Spectroscopic and Computational Studies of Co³⁺-Corrinoids:  Spectral and Electronic Properties of the B₁₂Cofactors and Biologically Relevant Precursors

Abstract

The B(12) cofactors methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl) have long fascinated chemists because of their complex structures and unusual reactivities in biological systems; however, their electronic absorption (Abs) spectra have remained largely unassigned. In this study, we have used Abs, circular dichroism (CD), magnetic CD (MCD), and resonance Raman spectroscopic techniques to probe the electronic excited states of Co(3+)Cbl species that differ with respect to their upper axial ligand, including MeCbl, AdoCbl, aquacobalamin (H(2)OCbl(+)), and vitamin B(12) (cyanocobalamin, CNCbl). Also included to probe the effect of the lower axial ligand on the electronic properties of Cbls is Ado-cobinamide (AdoCbi(+)), an AdoCbl derivative that lacks the tethered base 5,6-dimethylbenzimidazole (DMB) and instead binds a water molecule in the lower axial position. Spectroscopic data for each species are analyzed within the framework of time-dependent density functional theory (TD-DFT) to assign the major spectral features (the so-called alpha/beta, D/E, and gamma bands) and to generate experimentally validated electronic-structure descriptions. These studies reveal that the "unique" Abs spectra of MeCbl and AdoCbl, which differ considerably from the "typical" Abs spectra of H(2)OCbl(+) and CNCbl, reflect the high degree of sigma-donation from the alkyl ligand to the Co center and the consequent destabilization of all Co 3d orbitals. They reveal further that with increasing sigma-donor strength of the upper axial ligand, the contribution from the formally unoccupied Co 3d(z(2)) orbital to the HOMO increases, which induces a strong Co[bond]N(DMB) sigma-antibonding interaction, consistent with the experimentally observed lengthening of this bond from H(2)OCbl(+) to CNCbl and MeCbl. Alternatively, our spectroscopic and computational data for MeCbl and MeCbi(+) reveal that substitution of the DMB by a water molecule in the lower axial position has negligible effects on the Co[bond]C. A simple model is presented that explains why the identity of the upper axial ligand has a major effect on the Co[bond]N(ax) strength, whereas the lower axial ligand does not appreciably modulate the nature of the Co[bond]C. Implications of these results with respect to enzymatic Co[bond]C activation are discussed.

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

“…26,64,69,70 This lack of a significant trans influence is readily understood upon inspection of the qualitative axial ligand MO diagram in Figure 7, which reveals that even complete loss of the water ligand should not affect the Co-C bond order (this prediction is fully consistent with the conclusion drawn from our earlier work on Me-Co 3+ corrinoids possessing different lower ligands). 24 Collectively, these results indicate that the nature of the Co-C bond is insignificantly affected by changes in the trans axial ligand position and/or identity; thus, protein induced distortions at the lower face of the corrinoid are not expected to affect the strength of the Co-C bond to any significant degree.…”

“…54 All spectra were red-shifted by 3000 cm −1 to compensate for the fact that transition energies for Co 3+ -corrinoids and similar systems are consistently overestimated by the B3LYP TD-DFT method. [24][25][26][27]49,53,[55][56][57] …”

“…24 The appearance of this band at 450 nm (22,200 cm −1 ) in the protein spectrum eliminates the possibility that the methoxybenzimidazolyl (MBI) moiety of Factor III m directly coordinates to the cobalt center, as the α-bands of alkylated corrinoids with N-donor ligands in the lower axial position consistently peak between 535 and 515 nm (18,700-19,500 cm −1 ). While this result suggests that the MBI ligand is displaced by a water molecule in the enzyme-bound cofactor, the α-band in the Me-Co 3+ CFeSP Abs spectrum is blue-shifted by 8 nm (390 cm −1 ) relative to that of free AdoCbi + (Figure 4), signaling, perhaps, a previously unknown mode of cofactor binding.…”

“…Importantly, however, lower ligand bond deformation is predicted to have a negligible effect on the trans axial Co-C bond (Figure 7), consistent with previous experimental studies that have shown ν Co-C to be virtually unaffected by axial ligand substitution and/or bond elongation. 24,64,70 Currently, the motivation for this enzyme-induced Co-OH 2 bond lengthening with respect to the methyl group transfer mechanism is unclear. However, kinetic and thermodynamic studies of alkylated corrinoid species have shown that the nature of the lower ligand has a profound influence on the mode of Co-C bond breaking.…”

DFT Analysis of Co−Alkyl and Co−Adenosyl Vibrational Modes in B12-Cofactors

“…12 Co-alkyl stretching vibrations have been detected by infrared and Raman spectroscopy, [13][14][15][16] and give an indirect indication of the Co-C bond strength. 17,18 Resonance Raman (RR) spectroscopy can provide access to B 12 -containing proteins, [19][20][21] since laser tuning can be used to selectively excite cobalamin vibrations.…”