Modular Artificial Cupredoxins

Abstract

Cupredoxins are electron transfer proteins that have active sites containing a monomeric Cu center with an unusual trigonal monopyramidal structure (Type 1 Cu). A single Cu-Scys bond is present within the trigonal plane that is responsible for its unique physical properties. We demonstrate that a cysteine-containing variant of streptavidin (Sav) can serve as a protein host to model the structure and properties of Type 1 Cu sites. A series of artificial Cu proteins are described that rely on Sav and a series of biotinylated synthetic Cu complexes. Optical and EPR measurements highlight the presence of a Cu–Scys bond and XRD analysis provide structural evidence. We further provide evidence that changes in the linker between the biotin and Cu complex within the synthetic constructs allows for small changes in the placement of Cu centers within Sav that have dramatic effects on the structural and physical properties of the resulting artificial metalloproteins. These findings highlight the utility of the biotin-Sav technology as an approach for simulating active sites of metalloproteins.

Peroxide Activation Regulated by Hydrogen Bonds within Artificial Cu Proteins

“…Representation of biotinylated Cu complex [Cu II (biot-et-dpea)(H 2 O) 2 ] 2+ (A), the molecular structure of 1 (B, PDB 5K49), 26 and the reaction conditions to form 2 (C). The dashed lines indicate H-bonds.…”

“…Its molecular structure (PDB 5WBC) revealed an H-bonding network involving the aqua ligand containing O3, the structural water molecule (O1), the side chain O-atom of residue S112, and the backbone carbonyl O-atom from A86. 26 The placement of O1 is noteworthy as it is present in many structures of Sav WT that we have studied and is positioned to H-bond to ligands coordinated to [Cu II (biot-et-dpea)] 2+ . Treating 1 with excess H 2 O 2 in buffer A (Figure 2C & 3) produced changes in the absorbance spectrum that are, again, indicative of formation of a Cu II –OOH species, that is [Cu II (biot-et-dpea)(H 2 O)(OOH)] + ⊂Sav WT ( 2 ).…”

Artificial Metalloproteins Containing Co4O4 Cubane Active Sites

“…14 Figure 1a shows the structure of biot-β-Ala- 1 ⊂ 2×m-Sav to 1.46 Å resolution. Some ligand positions about the Co III centers could not be modeled due to insufficient electron density likely caused by ligand rotation (about the Co—N py axis) or exchange processes (at fluxional Co—OH 2 sites).…”

Coordination chemistry within a protein host: regulation of the secondary coordination sphere

“…21 This work established design principles to produce active sites that contain metal complexes with unusual primary coordination spheres. We have also established that Sav can be used to stabilize reactive Cu II –OOH species through H-bonds within the local environment provided by Sav WT.…”

“…1) ArM that we previously showed contains mononuclear Cu(II) complexes within each subunit of Sav. 21 The molecular structure of 1 (PDB 5WBC) shows the Cu(II) center with nearly trigonal bipyramidal coordination geometry (Fig. S1 and Table S1, ESI † ) with three N-atom donors from the dpea ligand.…”