Oxygen Reactivity of the Biferrous Site in the de novo Designed Four Helix Bundle Peptide DFsc: Nature of the “Intermediate” and Reaction Mechanism

Abstract

The DFsc and DFscE11D de novo designed protein scaffolds support biomimetic diiron cofactor sites that react with dioxygen forming a 520 nm "intermediate" species with an apparent pseudo-first-order formation rate constant of 2.2 and 4.8 s-1, respectively. Resonance Raman spectroscopy shows that this absorption feature is due to a phenolate-to-ferric charge transfer transition arising from a single tyrosine residue coordinating terminally to one of the ferric ions in the site. Phenol coordination could provide a proton to promote rapid loss of a putative peroxo species.

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

“…46 No prominent 520 nm bands appear in the UV–vis absorption in the O 2 reactivity of the biferrous form of any of the 4A → 4G variants studied here. The higher coordination of these variants likely protects the ferric sites from tyrosine binding.…”

“…Neither of these types shows a prominent 520 nm UV–vis absorption band in contrast to DFsc, which forms a tyrosine-bound biferric species upon reacting with O 2 . 46 …”

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.…”

Artificial Diiron Enzymes with a De Novo Designed Four‐Helix Bundle Structure

“…Furthermore, these substitutions also minimized the formation of an off-pathway tyrosinate–iron complex that occurred in previous versions of the designed protein. [170] However, substitution of four Gly residues into the DFsc scaffold resulted in an apo form slightly less α-helical than the holo form. In addition, greater active-site accessibility increases the exposure of the iron atoms to the aqueous solvent, which renders them prone to hydrolysis.…”