Rational Design of Artificial Metalloproteins and Metalloenzymes with Metal Clusters

Abstract

Metalloproteins and metalloenzymes play important roles in biological systems by using the limited metal ions, complexes, and clusters that are associated with the protein matrix. The design of artificial metalloproteins and metalloenzymes not only reveals the structure and function relationship of natural proteins, but also enables the synthesis of artificial proteins and enzymes with improved properties and functions. Acknowledging the progress in rational design from single to multiple active sites, this review focuses on recent achievements in the design of artificial metalloproteins and metalloenzymes with metal clusters, including zinc clusters, cadmium clusters, iron-sulfur clusters, and copper-sulfur clusters, as well as noble metal clusters and others. These metal clusters were designed in both native and de novo protein scaffolds for structural roles, electron transfer, or catalysis. Some synthetic metal clusters as functional models of native enzymes are also discussed. These achievements provide valuable insights for deep understanding of the natural proteins and enzymes, and practical clues for the further design of artificial enzymes with functions comparable or even beyond those of natural counterparts.

Keywords: metalclusters; metalloenzymes; metalloproteins; protein design; synthetic models.

Figure 1

Artificial metalloproteins and metalloenzymes with diverse metal clusters shown in the periodic table.

Figure 2

(a) Cage structure of three domain-swapped Cytcb₅₆₂ dimers (PDB code 5AWI), and a close-up view of the Zn²⁺ and SO₄²⁻ ions in the internal cavity. Reprinted with permission from Ref. [59], Copyright 2016 The Royal Society of Chemistry; (b) Crystal structure of 4DH1 (PDB code 5WLL), and a close-up view of the designed tetranuclear zinc cluster. Reprinted with permission from Ref. [63], Copyright 2018 American Chemical Society.

Figure 3

(a) X-ray crystal structure of a tetranuclear Cd–thiolate cluster (PDB code 4G1A), and the coordination structure. Reprinted with permission from Ref. [67], Copyright 2013 Elsevier; (b) X-ray crystal structure of apo-L161C/L165C-Fr with Cd ions bound (PDB code 6JEE), and a close-up view of the fourfold axis channel. Reprinted with permission from Ref. [70], Copyright 2019 The Royal Society of Chemistry; (c) X-ray crystal structure of tetrameric hsALR (PDB code 3R7C), and a close-up view of the Cd₂Cl₄O₆ cluster. Reprinted with permission from Ref. [71]. Copyright 2012, International Union of Crystallography; (d) X-ray crystal structure of nvPizza2-S61H58 (PDB code 5CHB), and a close-up view of the Cd₇Cl₁₂ cluster. Reprinted with permission from Ref. [72], Copyright 2015 Wiley-VCH.

Figure 4

(a) Pd(allyl) complexes binding to the threefold channel of apo-Fr, which form a trinuclear Pd-cluster as a result of H114A mutation. Reprinted with permission from Ref. [76], Copyright 2008 American Chemical Society. (b) X-ray crystal structures Au-loaded apo-Frs in the absence and presence of different concentrations of NaBH₄, showing the process of formation of an Au-cluster at the threefold channel of apo-Fr [77].

Figure 5

(a) X-ray structure of Zr₃–nFbp showing the coordination geometry. Reprinted with permission from Ref. [79]. Copyright 2004 Wiley–VCH; (b) Structure of the Mo/WSto protein of A. vinelandii, and a close-up view of the oxo-W₃-cluster. Reprinted with permission from Ref. [80], Copyright 2007 Wiley-VCH; (c) X-ray structure of a dimer of the metal-binding domain ofcellular copper efflux protein ATP7B (WLN4), and a close-up view of a cluster Mo₂S₆O₂ at the interface [81].

Figure 6

(a) Rational design of two [4Fe-4S] clusters in domain-swapped dimer(DSD)-Fdm that transfer electrons to ferric Cyt c. Reprinted with permission from [86]. Copyright 2014 American Chemical Society; (b)X-ray structure of BMC-T1-S55C and a close-up view of the [4Fe-4S] cluster (PDB code 5DII). Reprinted with permission from [89]. Copyright 2014 American Chemical Society; (c) Rational design of a [4Fe-4S] cluster in cytochrome c peroxidase (CcP) that closely mimics the active site of native sulfite reductase (SiR). Reprinted with permission from [98]. Copyright 2018 AAAS.

Figure 7

(a) An artificial metalloenzyme constructed by replacement of the M-cluster with a synthesized [Fe₆S₉(SEt)₂]⁴⁻ cluster in nitrogenase scaffold. Reprinted with permission from [102]. Copyright 2015 Wiley-VCH; (b) The structure of [4Fe-4S(SCH₂CH₂OH)₄]²⁻ cluster (left), and its catalysis of CO₂/CO reduction in the presence of europium(II) diethylenetriaminepentaacetic acid (Eu^II-DTPA) (right) [104]. Copyright 2018 Nature press; (c) The proposed mechanism of l-cluster assembly in nitrogenase, followed by the maturation of M-cluster. SAM: S-adenosyl-l-methionine; NifH: the reductase component of Mo-nitrogenase;HC:homocitrate. Reprinted with permission from [103]. Copyright 2018 Macmillan Publishers.

Figure 8

(a) The X-ray structure of the Cuz center in N₂OR from P. nautica (PDB code 1QNI [112]); (b) The coordination geometry of the Cuz center in native N₂OR; (c–e) The structures of the synthetic functional models of the native Cuz center [117,118,119].

Figure 9

(a) The X-ray crystal structure of native oxygen-evolving center (OEC) showing the core Mn₄CaO₅ cluster and the ligands; (b) The X-ray crystal structure of a synthesized Mn₄CaO₄ cluster; Reprinted with permission from [121], Copyright 2015 AAAS; (c) The chemical structure of biotinylated Co₄O₄ cluster, biot-β-Ala-1; (d) The X-ray crystal structure of 2xm-S112Y-Sav with biot-β-Ala-1 bound (PDB code 6AUE); (e–f) Proposed mechanisms for the multi-e⁻/multi-H⁺ reactivity of the designed enzyme at pH <9.5 or pH >9.5.Reprinted with permission from [123]. Copyright 2018 American Chemical Society.