De novo Designed Metallopeptides with a Type 2 Copper Center: A Structural and Functional Model for Copper Nitrite Reductase.

Abstract

The de novo design of three generations of copper-peptides as structural and functional models for the type 2 copper center (T2Cu) in copper nitrite reductase (CuNiR) is described. Using an α-helical coiled coil scaffold, a tris-histidine site is introduced in the TRI peptide, yielding TRIW-H. While the protein scaffold is completely different from that in native CuNiR, copper binds with high affinity in both oxidation states, with a three-coordinate cuprous form [Cu(I)(His)3+] and likely a five-coordinate cupric form [Cu(II)(His)3(OH2)1–22+]. Redox chemistry is demonstrated for the first time in a de novo designed copper-peptide system. Although the reduction potential of Cu(II)/(I)(TRIW-H)32+/+ is 150–200 mV higher than that of the native T2Cu in CuNiR, it is still capable of catalyzing nitrite reduction in the presence of sodium ascorbate as the reductant with multiple turnovers. The first generation model establishes the minimal requirements to confer NiR activity in this system. To understand the fundamental structure-function relationship of this Cu(His)3 center with the ultimate goal of improving the catalytic activity, two more generations of models are described, with designs based on the modifications of the charged residues at the helical interface (2nd) and the interior (3rd) of the helical coiled coils, respectively. The second generation peptides have a key glutamate residue replacing the lysine residue at the 22nd position. By systematically modifying the charged residues to the C-terminus of the copper site (the 24th and 27th positions), a series of peptides with modifications at the helical interface are designed. These changes lead to modulated copper-binding affinities, deprotonation equilibria, and reduction potentials. The NiR rate is influenced to a small extent (4–fold at pH 5.8) with these modifications. Two strategies are used for the third generation models: mutating the leucine at 19th position into an alanine and changing the histidine to N-methyl histidine. The interior modifications, with a maximum 1300-fold observed rate enhancement, are far more effective in tuning the NiR rates than the 2nd generation alterations. This thesis lays the groundwork for the incorporation of metal centers in de novo designed peptide scaffolds to carry out redox catalysis.