Substitution of an amino acid residue axially coordinating to the heme molecule in hexameric tyrosine-coordinated hemoprotein to enhance peroxidase activity

Article abstract of DOI:10.1142/S1088424617500936

To convert an originally tyrosine-coordinated heme to histidine-coordinated heme in hexameric tyrosine-coordinated hemoprotein, HTHP, Tyr45, a residue coordinating to the heme cofactor, and Arg25 located in the distal site are replaced with Phe45 and His25, respectively in each of the subunits of the protein. The obtained HTHP mutant (HTHP^R25H/Y45F) was characterized by SDS-PAGE, ESI-TOF MS, dynamic light scattering measurements and size exclusion chromatography. These analyses indicate that HTHP^R25H/Y45F maintains its stable hexameric structure with the altered ligation of each of the heme cofactors. Comparison of UV-vis absorption spectra of the ferric-, ferrous-, CO-and CN-forms of HTHPR25H/Y45F with those of several well-known His-ligated hemoproteins indicates that heme is coordinated by the His25 residue. The reaction of HTHP^R25H/Y45F with cumene hydroperoxide produces both cumyl alcohol and acetophenone in a 2.3:1 ratio, indicating that heterolytic O-O bond cleavage dominantly occurs to form the two-electron oxidized species known as compound I. Peroxidase activity of HTHPR25H/Y45F is found to follow Michaelis-Menten kinetics. The k_cat values of HTHP^R25H/Y45Ffor H2O2-dependent oxidation of ABTS and guaiacol are 10-and 100-fold higher, respectively, than those of wild type HTHP (HTHP^WT). The k_cat/_Km values of HTHP^R25H/Y45F for both substrates are increased 30-fold relative to that of HTHPWT. Moreover, HTHPR25H/Y45F is capable of promoting catalytic sulfoxidation of thioanisole with H₂O₂ with a turnover number ca. 2-fold higher than that of HTHPWT. The present findings demonstrate that proximal His ligation to the heme is significantly effective to increase the peroxidase activity in the HTHP matrix.

Full text of DOI:10.1142/S1088424617500936

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2017; 21: 1–8
DOI: 10.1142/S1088424617500936
Published at http://www.worldscinet.com/jpp/
Substitution of an amino acid residue axially coordinating
to the heme molecule in hexameric tyrosine-coordinated
hemoprotein to enhance peroxidase activity
Tsuyoshi Mashimaa, Koji Oohora*a,b,c◊ and Takashi Hayashi*a◊
a Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan
b Frontier Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University,
2-1 Yamadaoka, Suita, 565-0871, Japan
cPRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi 332-0012, Japan
Received 22 November 2017
Accepted 3 December 2017
ABSTRACT: To convert an originally tyrosine-coordinated heme to histidine-coordinated heme
in hexameric tyrosine-coordinated hemoprotein, HTHP, Tyr45, a residue coordinating to the heme
cofactor, and Arg25 located in the distal site are replaced with Phe45 and His25, respectively in each
of the subunits of the protein. The obtained HTHP mutant (HTHPR25H/Y45F) was characterized by SDS-
PAGE, ESI-TOF MS, dynamic light scattering measurements and size exclusion chromatography. These
analyses indicate that HTHPR25H/Y45F maintains its stable hexameric structure with the altered ligation of
each of the heme cofactors. Comparison of UV-vis absorption spectra of the ferric-, ferrous-, CO- and
CN-forms of HTHPR25H/Y45F with those of several well-known His-ligated hemoproteins indicates that
heme is coordinated by the His25 residue. The reaction of HTHPR25H/Y45F with cumene hydroperoxide
produces both cumyl alcohol and acetophenone in a 2.3:1 ratio, indicating that heterolytic O–O bond
cleavage dominantly occurs to form the two-electron oxidized species known as compound I. Peroxidase
activity of HTHPR25H/Y45F is found to follow Michaelis–Menten kinetics. The kcat values of HTHPR25H/Y45F
for H2O2-dependent oxidation ofABTS and guaiacol are 10- and 100-fold higher, respectively, than those
of wild type HTHP (HTHPWT). The kcat/Km values of HTHPR25H/Y45F for both substrates are increased 30-
fold relative to that of HTHPWT. Moreover, HTHPR25H/Y45F is capable of promoting catalytic sulfoxidation
of thioanisole with H2O2 with a turnover number ca. 2-fold higher than that of HTHPWT. The present
findings demonstrate that proximal His ligation to the heme is significantly effective to increase the
peroxidase activity in the HTHP matrix.
