The coordination of peptides Ser-Pro-His-His-Gly-Gly (SPHHGG) and (His)(6) (HHHHHH) to [Pd-II(mida)(D2O)] (mida(2-) = N-methyliminodiacetate) was studied by H-1 NMR as model reactions for Cu-II(iminodiacetate)immobilized metal affinity chromatography (IMAC) sites. This is the first direct physical description of peptide coordination for IMAC. A three-site coordination is observed which involves the first, third, and fourth residues along the peptide chain. The presence of proline in position 2 of SPHHGG achieves the best molecular mechanics and bonding angles in the coordinated peptide and enhances the interaction of the serine amino nitrogen. Histidine coordination of H-1, H-3, and H-4 of (His)(6) and H-3 and H-4 of SPHHGG was detected by H-1 NMR contact shifts and H/D exchange of histidyl protons. The EPR spectra of SPHHGG and HHHHHH attached to the [Cu-II(mida)] unit were obtained for additional modeling of IMAC sites. EPR parameters of the parent [Cu(mida)(H2O)(2)] complex are representative: g(u) = 2.31; g(yy) = 2.086; g(xx) = 2.053; A(ll) = 161G; A(N) = 19G (three line, one N coupling). Increased rhombic distortion is detected relative to the starting aqua complex in the order of [Cu(mida)L] for distortion of HHHHHH > SPHHGG > (H2O)(2) The lowering of symmetry is also seen in the decrease in the N-shf coupling, presumably to the imino nitrogen of mida(2-) in the order 19 G (H2O), 16 G (SPHHGG) and 11 G (HHHHHH). Visible spectra of the [Cu(mida)(SPHHGG)] and [Cu(mida)(HHHHHH)] as a function of pH indicate coordination of one histidyl donor at ca. 4.5, two in the range of pH 5-7, and two chelate ring attachments involving the terminal amino donor for SPHHGG or another histidyl donor of HHHHHH in the pH domain of 7-8 in agreement with the [Pd-II(mida)L] derivatives which form the two-chelate-ring attachment even at lower pH as shown by the H-1 NMR methods.