Palladium(II) and platinum(II) bind strongly to an engineered blue copper protein

Abstract

Studies of palladium(II) and platinum(II) binding to well-characterized proteins contribute to understanding the influence of these metals in the environment and the body. The well-characterized apo-protein of azurin has a soft-metal binding site that may be exposed to solvent by mutation of a coordinating histidine-117 residue to glycine. Palladium(II) and platinum(II) form strong 1:1 adducts with the apo form of H117G azurin. A combination of UV-visible, CD, and ICP-MS techniques suggests that the metal binds specifically at His-46 and Cys-112 of the protein.

Platinum-group elements (PGEs) play an integral role in modern chemistry, catalysis, and chemotherapeutic treatments. Because of their heavy use, they have entered our environment, particularly from leaching of catalyst from the catalytic converters in automobiles.^1,2 Catalytic converters emit PGEs in inhalable and commutable particle sizes (0.1–20 nm) that are rapidly complexed into soluble and mobile species after deposition.^3–5 As a result, PGEs are highly bioavailable and known to bioaccumulate in plant and aquatic life.^6,7 Human exposure to PGEs is an increasing concern, specifically following treatment with platinum for battling cancer.^8–10 Exposure to cisplatin and its derivates leads to highly elevated and long-term persistent in vivo exposure to bioreactive Pt.¹¹ Thus it is important to understand the chemistry underlying PGE interactions with biomolecules.

Though there has been progress on understanding the interactions of these metals with nucleic acids,¹² there remains a need for information on the binding of Pd²⁺ and Pt²⁺ to proteins. Interactions of Pd²⁺ and Pt²⁺ with proteins have been studied, but the precise location of metal binding is rarely known.^13–22 There is also interest in Pd²⁺-protein adducts as potential scaffolds for catalysis^23–25 including hydrolytic cleavage of peptides.^26–31 Pd²⁺ salts also influence amyloid fibril formation.^32,33

This contribution describes studies utilizing the apo-protein of azurin, a type 1 copper metalloprotein with a characteristic deep blue color. The canonical copper-binding site of Pseudomonas aeruginosa (Pa) azurin consists of four amino acid residues: His-46, Cys-112, His-117, and Met-121 (Figure 1a).³⁴ Pa azurin has been used extensively in the study of protein-metal interactions because it provides a common mononuclear binding site for Zn²⁺, Ni²⁺, Co²⁺, Mn²⁺, Cd²⁺, Au⁺, Ag⁺, and Hg^2+.35–43 Another advantage of azurin is that, as a small protein that is amenable to expression on a large scale from recombinant E. coli, it may easily be mutated to modify the coordination environment of copper^44–47 or other metals^48,49 in its metal binding site. Among mutations to the primary coordination sphere of the copper site, the H117G mutation is special because it is a small change that allows significant solvent access to the metal, enabling exogenous ligands to bind to copper(II) in place of the missing His-117 residue.^46,50,51 For example, addition of copper(II) and an excess of N-methylimidazole to the apo form of H117G apo-azurin results in a characteristic 630 nm absorbance band and a coordination environment similar to the wild type protein.⁵⁰ Figure 1b illustrates the binding of N-methylimidazole to the H117G copper(II) azurin. We anticipated that the compiled knowledge of H117G azurin would offer a way to unambiguously characterize Pd²⁺ and Pt²⁺ binding to native protein residues.

Figure 1.

The copper(II) sites of (a) azurin, (b) H117G azurin with added N-methylimidazole (Me-im).

Upon the addition of 1 equiv of MCl₄²⁻ to a 0.48 mM solution of the apo-form of H117G azurin (buffered at pH 7.0 with 5 mM of 3-(N-morpholino)propanesulfonic acid (MOPS)), a new broad absorbance developed in the UV-vis spectrum for M = Pd (ε = 2.6 mM⁻¹cm⁻¹ at 324 nm) and M = Pt (ε= 0.54 mM⁻¹cm⁻¹ at 330 nm). The spectra are shown in the Supporting Information. Addition of these metal salts to wild type protein does not yield any significant new absorbance, showing that the mutation is essential for PGE binding. The new absorptions in the Pd²⁺ and Pt²⁺ adducts of the apo-H117G azurin are reminiscent of those seen in Pd²⁺ and Pt²⁺ complexes with anionic sulfur donors, suggesting coordination to cysteinate.^52,53 The dependence of the absorbance on added metal concentration (Figure 2) fits to a standard binding curve (eq 1), yielding association constants of 2.1(5) × 10⁴ M⁻¹ for Pd²⁺ and 1.2(7) × 10⁵ M⁻¹ for Pt²⁺. Thus, both PGEs bind strongly, and Pt²⁺ binds roughly 6 times more strongly than Pd²⁺.