KEYWORDS: hemoprotein, peroxidase, hexamer.
impose a range of physicochemical properties to induce
INTRODUCTION
specific reactivities which support these sophisticated
Hemoproteins are responsible for several important
functions. In other words, the reactivity of a given heme
functions in biological systems such as oxygen
cofactor within a hemoenzyme is essentially controlled
storage and transport, gas sensing, electron transfer,
by the protein matrix. Axially coordinated amino acid
and metabolism via oxidation and oxygenation [1–3].
Despite using the same cofactor, the protein matrices
residues in the heme pocket are a prime example of such
protein matrix control [4, 5]. Wide ranging investigations
of hemoprotein chemistry over the past half century
◊ SPP full member in good standing
have indicated that the functional groups of histidine,
cysteine, and tyrosine are the primary axial heme ligands
[6]. Histidine is a common residue coordinating to heme
in peroxidases as well as oxygen-binding hemoproteins
*Correspondencence to: Koji Oohora, email: oohora@chem.
eng.osaka-u.ac.jp; Takashi Hayashi, email: thayashi@chem.
eng.osaka-u.ac.jp.
Copyright © 2017 World Scientific Publishing Company

2
T. MASHIMA ET AL.
such as myoglobin (Mb) and hemoglobin [7–9]. In the
case of horseradish peroxidase, the coordinated imidazole
functional group of the histidine residue forms a hydrogen
bond with an aspartic acid group to afford imidazolate-
like coordination, which induces efficient formation of
the high valent reactive intermediate, compound I species
(FeIV-oxo porphyrin p-cation radical) in a reaction with
hydrogen peroxide [10, 11]. Cytochrome P450s, which
are heme-dependent monooxygenases, employ cysteine
as the axial ligand [12, 13]. The strong push-effect and the
radical character in the compound I species collectively
enable highly efficient activation of inert C–H bonds.
Tyrosine is axially coordinated to heme in catalase,
which promotes the disproportionation of hydrogen
peroxide [14, 15]. The phenolate-type coordination to the
heme provided by the tyrosine side chain also appears
to provide a strong push-effect for the formation of a
compound I species. Thus, the axial ligand is extremely
important for regulating the reactivity of high-valent
catalytic species, and mutations focusing on axial ligands
in hemoproteins have been investigated [16–23].
Hexameric tyrosine-coordinated heme protein,
HTHP, from the marine bacterium Silicibacter pomeroyi
is a thermally stable homo-hexameric hemoprotein
composed only of a helices (Fig. 1a) [24]. HTHP has
a heme molecule in each subunit which is coordinated
by Tyr45 as an axial ligand to form a five-coordinate
structure (Fig. 1b). In the distal site of the heme pocket,
Arg25 interacts with heme via a p-cation interaction. The
peroxidase and catalase activities of HTHP are relatively
low compared with typical peroxidase and catalase
activities. In contrast to these enzymes, the heme in
HTHP is highly exposed to solvent [24]. Although the
native function of HTHP remains unknown, its rigid and
highly-symmetric structure is attractive for engineering
of protein functions [25]. In the present work, we focus
on proximal ligation of heme for modification of HTHP
and demonstrate that substitution of the axial ligand from
tyrosine to histidine enhances the peroxidase activity of
HTHP (Fig. 1b).
