Use of apomyoglobin to gently remove heme from a H 2 O 2-dependent cytochrome P 450 and allow its reconstitution †

The heme of hydrogen peroxide-dependent cytochrome P450BSb (P450BSb) was removed by apomyoglobin under mild conditions to give apo-P450BSb without the need for acidic conditions and organic solvents. The circular dichroism spectrum of the apo-P450BSb was essentially identical to that of holo-P450BSb, showing a small structural change resulting from the removal of heme using apomyoglobin. The apo-P450BSb was reconstituted with hemin or manganese protoporphyrin IX (MnPPIX), and the resulting reconstituted P450BSb catalyzed the one-electron oxidation of guaiacol using hydrogen peroxide as an oxidant. A higher catalytic activity was observed for P450BSb reconstituted with MnPPIX when meta-chloroperoxybenzoic acid (mCPBA) was used as the oxidant.


Introduction
Heme, Fe-protoporphyrin IX, is one of the most important metal complexes in nature, serving as the prosthetic group for hemoproteins that perform diverse functions, including oxygen storage and transport, gas sensing, electron transfer, and catalysis. 1,2 The properties of hemoproteins largely depend on the nature of heme; therefore, replacement of the heme with heme analogues are expected to drastically change the properties of hemoproteins. 3 A variety of reconstituted hemoproteins containing heme analogues, and heme derivatives including different metal ions, chemically modified hemes, and synthetic metal complexes have been constructed with the aim of developing synthetic proteins with (improved) catalytic activity or entirely different functions. 4,5 To allow the reconstitution of hemoproteins with synthetic metal complexes, the tightly bound heme must be removed to give apoproteins. Noncovalently bound hemes (b-type hemes) can be extracted from hemoproteins using organic solvents such as butan-2-one and acetone under acidic conditions (pH 2-4). Teale et al. first reported the removal of heme from hemoproteins utilizing acid-butanol (pH = 2) to prepare apo forms of horse heart and skeletal muscle myoglobin (Mb). 6 Several methods based on a slight modification of this acid-butanol method have been reported for the preparation of apo forms of hemoproteins such as HRP and P450 cam (see Fig. S1 and S2 for these different heme enzyme structures, ESI †). 7,8 However, these methods are not suitable if the hemoproteins as well as their apo-forms are not sufficiently stable in organic solvents and/or under acidic conditions. Although the preparation of apoproteins in bacterial cells has been developed as an alternative, their reconstitution was limited to metal complexes possessing a protoporphyrin IX framework. To avoid the use of harsh chemical treatments in the preparation of apoproteins, and to expand the range of applicable metal complexes to include various synthetic metal complexes, it is important to develop a simple strategy for removing the heme of hemoproteins without the need for organic solvents and acidic conditions. Cytochrome P450 BSb (P450 BSb ) is a hemoprotein that is irreversibly denatured during the preparation of apoprotein under the methods described above. Therefore, it has been believed to be impossible to prepare reconstituted P450 BSb . However, P450 BSb is regarded as a promising candidate for the construction of a biocatalyst because P450 BSb and its unique family, the hydrogen peroxide-dependent P450s, efficiently utilize hydrogen peroxide to catalyze various C-H bond hydroxylations and some other types of oxidation reactions. 9,10 If the heme of P450 BSb could be replaced with synthetic metal complexes, the properties of the enzymatic reaction catalyzed by P450 BSb could be altered to allow the development of a versatile biocatalyst.
Here we report a simple and useful method for the preparation of apo-P450 BSb using apo-Mb under very mild conditions (Scheme 1). We decided to employ apo-Mb as a heme scavenger because it binds strongly to heme and can be readily prepared by the aforementioned acid-organic solvent-methods; the dissociation constant of apo-Mb for heme is reported to be 10 À14 M À1 . 11 We also demonstrate that the resulting apo-P450 BSb can be reconstituted with manganese protoporphyrin IX (MnPPIX) and that the resulting reconstituted P450 BSb exhibits improved one-electron oxidation activity when mCPBA was employed as an oxidant.

