Design of metal cofactors activated by a protein–protein electron transfer system

Ueno et al. 10.1073/pnas.0510968103.

Supporting Information

Files in this Data Supplement:

Supporting Text
Supporting Table 4
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Supporting Figure 5

Fig. 5. Spectroelectrochemical titration of Fe(Schiff-base)•HO composites. (300 mM in 10 mM Tris•HCl buffer, pH 7.3 at 15°C). (A) 1•HO. (B) 2•HO. (C) 3•HO. The spectra were collected over a potential range of –500 to 50 mV vs. Ag/AgCl. (Inset) Nernst plots of reductive and oxidative titration. The fraction of the ferric form was plotted as a function of the applied potential from the change in absorbance at 387, 395, and 363 nm of 1•HO, 2•HO, and 3•HO, respectively.

Supporting Figure 6

Fig. 6. Liner fitting to observed pseudofirst rate constants versus square root of ditionite concentration. 1•HO, blue; 2•HO, green; 3•HO, black; Heme•HO, red.

Supporting Figure 7

Fig. 7. Absorption changes of Fe(Schiff-base)•HO composite at 400 nm by dithionite (250 mM). (A) 1•HO. (B) 2•HO. (C) 3•HO. (D) Heme•HO.

Supporting Figure 8

Fig. 8. k_obs for the iron reduction of Fe^III(Schiff-base)•HO composites by CPR. 1•HO, blue; 2•HO, green; 3•HO, black; Heme•HO, red.

Supporting Figure 9

Fig. 9. Absorption changes of Fe(Schiff-base)•HO composite at 415 nm by CPR. CPR concentrations are 0.05 mM (red), 0.10 mM (green), 0.20 mM (light blue), and 0.30 mM (blue). (A) 1•HO. (B) 2•HO. (C) 3•HO. (D) Heme•HO.

Supporting Figure 10

Fig. 10. Catalytic O₂ reduction measurements. (A) NADPH consumption rates were observed by UV-vis spectral changes caused at 360 nm. (B) Oxygen consumption rates were measured by oxygen electrode. Each measurement was started with the addition of NADPH solution (final concentration, 100 mM) into the protein mixture solution [final concentrations of Fe(Schiff-base)•HO composite, CPR, and SOD are 2 mM, 1 mM and 100 units, respectively] in 10 mM Tris•HCl buffer (pH 7.4) at 15°C, and catalytic rates of NADPH and O₂ consumption were determined at the beginning of reaction. 1•HO, solid line; 2•HO, dotted line; 3•HO, broken line.

Table 4. Summary of x-ray data from the crystals of 1•HO, 2•HO, and 3•HO

Supporting Text

General Procedure and Materials. Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification. 3-(3,4-diaminophenyl)propionic methylester (1) and Fe[N,N'-bis(salicylidene)phenylenediamine] (3•Cl) were prepared according to published procedures (2). Redox potentials, and all reduction rate constants were determined under argon.

Synthesis of Fe[N,N'-bis(salicylidene)-3,4-diamino benzenepropanoic acid]•Cl (1•Cl) 3,4-diamino-benzenepropanonic acid. To a 1 M HCl aqueous solution (1.0 ml) was added 3-(3,4-diaminophenyl) propionic methylester (500 mg, 2.57 mmol), and then the mixture was refluxed for 6 h and the solution was neutralized by 1 M sodium hydroxide aqueous solution. After removal of the solvent, the residue was recrystallized from hot ethanol to give this compound (144 mg): Yield: 31.1%. ¹H NMR (270 MHz, CDCl₃): d6.40 (s, 1H), 6.35 (d, J = 7.0 Hz, 1H), 6.21 (dd, J = 7.6, 1.9 Hz, 1H), 2.54 (t, J = 7.8 Hz, 2H), 2.38 (t, J = 7.6 Hz, 2H).