EXPERIMENTAL METHODS
Instruments
UV-vis spectral measurements were carried out with
a UV-2700 double-beam spectrophotometer (Shimadzu),
a V-670 UV-vis-NIR spectrophotometer (JASCO) or a
BioSpec-nano spectrophotometer (Shimadzu). ESI-TOF
MS analysis was performed with a Bruker Daltonics
micrOTOF-II mass spectrometer. The pH measurements
were made with an F-52 Horiba pH meter. Size exclusion
chromatography was performed using an ÄKTA purifier
system equipped with a Superdex 200 Increase 10/300
GL column (GE Healthcare) at 4°C. Dynamic light
scattering was measured using a Malvern Zetasizer mV
light scattering analyzer with an 830 nm laser at 25°C.
HPLC analysis was conducted with a Shimadzu HPLC
Prominence system equipped with a YMC-Triart C18
column (150 × 4.6 mm I.D.). The measurements for
evaluation of peroxidase activity were conducted with a
rapid scan stopped-flow system (Unisoku) using a Xe or
halogen light source. The GC/FID measurements were
made with a Shimadzu GC-2010 gas chromatography
system.
Materials
Ultrapure water (Milli-Q) was prepared using a
Millipore Integral 3 apparatus. Other all chemicals were
of the highest guaranteed grade commercially available
and were used as received unless otherwise indicated.
Mb and horseradish peroxidase (HRP), were purchased
from Sigma Aldrich and Wako, respectively.
Amino acid sequence of the monomers
of HTHPWT and HTHPR25H/Y45F
HTHPWT
;
SETWLPTLVTATPQEGFDLAVKLSRIAVKKTQPD-
AQVRDTLRAVYEKDANALIAVSAVVATHFQTIAA-
ANDYWKD
HTHPR25H/Y45F
;
SETWLPTLVTATPQEGFDLAVKLSHIAVKKTQPDA-
QVRDTLRAVFEKDANALIAVSAVVATHFQTIAAA-
NDYWKD
Expression and purification of HTHPR25H/Y45F
Fig. 1. (a) Crystal structure of HTHPWT (PDB ID: 2OYY).
(b) Schematic heme pocket structure of HTHPWT and
HTHPR25H/Y45F
The gene expression system for HTHPWT was reported
in our previous paper [25]. Site-directed mutagenesis was
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SUBSTITUTION OF AN AMINO ACID RESIDUE AXIALLY COORDINATING TO THE HEME MOLECULE IN HEXAMERIC
3
performed using the polymerase chain reaction (PCR)
and the LA PCR in vitro Mutagenesis Kit (Takara)
according to the manufacturer’s protocol. The HTHP
gene cloned into pDEST14 was used as a template to
introduce mutations into the HTHP matrix with primers
(i). After PCR, the template DNA plasmids were
digested with Dpn I (Thermo Fisher Scientific). E. coli
DH5a competent cells were transformed with the PCR
products. After cultivation, the plasmids were purified
with PureLink™ Quick Plasmid Miniprep Kit (Thermo
Fisher Scientific). The obtained plasmid was further
used as a template for site-directed mutagenesis with the
primers (ii). The desired plasmid containing the gene for
the double mutant of HTHP (R25H/Y45F) was obtained
using the same method.
6.0. The fractions with Rz > 2 (Rz is a ratio of absorbance
values at 402 nm and 280 nm) were collected and loaded
on a Hitrap Q HP 5 mL (GE Healthcare) anion-exchange
column pre-equilibrated with 100 mM potassium
phosphate buffer at pH 6.0. The fraction of the target
protein was eluted with 100 mM potassium phosphate
buffer at pH 6.0 containing 0.35 M NaCl. The obtained
HTHP mutant (HTHPR25H/Y45F) was characterized by
SDS-PAGE (Fig. 2), ESI-TOF MS (Fig. 3), analytical
size exclusion chromatography (SEC, Fig. 4), and
UV-vis spectroscopy (Fig. 5a), and stored at -80°C. The
concentration of heme was determined by characteristic
absorbance as a pyridine hemochrome complex (e556
=
=
34.4 mM-1 ·cm-1). The extinction coefficient (e405
111 mM-1 ·cm-1) of HTHPR25H/Y45F was determined from
the plots of the absorbance at 405 nm of HTHPR25H/Y45F
for the concentration of heme (Fig. S1).
