonding interactions to a much greater extent than a ring distortion along the LApolarized s(#3) mode, which entails N21–C4 and N24–C16 bond elongations. It may well be for this reason that laser excitation in resonance with the “-band-type” transition, formally corresponding to the corrin 7  8* transition, gives rise to predominant rR enhancement of the s(#3) mode for Co1+Cbl but of the s(#1) mode for CNCbl, where the corrin 8*-based acceptor MO contains a much smaller Co 3dyz orbital contribution. Similarly, the variable amount of Co 3dxz orbital character in the corrin 9*-based MO may be responsible for the fact that the “-band-type” transition, formally the corrin 7  8* transition, gives rise to a large rR enhancement of the s(#1) mode for Co1+Cbl, while in the case of CNCbl it is the s(#2) mode that most strongly couples to this transition. Although our computational approaches quite successfully reproduce the key experimental trends regarding the frequencies and relative rR intensities of the corrin-based vibrational modes of vitamin B12 as a function of the Co oxidation state, electronic excitations not explicitly accounted for in these calculations will likely influence the rR enhancement profiles of the corrrin-based s modes. In particular, the TDDFT computational results obtained in this study suggest that the electronic transitions associated with the dominant Abs bands of Co2+Cbl and Co1+Cbl also contain


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27

contributions from excitations between the corrin /Co 3d-based and corrin *-based MOs and between the Co 3d-based and corrin *-based MOs (Table 3). A visual representation of the changes in electronic structure accompanying these transitions is provided by electron density difference maps (EDDMs). Figure 8 shows the EDDMs for the “band-type” transitions of CNCbl, Co2+Cbl, and Co1+Cbl. While in the case of CNCbl electron density is redistributed almost exclusively within the corrin  system, an additional flow of electron density from the cobalt ion to the corrin  system is observed in the reduced cofactor forms. Most notably, for Co1+Cbl the “-band-type” transition induces a substantial shift of electron density from the Co 3dxz-based MO #100 to the corrin 10*-based MO #105. As a result, the -antibonding interaction between the Co 3dxz and N(corrin) 2p orbitals will decrease, facilitating geometric relaxation along the corrin LA. This scenario is consistent with the experimental observation that the s(#3) mode, rather than the s(#1) mode, is predominantly enhanced in the rR spectrum of Co1+Cbl obtained with laser excitation in resonance with the “-band-type” transition (Figure 3 and Table 4).

5. Conclusion We have shown that the relative rR enhancements of the corrin-based s mode modes of vitamin B12 vary strongly as a function of the Co oxidation state. Our computational data suggest that this variation reflects large differences in the degree of mixing between the occupied Co 3d orbitals and empty corrin * orbitals in CNCbl, Co2+Cbl, and Co1+Cbl. As a result, the corrinbased vibrational modes of corrinoids should not be assigned solely on the basis of their rR excitation profiles, because vibrations mainly involving stretching of conjugated C–C and C–N



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Page 28 of 46

28

bonds oriented along one axis of the corrin ring may, in fact, couple to a perpendicularly electronic transition.

Acknowledgment. This work was supported by the National Science Foundation Grant MCB-0238530.

Supporting Information Available: DFT optimized geometries of CNCbl, Co2+Cbl, and Co1+Cbl in their electronic ground states, and of CNCbl and Co1+Cbl in the relevant excited states. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1)

The Nomenclature of Corrinoids (1973 recommendations). IUPAC-IUB Commission on

Biochemical Nomenclature. Biochemistry 1974, 13, 1555-1560. (2)

Hodgkin, D. C.; Kamper, J.; Mackay, M.; Pickworth, J.; Trueblood, K. N.; White, J. G. Structure of

Vitamin B12. Nature 1956, 178, 64-66. (3)

Hodgkin, D. C. P.; Robertson, J. H.; Trueblood, K. N.; Prosen, R. J.; White, J. G. Structure of

Vitamin B12. Nature 1955, 176, 325-328. (4)

Bouquiere, J. P.; Finney, J. L.; Lehmann, M. S.; Lindley, P. F.; Savage, H. F. J. High-resolution

