Structure of cobamide coenzymes: influence of pH on absorption spectra and ionophoretic mobilities.

The absorption spectra of the 5,6-dimethylbenaimidazolylcobamide coenzyme (coenzyme Blz) and its benzimidaeolecontaining analogue (benzimidazolylcobamide coenzyme) are different from that of the adenine-containing analogue (adeninylcobamide’ coenzyme (1, 2)). The most conspicuous difference is in the wave length of the absorbancy maximum in the visible region. The spectra of the two benzimidazole-containing analogues have an absorbancy maximum at approximately 520 rnp, whereas the corresponding maximum of the purine-containing analogue is at 458 mp. An explanation for this difference was not immediately apparent, particularly because the spectra of the corresponding cyanocobamides are very similar in the wave length region above 340 rnp. Clues to the origin of the spectral differences were provided by two observations: (a) Removal of the nucleotide adenine from the adeninylcobamide coenzyme does not cause much change in its spectrum above 300 rnp (3) ; and (b) the 5,6-dimethylbenzimidazolylcobamide coenzyme is red-orange in neutral solution and becomes yelloworange in acid solution; the latter color is similar to that of the adeninylcobamide coenzyme in both acid and neutral solution. These observations indicate that the adenine in the nucleotide of the adeninylcobamide coenzyme has little influence on the position of the visible absorbancy maximum, whereas the dimethylbenzimidazole of the 5,6-dimethylbenzimidazolylcobamide coenzyme is responsible for the modification of the spectrum in neutral solution. They suggest that protonation of the benzimidazole moiety may be responsible for the shift in the position of the absorbancy maximum and the change in color in acid solution. We have investigated the relation between the charge and color of the adeninylcobamide, benzimidazolylcobamide, and 5,6-dimethylbenzimidazolylcobamide coenzymes more fully by determining their absorption spectra and ionophoretic mobilities as a function of pH. The results indicate that striking differences exist with respect to the strength of attachment of the nucleotide base to the cobalt of the corrinoid structure, not only