DNA sequencing was performed to verify each correct
mutation in the gene sequence.
The primer sequences used to generate the mutant
were:
Analytical size exclusion chromatography
For SEC analysis, 100 mM potassium phosphate
buffer at pH 7.0 was used as an eluent. The analysis
was performed at 4°C and a flow rate of 0.5 mL·min-1
with monitoring of the absorbance at 280 and 400 nm
for detection. The Superdex 200 column was calibrated
using the following reagents: ferritin (400 kDa), albumin
(67 kDa), ovalbumin (40 kDa), and chymotrypsinogen
(25 kDa).
(i) R25H: (5′-GGCCGTGAAACTGTCGCACATTGC-
GGTCAAGAAAAC-3′) and the complementary
primer;
(ii) Y45F: (5′-GCGATACTCTCCGTGCTGTGTTCGA-
GAAAGATGCGAATGCGCTG-3′) and the comple-
mentary primer.
Theresultingexpressionplasmidwasusedtotransform
E. coli BL21 (DE3). LB medium (4 L) containing
ampicillin (400 mg) was inoculated with 50 mL of the
culture (OD = 0.5) of the transformed cells. After the
cells were grown aerobically with vigorous shaking at
37°C until OD600 was reached ~0.5, isopropyl-b-D-
1-thiogalactopyranoside (IPTG, final concentration:
1 mM) was added to the culture for induction of
protein expression. The culture was continued at 37°C
overnight. The cells were harvested by centrifugation
at 4000 × g for 10 min. The harvested cells from 4 L
of culture were re-suspended in ca. 50 mL of a 10 mM
Tris-HCl buffer, pH 8.0, and lysed by freeze-thaw cycles
with subsequent sonication for 30 s × 10 times at 4°C.
The lysate was then centrifuged and the supernatant was
collected. The solution was diluted 10-fold with 100 mM
potassium phosphate buffer at pH 7.0 and loaded through
a CM Fast Flow (GE Healthcare) cation-exchange
column pre-equilibrated with 100 mM potassium
phosphate buffer at pH 7.0. The flow-through solution
was loaded onto a DEAE Fast Flow (GE Healthcare)
anion-exchange column pre-equilibrated with 100 mM
potassium phosphate buffer at pH 7.0. The fraction of the
target protein was collected with a 100 mM potassium
phosphate buffer at pH 7.0 containing 0.3 M NaCl. The
obtained solution was concentrated using an Amicon
stirred ultrafiltration cell with a 30-kDa molecular weight
cut-off membrane (Millipore). The concentrated solution
was passed through a Superdex 200 Increase 10/300 GL
column with 100 mM potassium phosphate buffer at pH
Preparation of ferrous-, CO- and cyanide-forms
of HTHPWT and HTHPR25H/Y45F
To obtain the ferrous-forms of HTHPWT and
HTHPR25H/Y45F, a small excess amount of Na2S2O4 was
added to the protein solution. The CO-forms were pre-
pared by bubbling the ferrous-form solutions with CO
gas for 2 min. To solutions of ferrous-HTHPWT and ferric
HTHPR25H/Y45F in 100 mM potassium phosphate buffer
at pH 7.0 potassium cyanide solution was added (final
concentration: 10 mM), giving cyanide-ferrous HTHPWT
and cyanide-ferric HTHPR25H/Y45F, respectively.
Reaction of HTHPWT and HTHPR25H/Y45F
with cumene hydroperoxide
A cumene hydroperoxide, CHPO, solution was added
to the protein solution ([protein] = 20 mM, [CHPO] =
2 mM, 100 mL) in 100 mM potassium phosphate buffer
at pH 7.0 and 25°C for 2 h. The reaction mixture was
filtered using a Millix®-GV filter (0.22 mm), and the
filtrate was analyzed using an HPLC system equipped
with a C18 column to determine the amounts of produced
acetophenone and cumyl alcohol.