Materials
All chemicals were purchased from commercial sources and used without further purification unless otherwise indicated. Mn(III) protoporphyrin IX chloride was obtained from Frontier Scientific, Inc. (Logan, UT, USA) and used after exchanging its Cl À for BF 4 À by treating with AgBF 4 . Hydrogen peroxide, dithiothreitol (DTT), ethylene glycol, sodium dodecyl sulfate (SDS), isopropyl-b-D-1-thiogalactopyranoside (IPTG), and kanamycin sulfate were obtained from WAKO Pure Chemical Industries, Ltd (Osaka, Japan). Glycerol, potassium chloride, urea, hydrochloric acid, potassium phosphate, ampicillin sodium salt and phenylmethylsulfonyl fluoride were purchased from Nacalai Tesque Inc. (Kyoto, Japan). Horse skeletal muscle Mb was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Concentrations of chemicals described in procedures are the final concentrations.

Measurements
UV-visible (UV-Vis) absorption spectra were recorded on a UV-2600 PC spectrophotometer (Shimadzu Corporation) or an Agilent 8453 equipped with an 89090A thermal controller (Agilent Technologies, Inc.), and the data were collected using screw-capped quartz cuvettes of 1 cm path length. Circular dichroism (CD) spectra were recorded with quartz cuvettes of 0.1 cm path length using a J-720WI spectropolarimeter (Jasco Corporation) equipped with a temperature controller, and the data were recorded from 200 to 260 nm at 24 1C and 20 nm min À1 scan speed, and an average spectrum was produced from four independent consecutive scans. Size distributions were recorded by dynamic light scattering (DLS) using a Nano ZS (Malvern Instruments Ltd) with disposable plastic cuvettes and caps. Inductively coupled plasma-optical emission spectrometer (ICP-OES) measurements were performed on a Varian Vista-Pro ICP Spectrometer (Varian Inc.). The purification procedures described below were carried out using a Bioassist eZ system (Tosoh Corporation). The protein figures were generated using the PyMOL Molecular Graphics System (version 1.8; DeLano Schrödinger, LLC.).

Protein expression and purification
For the expression of recombinant P450 BSb , 6Â histidine-tagged (6Â His-tagged) P450 BSb was expressed in the transformed Escherichia coli strain M15. Cells were cultivated at 27 1C in Luria-Bertani medium supplemented with 100 mg mL À1 of ampicillin and 25 mg mL À1 of kanamycin. Once the culture reached an optical density at 600 nm (OD 600 ) of 0.7-0.8, heme precursor d-aminolevulinic acid (0.5 mM) was added, and the culture was incubated for an additional 30 min. At OD 600 = 1.0-1.2, gene expression was induced by addition of IPTG (0.1 mM), and cultivation was followed by culturing at 20 1C for 20 h rotating at 80 rpm. Cells were then harvested by centrifugation and lysed in 0.1 M potassium phosphate buffer (pH 7.0) with 0.3 M potassium chloride, 50 mM imidazole, and 20% (v/v) glycerol.
Purification of 6Â His-tagged P450 BSb was performed by Ni-chelate affinity chromatography (GE Healthcare, Little Chalfont, UK). The protein was further purified using S-200 Sephacryl (GE Healthcare) size exclusion chromatography. An A Soret /A 280 absorbance ratio Z1 indicated the high purity of the protein, which was also confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Reduced CO-difference spectra were used to determine the concentration of P450 by subjecting the purified recombinant protein to sodium dithionite and CO gas. 12 Protein samples were stored at À80 1C for further use.

