H2O2-dependent substrate oxidation by an engineered diiron site in a bacterial hemerythrin

The O2-binding carboxylate-bridged diiron site in DcrH-Hr with an engineered His residue in place of Ile119 promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2.


Materials and Instruments
Oligonucleotides were obtained from Invitrogen, Inc. Restriction enzymes were obtained from Takara Bio Inc. Nucleotide sequences were determined by FASMAC Co., Ltd. or the University of Texas Health Science Center, San Antonio Nucleic Acids Core Facility. All reagents of the highest guaranteed grade were purchased and used as received unless otherwise noted. Sodium azide-1-15 N was purchased from Isotech Laboratories Inc. Distilled water was demineralized by a Barnstead NANOpure DIamond apparatus. Purification of the proteins was performed using a GE Healthcare ÄKTA Purifier system at 4 ºC. The UV−vis experiments were conducted using a Shimadzu UV-3150 double-beam spectrophotometer equipped with a thermostated cell holder with a 0.1 ºC deviation or Shimadzu BioSpec-nano. The FTIR experiments were conducted using a Jasco FT/IR 6100. During FTIR measurements, the sample temperature was maintained at 5 K by an Oxford Instruments continuous liquid helium cryostat equipped with a turbo pump to lower the vapor pressure of the liquid helium. The pH values were monitored with a Horiba F-52 pH meter.

Protein Overexpression and Purification
The expression plasmids of His-tagged and tag-free recombinant DcrH-Hr were prepared by the previously described procedures. S1 The plasmid containing the gene encoding the I119H variant was constructed by a round-the-horn site-directed mutagenesis protocol using the plasmids for the wild-type as a template. The forward primer overlapping the Ile119 codon, 5'-CATCATATGAAGGAGGACAAGAAGTACGAGGCGTACTTGCGCG-3', where the variant codons are underlined, and reverse primer, 5'-GTTCACCAGCCAGTCGACGAGTCCG-3', were used for the tag-free I119H variant. In a similar way, the forward primer, 5'-GGCTGGTCAACCACCACATGAAAGAAGACAAAAAATATGAAGCG-3' and the reverse primer, 5'-AGTCAACCAGACCGCGCAGCGTGGTC-3', were used for the His-tagged I119H.  The as-purified DcrH-Hr variants were oxidized by addition of at least 10 eq of potassium ferricyanide at 4 ºC for 16 h, and the resulting samples were purified using a HiTrap desalting column (GE Healthcare) equilibrated with 50 mM HEPES (pH 7.0).

Reaction of Reduced I119H with O 2
The met form of I119H (met-I119H) (50 M) was anaerobically reduced to the deoxy form by the addition of 1 eq of sodium dithionite at 25 ºC. The solution was incubated for 30 min in a processed using CCP4 package. S4 The reported structure of the met form of WT (met-WT) (PDB code 3AGT) was used as a starting model for rigid-body refinement by REFMAC5. S5 The model was refined with multiple rounds of manual rebuilding using Coot, S6 and crystallographic refinement at a 1.9 Å resolution. The data collection and refinement statistics are listed in Table S1. Figs depicting the structure were prepared with PYMOL (http://www.pymol.org). The atomic coordinates and structure factors (PDB code 3WHN) have been deposited into the Protein Data Bank, http://www.rcsb.org/.

Computational Procedure
The broken-symmetry DFT calculations were performed on the models of I119H, in which H118 or H119 coordinates to Fe1, with Gaussian09 program package. S7 In the H118 coordination model (H118on/H119off model), the atomic coordinates of the active site, which is composed of two iron atoms, the bridging E63 and D123, the five His residues coordinating to the iron atoms (H23, H59, H78, H82, H118), I119, and a chloride ion were extracted from the three-dimensional atomic structures of Cl − -bound met-WT determined at 1.4 Å resolution (PDB code: 3AGT), S1 and I119 was replaced with His. The H119 coordination model (H118off/H119on model) was constructed using the three-dimensional atomic structures of the two iron atoms, E63, D123, H23, H59, H78, H82, H119, M120, and a chloride ion of the three-dimensional atomic structures of the Cl − -bound met-I119H mutant determined at 1.9 Å resolution (PDB code: 3WHN). S1 In both models, the C atom is replaced with a CH 3 group.
The environmental effect inside the protein was computed with a polarized continuum model (PCM) S17 using UAKS cavity S18 with a dielectric constant of 4.0. S19 The C and C atoms of the coordinating residues were fixed during geometry optimization to preserve the structure and emulate the partially constraining effect of the protein environment.
The interaction energies (E int ) were evaluated between the first coordination sphere, which is composed of the two iron atoms, the chloride ion, the bridging residues, and five His residues coordinating to the iron atoms, and His119 for the H118on/H119off model or Met120 for the , where E model denotes the total energy of the H118on/H119off model or the H118off/H119on model, E 1st represents the total energy of the first coordination sphere, and E X is the total energy of His119 in the H118on/H119off model or Met120 in the H118off/H119on model. The geometries of the first coordination sphere, His119, and Met120 were extract from the optimized structures of the H118on/H119off and H118off/H119on models.

Resonance Raman Spectroscopy
Resonance Raman scattering was excited at 488.0 nm with an Ar + laser (Spectra Physics, 2017) and detected with a CCD (Princeton Instruments) attached to a triple polychrometer (JACSO, NR-1800). The slit width was set to 200 m. The laser power was 100 mW at the sample point. The spectra were collected at 25 ºC with a spinning cell. The concentration of all samples was controlled to be 1 mM. Toluene and acetone were used as references. Accuracy of the peak positions of the Raman bands was ±1 cm −1 .

FTIR Spectroscopy
The azide adduct of met-WT and met-I119H were prepared by mixing protein solution (1 mM) in 50 mM HEPES (pH 7.0) with 0.9 eq of sodium azide at 4 ºC for 12 h. The formation of the azide adduct was confirmed by increase of the absorption at 443 nm for met-WT and 425 nm for met-I119H, respectively. A 10 L droplet of the solution of the azide adduct was sandwiched between two CaF 2 windows with a 25 m polypropylene spacer. The cryostat was installed in the FTIR sample compartment and kept in the temperature dropped to 5 K. A series of FTIR spectra of 512 scan accumulations were collected with a 4 cm −1 resolution using a FT/IR-6100 type A (JASCO) equipped with liquid N 2 cooled MCT detector.

H 2 O 2 Consumption
The amount of unreacted H 2 O 2 was determined by titration with iodide ion after mixing 2 L of 500 mM H 2 O 2 with 200 L of 100 M protein solution in 50 mM HEPES (pH 7.0). Each 10 L of the reaction mixture was added to 1 mL of 1 M NaI aq after 1, 3, 5, 10, 15, 30, 60, 120, and 180 min.
The amount of I 3 − was determined by the absorption ( max 350 nm,  = 3.1 × 10 4 M −1 cm −1 ).   a The distances and the angle of met-WT, met-Hr, and met-Hr(OH) were obtained from the reported structures in PDB entries 3AGT, 1A7D, and 1A7E, respectively. The values of met-I119H are from this work. b X is Cl for met-WT and met-Hr, and OH for met-Hr(OH). c H118 for met-WT and H119 for met-I119H.