Peroxidase Activity of Myoglobin Variants Reconstituted with Artificial Cofactors

Abstract Myoglobin (Mb) can react with hydrogen peroxide (H2O2) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo‐peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo‐Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands. These Mb variants were fully characterised by UV‐Vis spectroscopy, circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP‐MS) and native mass spectrometry (nMS). The steady‐state kinetics of guaiacol oxidation and 2,4,6‐trichlorophenol (TCP) dehalogenation catalysed by Mb variants were determined. Mb variants with iron chlorin e6 (Fe−Ce6) and manganese chlorin e6 (Mn−Ce6) cofactors were found to have improved catalytic efficiency for both guaiacol and TCP substrates in comparison with wild‐type Mb, i. e. Fe‐protoporphyrin IX‐Mb. Furthermore, the selected cofactors were incorporated into the scaffold of a Mb mutant, swMb H64D. Enhanced peroxidase activity for both substrates were found via the reconstitution of Fe−Ce6 into the mutant scaffold.


Materials and Instruments
The commercial Mb was purchased from Sigma-Aldrich, the E.coli Top 10 and BL21 (DE3) cells and the plasmid pET-30(a)-eYFP was a gift from the Clark lab (Department of Chemical Engineering, University of California, Berkeley) [1] , the sperm whale myoglobin (swMb) gene was synthesised by GenScript with codon optimization. High-fidelity polymerase (Phusion) was purchased from Thermo Fisher, the T4 ligase was purchased from New England Biolabs.
The yeast extraction and tryptone for culture was from BD Biosciences. Isopropyl β-D-1thiogalactopyranoside (IPTG), δ-aminolevulinic acid (5-ALA) and kanamycin were ordered from Sigma-Aldrich. Other chemicals and solvents used were used as received from commercial supplier without further purification. Lab ultrapure water was from a Milli-Q integral 15 water purification system. All expression media were sterilized by either autoclave (45 min, 121 °C, Astell AMB440) or a sterile syringe filter (0.22 µm). To maintain sterile conditions, all the related experiments were manipulated in a class II biosafety cabinet (Thermo Scientific S2020 1.2). PCR reactions were performed with a SimpliAmp thermal cycler (Applied Biosystems), E.coli Top10 and BL21 (DE3) competent cells were prepared following the Hanahan method [2] , cell lysis was carried out by sonication (120 W, 20 kHz, Fisherbrand Model 120 Sonic Dismembrator, Fisher Scientific), UV-Vis spectra were recorded on Cary 60 (Agilent Technologies), CD spectra were recorded on Jasco-J810 spectropolarimeter using quartz cuvettes. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was performed on an Agilent 7700 instrument. Protein purification was carried out on ÄKTA pure chromatography system equipped with columns from GE Healthcare. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was run on a Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad).
Sodium Phosphate (NaPi) and PBS buffer was prepared based on the recipe from Cold Spring Harbor protocols. [3] PBS-Br buffer was modified from common PBS buffer as 100 mM NaBr, 10 mM Na2HPO4, 2 mM KH2PO4.

Mb Mutagenesis Protocol
The primers were designed according to guidelines (Agilent QuickChange II site-directed mutagenesis kit). Their melting temperature (Tm) were estimated with the formula: where N is the primer length in bases. Ideal Tm should be higher than 78 °C.

Protocol for Native Mb Purification
To maximize heme occupancy, swMb was reconstituted with heme chloride. Briefly, the lysate solutions were rapidly mixed with 0.2 mM of heme chloride (20 mM stock solution in 10 mM NaOH) and incubated at 4 °C for 10 min. The lysate was centrifuged at 7500 g for 20 min, the supernatant was transferred to another Falcon tube and the same centrifuge was applied again. After this, the supernatant was filtered through a 0.25 μm pore size membrane filter before being subjected to Ni-NTA agarose chromatography. The protein solution was concentrated with an Amicon Ultra-15 (10 kDa cut-off) centrifugal filter device (Merck Millipore). The concentration of native Mb was determined by UV-Vis absorbance measurements at 407 nm using an extinction coefficient 188 mM -1 •cm -1 . [4]