Neutron Study of Vitamin B12 Coenzyme at 15 K - Structure Analysis and Comparison With the Structure at 279 K. Acta Crystallogr. B. 1993, 49, 79-89. (5)

Randaccio, L.; Furlan, M.; Geremia, S.; Slouf, M.; Srnova, I.; Toffoli, D. Similarities and

Differences between Cobalamins and Cobaloximes. Accurate Structural Determination of Methylcobalamin


Page 29 of 46

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


29 and of LiCl- and KCl-Containing Cyanocobalamins by Synchrotron Radiation. Inorg. Chem. 2000, 39, 34033413. (6)

Banerjee, R.; Ragsdale, S. W. The Many Faces of Vitamin B12: Catalysis by Cobalamin-Dependent

Enzymes. Annu. Rev. Biochem. 2003, 72, 209-247. (7)

Buan, N. R.; Suh, S. J.; Escalante-Semerena, J. C. The EutT Gene of Salmonella enterica Encodes

an Oxygen-Labile, Metal-Containing ATP : Corrinoid Adenosyltransferase Enzyme. J. Bacteriol. 2004, 186, 5708-5714. (8)

Chowdhury, S.; Banerjee, R. Thermodynamic and Kinetic Characterization of Co-C Bond

Homolysis Catalyzed by Coenzyme B12-Dependent Methylmalonyl-CoA Mutase. Biochemistry 2000, 39, 7998-8006. (9)

Johnson, C. L. V.; Pechonick, E.; Park, S. D.; Havemann, G. D.; Leal, N. A.; Bobik, T. A.

Functional Genomic, Biochemical, and Genetic Characterization of the Salmonella pduO Gene, an ATP:Cob(I)alamin Adenosyltransferase Gene. J. Bacteriol. 2001, 183, 1577-1584. (10)

Marsh, E. N. G.; Ballou, D. P. Coupling of Cobalt-Carbon Bond Homolysis and Hydrogen Atom

Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase. Biochemistry 1998, 37, 11864-11872. (11)

Thoma, N. H.; Evans, P. R.; Leadlay, P. F. Protection of Radical Intermediates at the Active Site of

Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase. Biochemistry 2000, 39, 9213-9221. (12)

Brown, K. L.; Zou, X. Thermolysis of Coenzymes B12 at Physiological Temperatures: Activation

Parameters for Cobalt-Carbon Bond Homolysis and a Quantitative Analysis of the Perturbation of the Homolysis Equilibrium by the Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii. J. Inorg. Biochem. 1999, 77, 185-195. (13)

Gerfen, G. J.; Licht, S.; Willems, J. P.; Hoffman, B. M.; Stubbe, J. Electron Paramagnetic

Resonance Investigations of a Kinetically Competent Intermediate Formed in Ribonucleotide Reduction: Evidence for a Thiyl Radical-Cob(II)alamin Interaction. J. Am. Chem. Soc. 1996, 118, 8192-8197.



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

Page 30 of 46

30 (14)

Matthews, R. G. Cobalamin-dependent Methyltransferases. Acc. Chem. Res. 2001, 34, 681-689.

(15)

Banerjee, R. Radical Carbon Skeleton Rearrangements: Catalysis by Coenzyme B 12-Dependent

Mutases. Chem. Rev. 2003, 103, 2083-2094. (16)

Giorgetti, M.; Ascone, I.; Berrettoni, M.; Conti, P.; Zamponi, S.; Marassi, R. In Situ X-ray

Absorption Spectroelectrochemical Study of Hydroxocobalamin. J. Biol. Inorg. Chem. 2000, 5, 156-166. (17)

Perry, C. B.; Marques, H. M. Fifty Years of X-ray Crystallography of Vitamin B12 and Its

Derivatives. S. Afr. J. Sci. 2004, 100, 368-380. (18)

Wirt, M. D.; Sagi, I.; Chance, M. R. Formation of a Square-planar Co(I) B12 Intermediate-

Implications for Enzyme Catalysis. Biophys. J. 1992, 63, 412-417. (19)

Roth, J. R.; Lawrence, J. G.; Bobik, T. A. Cobalamin (Coenzyme B12): Synthesis and Biological

Significance. Annu. Rev. Microbiol. 1996, 50, 137-181. (20)

Warren, M. J.; Raux, E.; Schubert, H. L.; Escalante-Semerena, J. C. The Biosynthesis of

Adenosylcobalamin (Vitamin B12). Nat. Prod. Rep. 2002, 19, 390-412. (21)

Geno, M. K.; Halpern, J. Why Does Nature Not Use the Porphyrin Ligand in Vitamin B12. J. Am.

