Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

Article abstract of DOI:10.1016/j.jinorgbio.2010.07.008

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25°C. The activity constants (k_cat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25°C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~70°C. The rate of catalysis at temperatures above ~70°C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.

Full text of DOI:10.1016/j.jinorgbio.2010.07.008

Journal of Inorganic Biochemistry 104 (2010) 1185–1194
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Modification of the heme active site to increase the peroxidase activity of
thermophilic cytochrome P450: A rational approach
1
1
Rabindra Kumar Behera , Sandeep Goyal , Shyamalava Mazumdar ⁎
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce
residues that could act as acid–base catalysts near the active site to enhance the peroxidases activity. The
Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that
is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of
CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen
bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of
the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared
to that of the wild type (WT) CYP175A1 enzyme at 25 °C. The activity constants (kcat) for the L80H and L80Q
mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25 °C. The
optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~70 °C. The rate
of catalysis at temperatures above ~70 °C was higher for L80Q mutant of CYP175A1 compared to that of the
well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The
peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH
Received 10 March 2010
Received in revised form 13 July 2010
Accepted 15 July 2010
Available online 23 July 2010
Keywords:
Peroxidase activity
Rational protein design
Thermus thermophilus
CYP175A1
Heme active site
Site directed mutagenesis
~
5. The results have been discussed in the light of understanding the structure–function relationship of the
peroxidase properties of these thermostable heme proteins.
©
2010 Elsevier Inc. All rights reserved.
1
. Introduction
the formation of the high valent reactive intermediates (Scheme 1
known as the compound I (oxo-ferryl porphyrin(P) π-cation radical,
(P+•)Fe(IV) O, CpdI) and the compound II (oxo-ferryl porphyrin, (P)Fe
(IV) O, CpdII) that are responsible for their catalytic activity [12,14,15].
Heme proteins play diverse roles in biological systems [1,2]. These
proteins having the same heme prosthetic group are responsible for
many different types of functions such as oxygen storage and
transport, electron transfer, oxidation and oxygenation of different
substrates etc. The functional diversity of the heme in different heme
proteins has been attributed partly to the differences in the amino
acid environment around the distal heme pocket [1]. It however, still
remains a great challenge to understand the molecular basis of the
specific function of the heme in a particular type of heme protein
environment [3,4]. The most common strategy for the understanding
of the structure–function relationship for an enzyme involves the
substitution of amino acid residues near the active site by site-
directed mutagenesis followed by investigations of the effect of the
substitution on the function of that enzyme [3,5–11].
•
The reducing substrate (AH) is generally converted into a radical (A ),
which subsequently forms dimeric or polymeric products. These
reactive intermediates are suggested to be generated by the heterolytic
cleavage of the O–O bond of hydroperoxy ferric heme iron. The catalytic
cycle and the peroxide shunt pathway of the heme monooxygenases
(P450 enzymes) have also been proposed to involve the highly reactive
intermediate (compound I) [14]. The X-ray crystal structure analyses of
these heme containing enzymes such as chloroperoxidase (CPO, PDB:
1CPO.pdb), horseradish peroxidase (HRP, PDB: 1ATJ.pdb), cytochrome c
peroxidase (CcP, PDB: 2CYP.pdb) and other peroxygenases (some P450s
like P450bsβ) suggest the presence of certain key residues at the distal
heme pocket, which enhance the generation of compound I species by
acting as acid–base catalysts and by stabilizing it as well by forming
hydrogen bond with the ferryl oxygen [4,16]. A histidine is one of such
important amino acid residue around the distal heme pocket that is
found to be highly conserved in many heme-peroxidases including
HRP [3]. The His42 and Arg38 present near the distal side of the heme in
HRP are reported to be responsible for the very high rate of compound I
formation as well as for the high activity of the enzyme [17–20]. In
contrast to HRP, the distal Glu183 in CPO is shown to be the crucial
residue to act as the acid base catalyst [19,21,22]. The cytochrome P450,
Peroxidases are heme-enzymes catalyzing the one electron oxida-
tions that use hydrogen peroxide as the electron acceptor and have
interesting biocatalytic properties with potential use in biosensing and
immunoassays [12,13]. The peroxidase catalytic cycle generally involves
⁎
Corresponding author. Tel.: +91 22 22782363; fax: +91 22 2280 4610.
E-mail address: shyamal@tifr.res.in (S. Mazumdar).
URL: http://www.tifr.res.in/~shyamal (S. Mazumdar).
These two authors contributed equally to the work.
