Spectroscopic Definition of the Biferrous and Biferric Sites in de Novo Designed Four-Helix Bundle DFsc Peptides: Implications for O₂ Reactivity of Binuclear Non-Heme Iron Enzymes

Abstract

DFsc is a single chain de novo designed 4-helix bundle peptide that mimics the core protein fold and primary ligand set of various binuclear non-heme iron enzymes. DFsc and the E11D, Y51L and Y18F single amino acid variants have been studied using a combination of near-IR circular dichroism (CD), magnetic circular dichroism (MCD), variable temperature variable field MCD (VTVH MCD) and x-ray absorption (XAS) spectroscopies. The biferrous sites are all weakly antiferromagnetically coupled with μ-1,3 carboxylate bridges and one 4-coordinate and one 5-coordinate Fe, very similar to the active site of Class I ribonucleotide reductase (R2) providing open coordination positions on both irons for dioxygen to bridge. From perturbations of the MCD and VTVH MCD the iron proximal to Y51 can be assigned as the 4-coordinate center and XAS results show that Y51 is not bound to this iron in the reduced state. The two open coordination positions on one iron in the biferrous state would become occupied by dioxygen and Y51 along the O2 reaction coordinate. Subsequent binding of Y51 functions as an internal spectral probe of the O2 reaction and as a proton source that would promote loss of H2O2. Coordination by a ligand that functions as a proton source could be a structural mechanism used by natural binuclear iron enzymes to drive their reactions past peroxo biferric level intermediates.

Systematic Perturbations of Binuclear Non-heme Iron Sites: Structure and Dioxygen Reactivity of de Novo Due Ferri Proteins

“…38,40 Previous studies have suggested that incorporation of Ala to Gly mutations along the active site channel in DF proteins would decrease steric hindrance of solvent and substrate active site accessibility. One such study 40 showed that the perturbation of DFsc with four Ala to Gly mutations (A10G, A14G, A43G, and A47G), G4DFsc, altered protein reactivity.…”

“…Moreover, the resulting biferric form oxidizes substrates, as in natural diiron proteins, but not at appreciable rates. 38,39 By contrast, the biferrous form of G4DFsc catalyzes the O 2 -dependent two-electron oxidation of 4-amino-phenol (4AP). 40 However, the oxidation of 4-aminophenol by G4DFsc appears to be much slower than in the 4-Ala version of the protein, despite the fact that G4DFsc has a less sterically encumbered binding site.…”

Protein Design: Toward Functional Metalloenzymes

“…985,986 While rapid oxidation of the diferrous center was observed, it was due to an off-pathway iron-tyrosinate complex and not the important diferric intermediate observed for the other diferrous DF proteins. 987,988 Substitution of the four Ala residues to Gly (G4-DFsc) led to the minimization of this complex and a scaffold, which could also catalyze 4-aminophenol oxidation on the same order of magnitude as DFsc was obtained. 989 Most recently, the DeGrado group has shown that a single mutation (from Ile) provides an additional coordinating His residue at the dimetal center of G4-DFsc and results in a protein that can now catalyze the N -hydroxylation of arylamines such as p -anisidine, an activity not detected for G4-DFsc.…”

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

“…This strategy has successfully produced both a Zn-binding carbonic anhydrase mimic with activity that approaches that of the native system, as well as a Cu-binding nitrite reductase (CuNiR) mimic. [8] Other successful non-heme redox active de novo constructs include the Due Ferri system [9, 10] and 4Fe-4S clusters. [11, 12] …”