Chem. Soc. 1987, 109, 1238-1240. (22)

Randaccio, L.; Geremia, S.; Nardin, G.; Wuerges, J. X-ray Structural Chemistry of Cobalamins.

Coord. Chem. Rev. 2006, 250, 1332-1350. (23)

Halpern, J. Mechanisms of Coenzyme B12-Dependent Rearrangements. Science 1985, 227, 869-875.

(24)

Hay, B. P.; Finke, R. G. Thermolysis of the Co-C Bond in Adenosylcorrins. 3. Quantification of the

Axial Base Effect in Adenosylcobalamin by the Synthesis and Thermolysis of Axial Base-Free Adenosylcobinamide - Insights into the Energetics of Enzyme-Assisted Cobalt Carbon Bond Homolysis. J. Am. Chem. Soc. 1987, 109, 8012-8018.


Page 31 of 46

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


31 (25)

Pett, V. B.; Liebman, M. N.; Murrayrust, P.; Prasad, K.; Glusker, J. P. Conformational Variability of

Corrins - Some Methods of Analysis. J. Am. Chem. Soc. 1987, 109, 3207-3215. (26)

Giannotti, C. Electronic Spectra of B12 and Related Systems. In B12; Dolphin, D., Ed.; Wiley-

Interscience: New York, 1982. (27)

Liptak, M. D.; Brunold, T. C. Spectroscopic and Computational Studies of Co1+cobalamin: Spectral

and Electronic Properties of the "Superreduced" B12 Cofactor. J. Am. Chem. Soc. 2006, 128, 9144-9156. (28)

Stich, T. A.; Brooks, A. J.; Buan, N. R.; Brunold, T. C. Spectroscopic and Computational Studies of

Co3+corrinoids: Spectral and Electronic Properties of the B12 Cofactors and Biologically Relevant Precursors. J. Am. Chem. Soc. 2003, 125, 5897-5914. (29)

Stich, T. A.; Buan, N. R.; Brunold, T. C. Spectroscopic and Computational Studies of Co2+

corrinoids: Spectral and Electronic Properties of the Biologically Relevant Base-on and Base-off Forms of Co2+ cobalamin. J. Am. Chem. Soc. 2004, 126, 9735-9749. (30)

Dong, S. L.; Padmakumar, R.; Banerjee, R.; Spiro, T. G. Co-C Bond Activation in B12-Dependent

Enzymes: Cryogenic Resonance Raman Studies of Methylmalonyl-Coenzyme A Mutase. J. Am. Chem. Soc. 1999, 121, 7063-7070. (31)

Dong, S. L.; Padmakumar, R.; Maiti, N.; Banerjee, R.; Spiro, T. G. Resonance Raman Spectra show

that Coenzyme B12 Binding to Methylmalonyl-Coenzyme A Mutase Changes the Corrin Ring Conformation but leaves the Co-C Bond Essentially Unaffected. J. Am. Chem. Soc. 1998, 120, 9947-9948. (32)

Huhta, M. S.; Chen, H. P.; Hemann, C.; Hille, C. R.; Marsh, E. N. G. Protein-Coenzyme Interactions

in Adenosylcobalamin-Dependent Glutamate Mutase. Biochem. J. 2001, 355, 131-137. (33)

George, W. O. Resonance Raman Spectrum of Cyanocobalamin (Vitamin B12). Appl. Spectrosc.

1973, 27, 390-391. (34)

Mayer, E.; Gardiner, D. J.; Hester, R. E. Resonance Raman Spectra of Vitamin B12 and Some Cobalt

Corrinoid Derivatives. J. Chem. Soc., Faraday Trans. 1973, 69, 1350-1358.



