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

Abstract

De novo proteins provide a unique opportunity to investigate the structure-function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O(2)-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling 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 structural changes resulted in at least a 10(6)-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

Figure 1

Important structural features of and amino-acid sequences for the original and redesigned DFsc proteins. (a) Surface models of DFsc (top) and G4DFsc (bottom) based on the initial DFsc computational design. The four Ala to Gly substitutions (shown in white) significantly open the substrate access channel. (b) Structure of 3His-G2DFsc variant (PDB 2LFD) highlighting the added active-site His residue (H100) and supporting mutations (I37N and L81H). The helix closest to the viewer is shown as transparent to allow for viewing of the ligands. Note that the structure shown is for a variant with two Ala and two Gly residues along the substrate access channel which proved more stable during the extended data collection times required for the structure determination. This variant still exhibited N-oxygenase activity, but to a lesser extent than 3His-G4DFsc. (c) Amino acid sequences for DFsc, G4DFsc, and 3His-G4DFsc. Metal-binding residues are bolded and mutations introduced are underlined.

Figure 2

Folding and metal-binding characterizations of G4DFsc and 3His-G4DFsc. (a) UV-CD spectra of metal-free, Co(II)-bound, and Zn(II)-bound G4DFsc (top) and 3His-G4DFsc (bottom). Each sample contains 20 μM protein and 100 mM metal ions (where applicable). (b) Co(II) titration monitored by visible absorption spectroscopy for 100 μM G4DFsc (top) and 100 μM 3His-G4DFsc (bottom). The red lines are linear regressions of the initial and final linear regions of the curves to determine the stoichiometric ratio of Co(II) to protein. For these individual trials, the lines intersect at 225 μM for G4DFsc and 247 μM for 3His-G4DFsc, versus the theoretical value of 200 μM. The titrations have been performed in triplicate at slightly different protein concentrations, resulting in calculated Co(II):protein stoichiometries of 1.90 ±0.33 and 2.65±0.15. Insets display a representative Abs spectrum collected in the presence of 2 equivalents of Co(II) ions. Extinction coefficients at the peak maxima are 157 M⁻¹ cm⁻¹ for G4DFsc and 94 M⁻¹ cm⁻¹ for 3His-G4DFsc per Co(II) ion. (c) Fe(II) titration monitored by near-IR CD spectroscopy for G4DFsc (top) and 3His-G4DFsc (bottom). For both proteins, the titrations saturate at 2 equivalents of Fe(II).

Figure 3

Ferroxidase activity of G4DFsc (top) and 3His-G4DFsc (bottom) indicated by the formation of a strong oxo-to-ferric charge transfer band near 360 nm. The saturation of this feature occurs much faster for G4DFsc than 3His-G4DFsc (see insets), but is more intense for 3His-G4DFsc (~4600 M^-1 cm^-1 per di-iron site) than G4DFsc (~2300 M⁻¹ cm⁻¹ per di-iron site). These molar absorptivity values are in range with those observed for natural oxo-bridged di-iron proteins.

Figure 4

Hydroquinone oxidase activity of G4DFsc (top) and 3His-G4DFsc (bottom). Only G4DFsc reacts to an appreciable extent with 4-aminophenol to form the corresponding quinone (as monitored through a coupling reaction with m-phenylenediamine that produces a species with λ_max=486 nm).

Figure 5

The oxygenation of p-anisidine by G4DFsc (top) and 3His-G4DFsc (bottom) in the presence of two equivalents of Fe(II). No reaction is observed in the presence of G4DFsc, but in the 3His-G4DFsc reaction, an absorption feature at 360 nm is observed to grow in and then decay (inset, black triangles) followed by the appearance of a strong absorption feature at 445 nm (inset, red circles) arising from the formation of 4-nitrosodiphenylamine.

Figure 6

N-hydroxylation of p-anisidine by 3His-G4DFsc. (a) HPLC chromatograms (monitoring Abs at 220 nm) for the reaction mixture of 0.25 mM 3His-G4DFsc with 5 mM p-anisidine at 0 (black), 2 (red), and 14 (blue) hours after mixing. Products were identified by LC-MS/MS (m/z values indicated in panel b) and comparison with authentic samples. (b) Proposed reaction scheme depicting the oxidation of p-anisidine to p-nitrosoanisole and subsequent nucleophilic aromatic substitution with unreacted p-anisidine to form 4-nitrosodiphenylamine that gives rise to a visible absorption features at 445 nm.