The distal nickel site of acetyl-CoA synthase (Ni-d-ACS) and reduced nickel superoxide dismutase (Ni-SOD) display similar square-planar (NiN2S2)-N-II coordination environments. One difference between these two sites, however, is that the nickel ion in Ni-SOD contains a mixed amine/amidate coordination motif while the Ni-d site in Ni-ACS contains a bisamidate coordination motif. To provide insight into the consequences of the different coordination environments on the properties of the Ni ions, we systematically examined two square-planar (NiN2S2)-N-II complexes, one with bisthiolatebisamidate ligation (Et4N)(2)(Ni(L1))center dot 2H(2)O (2) [H(4)L1 = N-(2-mercaptoacetyI)-/V-(2-mercaptoethyl)glycinamide] and another with bisthiolate-amine/amidate ligation K(Ni(HL2)) (3) [H(4)L2 = N-(2 ''-mercaptoethyl)-2-((2'-mercaptoethyl)amino)acetamide]. Although these two complexes differ only by a single amine versus amidate ligand, their chemical properties are quite different. The stronger in-plane ligand field in the bisamidate complex (Ni-II(L1))(2-) (2) results in an increase in the energies of the d d transitions and a considerably more negative oxidation potential. Furthermore, while the bisamidate complex (Ni-II(L1))(2-) (2) readily forms a trinuclear species (Et4N)(2)({Ni(L1)}(2)Ni)(2)- H2O (1) and reacts rapidly with 02, presumably via sulfoxidation, the mixed amine/amidate complex (Ni-II(HL2)) (3) remains monomeric and is stable for days in air. Interestingly, the Nil" species of the bisamidate complex formed by chemical oxidation with 12 can be detected by electron paramagnetic resonance (EPR) spectroscopy while the mixed amine/amidate complex immediately decomposes upon oxidation. To explain these experimentally observed properties, we performed S K-edge X-ray absorption spectroscopy and low-temperature (77 K) electronic absorption measurements as well as both hybrid density functional theory (hybrid-DFT) and spectroscopy oriented configuration interaction (SORCI) calculations. These studies demonstrate that the highest occupied molecular orbital (HOMO) of the bisamidate complex (Ni-II(L1))2 (2) has more Ni character and is significantly destabilized relative to the mixed amine/amidate complex (Ni-II(HL2)) (3) by similar to 6.2 kcal mol(-1). The consequence of this destabilization is manifested in the nucleophilic activation of the doubly filled HOMO, which makes (Ni-II(L1))(2-) (2) significantly more reactive toward electrophiles such as O-2.