Peroxidase activity
A mixture of protein (2 mM) and substrate dissolved
in 100 mM potassium phosphate buffer at pH 7.0 was
incubated at 25°C for 10 min. UV-vis spectral changes
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4
T. MASHIMA ET AL.
were recorded upon addition of H2O2aq (10 mM). The
final concentrations of guaiacol or ABTS as a substrate
were from 0.05 to 1.5 mM. After mixing the same volume
(125 mL) of these two solutions, absorbance changes
were recorded and the initial rates within 1 s were
determined using the extinction coefficients of the
oxidized products: e470 = 26.6 mM-1 ·cm-1 for guaiacol
and e728 = 15.0 mM-1 ·cm-1 for ABTS. The plots of initial
rates, v, per protein against substrate concentration were
fitted with the following Michaelis–Menten equation
to estimate the kinetic parameters for the oxidation
reactions.
kcat [substrate]
υ
=
Fig. 2. SDS-PAGE electrophoretogram of HTHPR25H/Y45F (left
lane) and several protein standard markers (right lane)
[protein] Km +[substrate]
Sulfoxidation of thioanisole
The reactions were carried out in 100 mM potassium
phosphate buffer at pH 7.0 and 25°C. A buffer solution
of protein and thioanisole was incubated prior to the
addition of H2O2 to initiate the reaction. The final concen-
trations of each component was follows: [protein] =
10 mM, [thioanisole] = 2.0 mM and [H2O2] = 1 mM. After
the reaction was complete at 12 h, benzyl alcohol as an
internal standard and ether were added, and the reaction
mixture was vigorously shaken using a vortex mixer to
extract the organic materials. The separated organic phase
was evaporated by N2 gas flow, dissolved in CH3CN and
analyzed with a GC/FID system equipped with a DB-1
column.
Fig. 3. ESI-TOF mass spectrum of the monomer of HTHPR25H/Y45F
.
The sample was dissolved in 10 mM NH4OAc aqueous solution
at pH 6.9. Multiply ionized species were observed: found
m/z = 2732.5 and 2938.3; calcd m/z = 2732.4 (z = 3+, apo-form)
and 2938.2 (z = 3+, holo-form). Inset: The simulated (red,
upper) and experimental (black, lower) isotopic distribution
RESULTS AND DISCUSSION
Mutant design
3+
patterns for [C370H582N98O112
]
+ 3H+ (z = 3+, monomer of
apo-form of HTHPR25H/Y45F
)
The crystal structure of HTHPWT suggests that direct
replacement of Tyr45, an axial ligand to the heme
molecule in the protein, with His45 is inappropriate for
constructing a His-coordinating hemoprotein, because
the distance between the heme Fe ion and the a-carbon
of the axially coordinated residue Tyr in the protein is
significantly longer than the analogous distance observed
in a series of His-coordinated hemoproteins. In contrast,
it is found that Arg25 in the distal site of the heme pocket
is relatively close to the heme iron: the distances from Ca
(Tyr45) or Ca (Arg25) to Fe (heme) in HTTPWT are 8.5
and 5.8 Å, respectively (PDB ID: 2OYY [24]), whereas
the distances from Ca (His93) or Ca (His64) to Fe
(heme) in Mb are 6.6 and 8.6 Å, respectively (PDB ID:
1YMB [26]). Therefore, we prepared a double mutant
HTHPR25H/Y45F where Tyr45 and Arg25 were replaced
with Phe45 and His25, respectively. As a result, the
engineering allows swapping of proximal and distal sites
in this hemoprotein as shown in Fig. 1b.