Figure 2.

Development of UV-visible intensity upon the addition of (a) Pd²⁺ and (b) Pt²⁺ to 0.48 mM apo H117G azurin in 5 mM MOPS buffer, pH = 7.0. The lines represent fits to binding curves using equation 1.

$$[\text{ML}]=\frac{({[\text{M}]}_0+{[\text{L}]}_0+\frac{1}{{\text{K}}_{\text{eq}}})-{\{{({[\text{M}]}_0+{[\text{L}]}_0+\frac{1}{{\text{K}}_{\text{eq}}})}^2-4{[\text{L}]}_0{[\text{M}]}_0\}}^{1/2}}{2}$$ (1)

The development of the new absorbance saturated in each case at approximately 1 equiv of Pd²⁺ or Pt²⁺ relative to protein (Figure 2). Exchanging the buffer after addition of the metal salt gave no significant change to the UV-vis spectra, indicating low k_off rates. After buffer exchange and dilution, the PGE loaded proteins were digested with acid followed by analysis with inductively coupled plasma spectroscopy (ICP). The metal analysis indicated that the proteins incorporated 95% Pd²⁺ and 98% Pt²⁺ into the protein. Since there are no alternative metal binding sites known in azurin,⁴⁶ and the new UV absorbance is consistent with a thiolate-to-metal charge transfer transition,^52–54 these results suggest that the heavy metals bind at the copper site with Cys and His donors.

In order to further support the location of metal binding, we took advantage of the fact that Cu²⁺-bound H117G azurin develops a intense blue color upon binding of N-methylimidazole to regenerate the N₂S₂ coordination environment of the wild-type copper(II) protein.⁵⁰ This property enabled us to query the status of the copper binding site as shown in Scheme 1. Addition of copper(II) and an excess of N-methylimidazole to the apo form of H117G azurin resulted in the expected copper-based LMCT absorbance at λ_max = 630 nm, with a ratio of absorbances A₆₃₀/A₂₈₀ = 0.48. In constrast, addition of Cu²⁺ and N-methylimidazole to the Pd²⁺ or the Pt²⁺ substituted protein did not result in appearance of the 630 nm absorbance band. The inability of the Pd²⁺ and Pt²⁺ substituted proteins to bind copper suggests that the heavy metals bind at the copper site, but does not preclude the possibility that the protein is somehow damaged by the heavy metals in a manner that prevents copper binding. We tested for protein damage by incubating the Pd²⁺ and Pt²⁺-loaded proteins in an excess of the soft ligand 2-mercaptoethanol for 12 hours to chelate the PGE salt, followed by buffer exchange. After this treatment to remove the heavy metal, the copper binding ability of the protein was completely restored (A₆₃₀/A₂₈₀ = 0.48). These results indicate that the protein had undergone no irreversible change, and provides additional support for Pd²⁺ and Pt²⁺ binding specifically at the H46/C112 site of the engineered azurin.

Scheme 1.

Using the Cu²⁺ binding ability of H117G azurin to query M²⁺ binding at the copper site.

To evaluate whether the addition of Pd²⁺ and Pt²⁺ changed the secondary structure of H117G azurin, circular dichroism (CD) experiments were performed after the addition of either Pd²⁺ or Pt²⁺ to the apo form of H117G azurin. The CD spectrum, which matches the literature spectrum for the azurin protein,⁵⁵ was unchanged by the H117G mutation or by the addition of either metal (Figure 3). These results suggest that Pd²⁺ and Pt²⁺ binding to the protein does not induce any significant conformational changes. Thus, our experiments argue against a change in the structure of the H117G azurin apo-protein upon PGE binding, and clearly point toward PGE binding at the vacant copper-binding site of H117G apo-azurin.

Figure 3.

Circular dichroism spectra of H117G azurin in the apo form and after the addition of Pd²⁺ and Pt²⁺. Samples have 0.22 mM protein in 5 mM MOPS buffer, pH = 7.0.

In conclusion, the copper-binding apo-protein of azurin may be engineered so that it coordinates a single Pd²⁺ or Pt²⁺ ion. Each ion binds with K_assoc > 20,000 M⁻¹, and binding to Pt²⁺ is stronger than to Pd²⁺. Our evidence indicates that the metal binds to the cysteine at the vacant copper site, and thus we suggest that the metals have square-planar NSCl₂ coordination that includes His-46 and Cys-112 of the protein.