N,N'-bis(salicylidene)-3,4-diamino benzenepropanoic acid (10-CH₂CH₂COOH-salophen-H₂). To an ethanol solution of 3,4-diamino benzenepropanoic acid (71 mg, 0.39 mmol, 5.0 ml) was added salicylaldehyde (140 ml, 1.3 mmol). The reaction mixture was refluxed for 2 h and stood at room temperature. It was obtained as yellow powder. Yield: 76%, ¹H NMR (270 MHz, D₂O); d 8.56 (d, J = 3.5 Hz, 2H) 7.30 (m, 4H), 7.12 (s, 2H), 7.02 (s, 1H), 6.97 (m, 2H), 6.86 (m, 2H), 2.96 (t, J = 8.0 Hz, 2H), 2.69 (t, J = 7.2 Hz, 2H); Analysis: calculated for C₂₃H₂₀N₂O, C (71.12), H (5.19), N (7.21), found for: C (71.12), H (5.15), N (7.12).

Fe(10-CH₂CH₂COOH-salophen)•Cl (1•Cl). To a acetonitrile solution of 10-CH₂CH₂COOH-salophen-H₂ (26 mg 0.07 mmol, 1.0 ml) was added FeCl₃•6H₂O (23 mg, 0.1 mmol), and the mixture was refluxed for 4 h. 1•Cl was obtained as black microcrystals (15 mg). Yield 48%, UV/Vis: l_max(EtOH)433 nm (e/m^-1cm^-1 7800), 370 nm (16000), 334 nm (22000), 299 nm (30000).

Fe[N,N'-bis(salicylidene)-3,4-diamino benzoic acid]•Cl (2•Cl) N,N'-bis(salicylidene)-3,4-diamino benzoic acid (10-COOH-salophen-H₂). Salicylaldehyde (330 ml, 3.1 mmol) was added to an ethanol solution of 3,4- diaminobenzoic acid (237 mg, 1.55 mmol, 5.0 ml). The reaction mixture was refluxed for 2 h and stand at room temperature. The product was obtained as orange powder. Yield 45.0%, ¹H NMR (270 MHz, DMSO): d 12.82 (s, 1H), 12.60 (s,1H), 9.05 (s,1H), 8.98 (s,1H),7.99-7.94 (m, 2H), 7.75-7.70 (m, 2H), 7.55-7.40 (m, 3H), 7.00-6.95 (m, 4H).

Fe(10-COOH-salophen)•Cl (2•Cl). 2•Cl was synthesized according to the method of 1•Cl. Yield: 16%, UV/Vis: l_max(EtOH)431 nm (e /m^-1cm^-1 10000), 379 nm (16000), 335 nm (25000), 300 nm (33000); ESI-TOFMS (m/z):[M]⁺ calcd for C₂₁H₁₆N₂O₄NaFe, 437.02; found, 437.04.

Physical Measurement. UV-vis spectra were recorded on a Shimadzu UV-2400PC UV-vis spectrometer. ¹H NMR spectra were recorded on a JEOL JNM-ECP500 and JNM-GSX270. Concentration of iron ion was determined by a Polarizing Zeeman-effect atomic absorption spectrometer Z-5710 (Hitachi, Tokyo) opening in graphite furnace mode using a Fe hollow cathode lamp.

Thermal Stability. Melting points (T_m) of the composites were determined according to a reported procedure (3). The conditions are described below. Sample concentration: 2.5 mM in 10 mM Tris•HCl buffer at pH 7.4 and 4°C and temperature range: 20–60°C (heating rate: 50°C/h).

