A De Novo‐Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity

Abstract

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di‐copper site and mimicking the Type 3 (T3) copper‐containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di‐metal coordination spheres to engineer the di‐copper site into a simple four‐helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2‐dependent oxidation of catechols to o‐quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four‐helix bundle protein.

Experimental Evidence and Mechanistic Description of the Phenolic H-Transfer to the Cu2O2 Active Site of oxy-Tyrosinase

“…In summary, this study (i) shows that the rate-limiting TS of monophenol monooxygenation in Ty is tuned by the substrate para -group (TS1 for EDGs versus TS2 for EWGs; see Figure A) and (ii) provides direct experimental evidence and a detailed mechanistic description of the phenolic H-transfer to the peroxide of the μ-η 2 :η 2 -Cu­(II) 2 (O 2 2– ) site of oxy-Ty. Together with our previous study on the subsequent aromatic hydroxylation step, our results define a complete mechanistic framework for the monophenol monooxygenation reactivity of oxy-Ty, which is fundamentally different from previous proposals and revises our understanding of O 2 activation and reactivity in biological CBC sites. ,, These new mechanistic insights into the monooxygenation reaction of oxy-Ty provide the basis for understanding the elusive structure–function correlations between the different families of CBC enzymes, ,, as well as for engineering CBC active sites in de novo enzymes and homogeneous and heterogeneous catalysts. ,,, …”

“…Coupled binuclear copper (CBC) active sites are found in a diverse set of catalysts, ranging from native and artificial metalloenzymes to heterogeneous materials, including zeolites and metal–organic frameworks. − These CBC sites have attracted significant interest, as they activate O 2 to catalyze a wide array of challenging oxidative transformations. In biology, the prototypical O 2 -activating CBC enzyme tyrosinase (Ty) catalyzes the regioselective hydroxylation of para -substituted monophenols to catechols, including the conversion of l -tyrosine to l -3,4-dihydroxyphenylalanine ( l -DOPA), which constitutes the initial step in melanin biosynthesis across a wide range of organisms from soil bacteria to humans. , In its fully reduced form (deoxy-Ty; center panel in Figure ), its dicopper­(I) active site binds and activates O 2 to form a well-characterized μ-η 2 :η 2 -peroxide dicopper­(II) intermediate (oxy-Ty; Figure ) − that regioselective hydroxylates para -substituted monophenols to catechols.…”

“…Together with our previous study on the subsequent aromatic hydroxylation step, 12 our results define a complete mechanistic framework for the monophenol monooxygenation reactivity of oxy-Ty, which is fundamentally different from previous proposals and revises our understanding of O 2 activation and reactivity in biological CBC sites. 1,5,19 These new mechanistic insights into the monooxygenation reaction of oxy-Ty provide the basis for understanding the elusive structure−function correlations between the different families of CBC enzymes, 16,17,26 as well as for engineering CBC active sites in de novo enzymes 2 and homogeneous and heterogeneous catalysts. 3,4,33,34 ■ ASSOCIATED CONTENT…”

Designed Rubredoxin miniature in a fully artificial electron chain triggered by visible light

“…De novo design provides a remarkable alternative, which allows incorporating all the desired mutations at once, and generating the most suited structural arrangement for testing and refining folding and functions, such as redox potential 55 . Design of synthetic metalloproteins by miniaturization is particularly helpful, limiting the metal surroundings to only a few crucial residues 56,57 . Therefore, it was of considerable interest to develop by design and miniaturization a synthetic Rd, METPsc1, and show that it is capable of keeping the intended structural and functional properties in a small 28-residue peptide.…”

Revving up a Designed Copper Nitrite Reductase Using Noncoded Active Site Ligands

“…In principle, designing metalloproteins from the ground up allows one to simplify the metalloenzyme’s structure, yet capture important properties of the native enzyme such as those around the first and second coordination sphere in an entirely different fold. Modifying the primary coordination sphere in artificial, redox-active metalloenzymes with unnatural heterocycles has been exploited to make new biologically inspired catalysts and to learn more about existing enzymes. − A key goal of de novo metalloprotein design is building protein models that share a similar metal environment of a native enzyme in a very dissimilar scaffold, removing any evolutionary baggage that is unnecessary for catalysis. − However, evolution has selected different N-isomers of histidine to bind copper, depending on the function of the protein. While there are rare exceptions, CuN ε sites typically catalyze oxidase or oxygenase reactions while CuN δ sites participate in electron transfer. − Herein, we have altered the orientation of the Cu-active site by incorporating two different pyridine ligands into a simple parallel 3SCC composed of the repeating heptad [Ac-G­(L a K b A c L d E e E f K g ) 4 G-NH 2 )] (TRIW) to explore the NiR activity of unnatural T2Cu centers (Figure ).…”