Embedding the Ni-SOD Mimetic Ni-NCC within a Polypeptide Sequence Alters the Specificity of the Reaction Pathway

Abstract

The unique metal abstracting peptide (MAP) asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to DLD-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion. Characterization of the NCC sequence within longer peptides with absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies and mass spectrometry (MS) shows that the geometry of metal coordination is maintained, though the electronic properties of the complex are varied to a small extent due to bis-amide, rather than amine/amide, coordination. In addition, both the Ni-tripeptides and Ni-pentapeptides have a −2 charge. The study here demonstrates that the chiral inversion chemistry does not occur when NCC is embedded in a longer polypeptide sequence. Nonetheless, the superoxide scavenging reactivity of the embedded Ni-NCC module is similar to that of the chirally inverted tripeptide complex, which is consistent with a minor change in reduction potential for the Ni-pentapeptide. Together, this suggests that the charge of the complex could affect the SOD activity as much as a change in primary coordination sphere. In Ni-NCC and other Ni-SOD mimics, changes in chirality, superoxide scavenging activity, and oxidation of the peptide itself all depend on the presence of dioxygen or its reduced derivatives (e.g., superoxide), and the extent to which each of these distinct reactions occurs is ruled by electronic and steric effects that emenate from the organization of ligands around the metal center.

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N2S2 Synthetic Models of NiSOD

“…Questions concerning the enzyme's unusual N/S coordination sphere, particularly its stability toward O 2 and reactive oxygen species (ROS), in light of the reactive Ni–S(Cys) bonds, 13 and the catalytic mechanism have prompted researchers to develop maquette-based, 14–20 tripeptide, 21–24 and low molecular weight 25–38 analogues that seek to replicate NiSOD's structure and/or function. 39,40 Indeed, several experimental and theoretical efforts have suggested that the mixed peptide-N/amine-N ligation serves as one key factor to promote greater Ni-character in the redox-active molecular orbital of NiSOD, in particular for the Ni(II) or NiSOD red state, thus preventing unwanted S-oxidation/oxygenation.…”

claMP Tag: A Versatile Inline Metal-Binding Platform Based on the Metal Abstraction Peptide

“…21 In the EGF-Ni- cla MP spectrum, there are broad features present in the visible region that reflect the structure of the unique metal complex, as seen in Figure 7a. These features are absent from the control spectrum of native EGF, which confirms they are due to correct Ni- cla MP formation and not nonspecific binding of Ni(II) to EGF.…”

Stability Analysis of an Inline Peptide-based Conjugate for Metal Delivery: Nickel(II)-claMP Tag Epidermal Growth Factor as a Model System

“…Here, cla MP‐Tagged EGF was used as a model system to analyze the stability of the cla MP Tag in the context of a protein system and verify that incorporation of the cla MP Tag does not substantially adversely affect protein stability. The tag rapidly becomes fully occupied in the presence of the metal transfer agent, efficiently yielding a highly stable single species in solution . Nonetheless, in the event of incomplete loading, the metal‐occupied cla MP‐Tagged protein may be easily separated from any residual unreacted proteins using ion‐exchange chromatography because the correctly formed Ni(II) complex develops a 2 − charge, differentiating it from the unoccupied form.…”

“…To investigate changes in the Ni– cla MP complex, such as loss of metal or alteration of the ligands around the metal center, absorption spectroscopy, and charge‐based separation was used. Incorporation of Ni(II) into the cla MP Tag was used to perform this initial stability study because the absorption spectrum of this complex is rich in structural detail, and any chemical or structural transformation of the Ni– cla MP complex would alter the features in the UV–vis absorption spectrum and the unique 2 − charge of the complex. The UV–vis absorption data show that EGF–Ni– cla MP is stable over a 12‐week period, as no decreases in peak height were observed for the protein and Ni– cla MP features (Fig.…”

“…) and unusual binding and release properties . Incorporation of the NCC module into longer peptide sequences has been shown to have no discernable effect on complex formation . Therefore, the cla MP Tag can be biosynthetically encoded into a protein to generate a linker‐less bioconjugate, which eliminates the need to chemically link to a chelating moiety.…”