1
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.07.008

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R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
sodium cholate, guanidine hydrochloride (GdnHCl), ampicillin sodi-
um salt, isopropyl β-D-thiogalacto-pyranoside (IPTG), lysozyme,
phenyl methyl sulphonyl fluoride (PMSF), oligonucleotide primers,
guaiacol, ABTS were purchased from Sigma Chemicals Co. Restriction
enzymes was purchased from New England Biolabs (NEB). DNAse I
was purchased from Boehringer Mannheim, Germany. Amplex Red
Scheme 1. Formation of high valent reactive intermediates in peroxidase catalytic cycle.
P: corresponds to protoporphyrin IX inside the protein matrix. P+•: indicates porphytin
cation radical. AH is reducing substrate and A• is substrate radical. CpdI and CpdII refer
to compound I and compound II redox intermediates.
was purchase from Invitrogen. Hydrogen peroxide (H
purchased from Qualigen, India. The H stock solution was freshly
prepared every time and its concentration was quantified spectro-
photometrically (ε240 =39.4 M cm ) [36]. All buffer components,
solvents and other reagents were of analytical grade. Deionized water
(Millipore) was used in the preparation of all the buffers.
2 2
O ) was
2 2
O
−
1
−1
whose natural function is monooxygenation of substrates, lacks such
architecture of the distal pocket [14]. The kinetics of generation of
compound I by hydrogen peroxide in cytochrome P450 was proposed to
be very slow possibly because of the absence of appropriate amino acids
at the distal pocket of the heme that could act as acid–base catalyst in
stabilizing this reactive intermediate [23,24].
Extensive studies on the peroxidase activities of different heme
proteins have been reported in literature and modification of certain
distal residues have been shown to enhance the peroxidases activity of
hemeproteins such as myoglobin, cytochrome c etc. [8,9,20,25,26]. One of
the interests in the study of the peroxidase activity of different heme
enzymes also stems from the extensive use of peroxidases as biocatalysts
for food processing and bioremediation [13,27]. The low thermal stability
is one of the disadvantages of many such commercially available enzymes
in the industry. Therefore, the peroxidases with high thermal stabilities
are highly preferred over the mesophilic enzymes. Several peroxidases
isolated from thermophilic organism showed very high thermostability
2.2. Mutation of recombinant CYP175A1
The pKK-223 plasmid, encoding the wild type CYP175A1 enzyme
was a kind gift from Prof. Vlada B. Urlacher (University of Stuttgart,
Germany). Site-directed mutagenesis was carried out using Quik-
change™ mutagenesis kit (Stratagene Co.). The forward and reverse
oligonucleotide primers were GAGGGGtCTC-XXX-ACCGACTGGGGGG
and CCCCCCAGTCGGT-YYY-GAGaCCCCTC respectively. Where XXX
and YYY were the codons for the 80th residue in forward and reverse
primer. The mutants were designed by changing the sequences of
XXX and YYY in the primers, e.g., with XXX=CaC for L80H, Cag for
L80Q, gaC for L80D, gag for L80E, atC for L80I, and with YYY=GtG for
L80H, ctG for L80Q, Gtc for L80D, ctc for L80E, Gat for L80I mutation.
These primers introduced a BsaI restriction site at 239 bp position of
the structural gene along with the desired mutation.
2 2
but they were shown to lose the activity in the presence of H O [28–30].
In the recent years, efforts have been made to improve the peroxidase
activity in both mesophilic and thermophilic proteins by rational protein
engineering but most of them often fail to retain their activity at higher
temperatures [3,5,8,26]. Efforts have also been made to enhance the
thermostability of HRP [31,32] by modification of the enzyme.
The CYP175A1 is a 44-kDa soluble thermostable cytochrome P450
from Thermus thermophilus HB27 [33]. This enzyme was shown to
hydroxylate β-carotene at 3- and 3′-positions, resulting in β-
cryptoxanthin and zeaxanthin, although its natural substrates are
still not known [34,35]. The crystal structure of CYP175A1 (PDB:
2.3. Expression and purification of wild type and variant CYP175A1
The wild type, L80I, L80H and L80Q mutant CYP175A1 proteins were
expressed and purified by a small modification of the reported method
[34]. pKK-223 plasmid having structural gene for the expression of
CYP175A1 was transformed in the BL21 (DE3) codon plus RP cells and
grown overnight in 5 ml 2xYT medium containing 100 μg/ml ampicillin
and 50 μg/ml chloramphenicol. The overnight culture was used to
inoculate 700 ml 2xYT medium keeping the same concentration of
antibiotics and wasfollowed by further incubation at 37 °C and 170 rpm.
Induction was done by adding 1 mM IPTG to the cell culture after
reachingOD600nm ~0.8–1 andincubated for 50 h at30 °Cat 130 rpm. The
cell pellets were harvested by centrifugation 6000 rpm for 15 min.