Preparation of apo-Mb
A heme moiety was extracted from horse skeletal muscle Mb by the butan-2-one extraction according to a published procedure 6 with slight modifications. The pH of a Mb solution (100 mg Mb in 40 mL Milli-Q water) was adjusted to 2.5 in an ice-water bath using chilled 0.1 M HCl (aq) . The solution was transferred to a separation funnel, and an equal volume of 2-butanone was added. The mixture was gently shaken for 30 s and then allowed to stand for 10 min at 4 1C. The colorless aqueous phase was separated and dialyzed for 24 h at 4 1C against 5 L of Milli-Q water, and then for 24 h against PBS buffer (pH 7.3). Next, the solution was passed through a 0.22 mm filter and stored at 4 1C for further use. The concentration of prepared apo-Mb was spectrophotometrically determined using its extinction coefficient of 15 700 M À1 cm À1 at 280 nm. 13 Preparation of apo-P450 BSb The removal of heme from P450 BSb to prepare apo-P450 BSb was performed by treating P450 BSb with apo-Mb. The reaction was Five equivalents of apo-Mb (25 mL, 42.5 mM) was added to the reaction buffer, and the final concentration of glycerol was adjusted to 5% using additional PBS buffer. The reaction was maintained in a gas-tight flask for 24 h without stirring. Before purification, 0.02% of Triton X-100 was added to aid the separation of P450 BSb and Mb. Purification of 6Â His-tagged apo-P450 BSb was performed by Ni-chelate affinity chromatography to remove holo-Mb and excess apo-Mb. This is the same purification procedure used for recombinant P450 BSb . Sample purity was checked by SDS-PAGE. Pure apo-P450 BSb was stored at 4 8C for further use or stored at À80 8C for long-term preservation.

Reconstitution of apo-P450 BSb
Reconstitution of apo-P450 BSb was performed by adapting the procedure for apo-P450 cam 14 at 25 1C as well as subsequent protein purification at 4 1C. After the preparation of protoporphyrin complex solution, it should be added to apo-P450 BSb buffer immediately. Any delay in the addition of protoporphyrin complex solution to apo-P450 BSb led to incomplete recombination due to its low solubility in water solution. 13 rFe-P450 BSb . A 7 mL apo-P450 BSb (6 mM) solution at pH 7.0 under an argon atmosphere was combined with urea (2 M), DTT (10 mM), histidine (10 mM), and Triton X-100 (0.02%). The solution was then left to stand for a further 15 min at room temperature. A 50 mL KOH solution (0.1 M) containing hemin (2.5 eq., 2.1 mM) and myristic acid (0.2 mM), diluted tenfold with Milli-Q water, was added dropwise to the solution of apoprotein. The whole reaction was performed in a gas-tight flask for 24 h without stirring. Before purification, an Amicon ultracentrifugal filter was used to remove excess hemin and other chemicals. Purification of 6Â His-tagged rFe-P450 BSb was performed by Ni-chelate affinity chromatography to remove free hemin by the same procedure used for the preparation of recombinant P450 BSb . The final purified rFe-P450 BSb gave a single band on SDS-PAGE.
rMn-P450 BSb . The insertion of MnPPIX into the apoprotein was carried out using a 7 mL apo-P450 BSb (6 mM) solution at pH 7.0 under an argon atmosphere. The apoprotein solution was combined with urea (2 M), DTT (10 mM), histidine (10 mM), and Tritont X-100 (0.02%), and then left to stand for 15 min at room temperature. A 210 mL KOH solution (0.1 M) containing MnPPIX(BF 4 ) (2.5 eq., 0.5 mM, the concentration determined using a e 462nm of 25 000 M À1 cm À1 ), 15 diluted tenfold with Milli-Q water, was added dropwise to the solution of apoprotein. The whole reaction was then incubated in a gas-tight flask wrapped in aluminum foil for 24 h without stirring. Before purification, an Amicon ultracentrifugal filter was used to remove excess MnPPIX and other chemicals. Purification of 6Â His-tagged rMn-P450 BSb was performed by Ni-chelate affinity chromatography to remove free MnPPIX by the same procedure as that used for recombinant P450 BSb . The final purified rMn-P450 BS gave a single band on SDS-PAGE, and the A Soret(381) /A 280 absorbance ratio was 0.7.

