His/His motif is crucial to CuII binding of multi-His-containing metal sites in proteins and peptides, and thus receives much research attention. However, how CuII -His/His interactions are affected by molecular dynamic context remains elusive. Herein a study of heterogeneity in CuII-His/His interactions is demonstrated using a synthetic ethylenediaminetetraacetate (EDTA)-based glycinamide-type N, O-ligand, namely, EDTA-bis(L-histidine methyl ester) (P), complemented by its analog EDTA-bis(histamine) (P'). CuII binding feature was dissected by means of electrospray ionization-high resolution mass spectrometry (ESI-HRMS) which established a simultaneous formation and coexistence of mono-CuII (1), di-CuII (2) and decarboxylated di-CuII complex (3). Comprehensive ESI-HRMS titration revealed metal-concentration-dependent interaction modes, pH-dependence in ligating-group competition and cooperation, and substituent effect on the consecutive CuII binding. Further, ESI-HRMS allowed the associated innate proton affinity (pKa ~6.46 [1 + H+], 7.82 [2 + H+], and 8.77 [3 + H+], and Na+ (K+) uptaking ability of the Cu-complexes to be determined to substantiate the above putative structural elucidation. In parallel, external-ligand (Tris, PO42-) competition experiments permitted capturing and distinguishing between the two types of di-nuclear coordination modes (anti-Cu/Cu independent vs syn-Cu/Cu mutual-dependent modes) pertaining to the di-CuII-centers in 2 and 3, respectively; the latter being Cu/Cu inter-dependent di-Cu complex, which apparently resisted external-ligand capturing, was conceivably resulted from decarboxylation of the accompanying syn-isomer of 2 (anti). Moreover, circular dichroism (CD) titration enabled an estimation that the first CuII-binding is three-fold stronger than the second CuII at neutral and basic pH, but nearly similar to the second CuII at acidic pH. Furthermore, 13C NMR assisted in illuminating that both amidate and Im groups strongly participated in CuII binding at pH 7.10. Consequently, a two-dimensional landscape (variation of pH and CuII concentration) for binding was suggested, and from which an internally non-static binding mode in the mono-CuII complex (1) was extracted to account for a dynamic transition from mono-CuII to two types of di-CuII species, due to carboxylate-group scrambling on CuII ions together with backbone Δ-Λ conformation change. It is concluded that the pH-dependent on-off Im-CuII, amidate-CuII, carboxylate-CuII, amide oxo-CuII and amino-CuII binding and backbone dynamics act synergistically in the mono- vs di-CuII binding.