Alteration of the oxygen-dependent reactivity of de novo Due Ferri proteins

Abstract

De novo proteins provide a unique opportunity for investigating the structure-function relationships of metalloproteins in a minimal, well-defined, and controlled scaffold. Herein, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the due ferri family. Originally created to catalyze O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyze the selective N-hydroxylation of arylamines by remodeling the substrate access cavity and introducing a critical third His ligand to the metal binding cavity. Additional second-and third-shell modifications were required to stabilize the His ligand in the core of the protein. These changes resulted in at least a 106 –fold increase in the relative rates of the two reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

De Novo Design of Tetranuclear Transition Metal Clusters Stabilized by Hydrogen-Bonded Networks in Helical Bundles

“…55,56 In each case, the crystallographic and NMR studies showed the proteins folded into their designed conformations, which recapitulated the main structural features of much larger and more complex diiron proteins. 8–10,57 A highly symmetrical starting protein was used in the initial design, but this symmetry was eliminated in subsequent functional studies in which the coordination environment of each metal ion was systematically varied. 10–12,57–59 The DF scaffold was ultimately exploited to examine the structural requirements for two-electron and four-electron reduction of dioxygen with concomitant substrate oxidation.…”

Predictive methods for computational metalloenzyme redesign – a test case with carboxypeptidase A

“…26 The addition of an extra metal-binding residue and adjusting the substrate cavity switch a due ferri protein originally intended to oxidize hydroquinones to catalyze the selective N-hydroxylation of arylamines. 27 While design efforts focused on metal-free enzymes have yielded many useful in silico tools, these tools are insufficient for metal-containing enzymes. [28][29][30] When designing metalloenzymes, it is critical to properly treat the electronic structure of the metal.…”

Protein design with a comprehensive statistical energy function and boosted by experimental selection for foldability

“…These progresses have been driven by underlying computational or rule-based design methods, which can be stringently calibrated through the de novo design of amino acid sequences that fold into desired three-dimensional structures 4,[8][9][10] . In this regard, rule-based designs have attained some remarkable successes 7,11,12 , albeit limited to particular types of target structures or structure motifs. More general methods such as RosettaDesign 13 are based on minimizing an effective energy function, which has been, for large parts, derived from molecular mechanics force fields 4 .…”

“…I n recent years, protein design has achieved some milestone successes with profound implications in biosciences and biotechnology [1][2][3] , including the designs of a novel protein fold 4 , new biomolecular interactions and regulations 5 , as well as new biocatalysts 6,7 . These progresses have been driven by underlying computational or rule-based design methods, which can be stringently calibrated through the de novo design of amino acid sequences that fold into desired three-dimensional structures 4,[8][9][10] .…”