Determination of P450 concentration
The concentration of the recombinant P450 BSb was measured by reduced CO-difference spectroscopy. 12 In the case of rFe-P450 BSb with a higher content of P420 isoform (Fig. S4, ESI †), the concentration was measured by reduced pyridine-hemochrome spectroscopy. 16 The concentrations of holo-and apo-P450 for CD spectroscopy measurements were determined by the Bradford protein assay. 17  . Initial oxidation rates were determined by monitoring the absorption change at 470 nm relative to the tetramer of guaiacol with an extinction coefficient of 26 600 M À1 cm À1 . 18 All initial turnover numbers (TONs) are the average of at least three measurements, and are expressed as nmol product per nmol P450 per min AE standard deviation.
Using mCPBA as an oxidant. The reaction conditions were similar to those described above, except that the oxidant was changed to mCPBA and the reaction mixture does not contain n-heptanoic acid. The reactions were started by adding 4 mM mCPBA (70 mL of 60 mM ethanol solution) and monitored by the absorption change at 470 nm. The oxidation products were also determined quantitatively, as described previously.

ICP-OES
The iron and manganese contents in rP450 BSb were determined by ICP-OES. Concentrated samples of purified rFe-P450 BSb and rMn-P450 BSb protein were diluted with 0.2 M HNO 3 to 2 mL of 1.5, 3.0, 4.5 and 6.0 mM for the determinations. The solution was placed in a new glass vial and subjected to ICP-OES via a peristaltic pump. The sample was aspirated with argon, passed over plasma, and analyzed for iron and manganese. A control experiment was performed using Milli-Q water as a sample. Commercial calibration standards and multi-element standards (Wako Pure Chemical Industries, Ltd) were used.

