Redesign of a Copper Storage Protein into an Artificial Hydrogenase

Abstract

We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H 2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H + reduction to H 2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H 2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.

A De Novo‐Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys

“…The turn over frequency (TOF) follows a bell‐shape pH profile with a maximum TOF of 32 h −1 mol −1 (Figure 4C) at pH 5.6, corresponding to a turn over number (TON) of about 44 over 2 h. Ni‐rubredoxin showed a TON of 32 photocatalytically [8a] at similar concentrations (25 μ m ). Our previously reported ArH, NBP had a TON of 115 for light‐induced H 2 production [8d] . In other reported examples where the well‐known DuBois catalyst has been encapsulated in photosystem I directly or via flavodoxin, the TON for photogenerated H 2 was 1.87×10 3 and 2.8×10 3 , respectively [5a] .…”

Remarkable stability of a molecular ruthenium complex in PEM water electrolysis

“…29,30 They contain polypyridyl, 14,30 bipyridine 31,32 or porphyrinoid-type 29 ligands and can be considered as artificial metalloproteins. 33,34 Some dinuclear ruthenium complexes have been used as [Fe–Fe] hydrogenase mimics, 35,36 but only one complex has been used as an electrocatalyst for the splitting of H 2 into protons and electrons. 37 Even though many (electro)catalysts for hydrogen production are known in the literature, very few of them have actually been employed as immobilized catalysts in a full PEM electrolyser setup.…”

Biosynthetic Approaches towards the Design of Artificial Hydrogen‐Evolution Catalysts

“…We have reported the redesign of a naturally existing copper storage protein (Csp1) into a mononuclear nickel binding protein (NBP) featuring a NiS 4 active site as a HER catalyst . Csp1 is a four‐helix bundle that binds 13 equivalents of Cu I to 13 Cys residues along the protein core .…”