The cell pellets were suspended in 10 volumes of lysis buffer
(50 mM Tris–HCl: pH 7.5, 200 mM KCl, 1 mM EDTA, 0.2 mM PMSF,
1 mg/ml lysozyme, DNase I: 40 Units/ml and 2–3 mM MgCl ) and
2
stirred for 45 min at 4 °C. The cell suspension were sonicated and then
centrifuged at 19,000 rpm for 90 min. The supernatant was treated
4 2 4
with slow addition of solid (NH ) SO to reach 35% saturation with
stirring at 4 °C for 45 min followed by centrifugation at 18,000 rpm for
45 min. The collected supernatant was further proceeded to reach 50%
1
N97.pdb) shows absence of suitable amino-acid residues in the distal
heme pocket that could act as acid–base catalysts as in the heme
peroxidases such as HRP and CPO. The aim of the present project is to
modify the active site of this thermostable P450 to introduce a residue
at the distal heme pocket that could act as an acid–base catalyst and
thus enhance the peroxidase activity in the mutant enzyme. This
could potentially lead to the creation of a thermally stable artificial
peroxidase. In this project we have carried out molecular modeling
studies and identified that the Leu80 residue in the CYP175A1 could
be suitably mutated to enhance the peroxidase activity of the mutant
m
enzyme. The high thermostability (T =87 °C) of CYP175A1 has the
advantage to engineer this protein to create an artificial thermostable
enzyme [33]. We have studied the peroxidase activities of L80H and
L80Q mutants of the enzyme at different temperatures and pH to
determine the optimum conditions and results were compared with
those of the archetype peroxidase, HRP. The substitution of the
leucine with histidine or glutamine was found to enhance the
peroxidase activity of CYP175A1, which is more prominent at high
temperature.
4 2 4
(NH ) SO saturation followed by centrifugation at the same
condition (at 18,000 rpm for 45 min). After ammonium sulfate
fractionation, the pellet was dissolved in minimum volume of
50 mM Tris–HCl buffer (pH 7.5) which was centrifuged and filtered
(0.4 μm). The filtrate was loaded to a phenyl sepharose column, pre-
equilibrated with 35% saturated (NH
(pH 7.5). The column was washed with the same buffer as above and
then subjected to a linear gradient (35–0%) of (NH SO in 50 mM
4 2 4
) SO in 50 mM Tris–HCl buffer
4
)
2
4
Tris–HCl buffer (pH 7.5). Elution was continued with 50 mM Tris–HCl
2
. Experimental procedures
buffer (pH 7.5) having 1% sodium cholate. The protein fractions
having R (A418/A280)≥1 were combined, concentrated with centri-
z
2
.1. Materials
con tube (Millipore) and loaded onto a hydroxyapatite column.
CYP175A1 enzyme was eluted from the column by applying linear
gradient of 50–300 mM potassium phosphate (pH 7.4) containing 5%
glycerol. The L80H mutant protein was again purified by gel filtration
chromatography (Superdex 75 prep grade, Pharmacia Biotech).
Phenyl sepharose and Sephadex G-25 columns were from GE
Healthcare Bio-Sciences AB, Uppsala. Hydroxyapatite material was
from Biorad Laboratories Inc, Hercules, CA., ammonium sulphate,

R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
1187
Purified protein with R
z
≥1.5 was collected and concentrated by
2.8. Temperature dependent peroxidase activity
centricon (Amicon Millipore, Ireland) and then finally stored in small
aliquots in 35% (v/v) glycerol at −30 °C. The concentrations of
purified enzymes were quantified by using extinction coefficients
The initial rate of peroxidation of Amplex Red (10 μM) was used to
investigate the optimum temperature of the wild type, L80H, L80Q and
HRP in 50 mM potassium phosphate buffer. The formation of Amplex
Red oxidation product (resorufin, ε571=5.4×10 M cm ) was
monitored at 571 nm. The concentration of the enzyme and the H
−
1
−1
−1
−1
(
wild type: ε417 =104 mM cm , L80I: ε417 =100 mM cm
,
L80H: ε424 =114 mM cm−1 and L80Q: ε420 =108 mM cm
−
1
−1
−1
)
4
−1
−1
which were determined by pyridine–hemochrome method [37].
2 2
O
used for this experiment were 0.2 μM and 4 mM respectively. The
protein samples were incubated at the required temperature for about
2
.4. UV–visible absorption and circular dichroism study
1
0 min. The temperature dependent peroxidase activities for all the
species were determined by using a Hi-Tech SF61MX stopped flow
spectrometer. The sample handling unit was mounted inside a thermo-
stated bath compartment and the temperature of the compartment was
maintained (within± 0.3 °C) using a circulating water bath.