Results and discussion
The removal of the heme from P450 BSb to prepare apo-P450 BSb was performed by treating P450 BSb with apo-Mb. When five equivalents of apo-Mb were added to a solution of histidinetagged P450 BSb at 25 1C, apparent shifts of the Soret absorption band and Q-band were observed. ‡ The absorption peak of P450 BSb at 417 nm shifted to 409 nm, which is readily attributed to the formation of holo-Mb (Fig. 1A). After 24 h incubation, the resulting apo-P450 BSb was purified by Ni-chelate affinity chromatography, but a small amount of holo-P450 BSb was still observed. To bring the heme transportation to completion, P450 BSb was reduced using Na 2 S 2 O 4 because the dissociation rate constant of reduced heme (Fe II ) coordinated with cysteine is rapid compared with that of heme (Fe III ). 19 DFT calculations also showed a weaker binding of cysteine to ferroheme than to ferriheme. A clear shift of the absorption maxima of heme was again observed after the addition of apo-Mb, and no remarkable change was observed after 24 h (Fig. 1B).
Apo-P450 BSb purified by Ni-chelate affinity chromatography did not show any absorption assignable to heme, indicating that the heme of P450 BSb can be removed completely (Fig. S5, ESI †). Interestingly, the CD spectrum of apo-P450 BSb was essentially identical to that of the holo form, indicating a small structural change upon the removal of the heme (Fig. 2B), while the CD spectra of apoproteins produced by acid-organic solvent methods were generally significantly different from those of the holo form. 20 For example, apo-P450 cam prepared by the acid-butanone method gave a weaker CD signal, indicative of partial denaturation. The hydrodynamic diameters of holo-and apo-P450 BSb determined by DLS analysis were 4.36 and 5.05 nm, respectively ( Fig. 2C and Fig. S6, ESI †), which are consistent with the size of holo-P450 BSb (PDB ID: 3WSP), indicating that there is no appreciable denaturation or aggregation. These results clearly showed that apo-P450 BSb could be prepared using apo-Mb and the resulting apo-P450 BSb retained almost the same conformation as its holo form without denaturation.
With pure apo-P450 BSb in hand, we reconstituted it according to the method reported for preparing reconstituted P450 cam . 14 Initially, apo-P450 BSb was reconstituted with heme to confirm that it is possible to reinsert heme into an appropriate position in P450 BSb and to fully recover its catalytic activity. A solution of apo-P450 BSb was added with 2.5 equivalents of hemin in the presence of 10 mM DTT and 10 mM histidine, and then incubated for 24 h under argon. Reconstituted P450 BSb (rFe-P450 BSb ) could be purified by Ni-chelate affinity chromatography. The UV-Vis absorption spectrum showed a Soret peak at 417 nm. The CD spectrum of reconstituted P450 BSb remained similar to those of recombinant P450 BSb and a-helical proteins (Fig. S7, ESI †).
rFe-P450 BSb exhibited catalytic activity similar to that of native P450 BSb for the one-electron oxidation of guaiacol with the assistance of n-heptanoic acid, which accelerates the generation of active species of P450 BSb because the carboxylate of n-heptanoic acid serves as an acid-base catalyst. 21 The initial TON (nmol product per nmol P450 per min) of one-electron oxidation of guaiacol by rFe-P450 BSb with heptanoic acid was estimated to be 3150, which is almost the same as that of P450 BSb under similar conditions (Table 1). 22 These results indicate that apo-P450 BSb prepared using apo-Mb can be reconstituted with heme and that the reconstituted rFe-P450 BSb has a catalytic activity comparable to that of recombinant P450 BSb .
Given that apo-P450 BSb could be reconstituted with heme, we have attempted to reconstitute apo-P450 BSb with MnPPIX in a similar manner, and obtained P450 BSb reconstituted with MnPPIX (rMn-P450 BSb ). The CD spectrum of rMn-P450 BSb confirmed that rMn-P450 BSb had the same protein structure as P450 BSb (Fig. 3 and Fig. S7, ESI †). The Soret absorption band of rMn-P450 BSb was observed at 381 nm, which corresponded to that of P450 cam reconstituted with MnPPIX (Fig. 3). 23,24 ICP-OES showed that 70% of the apo-P450 BSb was reconstituted with MnPPIX. The UV-Vis absorbance ratio (A Soret(381) /A 280 ) of rMn-P450 BSb was consistent with 70% reconstitution.
The catalytic activity of rMn-P450 BSb for one-electron oxidation of guaiacol using hydrogen peroxide as an oxidant was examined, but the TON was very low at 40 min À1 . In contrast, when mCPBA was used as the oxidant, the catalytic activity was improved (2850 min À1 ) compared with that of recombinant P450 BSb (Table 1). On the other hand, hydroxylation of cyclohexane was not catalyzed by neither recombinant P450 BSb nor rFe-P450 BSb .
We also examined the hydroxylation of cyclohexane by rMn-P450 BSb with mCPBA, but no product was detected, suggesting either the Mn IV QO, not the Mn V QO, species generated by homolytic cleavage of Mn III -OOR or very rapid reduction of Mn V QO under the conditions and eventually Mn IV QO is responsible for oneelectron oxidation by rMn-P450 BSb in the presence of mCPBA (Scheme S2, ESI †). 25

Conclusions
We have demonstrated that apo-P450 BSb can be prepared using apo-Mb as a heme scavenger without subjecting the proteins to harsh and denaturing conditions. Although the preparation of apo-P450s and their reconstitution is generally difficult, the heme of P450 BSb was completely removed by apo-Mb to give pure apo-P450 BSb that could be reconstituted with MnPPIX. The holo-, apo-, rFe-and rMn-P450 BSb remained as analogous a-helical structures, in contrast to other reported reconstituted P450s and their apo-P450s prepared using the acid-butanone method 20 or incubation with hydrogen peroxide. 26 Although we focused on the preparation of apo-P450 BSb , other P450s whose apo-forms are difficult to prepare by conventional acid-organicsolvent methods may also be potent targets for our methodology. Furthermore, we believe that this method is useful to selectively remove the heme of hemoproteins containing other cofactors such as flavin derivatives or iron-sulfur clusters.