Purification and characterization of HTHPR25H/Y45F
HTHPR25H/Y45F was expressed in a recombinant E. coli
system and purified by ion exchange chromatography
and SEC. The purity of HTHPR25H/Y45F isolated from
cultured cells was confirmed by SDS-PAGE (Fig. 2). The
band of HTHPR25H/Y45F was only at about 6 kDa, which
is consistent with the molecular weight of the monomer
of HTHPR25H/Y45F. This is an expected result because
HTHPR25H/Y45F disassembles to the apo-form monomer
in the presence of SDS. ESI-TOF MS (Fig. 3) shows
the desired mass numbers of the multiply-ionized apo-
and holo-forms of the monomer: found m/z = 2732.5
and 2938.3; calcd m/z = 2732.4 (z = 3+, apo-form) and
2938.2 (z = 3+, holo-form). The observed isotopic pattern
of the apo-form (z = 3+) matched well with the simulated
pattern. Dynamic light scattering measurements revealed
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SUBSTITUTION OF AN AMINO ACID RESIDUE AXIALLY COORDINATING TO THE HEME MOLECULE IN HEXAMERIC
5
of HTHPR25H/Y45F at 14.9 mL indicates a molecular weight
of approximately 50 kDa. Furthermore, the SEC trace
for HTHPR25H/Y45F monitored by measuring absorbance
at 280 nm (for protein matrices) is clearly consistent
with that monitored at 400 nm (for heme), indicating
that the heme molecules are bound to the matrix of
HTHPR25H/Y45F
.
UV-vis spectroscopic study
The UV-vis absorption spectra of several states of
HTHPR25H/Y45F and HTHPWT are shown in Fig. 5. The
spectrum of as-isolated HTHPR25H/Y45F is almost identical
to that of HTHPWT, although the peak of the Q-band
at 528 nm in HTHPWT is absent in the spectrum of
HTHPR25H/Y45F. Interestingly, a similar result has also been
observed in the engineered heme-binding human serum
albumin (HSAI142H/Y161L) where the proximal and distal
sites are swapped in a double mutation (Table 1) [19, 27].
These findings support the evidence indicating that the
heme pocket of HTHPR25H/Y45F provides a coordination
environment which is similar to that of His-ligated heme-
binding HSAI142H/Y161L. Additionally, the characteristic
features of the spectrum of HTHPR25H/Y45F are almost
identical to those of wild type met-Mb with high-spin
ferric heme, which has a His residue as an axial ligand.
Reduction of HTHPR25H/Y45F upon addition of a small
Fig. 4. Analytical SEC traces of HTHPR25H/Y45F and various
protein standards. Traces of HTHPR25H/Y45F were observed
at 400 and 280 nm, and traces of the protein standards were
monitored at 280 nm
a monodispersed species with a hydrodynamic diameter
of 6.26 nm, which is consistent with the value expected
from the reported crystal structure of HTHPWT [24].
Support for the formation of the hexameric structure in
solution is also provided by analytical SEC with several
protein standards as shown in Fig. 4: the elution volume
Fig. 5. UV-vis absorption spectra of ferric, ferrous, CO- and CN-forms of (a) HTHPR25H/Y45F and (b) HTHPWT in 100 mM potassium
phosphate buffer at pH 7.0 and 25°C
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T. MASHIMA ET AL.
Table 1. lmax in the UV-vis absorption spectra of HTHPWT, HTHPR25H/Y45F and reported
hemoproteins
Protein
State
Fe3+
Fe2+
Fe2+ + CO
Fe2+ + CN
Fe3+
l
max at Soret band (nm)
lmax at Q-band (nm)
HTHPWT
402
431
420
413
405
432
421
420
405
419
416
402
426
419
409
434
423
501, 528, 616
560, 592
535, 566
523, 553, 591
495, 620
HTHPR25H/Y45F
Fe2+
554, 589(sh),
539, 568, 615 (sh)
542
Fe2+ + CO
Fe3+ + CN
Fe3+
Fe2+
Fe2+ + CO
Fe3+
Fe2+
Fe2+ + CO
Fe3+
Fe2+
Fe2+ + CO
heme-binding HSAWTa
heme-binding HSAI142H/Y161La
Mba
501, 534, 624
538, 559 (sh), 570
539, 568
533, 620
531 (sh), 559
538, 565
503, 548 (sh), 632
557
541, 579
a Reference[27].
