Glutathionylcobalamin (gamma -glutamylcysteinylglycinylcobalamin; gamma -GluCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B-12 coenzymes adenosylcobalamin and methylcobalamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the greater than or equal to6 x 10(4) fold less stable cysteinylcobalamin, Cys-Cbl. In order to determine which parts of the glutathione tripeptide contribute to the overall stability of glutathionylcobalamin, two cysteine-containing dipeptides, which are truncated versions of glutathione, were used to synthesize their corresponding cobalamins, specifically gamma -glutamylcysteinylCb1 (gamma -GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with glutathionylCbl, the dipeptide gamma -GluCys-Cbl forms a stable thiolatocobalamin. However and most interestingly, CysGly-Cbl is observed to be unstable much like Cys-Cbl. The results require that the extra stability of glutathionylcobalamin and its congeners, compared to cysteinylcobalamin and its analogues, must be derived from destabilization by the gamma -NH3+ group in cysteinylcobalamin, or stabilization by the gamma -NHC(=O)- amide linkage in glutathionylcobalamin, or both. To probe any ground-state structural basis for the possible stabilization in gamma -GluCys-containing cobalamins, gamma -GluCys-Cbl was crystallized and yielded the first X-ray structural determination of a true thiolalocobalamin, and only the second structure of a cobalamin containing a Co-S bond, the first example being Randaccio and co-workers' 1999 structure of the thioketone complex, thioureacobalamin, (NH2)(2)CSCbl. Key features of the structure of gamma -glutamylcysteinylcobalanlin include (i) a normal Co-S bond length of 2.267(2) Angstrom, (ii) a Co-N(axial) bond length of 2.049(6) Angstrom, (iii) two alternate conformations of the gamma -glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2 degrees, the highest degree of folding yet observed for a cobalamin. These results do not show any strong stabilization (e.g., no shortened Co-S bond), although it is not clear for certain what the effect is (stabilizing or destabilizing) of the elongated Co-N(axial) bond; instead, the crystallographic results suggest that the metastable Cys-Cbl probably has a Co-S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co-S bond). Overall, the results strongly suggest that placing a positive charge on the gamma -NH3+ stabilizes the Co-S bond cleavage transition state, thereby setting the stage for the needed full thermolysis product and kinetic studies-as a function of the axial-base on-off equilibrium-that will be required to understand in even greater detail the unique stability of glutathionyl- (gamma -glutamylcysteinylglycinyl-) and gamma -glutamylcysteinylcobalamins.