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

Abstract

DFsc (single-chain due ferri) proteins allow for modeling binuclear non-heme iron enzymes with a similar fold. Three 4A → 4G variants of DFsc were studied to investigate the effects of (1) increasing the size of the substrate/solvent access channel (G4DFsc), (2) including an additional His residue in the first coordination sphere along with three additional helix-stabilizing mutations [3His-G4DFsc(Mut3)], and (3) the three helix-stabilizing mutations alone [G4DFsc-(Mut3)] on the biferrous structures and their O2 reactivities. Near-infrared circular dichroism and magnetic circular dichroism (MCD) spectroscopy show that the 4A → 4G mutations increase coordination of the diiron site from 4-coordinate/5-coordinate to 5-coordinate/5-coordinate, likely reflecting increased solvent accessibility. While the three helix-stabilizing mutations [G4DFsc(Mut3)] do not affect the coordination number, addition of the third active site His residue [3His-G4DFsc(Mut3)] results in a 5-coordinate/6-coordinate site. Although all 4A → 4G variants have significantly slower pseudo-first-order rates when reacting with excess O2 than DFsc (~2 s−1), G4DFsc and 3His-G4DFsc(Mut3) have rates (~0.02 and ~0.04 s−1) faster than that of G4DFsc(Mut3) (~0.002 s−1). These trends in the rate of O2 reactivity correlate with exchange coupling between the Fe(II) sites and suggest that the two-electron reduction of O2 occurs through end-on binding at one Fe(II) rather than through a peroxy-bridged intermediate. UV–vis absorption and MCD spectroscopies indicate that an Fe(III)Fe(III)-OH species first forms in all three variants but converts into an Fe(III)-μ-OH-Fe(III) species only in the 2-His forms, a process inhibited by the additional active site His ligand that coordinatively saturates one of the iron centers in 3His-G4DFsc(Mut3).

Molecular-Level Insight into the Differential Oxidase and Oxygenase Reactivities of de Novo Due Ferri Proteins

“…This is confirmed where the rate of the O 2 reaction for G4DFsc+P-AN is 0.002 s −1 (Figure S14) (relative to ~0.02 s −1 without p -anisidine). 52 In contrast, the binding of p -anisidine to the 3His form results in only a small change in exchange-coupling (− J from 1–2 cm −1 to 2–3 cm −1 ), and the rate of obtaining steady state for nitroso production ( k ≈ 0.045 s −1 , Figure S15) is close to the O 2 reaction rate of the 3His form without substrate (~0.04 s −1 ). 52 …”

“…52 However, 3His-G4DFsc(Mut3) only formed the biferric paramagnetic species B due to the additional His coordinatively saturating one iron center. In our prior studies that showed oxidase reactivity for G4DFsc, but not for the 3His form, Fe(II) was added under aerobic conditions to a solution of apo-protein and 4-AP ( 1 ) in the presence of m -phenylenediamine ( 3 ), which couples to the oxidized product (quinone imine) ( 2 ) to yield an aminoindoaniline dye ( 4 ) with λ max = 486 nm ( ε = 13 200 cm −1 M −1 ) at pH 7 (Figure 2).…”

“…In our prior studies that showed oxidase reactivity for G4DFsc, but not for the 3His form, Fe(II) was added under aerobic conditions to a solution of apo-protein and 4-AP ( 1 ) in the presence of m -phenylenediamine ( 3 ), which couples to the oxidized product (quinone imine) ( 2 ) to yield an aminoindoaniline dye ( 4 ) with λ max = 486 nm ( ε = 13 200 cm −1 M −1 ) at pH 7 (Figure 2). 51 The conditions of the prior studies, which allowed for the simultaneous presence of the biferric (Species B and C) 52 and biferrous forms and may have included contributions from Fe(II) binding to the protein, complicated our ability to identify the catalytically active species. Using more controlled reaction conditions, the prior studies 51 were thus extended to identify the reactive species of G4DFsc responsible for catalyzing the oxidation of 4-AP, as well as address the apparent lack of activity in the 3His form.…”

“…The CD and MCD spectra for 3His-G4DFsc(Mut3) with excess 4-AP were also resolved into three bands (Figure 5B) that have the same signs and energies as those of the 3His form without 4-AP but with ~7 times greater intensity with substrate bound. 52 Note that the small shifts in energy and narrowing of the band shapes in the MCD spectra are due to the lower temperature of data collection. The three bands for the 3His form (7400(+), 9300(−), and 10 500(+) cm −1 for CD and 7700, 9400, and 10 300 cm −1 for MCD) also have the same sign and are at energies very similar to those for G4DFsc with 4-AP bound (G4DFsc+4-AP).…”

“…52 The transfer of the second electron from the remote Fe(II) site would then occur through the carboxylate bridges. In this model, the decrease in the carboxylate-bridge-mediated exchange-coupling of G4DFsc upon addition of p -anisidine would result in decreased O 2 reactivity.…”