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-d 3-Cbl...

Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri

“…The fact that a vibrational mode showing strong enhancement upon laser excitation into the “α-band” is sensitive to isotopic labeling at C10 implies that significant coupling occurs between corrin SA-stretching motion and an LA-polarized electronic transition. Although it had generally been assumed that the corrin-centered vibrational modes mainly involving SA-stretching motion should only couple to SA-polarized electronic transitions, 45,46 our recent DFT-based rR intensity calculations revealed that these modes can in fact also couple to LA-polarized electronic transitions. 48 …”

“…On the basis of these findings, the ~1500 and ~1550 cm −1 features were assigned as vibrational modes that primarily entail stretching motion of the conjugated C–C and C–N bonds oriented along the LA (the LA-stretching mode) and the SA (the SA-stretching mode), respectively. 45,46 In support of these assignments, the ~1550 cm −1 feature was shown to undergo a significant down-shift upon H/D exchange at C10, while the ~1500 cm −1 feature was largely unaffected by this isotopic substitution. 45 However, on the basis of a density functional theory (DFT)-assisted normal mode analysis for methylcobalamin (MeCbl), 47 Kozlowski and coworkers suggested that the frequency of the SA-stretching mode is in fact lower than that of the LA-stretching mode.…”

“…45,46 In support of these assignments, the ~1550 cm −1 feature was shown to undergo a significant down-shift upon H/D exchange at C10, while the ~1500 cm −1 feature was largely unaffected by this isotopic substitution. 45 However, on the basis of a density functional theory (DFT)-assisted normal mode analysis for methylcobalamin (MeCbl), 47 Kozlowski and coworkers suggested that the frequency of the SA-stretching mode is in fact lower than that of the LA-stretching mode. This finding raised questions regarding the validity of the normal mode assignments that were based on the rR enhancement patterns.…”

X-ray Structural Characterization of Imidazolylcobalamin and Histidinylcobalamin: Cobalamin Models for Aquacobalamin Bound to the B12 Transporter Protein Transcobalamin

“…20 Model studies and theoretical calculations also show that displacement of α-DMB trans to an alkyl ligand by imidazole-type ligands does not significantly increase the rate of homolytic cleavage or decrease the bond strength of the Co-C bond. [21][22][23][24] Furthermore, although initial X-ray diffraction results suggested that the Co-N(Im) bond is abnormally long for some B 12 -dependent enzymes (2.5 Å for MCM 7 and 2.3 Å for glutamate mutase 10 ), the structures may actually represent Co in mixed-ligand and oxidation states (Co III and Co II ), due to X-ray-induced photoreduction. 10,[25][26][27] In protein-free Cbls, α-DMB is difficult to displace from the α-axial site but can be displaced by cyanide, 28 by imidazole-type ligands, 29,30 and by protonation of the basic N of the imidazole moiety.…”

Thermodynamic and Kinetic Studies on Carbon−Cobalt Bond Homolysis by Ribonucleoside Triphosphate Reductase: The Importance of Entropy in Catalysis

“…Model studies have shown that a weaker donor ligand causes the carbon-cobalt bond to become longer and the bond dissociation energy to decrease (15)(16)(17). In contrast, FT-Raman (18) and resonance Raman (19) studies have shown that, for alkylcobalamins, carbon-cobalt bond stretching frequencies do not depend on the trans axial ligand. Thus the role of the axial ligand is more complex than simply modulation of the carbon-cobalt bond strength.…”