La Gatta, Salvatore (2023) De novo designed copper-containing metalloenzymes for oxidative chemistry. [Tesi di dottorato]

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Abstract

Copper proteins are involved in several key biological processes, such as electron-transfer, dioxygen binding and activation, pigmentation, methane monooxygenation, and denitrification. The copper’s ubiquity is mostly related to its abundance on the earth crust, the two accessible oxidation states, and its coordinative promiscuity. It is, in fact, found in a range of coordination environments, which are classified by their spectroscopic properties. Moreover, the protein matrix strictly controls selectivity and catalytic efficiency of the copper center by primary and outer sphere interactions. The most abundant copper sites are the so-called Type 1, 2, and 3. While the subtle interplay of bonding and non-bonding interactions has been exhaustively explored for simpler and closed-shell Type 1 proteins, Type 2 and 3 remain more elusive in their structure-function correlations. Among several approaches to study natural metalloenzymes, such as protein engineering and site-directed mutagenesis, de novo protein design represents a valuable strategy to transplant the target metal-binding-site in a simpler and possibly more stable small-sized scaffolds. This generally allows for direct evaluation of metal-protein interactions by a bottom-up approach, in which structural determinants for metal-binding are engineered from scratch. In this thesis, three copper-binding designed proteins are showcased: (i) DR1, a four-helical bundle hosting a Type 3 copper site; (ii) miniLPMO, a quite unnatural homodimeric four-helical bundle hosting a Type 2 copper site; (iii) dHisB, a heterodimeric three-helical bundle hosting a Type 2 copper site. DR1 (Due Rame, in Italian) is a newly designed protein that mimics polyphenol oxidases and contains a di-copper site. The first and second di-metal coordination spheres were hierarchically engineered in order to nest the di-copper site into a simpler scaffold made of a four-helix bundle. DR1 recapitulates the Type 3 copper site, supporting several copper redox states and being active in the O2-dependent oxidation of catechols to o-quinones, according to spectroscopic, thermodynamic, and functional analysis. Most importantly, DR1 is endowed with substrate recognition thanks to the careful design of the binding pocket residues, as confirmed by Hammett analysis and computational studies on substituted catechols. Moreover, deeper spectroscopic characterization prompted us to propose a kinetic model for dioxygen activation for this synthetic enzyme, which involves hydrogen peroxide as an intermediate. In parallel, the de novo design of a peptide-based model inspired by lytic polysaccharide monooxygenase (LPMO) proteins was undertaken. Motivated by the elusive nature of the histidine brace copper-binding site, as found in LPMO proteins, the design process was performed on two different scaffolds of increasing complexity. MiniLPMO is a C2-symmetric de novo designed miniprotein that homodimerizes to form a bisecting four-helix bundle, as confirmed by CD analysis. MiniLPMO binds copper with a histidine brace-like motif, as assessed by UV-Visible absorption and continuous wave/pulsed EPR spectroscopy and shows oxidase activity towards the model substrate 4-nitropheyl-beta-D-glucopyranoside. In a second approach, symmetry was released and a heterodimeric construct was designed, featuring a helix-loop-helix (alfa2 motif) that specifically recognizes a helical peptide (alfa chain), generating a heterodimeric three-helix bundle, called dHisB (designed Histidine Brace). The design process led to a single histidine brace site hosted on the N-terminal side of the alfa chain, facing toward the loop of the alfa2 motif that exerts secondary shell interactions. The desired folding and heterodimerization were assessed by size-exclusion chromatography, CD spectroscopy and direct quantification of peptide content by reversed-phase HPLC. Using a combination of continuous wave EPR and UV-Visible absorption spectroscopy over a range of pH values, it has been shown that the active site of dHisB can exist in different protonation states. These small models recapitulate the spectroscopic fingerprint of natural LPMOs, confirming the power of the design strategy. Interestingly, dHisB is able to cycle between both copper oxidation states under mild conditions and bind exogenous ligands like sodium azide. Finally, O2-activation has been shown by the Amplex Red assay with 3-fold apparent enhancement with respect to free copper in aqueous solution. Remarkably, the three models allowed a very fine spectroscopic characterization, not always attainable with natural proteins. The Type 2 copper-containing miniproteins allowed unraveling for the first time, to the best of our knowledge, the specific contribution to the EPR spectrum of the different protonation states of the histidine brace center over a wide range of pH (from pH 2 to 13). Noteworthy, all the designed models activate O2 or H2O2 in a similarly way to their natural counterparts. These simple models represent a milestone in the development of synthetic metalloenzymes for the degradation and conversion of biomass into second-generation fuels.

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