Ni^II Complex Formation and Protonation States at the Active Site of a Nickel Superoxide Dismutase‐Derived Metallopeptide: Implications for the Mechanism of Superoxide Degradation

Abstract

A small, catalytically active metallopeptide (Nim SOD, m SOD=ACDLAC), which was derived from the nickel superoxide dismutase (NiSOD) active site was employed to study the mechanism of superoxide degradation, especially focusing on the protonation states of the Ni donor atoms, the proton source, and the role of the N-terminal proton(s). Therefore, the Ni -metallopeptide was studied at various pHs and temperatures using UV/Vis and NMR spectroscopy. These studies indicate a strong reduction of the pK of the Ni -ligating donor atoms, resulting in a fully deprotonated Ni active-site environment. Furthermore, no titratable proton could be observed within a pH ranging from 6.5 to 10.5. This rules out a recently discussed adiabatic proton tunneling-like hydrogen-atom transfer process for the metallopeptides, not found in the native enzyme. Furthermore, variable-temperature H NMR measurements uncovered an extended hydrogen-bond network within the Ni active site of the metallopeptide similar to the enzyme. With respect to the deprotonated Ni active site, the residual N-terminal proton, which is a prerequisite for catalytic activity, cannot act as proton source. Most likely, it stabilizes the Ni -coordinated substrate in an end-on fashion, thus allowing for an inner-sphere electron transfer. Lastly, and unlike the enzyme, the catalytic rate constant of superoxide degradation by the metallopeptides was determined to be strongly pH dependent, suggesting bulk water to be directly involved in proton donation, which in turn strongly suggests the N-terminal histidine to be the respective proton donor in the enzyme.

Mononuclear Ni(II) Complexes with a S3O Coordination Sphere Based on a Tripodal Cysteine-Rich Ligand: pH Tuning of the Superoxide Dismutase Activity

“…This conclusion is in contrast to that arising from an investigation carried out on metallopeptide-based models for which it has been proposed that protonation of one of the cysteines occurs during the catalytic mechanism . However, this is perfectly consistent with a more recent study performed on similar maquettes that show that the cysteine residues are not protonated …”

“…26 However, this is perfectly consistent with a more recent study performed on similar maquettes that evidences that the cysteine residues are not protonated. 75 The SOD efficiency of NiL 3S (OH2) (IC50 ∼ 1 10 -6 M, pH 7.4) is lower than that of the NiSOD (IC50 = 4-8 10 -8 M per metallic site), 16 and the reaction is slower (kcat = 1.8 10 5 M -1 s -1 , pH 7.75) than that between O2 • -and NiSOD (kcat ∼ 1 10 9 M -1 s -1 , pH 6.0-8.0) 16 or peptide-based models (in the range 6 10 6 -6 10 8 M −1 s −1 , pH 8.0). However, the activity of the present Ni II SOD catalyst is expected to be enhanced at physiological pH, since the measured SOD activity at pH 7.75 contains a significant contribution of the inactive NiL 3S (OH) complex.…”

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

“…As stated above, NiSOD itself has been shown to possess at least one Ni-S(H + )-Cys moiety in its reduced form [15]. Although not without controversy [33], we have provided strong evidence based on sulfur K-edge X-ray absorption studies that like NiSOD itself, NiSOD metallopeptide based mimics possess a Ni-S(H + )-Cys moiety at physiological pH as well [18,22]. Studies have also suggested that the pK a of the Ni-S(H + )-Cys proton within these active-sites is~8.5, and can become reversibly deprotonated at high pH (>9.0) [22].…”

“…Taking advantage of the fact that all of the ligating residues to nickel are found within the first six residues from the protein N-terminus, we, and others, have prepared functional NiSOD metallopeptide based mimics utilizing the first 6-12 residues from the NiSOD N-terminal primary protein sequence. These metallopeptide based mimics reproduce the key structural and spectroscopic properties of the metalloenzyme [17,[26][27][28][29][30][31][32][33].Inorganics 2019, 7, x FOR PEER REVIEW 2 of 19 Chart 1. Representative structures of the active sites of cysteinate-ligated nickel containing metalloproteins.…”

A Bioinspired NiII Superoxide Dismutase Catalyst Designed on an ATCUN-like Binding Motif

“…Such a chemical environment is unexpected considering the well-known reactivity between O 2 · – , H 2 O 2 , and even O 2 with metal-bound thiolates. − Together with a poor understanding of the catalytic mechanism, especially due to the lack of characterized intermediate species, NiSOD has attracted the curiosity of the bioinorganic community. − To face this challenge, it was not only crucial to develop catalytic NiSOD models active in water but also to trap intermediate species to provide key mechanistic insights. In this context, the ATCUN motif (ATCUN for amino terminal Cu II and Ni II binding motif) is an ideal choice to stabilize a square planar coordination for Ni II , − which can be properly modulated with the introduction of a thiolate ligand through a cysteine residue to study the role and reactivity of the thiolate function.…”