Pro5 is not essential for the formation of ‘Ni-hook’ in nickel superoxide dismutase

The N-terminus of nickel-dependent superoxide dismutase (NiSOD) forms a structural motif known as the “Ni-hook,” where the peptide wraps around the metal to bring cysteine-2 and cysteine-6 into spatial proximity, allowing these residues to coordinate in a cis-geometry. A highly conserved proline-5 residue in the Ni-hook adopts a cis-conformation that is widely considered important for its formation. Herein, we investigate this role by point mutation of Pro5 to alanine. The results obtained show that the variant exhibits wild-type-like redox catalysis and features a Ni(III) center very similar to that found in enzyme. Structural analysis using X-ray absorption spectroscopy of the nickel sites in as-isolated P5A-NiSOD reveals changes in the variant and are consistent with a six-coordinate Ni site with (N/O)4S2 coordination. These changes are attributed to changes in the Ni(II) site structure. Nickel-binding studies using isothermal titration calorimetry reveal two binding events with Kd = 25(20) nM, and 250(60) nM. These events are attributed to i) Ni(II) binding to a preformed Ni-hook containing cis-Pro5 and ii) the combination of trans- to cis- isomerization upon Ni(II) binding, respectively. The higher-affinity binding event is absent in P5A-NiSOD, an observation attributed to the low abundance of the cis-Ala5 isomer in the apo-protein. The role of proline5 in forming the active site of Ni-dependent superoxide dismutase is explored using a single point mutation. The resulting proline5alanine variant is an active enzyme and displays spectra similar to the wild-type enzyme, indicating that Ala5 adopts a cis-conformation. [Display omitted] •A proline5 to alanine variant of Ni-dependent superoxide dismutase is reported.•The variant is an active enzyme and exhibits wild-type-like spectra.•The Ni site in the variant is structurally perturbed as-isolated.•Ni-binding studies reveal two binding events for the enzyme but not for the variant.•The data are interpreted as cis-isomerization of Pro5 and the Ala5 peptide bond.