Introducing the penicillamine moiety into a metallopeptide mimicking the NiSOD enzyme: electronic and kinetic effects

Abstract

Multidisciplinary protein design is reported on a novel metallopeptide mimicking the binding loop of nickel containing SOD enzyme (NiSOD). D-Penicillamine, a natural decomposition product of penicillin, was introduced into the peptide chain yielding the H(Pen)DLPCGLY (wtPen) peptide. The nickel(II) binding ability of wtPen was characterized by thermodynamic (pH-potentiometry), spectroscopic (UV-Vis, CD, MS, NMR) and computational techniques (full DFT and Molecular Dynamics methods). Oxidation of the Ni(II) complex by KO₂ yields a square pyramidal Ni(III) species coordinated by the axial His-N in a well-defined α-helix folding state. The structure of the Ni(III) species was analyzed by EPR spectroscopy and theoretical methods confirming that the donor set involved in the metal ion coordination and the folding state are retained after oxidation. The complex exhibits superior SOD activity which was studied by sequential stopped-flow method. Thorough analysis of the data shows that the Ni(III) species rapidly accumulates in the nickel catalyzed decomposition of superoxide anion. Accordingly, the presence of the penicillamine moiety close to the catalytic center increases the life-time of the Ni(III) transient species. In contrast, Ni(III) exists only at relatively low concentration level in the dismutation reaction catalyzed by the native NiSOD enzyme fragment.