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

Page 32 of 46

32 (35)

Mayer, E.; Gardiner, D. J.; Hester, R. E. The Selective Enhancement of Band Intensities in

Resonance Raman Spectra of Cobalt Corrinoids. Mol. Phys. 1973, 26, 783-787. (36)

Salama, S.; Spiro, T. G. Visible and Near-Ultraviolet Resonance Raman Spectra of Photolabile

Vitamin B12 Derivatives with a Rapid-Flow Technique. J. Raman Spectrosc. 1977, 6, 57-60. (37)

Galluzzi, F.; Garozzo, M.; Ricci, F. F. Resonance Raman Scattering and Vibronic Coupling in

Aquo- and Cyano-cobalamin. J. Raman Spectrosc. 1974, 2, 351-362. (38)

Andruniow, T.; Zgierski, M. Z.; Kozlowski, P. M. Vibrational Analysis of Methylcobalamin. J.

Phys. Chem. A 2002, 106, 1365-1373. (39)

Puckett, J. M.; Mitchell, M. B.; Hirota, S.; Marzilli, L. G. Near-IR FT-Raman Spectroscopy of

Methyl-B12 and Other Cobalamins and of Imidazole and Imidazolate Methylcobinamide Derivatives in Aqueous Solution. Inorg. Chem. 1996, 35, 4656-4662. (40)

Blout, E. R.; Fields, M.; Karplus, R. Absorption Spectra. VI. The Infrared Spectra of Certain

Compounds Containing Conjugated Double Bonds. J. Am. Chem. Soc. 1948, 70, 194-198. (41)

Gavin Jr., R. M.; Rice, S. A. Correlation of Pi-Electron Density with Vibrational Frequencies of

Linear Polyenes. J. Chem. Phys. 1971, 55, 2675-2681. (42)

Beaven, G. H.; Holiday, E. R.; Johnson, E. A.; Ellis, B.; Petrow, V. The Chemistry of Anti-

pernicious Anaemia Factors: Part VI. The Mode of Combination of Component Alpha in Vitamin B12. J. Pharm. Pharmacol. 1950, 2, 944-955. (43)

Schindler, O. Reduktionsprodukte von Vitamin B12. Vesuche mit Vitamin B12, 2.Mitteilung. Helv.

Chim. Acta 1951, 34, 1356-1361. (44)

Hill, H. A. O.; Pratt, J. M.; Williams, R. J. P. Chemistry of Vitamin B12. 3. Proton Magnetic

Resonance Spectra of Some Cobalamins. J. Chem. Soc. 1965, 2859-2865.


Page 33 of 46

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


33 (45)

Kräutler, B.; Keller, W.; Kratky, C. Coenzyme-B12 Chemistry - the Crystal and Molecular Structure

of Cob(II)alamin. J. Am. Chem. Soc. 1989, 111, 8936-8938. (46)

ADF2008.01; SCM: Theoretical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.

(47)

Perdew, J. P.; Burke, K.; Ernzerhof, M. Generalized Gradient Approximation Made Simple. Phys.

Rev. Lett. 1996, 77, 3865-3868. (48)

Vosko, S. H.; Wilk, L.; Nusair, M. Accurate Spin-Dependent Electron Liquid Correlation Energies

for Local Spin-Density Calculations - a Critical Analysis. Can. J. Phys. 1980, 58, 1200-1211. (49)

Berces, A.; Dickson, R. M.; Fan, L. Y.; Jacobsen, H.; Swerhone, D.; Ziegler, T. An Implementation

of the Coupled Perturbed Kohn-Sham Equations: Perturbation Due to Nuclear Displacements. Comput. Phys. Commun. 1997, 100, 247-262. (50)

Guerra, C. F.; Snijders, J. G.; te Velde, G.; Baerends, E. J. Towards an Order-N DFT Method.

Theor. Chem. Acc. 1998, 99, 391-403. (51)

Jacobsen, H.; Berces, A.; Swerhone, D. P.; Ziegler, T. Analytic Second Derivatives of Molecular

Energies: A Density Functional Implementation. Comput. Phys. Commun. 1997, 100, 263-276. (52)

Velde, G. T.; Bickelhaupt, F. M.; Baerends, E. J.; Guerra, C. F.; Van Gisbergen, S. J. A.; Snijders, J.