Synthesis of Cofactors
Scheme S1. Structures of protoporphyrin IX and chlorine e6. Cu-Ce6/Co-Ce6/Mn-Ce6/Ni-Ce6/Ru-Ce6/Ru-PPIX were synthesised with the reaction conditions stated in the following Table. Reaction conditions of cofactor synthesis Under positive argon pressure, the reagents were dissolved in specific solvents and heated under reflux at the mentioned temperatures. After this, the solvent was removed under reduced pressure, the residue taken up in a small amount of MeOH, filtered and re-dissolved in CH2Cl2. The crude product was purified by silica column. Unreacted salt was first eluted with CH2Cl2, followed by unreacted free base. The cofactor product was collected by elution

Reconstitution of Mb Variants
In vitro reconstitution of artificial myoglobin with non-native cofactors was carried out in two steps. Firstly, the removal of the iron-protoporphyrin IX (Fe-PPIX, heme) was based on the Teale's method [5] : extraction of the cofactor via cold 2-butanone in acidic condition (pH 2.5) and followed by extensive dialysis against PBS buffer to refold the protein (apo-Mb). Second, an equal volume of cold solution of excess (10 eqiv.) cofactors in dimethylformamide (DMF) was added to 1 mL of the apo-Mb solution with slowly shaking at 4 °C. The molar ratio between the cofactor and apo-Mb was close to 5:1, while keeping the organic solvents lower than 2% v/v. The mixture was then dialyzed against a 100-fold volume of desired buffer (via 10 KDa cut-off dialysis tube) and purified via a Sephadex G-25 column to remove free cofactor.
Final protein solution was concentrated with an Amicon ultra centrifugal filter device to desired concentration. The myoglobin variants were maintained at 4 °C and used within 2 days.

Characterisation of Protein Samples by UV-Vis Spectroscopy
Before the sample measurement, the baseline was corrected with the blank buffer. The scan rate was set to 4800 nm min -1 with 1 nm interval. All the protein samples without specific mentions were measured in PBS buffer at 22 °C. Cofactor characterisation was measured in Milli-Q water containing 1% DMF. Soret peak, Q bands and A280 were collected to characterise Mb samples.

Characterisation of Mb Variants by CD Spectroscopy
The concentration of protein samples was firstly measured (Lowry method) and then adjusted to 0.1 μg•mL -1 . Samples were measured in 2 mm pathlength quartz cuvettes. The sample scan was from 260 to 185 nm with 1 nm resolution, 1 nm bandwidth, 100 mdeg sensitivity and 50 nm•min -1 scan speed. Spectra were averaged from three consecutive scans and smoothed over five data points. The measurement was carried out at 22 °C in 20 mM Tris-HCl buffer (pH 8.0). The spectra were smoothed by the means-movement method using Jasco Spectra Analysis software and subjected to secondary structure analysis using the analysis programme CDSSTR (Reference set 6) provided by DICHROWEB.
[6] Reference set 6 was selected because of its match with the wavelength range. It also contains myoglobin, and heamoglobin.

Sample Preparation for ICP-MS Analysis
Protein samples were firstly desalted with Milli-Q water using Amicon centrifugal filter device.
UV-Vis spectroscopy was used to measure the Soret peak absorbance and to estimate the

Characterisation of Mb Variants by SDS-PAGE
Separation gel (10%) and stacking gel were prepared based on the following order:

Characterisation of Mb Variants by Native Mass Spectrometry
Protein samples were desalted and buffer exchanged into 100 mM ammonium acetate with

Spectroelectrochemical Redox Potential Determination
The

Stopped-flow Colorimetric Peroxidase Assays
The peroxidase activity assay of Mb and its variants in PBS buffer (pH 7.4) was performed on a UV-Vis spectrometer equipped with a dual mixing stopped-flow unit (Applied Photophysics  (Table S6)   Here, koff represents the dissociation rate constant of the Mb-H2O2 complex. Figure S1. UV-Vis spectra of free PPIX and Ce6 in the absence of metal ions.                  Mean values and standard deviations of triplicates are shown.