Interaction of divalent metal ions with pyridoxal phosphate. I. Nuclear magnetic resonance studies of cobalt(II) binding

Abstract

The binding of Co(II) to pyridoxal phosphate (PLP)at neutral pH was investigated with 31P, 1H, and 13C nmr spectroscopy. The proton and phosphorus spin–lattice relaxation times of PLP were measured in the absence of Co(II) and with increasing amounts of the metal ion. The shifting and the relaxation enhancement of the phosphorus resonance in the presence of Co(II) have indicated the binding of the metal ion to the phosphate of PLP. The addition of metal ion also shifted and broadened the proton and carbon resonances. The shifting and broadening was the largest for the 4-CHO proton with other protons following the order 4-CHO [Formula: see text] 2-methyl > 5-methylene > 6-H. At neutral pH Co(II) shifted only the 5-methylene proton resonance of pyridoxamine phosphate (PMP) indicating binding of Co(II) only to the 5′-phosphate of PMP at this pH. Comparison of the shifting and broadening of the proton resonances of PLP with similar shifts in model compounds has indicated a structure for the 1:1 PLP:Co(II) complex in which the metal ion is simultaneously bound to the phosphate and the aldehyde oxygen. The proton spin–lattice relaxation times and the isotropic shifts of the 13C resonances in the 1:1 PLP:Co(II) complex support this conclusion.