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.

Pro5 is not essential for the formation of 'Ni-hook' in nickel superoxide dismutase / Basak, Priyanka; Zambelli, Barbara; Cabelli, Diane E; Ciurli, Stefano; Maroney, Michael J. - In: JOURNAL OF INORGANIC BIOCHEMISTRY. - ISSN 1873-3344. - ELETTRONICO. - 234:(2022), pp. 111858.1-111858.9. [10.1016/j.jinorgbio.2022.111858]

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

Zambelli, Barbara;Ciurli, Stefano;Maroney, Michael J
2022

Abstract

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.
2022
Pro5 is not essential for the formation of 'Ni-hook' in nickel superoxide dismutase / Basak, Priyanka; Zambelli, Barbara; Cabelli, Diane E; Ciurli, Stefano; Maroney, Michael J. - In: JOURNAL OF INORGANIC BIOCHEMISTRY. - ISSN 1873-3344. - ELETTRONICO. - 234:(2022), pp. 111858.1-111858.9. [10.1016/j.jinorgbio.2022.111858]
Basak, Priyanka; Zambelli, Barbara; Cabelli, Diane E; Ciurli, Stefano; Maroney, Michael J
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/904332
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