G.; Ziegler, T. Chemistry with ADF. J. Comput. Chem. 2001, 22, 931-967. (53)

Wolff, S. K. Analytical Second Derivatives in the Amsterdam Density Functional Package. Int. J.

Quantum. Chem. 2005, 104, 645-659. (54)

Jmol: an Open-Source Java Viewer for Chemical Structures in 3D.

(55)

Neese, F.; Petrenko, T.; Ganyushin, D.; Olbrich, G. Advanced Aspects of Ab Initio Theoretical

Optical Spectroscopy of Transition Metal Complexes: Multiplets, Spin-Orbit Coupling and Resonance Raman Intensities. Coord. Chem. Rev. 2007, 251, 288-327.



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

Page 34 of 46

34 (56)

Neugebauer, J.; Baerends, E. J.; Efremov, E. V.; Ariese, F.; Gooijer, C. Combined Theoretical and

Experimental Deep-UV Resonance Raman Studies of Substituted Pyrenes. J. Phys. Chem. A 2005, 109, 2100-2106. (57)

Neugebauer, J.; Baerends, E. J.; Nooijen, M. Vibronic Coupling and Double Excitations in Linear

Response Time-Dependent Density Functional Calculations: Dipole-Allowed States of N2. J. Chem. Phys. 2004, 121, 6155-6166. (58)

Neugebauer, J.; Baerends, E. J.; Nooijen, M. Vibronic Structure of the Permanganate Absorption

Spectrum from Time-Dependent Density Functional Calculations. J. Phys. Chem. A 2005, 109, 1168-1179. (59)

Nooijen, M. First-Principles Simulation of the UV Absorption Spectrum of Ketene. Int. J. Quantum.

Chem. 2003, 95, 768-783. (60)

ORCA - An Ab Initio, DFT and Semiempirical Electronic Structure Package; Version 2.6.4 ed., July

2007. (61)

Neese, F.; Olbrich, G. Efficient Use of the Resolution of the Identity Approximation in Time-

Dependent Density Functional Calculations with Hybrid Density Functionals. Chem. Phys. Lett. 2002, 362, 170-178. (62)

Baerends, E. J.; Ellis, D. E.; Ros, P. Self-Consistent Molecular Hartree-Fock-Slater Calculations - I.

The Computational Procedure. Chem. Phys. 1973, 2, 41-51. (63)

Dunlap, B. I.; Connolly, J. W. D.; Sabin, J. R. Some Approximations in Applications of X-Alpha

Theory. J. Chem. Phys. 1979, 71, 3396-3402. (64)

Eichkorn, K.; Treutler, O.; Ohm, H.; Haser, M.; Ahlrichs, R. Auxiliary Basis Sets to Approximate

Coulomb Potentials. Chem. Phys. Lett. 1995, 240, 283-289. (65)

Eichkorn, K.; Weigend, F.; Treutler, O.; Ahlrichs, R. Auxiliary Basis Sets for Main Row Atoms and

Transition Metals and Their Use to Approximate Coulomb Potentials. Theor. Chem. Acc. 1997, 97, 119-124.


Page 35 of 46

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


35 (66)

Kendall, R. A.; Fruchtl, H. A. The Impact of the Resolution of the Identity Approximate Integral

Method on Modern Ab Initio Algorithm Development. Theor. Chem. Acc. 1997, 97, 158-163. (67)

Vanalsenoy, C. Ab Initio Calculations on Large Molecules - The Multiplicative Integral

Approximation. J. Comput. Chem. 1988, 9, 620-626. (68)

Whitten, J. L. Coulombic Potential-Energy Integrals and Approximations. J. Chem. Phys. 1973, 58,

4496-4501. (69)

Godbout, N.; Salahub, D. R.; Andzelm, J.; Wimmer, E. Optimization of Gaussian-Type Basis-Sets

for Local Spin-Density Functional Calculations .1. Boron through Neon, Optimization Technique and Validation. Can. J. Chem. 1992, 70, 560-571. (70)