Crystallization, X-Ray Data Collection, and Crystallographic Refinement. 1•HO, 2•HO, and 3•HO were dialyzed against 20 mM Mes buffer (pH 7.0) and concentrated to 19, 18, and 12 mg/ml, respectively. Crystals were obtained by hanging drop vapor diffusion method from a drop of the solution of the composite (1 ml) and a reservoir solution (1 ml) at 20°C. Reservoir solutions (500 ml) for 1•HO contained Polyethylene Glycol 2000 monomethyl ester 30% (wt/vol) and ammonium sulfate (0.2 mM) in 100 mM Mes buffer (pH 6.5). These conditions for 2•HO and 3•HO were 37.5% (wt/vol), 0.16 mM, and pH 6.7. For cryogenic date collection, crystals were soaked into each reservoir solution containing 10% (vol/vol) glycerol and flash-frozen by liquid nitrogen.

Full data set of 1•HO was collected to 1.85-Å resolution with an R-AXIS VII imaging plate (Rigaku, Tokyo) using a CuKa radiation generated by Rigaku micromax 007 at the Hybrid Nano-Material Research Center of the Institute of Multidisciplinary Research for Advanced Materials (Tohoku University). The temperature around the crystals was maintained at 100 K throughout the data collection. The oscillation angle and camera range were 1° and 110 mm, respectively. Data were integrated, merged, and processed with HKL-2000 (4). Rigid-body refinement up to 3 Å was performed by CNS (5) using the structure of HmuO without heme from Corynebacterium diotheriae (6) as an initial model for the refinements. The refinement was carried out as described (6). The final R and R_free factors were dropped to 14.9 and 19.5, respectively. We used this model (1•HO initial) for initial model for refinement of more high-resolution structures of 1•HO, 2•HO, and 3•HO.

High-resolution diffraction data for 1•HO, 2•HO, and 3•HO were collected with ADSC Quantum 315 using 1-Å synchrotron radiation at BL5 of Photon Factory (Ibaraki, Japan). The temperature around the crystals was maintained at 100 K throughout the data collection. The oscillation angle, camera range, and exposure time were 1°, 150 mm, and 1 s, respectively. Data sets consisted of 180 frames. Data were integrated, merged, and processed with HKL-2000. Diffraction statistics are summarized in Table 4.

The structures were solved by using molecular replacement with the structure of 1•HOinitial as an initial model for the refinements. The structure was refined by same method with 1•HOinitial. Complete statistics are summarized in Table 4. Several residues located in N- and C-terminal regions were not determined due to their disorder. Drawing was made by PYMOL or DISCOVERY–STUDIO (Accelrys, Inc., San Diego).

Coordinates and structural factors have been deposited in the Protein Data Bank (PDB ID codes 1WZD, 1WZF, and 1WZG for 1•HO, 2•HO and 3•HO, respectively).

Redox Potential Measurements for Fe(Schiff-base)•HO Composites. All redox potentials were determined by a described method (7). Current voltage was controlled on an ALS electrochemical analyzer model 660A (BAS). UV-vis spectra were recorded on an OOBIBase32 instrument (Ocean Optics, Dunedin, FL). A working gold mesh electrode (40 ´ 9 ´ 0.7 mm) was immersed in the optical cell (path length: 0.5 mm). A platinum wire and Ag/AgCl (3 M KCl) electrodes were used as auxiliary and reference electrodes, respectively. Gold mesh and platinum wire were purchased from Nilaco (Tokyo). Final concentration; Fe(Schiff-base)•HO composite: 300 mM, sodium chloride: 100 mM; and electron mediators: 5 mM in 10 mM Tris•HCl buffer (pH 7.3) at 15°C. UV-vis spectral changes and Nernst plots of 1•HO, 2•HO, and 3•HO upon the reduction/oxidation are shown in Fig. 5. Redox potentials of 1•Cl, 2•Cl, and 3•Cl in N,N-dimethylformamide (DMF) were determined by cyclic voltammetry following a described method (8). Cyclic voltammetry were recorded on an ALS electrochemical analyzer model 660A (BAS). Concentrations of each complexes were 100 mM in 10 ml of DMF solution with 0.1 M tetra-n-butylammnonium tetrafluoroborate as the supporting electrolyte.

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