Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils

Abstract

One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in α-helical coiled coils, as a model for the Cu(T2) center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano- to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu(His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu(T2) site in the natural enzyme. The species Cu(II)(TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.

Fig. 1.

(A) Representation of the model of the metallopeptide Cu(I/II)(TRIL23H)₃^+/2+ based on the structure of Hg(II)_S[Zn(II)_N(H₂O)](CSL9CL23H)₃⁺ [PDB ID code 3PBJ (6)]. (B) View of the Zn(II)(H₂O)(His)₃ site along the pseudo-three-fold axis of Hg(II)_S[Zn(II)_N(H₂O)](CSL9CL23H)₃⁺ (light gray), overimposed to the type 2 Cu(II)(H₂O)(His)₃ site in R. sphaeroides NiR (PDB ID code 2DY2, dark gray). Light gray for Zn(II)(H₂O), dark gray for Cu(II)(H₂O). Coordinated water molecules are reported as spheres. (C) Side view of the two metal sites, as in B.

Fig. 2.

Visible spectra of Cu(II)(TRIL23H)₃²⁺. (A) Spectrum of a 0.34-mM solution of Cu(II)(TRIL23H)₃²⁺ in presence of 30 mM sodium nitrite in deoxygenated H₂O before (black line) and after (gray line) the addition of 1 eq of sodium ascorbate (200 mM buffer MES, pH 5.8). (B) Recovery of Cu(II)(TRIL23H)₃²⁺ absorbance. The spectra were collected every 3 min after ascorbate addition. (C) Absorbance values at 640 nm vs. time.

Fig. 3.

(A) UV spectra of a solution of sodium ascorbate and sodium nitrite (pH 5.9) collected every 5 min, containing Cu(II)(TRIL23H)₃²⁺ (0.180 mM) and apo-(TRIL23H)₃ (0.090 mM). (B) ■, decrease of [Asc⁻] vs. time in samples, as in A; ○, samples containing only apo-(TRIL23H)₃ (0.090 mM), pH 5.8.

Fig. 4.

(A) Values of k_first,Cu (X) and k_first,Asc (●) as a function of the pH. (B) Pseudo-first-order rate constants (k_first,Asc, ▲) values as a function of the pH.