Schafer, A.; Horn, H.; Ahlrichs, R. Fully Optimized Contracted Gaussian-Basis Sets for Atoms Li to

Kr. J. Chem. Phys. 1992, 97, 2571-2577. (71)

Hirata, S.; Head-Gordon, M. Time-Dependent Density Functional Theory within the Tamm-Dancoff

Approximation. Chem. Phys. Lett. 1999, 314, 291-299. (72)

Bergman, D. L.; Laaksonen, L.; Laaksonen, A. Visualization of Solvation Structures in Liquid

Mixtures. J. Mol. Graphics Modell. 1997, 15, 301-306. (73)

Laaksonen, L. A Graphics Program for the Analysis and Display of Molecular-Dynamics

Trajectories. J. Mol. Graphics 1992, 10, 33-34. (74)

Laaksonen, L. gOpenMol; 3.00 ed., 2005.

(75)

Andruniow, T.; Kozlowski, P. M.; Zgierski, M. Z. Theoretical Analysis of Electronic Absorption

Spectra of Vitamin B12 Models. J. Chem. Phys. 2001, 115, 7522-7533. (76)

Hirota, S.; Marzilli, L. G. Vibrational Spectroscopy of B12 and Related Compounds. In Chemistry

and Biochemistry of B12; Banerjee, R., Ed.; John Wiley & Sons, Inc.: New York, 1999; pp 239-260.



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

Page 36 of 46

36 (77)

Czernuszewicz, R. S.; Spiro, T. G. IR, Raman, and Resonance Raman Spectroscopy. In Inorganic

Electronic Structure and Spectroscopy; Solomon, E. I., Lever, A. B. P., Eds.; John Wiley & Sons, Inc.: New York, 1999; Vol. 1; pp 353-441. (78)

Shiang, J. J.; Cole, A. G.; Sension, R. J.; Hang, K.; Weng, Y. X.; Trommel, J. S.; Marzilli, L. G.;

Lian, T. Q. Ultrafast Excited-State Dynamics in Vitamin B12 and Related Cob(III)alamins. J. Am. Chem. Soc. 2006, 128, 801-808. (79)

Lodowski, P.; Jaworska, M.; Kornobis, K.; Andruniow, T.; Kozlowski, P. M. Electronic and

Structural Properties of Low-lying Excited States of Vitamin B12. J. Phys. Chem. B 2011, 115, 13304-13319. (80)

Toohey, J. I. A Vitamin B12 Compound Containing No Cobalt. Proc. Natl. Acad. Sci. USA 1965, 54,

934-942. (81)

Shorygin, P. P. Raman Scattering of Light Near and Far from Resonance. Sov. Phys. Usp. 1973, 16,

99-120. (82)

Jensen, K. P. Electronic Structure of Cob(I)alamin: The Story of an Unusual Nucleophile. J. Phys.

Chem. B 2005, 109, 10505-10512.


Page 37 of 46

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37

Table 1. Experimental and computed frequencies and isotope shifts for H/D exchange at C10 (in cm1) of the relevant corrin-based vibrational modes Species

Source

CNCbl

Exp. at pH 6 Exp. at low pH

2+

Co Cbl

a



Co Cbl

s(#2)

as

s(#3)

1505 (0)

1550 (–2)

1580 (0)

1606 (0)

1507 (0)

1551 (–4)

1583 (–6)

1615 (0)

Calc.

1498 (–4)

1540 (–3)

1577 (–4)

1598 (–1)

Calc.2b

1493 (–7)

1536 (–3)

1573 (–5)

1595 (–1)

Exp.

