The influence of amine/amide versus bisamide coordination in nickel superoxide dismutase

Nickel superoxide dismutase (NiSOD) is a mononuclear nickel-containing metalloenzyme that catalyzes the disproportionation of superoxide by cycling between NiII and NiIII oxidation states. In the reduced NiII 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 NiIII 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 [NiII(SODM1)] (SODM1 = H2N-HCDLPCGVYDPA-COOH) by exploring how acylation of the N-terminus (producing [NiII(SODM1-Ac)]) influences the properties of the metallopeptide. Titration results, GPC data, and mass-spectrometry data demonstrate that NiII coordinates to SODM1-Ac in a 1:1 ratio, while variable pH studies show that NiII coordination is strong at a pH of 7.5 and above but not observed below a pH of 6.2. This is higher than [NiII(SODM1)] by approximately 1.0 pH unit consistent with bisamide ligation. Ni K-edge XAS demonstrates that the NiII center is coordinated in a square-planar NiN2S2 coordination environment with Ni-N distances of 1.846(4) A and Ni-S distances of 2.174(3) A. Comparison of the electronic absorption and CD spectrum of [NiII(SODM1)] versus [NiII(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. [NiII(SODM1-Ac)] has a quasireversible NiII/NiIII redox couple of 0.49(1) V vs Ag/AgCl, which represents a -0.2 V shift compared with [NiII(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 [NiII(SODM1-Ac)] is less active than [NiII(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.