Nickel superoxide dismutase (NiSOD) is a mononuclear nickel-containing metalloenzyme that catalyzes the disproportionation of superoxide by cycling between Ni-II and Ni-III oxidation states. In the reduced Ni-II oxidation state, the metal center is ligated by two cysteinate sulfurs, one amide nitrogen, and one amine nitrogen (from the N-terminus), while in the oxidized Ni-III state, an imidazole nitrogen coordinates to the metal center. Herein, we expand on a previous report in which we described a functional metallopeptide-based NiSOD model compound [Ni-II(SODM1)] (SODM1 = H2N-HCDLPCGVYDPA-COOH) by exploring how acylation of the N-terminus (producing [Ni-II(SODM1-Ac)]) influences the properties of the metallopeptide. Titration results, GPC data, and mass-spectrometry data demonstrate that Ni-II coordinates to SODM1-Ac in a 1:1 ratio, while variable pH studies show that Ni-II coordination is strong at a pH of 7.5 and above but not observed below a pH of 6.2. This is higher than [Ni-II(SODM1)] by similar to 1.0 pH unit consistent with bisamide ligation. Ni K-edge XAS demonstrates that the Ni-II center is coordinated in a square-planar NiN2S2 coordination environment with Ni-N distances of 1.846(4) angstrom and Ni-S distances of 2.174(3) angstrom. Comparison of the electronic absorption and CD spectrum of [Ni-II(SODM1)] versus [Ni-II(SODM1-Ac)] in conjunction with time-dependent DFT calculations suggests a decrease in Ni covalency in the acylated versus unacylated metallopeptide. This decrease in covalency was also supported by DFT calculations and Ni L-edge XAS. [Ni-II(SODM1-Ac)] has a quasireversible Ni-II/Ni-III redox couple of 0.49(1) V vs Ag/AgCl, which represents a-0.2 V shift compared with [Ni-II(SODM1)], while the peak separation suggests a change in the coordination environment upon oxidation (i.e., axial imidazole ligation). Using the xanthine/xanthine oxidase assay, we determine that [Ni-II(SODM1-Ac)] is less active than [Ni-II(SODM1)] by over 2 orders of magnitude (IC50 = 3(1) x 10(-5) vs 2(1) x 10(-7) M). Possible reasons for the decrease in activity are discussed.