1482 (–6)

1529 (–3)

1555 (–9)

1587 (–2)

1487 (–5)

1530 (–2)

1557 (–6)

1585 (–1)

1487 (–4)

1503 (–2)

1530 (–1)

1567 (–2)

Calc. 1+

s(#1)

Exp.

c

Calc. 1478 (–6) 1519 (–1) 1526 (–7) 1562 (–1) In 0.5 mM HCl/H2O (0.5 mM DCl/D2O) solution, conditions under which CNCbl is in the base-off form. b Calculated for a hypothetical model whose hydrogen atoms were assigned masses equivalent to those of the side chains in the actual (i.e., complete) cofactor. c From ref (27). a

Table 2. Corrin fold angles and axial bond distances for CNCbl, Co2+Cbl, and Co1+Cblas determined by X-ray crystallography and DFT energy minimizations on truncated models Species

Method 5

a

b

Co–axial ligand

CNCbl

X-ray

18.3

3.3

2.041 ()/ 1.886 () Å

CNCbl

DFT (Fig. 4a)

14.1

1.0

2.136 ()/ 1.863 () Å

16.2

3.6

1.928 Å

10.4

8.0

2.213 Å

2+

Co Cbl 2+

Co Cbl

45

X-ray

DFT (Fig. 4b)

Co1+Cbl DFT (Fig. 4c) 2.2 0.0 a  corresponds to the LA fold angle, defined as the angle between the plane composed of N21, C4, C5, C6, and N22 and the vector that connects the Co center and C15. b corresponds to the SA fold angle, defined as the angle between the plane composed of N22, C9, C10, C11, and N23 and the vector that passes though the Co center and the midpoint between C1 and C19.



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Page 38 of 46

38

Table 3. TDDFT-calculated energies (E), oscillator strengths (f), polarizations (P), and contributions from dominant one-electron excitations (%) for the major electronic transitions of CNCbl, Co2+Cbl, and Co1+Cbl Species

State

E (cm1)

f

P

Excitation

%

CNCbl

1

20434

0.1188

LA

143  144

89

cor- (HOMO)

cor-* (LUMO)

21

30457

0.1683

SA

140  147

16

DMB-/Co 3dxz

Co 3dxy/3dz²

142  144

12

cor-/Co 3dyz

cor-* (LUMO)

143  145

10

cor- (HOMO)

cor-*/Co 3dz²/3dxy

143  146

9

cor- (HOMO)

cor-*/Co 3dz²/3dxy

134a  138a

14

cor-/Co 3dz²

cor-* (LUMO)

2+

Co Cbl

20

70 1+



Co Cbl

8 11 12

21

24104

34301 23479 26983 28882

34262

0.0371

0.3015 0.0167 0.0172 0.4276

0.1939

LA

SA LA SA LA

SA

donor MO

acceptor MO

136a  139a

15

Co 3dyz/cor-

cor-*/Co 3dxz

133b  138b

23

Co 3dx²y²

Co 3dz²

134b  137b

11

cor 

cor-*

134a  139a

13

cor-/Co 3dz²

cor-*/Co 3dxz

134b  139b

11

cor 

cor-*

99  103

51

cor 

cor Co 3dyz

100  105

47

Co 3dxz

cor 

99  104

39

cor 

cor /Co 3dxz

97  103

32

cor Co 3dyz/3dx²y²


100  105

43

Co 3dxz

cor 

99  103

32

cor 


102  106

10

Co 3dz²

Co 3dxy

100  107

29

Co 3dxz

cor 

99  104

24

cor 

cor /Co 3dxz

97  103

13

cor Co 3dyz/3dx²y²


98  105

12

Co 3dx²y²

cor 

Table 4. Experimental and computed relative rR intensities (In) of the three corrin-based stretching modes possessing a high degree of totally symmetric character in the parent C2v point group, s (#n), where n=1, 2, or 3 Species CNCbl

a

max

TDDFT excitation

(nm)

(Polarization)

Exp

Method 1

Method 2

Method 3

554.0

State 1 (LA)

100 : 9 : 5

98 : 100 : 14

100 : 46 : 21

100 : 50 : 14

I1 : I 2 : I 3

359.0

State 21 (SA)

50 : 100 : 11

13 : 100 : 59

0 : 100 : 5

3 : 100 : 4

Co2+Cbl

476.0

State 20 (LA)

56 : 9 : 100

NAa

NAa

100 : 41 : 35

Co1+Cbl

386.5

State 12 (LA)

