Near-IR FT-Raman Spectroscopy of Methyl-B₁₂ and Other Cobalamins and of Imidazole and Imidazolate Methylcobinamide Derivatives in Aqueous Solution

Abstract

Near-IR FT-Raman spectra of B12 imidazole derivatives and cobalamins in aqueous solution were compared since there is now strong evidence that, in human B12-dependent enzymes, the 5,6-dimethylbenzimidazole (DMBz) is replaced by imidazole from a histidine in the protein. Derivatives studied include methylcobalamin [MeCbl (DMBz base-on) and MeCbl+ (base-off by acidification to protonate the DMBz)], methylaquacobinamide (MeCbi+) [Cbi's have the DMBz-bearing nucleotide loop removed by hydrolysis], and Me(N-acetylhistidine)Cbi [coordinated through imidazole in Me(N-AcHis)Cbi at pH 10 and imidazolate in Me(N-AcHis)Cbi- in 1 M NaOH]. Several marker bands changed with changes in the axial ligand trans to the methyl group. The frequency of the Co−CH3 stretching mode at ∼505 cm-1 (assigned by isotopic shift using −CD3) was similar for all MeCbl and MeCbi species; thus, the trans ligand, including the very powerful electron-donating imidazolate species, has little effect on Co−C bond strength. In contrast, the peak height of the Co−CH3 band, relative to the corrin long-axis mode band at 1495 cm-1, consistently increased 2-fold upon coordination of an N-donor ligand to MeCbi+ and MeCbl+. This intensity change appears to be a useful means of assessing ligand replacement reactions. With increasing trans ligand donor ability, the frequency of the corrin in-phase double-bond stretching mode along the short axis at ∼1545 cm-1 increased, but the frequencies of the corresponding long-axis mode at ∼1495 cm-1 and the corrin band at ∼1570 cm-1 were unchanged. The frequency of a corrin band at ∼1600 cm-1 increased slightly to 1603 cm-1 from the base-on to the base-off MeCbl form; the band is also at 1603 cm-1 for MeCbi+, Me(N-AcHis)Cbi, and Me(N-AcHis)Cbi-. A decrease in the frequency of the ∼1570 cm-1 band coincident with acid-catalyzed H-to-D exchange at the corrin C10 in acidic D2O solutions (confirmed by 1H NMR spectroscopy) was evidence that it is a corrin band. However, the frequency and intensity of this band were relatively insensitive to the trans ligand. This diverse dependence of the corrin-band frequencies on changes in trans axial ligand bulk suggests that in these B12 derivatives there are no large structural distortions. Differences in electron donation by the trans ligands have the greater influence on the spectra. A band at ∼1315 cm-1 was found to be characteristic of unprotonated DMBz; it is absent when DMBz is removed (Cbi's) or protonated (MeCbl+) but present in all other Cbl's, including base-off (CN)2Cbl-. An ∼30 cm-1 shift to lower frequency of the overlapping amide I bands of cobalt corrinoids between H2O and D2O was caused by amide NH2 to ND2 exchange. The frequency shift of ∼10 cm-1 of this band between H2O and ethanol was consistent with a small redistribution of resonance forms of the amide group between solvents. Since the band for the CD3 symmetric stretch of the Co−CD3 group lies in a region of the spectrum (∼2105 cm-1) that is devoid of other bands, it may be useful in studies of enzyme-bound Me-d3-Cbl. In summary, our results show that the exchange of DMBz by imidazole has minimal influence on the methylcobalt(III) ground state. We suggest that the functional role of the imidazole occurs later in the catalytic cycles of B12 enzymes.