Nickel superoxide dismutase (Ni-SOD) catalyzes the disproportionation of superoxide to molecular oxygen and hydrogen peroxide, but the overall reaction mechanism has yet to be determined. Peptidebased models of the 2N:2S nickel coordination sphere of Ni-SOD have provided some insight into the mechanism of this enzyme. Here we show that the coordination sphere of Ni-SOD can be mimicked using the tripeptide asparagine-cysteine-cysteine (NCC). NCC binds nickel with extremely high affinity at physiological pH with 2N:2S geometry, as demonstrated by electronic absorption and circular dichroism (CD) data. Like Ni-SOD, Ni-NCC has mixed amine/amide ligation that favors metal-based oxidation over ligand-based oxidation. Electronic absorption, CD, and magnetic CD data (MCD) collected for Ni-NCC are consistent with a diamagnetic Ni(II) center bound in square planar geometry. Ni-NCC is quasi-reversibly oxidized with a midpoint potential of 0.72 (2) V (versus Ag/AgCl) and breaks down superoxide in an enzyme-based assay, supporting its potential use as a model for Ni-SOD chemistry.The radical species superoxide (O 2 •− ) and its reactive downstream products are known to cause oxidative damage to biological molecules, and the presence of superoxide in the body has been linked to many different diseases. 1 Superoxide dismutases (SODs) are oxidoreductases that catalyze the disproportionation of superoxide to hydrogen peroxide and molecular oxygen, therefore helping to protect biological systems from oxidative damage. These enzymes, classified by their metal center, include Cu/Zn-SODs, Ni-SOD is the most recently discovered form of the enzyme.3 , 4 Its active site contains a mononuclear nickel center 5 coordinated in square planar geometry at the N-terminus of the enzyme via two cysteine sulfurs (Cys-2 and Cys-6), an amine nitrogen from the N-terminus, and a deprotonated, anionic amide nitrogen from the peptide backbone (Cys-2). 6, 7 Ni-SOD reacts with superoxide in a two-step reaction, involving oxidized and reduced forms of the enzyme (Scheme 1). 3,[6][7][8] As part of the redox reaction, the geometry of the bound nickel switches from square planar Ni(II) to square pyramidal Ni(III). An additional ligand, an axial nitrogen donor, coordinates Ni(III) in the resting state, 3 and the X-ray crystal structure revealed this species to be the imidazole nitrogen of His-1. Upon reduction of the enzyme, the histidine imidazole rotates away from the active site, leaving Ni(II) coordinated in 2N:2S, square planar geometry.6 , 7 , 9While much has been learned about the structure of the enzyme, only recently have studies begun to reveal more about the influence of the secondary coordination sphere. 10,11 While 1Author to whom correspondence should be addressed. laurencj@ku.edu. Supporting Information Available: Experimental procedures and ESI-MS, pH titration, MCD, DFT, CV, and control peptide data. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptInorg Chem. Autho...

The metal abstraction peptide (MAP) tag is a tripeptide sequence capable of abstracting a metal ion from a chelator and binding it with extremely high affinity at neutral pH. Initial studies on the nickel-bound form of the complex demonstrate that the tripeptide asparagine-cysteine-cysteine (NCC) binds metal with 2N:2S, square planar geometry and behaves as both a structural and functional mimic of Ni superoxide dismutase (Ni-SOD). Electronic absorption, circular dichroism (CD), and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic NiII center. It is apparent from the CD signal of Ni-NCC that the optical activity of the complex changes over time. Mass spectrometry data show that the mass of the complex is unchanged. Combined with the CD data, this suggests that chiral rearrangement of the complex occurs. Following incubation of the nickel-containing peptide in D2O and back-exchange into H2O, incorporation of deuterium into non-exchangeable positions is observed, indicating chiral inversion occurs at two of the alpha carbon atoms in the peptide. Control peptides were used to further characterize the chirality of the final nickel-peptide complex, and DFT calculations were performed to validate the hypothesized position of the chiral inversions. In total, these data indicate Ni-SOD activity is increased proportionally to the degree of structural change in the complex over time, as cross-correlation between the change in CD signal and change in SOD activity reveals a linear relationship.

Synthetically generated metallopeptides have the potential to serve a variety of roles in biotechnology applications, but the use of such systems is often hampered by the inability to control secondary reactions. We have previously reported that the NiII complex of the tripeptide LLL-asparagine-cysteine-cysteine, LLL-NiII-NCC, undergoes metal-facilitated chiral inversion to DLD-NiII-NCC, which increases the observed superoxide scavenging activity. However, the mechanism for this process remained unexplored. Electronic absorption and circular dichroism studies of the chiral inversion reaction of NiII-NCC reveal a unique dependence on dioxygen. Specifically, in the absence of dioxygen, the chiral inversion is not observed, even at elevated pH, whereas the addition of O2 initiates this reactivity and concomitantly generates superoxide. Scavenging experiments using acetaldehyde are indicative of the formation of carbanion intermediates, demonstrating that inversion takes place by deprotonation of the alpha carbons of Asn1 and Cys3. Together, these data are consistent with the chiral inversion being dependent on the formation of a NiIII-NCC intermediate from NiII-NCC and O2. The data further suggest that the anionic thiolate and amide ligands in NiII-NCC inhibit Cα–H deprotonation for the NiII oxidation state, leading to a stable complex in the absence of O2. Together, these results offer insights into the factors controlling reactivity in synthetic metallopeptides.

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.