Table 3. Kinetics parameters of HTHPWT and HTHPR25H/Y45F
from the Michaelis-Menten equation for the oxidation of
guaiacola
Table 2. Kinetics parameters of HTHPWT and HTHPR25H/Y45F
from the Michaelis-Menten equation for the oxidation of
ABTSa
Protein
HTHPWT
HTHPR25H/Y45F 2.8
HRPb 5.5 × 102
a Reaction conditions: [protein] = 1 mM, [H2O2] = 5 mM,
[Guaiacol] = 0.05 to 1.5 mM in 100 mM potassium phosphate
buffer at pH 7.0 and 25°C. b Figure S2b.
kcat (s-1)
3.0 × 10-2
Km (mM) kcat/Km (mM-1 ·s-1)
Protein
kcat (s-1)
Km (mM) kcat/Km (mM-1 ·s-1)
0.11
0.34
6.0
0.27
8.1
92
HTHPWT
HTHPR25H/Y45F 5.2
HRPb
0.54
0.12
4.5
3.8 × 10-2
1.4 × 102
5.1 × 103
4.0 × 102 5.8
a Reaction conditions: [protein] = 1 mM, [H2O2] = 5 mM,
[ABTS] = 0.05 to 1 mM in 100 mM potassium phosphate
buffer at pH 7.0 and 25°C. b Figure S2a.
HTHPWT: the wild type ferric protein with the Tyr ligand
does not bind cyanide [24]. Taken together, these results
indicate that the introduced His25 residue undoubtedly
coordinates to the heme iron in the heme pocket of
HTHPR25H/Y45F, since HTHPR25H/Y45F does not have other
accessible His residues in the protein matrix.
excess of Na2S2O4 affords the characteristic red-shift of
the Soret band (Fig. 5) which is seen for Mb and heme-
binding HSAI142H/Y161L, indicating the formation of deoxy
HTHPR25H/Y45F which has a His-ligated five-coordinate
structure [27]. Next, bubbling of CO into the sample of
deoxy HTHPR25H/Y45F yields a sharp Soret band at 420 nm
and two peaks in the Q-band region (Fig. 5a and Table 1).
These are typical features of CO-ligated heme as seen
in heme-binding HSAI142H/Y161L and Mb. The addition of
potassium cyanide to ferric HTHPR25H/Y45F also produces
clear spectral changes of the Soret and Q-band as shown
in the formation of cyanide complex of met-Mb [28].
This behavior is sharply different from that observed for
Reaction with cumene hydroperoxide
To evaluate the reaction behavior of ferric
HTHPR25H/Y45F withhydroperoxide,theO–Obondcleavage
of cumene hydroperoxide (CHPO) was investigated. In
this reaction, it is known that two possible products, cumyl
alcohol and acetophenone, are produced after protein-
assisted heterolytic and homolytic cleavage of the O–O
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SUBSTITUTION OF AN AMINO ACID RESIDUE AXIALLY COORDINATING TO THE HEME MOLECULE IN HEXAMERIC
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Fig. 6. Michaelis-Menten plots for (a) ABTS and (b) guaiacol oxidations catalyzed by HTHPR25H/Y45F and HTHPWT. Conditions:
[protein] = 1 mM, [H2O2] = 5 mM in 100 mM potassium phosphate buffer at pH 7.0 and 25°C
bond, respectively. The product ratio (cumyl alcohol/
acetophenone)providedbyHTHPR25H/Y45Fwasdetermined
to be 2.3 0.3, whereas the ratio of Mb was reported to
be 3.3 [29]. The obtained lower ratio for HTHPR25H/Y45F
indicates that approximately 1/3 heme-peroxide provides
the oxoferryl species (Fe(IV)=O), compound II, via
O–O bond homolysis. These findings suggest that the
push-effect of the proximal His to the heme in
HTHPR25H/Y45F is lower than that of Mb. In contrast, it is
found that HTHPWT does not react with CHPO.
the tyrosine axial ligand with histidine clearly enhances
peroxidase activity of HTHPWT
.
