Single-crystal X-ray and in crystallo XANES studies resolve cobalt coordination in cobalamin and rule out radiation-induced ligand loss in human intrinsic factor.

Human intrinsic factor (IF) mediates the intestinal uptake of cobalamin (Cbl; vitamin B12). A previous crystal structure of the IF-cobalamin complex revealed the apparent absence of an upper axial ligand at the cobalt center, raising questions regarding radiation-induced cleavage or chemical modification during crystallization. Here, high-resolution single-crystal X-ray crystallography of cobalamin in three states, oxidized and two independently prepared chemically reduced crystals, resolves clear electron density for an intact axial cyanide ligand at the cobalt center in all cases, including a crystal previously subjected to X-ray exposure during spectroscopic measurement. In crystallo Co K-edge X-ray absorption near-edge structure (XANES) spectroscopy performed on the same crystals shows a shift in absorption-edge energy compatible with, but not independently diagnostic of, partial chemical reduction, while indicating the retention of six-coordinate cobalt geometry in both states. Correlation of the spectroscopic and crystallographic data shows that radiation-induced photolysis of the Co-CN bond is unlikely to explain the apparent absence of axial electron density previously reported for IF-bound cobalamin. These results establish in crystallo XANES as a useful complement to macromolecular crystallography for resolving ambiguities in metalloprotein active-site chemistry, while demonstrating that the crystallographic data alone provide the most direct evidence for ligand retention.