0 : 88 : 100b

22 : 4 : 100

19 : 0 : 100

28 : 3 : 100

c

457.0 State 11 (SA) 100 : 31 : 82 100 : 9 : 76 100 : 3 : 43 100 : 32 : 12 not available. b obtained with laser excitation at 363.8 nm. c obtained with laser excitation at 454.5 nm


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39

SA H2 NOC

CONH 2 CH 3

H2NOC

CH 3

5

A

H 3C H 18

24 19

Co

10

N

16

14

11

C 15

LA

23

N

CH 3

O

CONH2

9

22

D 17

8

N

21

1

7

B

CN N

H3 C

H 2NOC

6

4

3 2

CH3

12

CH3

13

CH3 CONH2

NH

B4 B3

H3C

H

CH 3 B5

B2

DMB

B6

O O

N

HO N B1 O

P

B7

CH 3

O O HO

Figure 1. Chemical structure of vitamin B12 (cyanocobalamin, CNCbl). Relevant atom numbers and the short axis (SA) and long axis (LA) of the corrin ring are indicated, and the four pyrrole rings are labeled A–D. Note that in AdoCbl and MeCbl, the CN ligand is replaced by an adenosyl moiety and a methyl group, respectively.



40

1000 800 40

wavelength (nm) 600 500 400

350



(a)

30 20



10 0 30

-1

-1

 (cm M )

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

Page 40 of 46

(b)

20 10 0 30

(c)

20 10 0 10000

15000

20000 25000 -1 wavenumbers (cm )

30000

Figure 2. Electronic absorption spectra of (a) CNCbl at 4.5 K, (b) Co2+Cbl at 4.5 K, and (c) Co1+Cbl– at 250 K. Arrows indicate the wavelengths used for collecting the rR spectra shown in Figure 3.


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41

Figure 3. rR spectra obtained at 77 K for (a) CNCbl with 514.5 nm (solid black) and 363.8 nm (dashed gray) laser excitation, (b) Co2+Cbl with 476.5 nm laser excitation, (c) Co1+Cbl with 514.5 nm (solid black), 454.5 nm (solid light-gray), and 363.8 nm (dashed dark-gray) laser excitation.

Figure 4. DFT-optimized models for (a) CNCbl, (b) Co2+Cbl, and (c) Co1+Cbl.



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42

s (#1)

s (#2)

as

 s (#3)

CNCbl

Co2+Cbl

Co1+Cbl

Figure 5. DFT-computed eigenvector representations of the relevant corrin-based stretching modes for CNCbl (top), Co2+Cbl (middle), and Co1+Cbl (bottom). s(#1~3) and as refer to modes that can be classified as totally symmetric and antisymmetric, respectively, in the parent C2v point group.


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43

Figure 6. Experimental (thick solid black) and TDDFT-computed (thin solid black) Abs spectra of (a) CNCbl, (b) Co2+Cbl, and (c) Co1+Cbl. Contributions from LA-polarized and SA-polarized transitions to the computed Abs spectra are shown by dark gray dashed and light gray solid lines, respectively. The computed Abs spectrum for Co1+Cbl was scaled by a factor of ×0.5 to facilitate a comparison with the experimental spectrum.



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44

Figure 7. Relevant portions of the calculated MO diagrams for CNCbl (left), Co2+Cbl (middle), and Co1+Cbl (right). For the paramagnetic Co2+Cbl model, which was treated with the spin-unrestricted formalism, only the spin-up MOs are shown. The MOs were shifted vertically to align the LUMOs and are arranged according to their calculated energies, with the occupied and unoccupied orbitals shown below and above, respectively the horizontal dashed line.


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45

Figure 8. EDDMs associated with the “-band-type” transitions of CNCbl (State 1, left), Co2+Cbl (State 20, middle), and Co1+Cbl (State 12, right) as obtained from TDDFT calculations. White and dark gray indicate gain and loss of electron density by 0.001 au, respectively.



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46

Table of Contents Image C o3+

 s(# 1 )

 s(# 1 )

 s(# 2 )

 s(# 2 )  a s s ( # 3 ) C o2+

 as

 s(# 3 )

C o1+

1 4 00

15 0 0    (c m

1600 -1

)


Combined Spectroscopic and Computational Analysis of the

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