Sulfoxidation activity of HTHPR25H/Y45F
To evaluate the two-electron oxidation ability of
HTHPR25H/Y45F and HTHPWT, thioanisole was employed as
a substrate for an H2O2-dependent sulfoxidation reaction.
The turnover numbers of HTHPR25H/Y45F and HTHPWT
were found to be 43 and 23, respectively, at pH 7.0. Over-
oxidation products such as sulfone were not detected after
12 h. These results indicate that axial-ligand substitution
in HTHP is also effective for promotion of catalytic
sulfoxidation.
Peroxidase activity of HTHPR25H/Y45F
The steady-state kinetics of ABTS and guaiacol
oxidation by HTHPR25H/Y45F and HTHPWT were evaluated
to understand the effects of the axial ligand substitution
on peroxidase activity.After addition of H2O2 to a solution
of HTHPR25H/Y45F or HTHPWT containing the substrate, the
absorbance changes derived from the resulting products
were observed. The initial reaction rates (u) were used
to determine the values of Michaelis–Menten kinetic
parameters, kcat and Km (Fig. 6 and Tables 2, 3).
CONCLUSION
HTHPR25H/Y45F, a double mutant of HTHP designed
to exchange the native Tyr axial ligand with His, was
prepared and characterized. Analytical size exclusion
chromatography and dynamic light scattering measure-
ments revealed that HTHPR25H/Y45F maintains its intrinsic
hexameric structure with bound heme molecules even
though swapping of the proximal and distal sites in
the protein matrix was carried out. His-ligation in
HTHPR25H/Y45F is clearly supported by UV-vis spectra of
as-isolated and reduced HTHPR25H/Y45F. HTHPR25H/Y45F
also has typical UV-vis spectra of CO- and CN-bound
hemoproteins with His-ligation.
It is known that axial-ligand exchange by mutagenesis
cancausesignificantperturbationsofthephysicochemical
properties and reactivities of hemoproteins. In the present
study, the His-ligating HTHPR25H/Y45F clearly shows higher
catalytic efficiency toward ABTS, guaiacol oxidation and
thioanisole sulfoxidation compared to that of the wild
type protein. These results indicate that hemoprotein
engineering focusing on axial ligand exchange will serve
as a useful strategy for development of new biocatalysts.
The kcat values of HTHPR25H/Y45F forABTS and guaiacol
oxidation are 10- and 100-fold higher than those of
HTHPWT, respectively. It is possible that the improvement
of the peroxidase activity is due to the substitution of
the axial ligand. However, the kcat value of HTHPR25H/
Y45F
was found to be much lower than that of HRP,
primarily because HTHPR25H/Y45F lacks the imidazolate-
formed proximal His and distal His supporting the H2O2
activation. The Km values for the ABTS and guaiacol
oxidation reactions with HTHPR25H/Y45F are only 3-fold
lower and higher than those of HTHPWT, respectively.
The present modification of HTHP does not significantly
affect the affinity between the substrates and protein.
The values of overall catalytic efficiency, kcat/Km, of
HTHPR25H/Y45F are 30-fold higher than that of HTHPWT
for both substrates, suggesting that the replacement of
Copyright © 2017 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2017; 21: 7–8

8
T. MASHIMA ET AL.
Acknowledgments
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This work was supported by Grants-in-Aid for
Scientific Research provided by JSPS KAKENHI Grant
Numbers JP15H05804, JP15H00873, JP16K14036,
JP16H06045, and JP16H00837. We appreciate the sup-
port from JST PRESTO (JPMJPR15S2), JST SICORP
and Osaka University International Joint Research
Promotion Program. T.M. appreciates support from the
JSPS Research Fellowship for Young Scientists (JSPS
KAKENHI Grant Number JP15J00743).
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