The UV–visible absorption spectra of the wild type and its variants
L80I, L80H and L80Q) CYP175A1 in 50 mM KPi (pH 7.4) were
(
recorded on a Perkin Elmer (Lamda 750-UV/Vis/NIR) spectropho-
tometer. The tertiary CD spectra of the wild type and mutants (L80I,
L80H and L80Q) CYP175A1 in 50 mM KPi (pH 7.4) were recorded with
cuvette having 1 cm pathlength on a JASCO J-810 spectropolarimeter
equipped with a Peltier cell temperature controller (± 0.2 °C). The
enzyme concentrations used for the absorption and tertiary CD
experiment were 10 and 25 μM respectively.
2.9. pH dependent peroxidase activity
The initial rate of peroxidation of ABTS (1 mM) was used to
investigate the optimum pH for peroxidase activity of wild type along
with L80H and L80Q mutants of CYP175A1 in universal buffer at 25 °C.
2 2
The concentration of the enzyme and H O used for this experiment
2
.5. Resonance Raman study
were 0.2 μM and 50 mM respectively.
Raman spectra were taken using a Horiba Yvon Raman Spectrom-
2.10. Hydrogen peroxide dependent guaiacol oxidation
eter T64000, with an inverted optical microscope adapted to a triple-
grating equipped with (1024×256) liquid N cooled CCD. A Spectra-
2
The H dependence peroxidase activities (guaiacol oxidation)
2 2
O
Physics model with Argon Ion laser provided the excitation at 488 nm.
The laser power at the sample was 5–6 mW and was focused using a
were performed to determine the apparent kinetic constants for wild
type along with L80H and L80Q mutants of CYP175A1 enzymes. The
steady-state kinetics was studied with 0.2 μM enzyme and 10 mM
guaiacol in 50 mM potassium phosphate buffer (pH 7.4) at 25 °C by
1
0× objective lens. Wave number calibration was carried out using
−
1
the 521 cm
scanning the 1300–1700 cm
line of the silicon wafer. Spectra were recorded by
−
1
region with a total acquisition time of
2 2
varying concentration of the H O . The initial reaction rates obtained
3
00 s and data were averaged over 5 scans. The concentrations of the
were fitted to the Michaelis–Menten equation by non-linear least-
proteins (wild type and mutants of CYP175A1) were 600 μM–800 μM
in 50 mM KPi buffer at pH 7.5. All measurements were done at room
temperature.
squares method by using ORIGIN software to determine the apparent
k
cat and K
m
values.
3
. Results and discussion
2
.6. Guaiacol oxidation study
3.1. Molecular modeling and design of the active site
The peroxidase activity for wild type CYP175A1 and its variants
The crystal structure of CYP175A1 (PDB: 1N97.pdb) shows that the
(
L80I, L80H and L80Q) were estimated from initial oxidation rate of
active site of the enzyme consists of the heme anchored to the protein
matrix through coordination with cysteine 336. The distal pocket of
the enzyme, analogous to all P450 systems consists of a cavity that
could accommodate the substrate of the enzyme. The crystal structure
of HRP (PDB: 1ATJ.pdb) shows that there are His42 (NE) and Arg38
4
−1
−1
the guaiacol. The product (tetraguaiacol, ε470 =2.6×10 M cm
)
formation was monitored at 470 nm. The steady-state kinetics was
studied with 0.2 μM enzyme in 50 mM potassium phosphate buffer
pH 7.4) at 25 °C. The concentration of H O was 50 mM and the
2 2
concentration of guaiacol was varied (0.05–12.0 mM). The initial
reaction rates obtained were fitted to the Michaelis–Menten equation
by non-linear least-squares method by using ORIGIN software to
m
determine the apparent kcat and K values. The control reactions were
also conducted by maintaining the same condition as above except
the addition of enzyme to the reaction mixture.
(
(
NE) that promote heterolytic cleavage of the O–O bond in the
enzymatic cycle are respectively at 5.6 Å and 4.6 Å from the Fe of the
heme. Analogously, in case of CPO (chloroperoxidase, PDB: 1CPO.
pdb), the Glu183 (OE) is ~5 Å from the Fe centre and proposed to
participate in the acid–base interaction with the peroxide leading to
formation of the compound I species. The active sites of CYP175A1
was superimposed with that of HRP and of CPO by overlapping the
heme centers of each of the enzymes and the relative dispositions of
the residues at the distal site of the heme were compared. It was found
that there is no suitable residue in CYP175A1 at the position
equivalent to His42 or Arg38 of HRP. However, we found that the
Leu80 in CYP175A1 residue is located at a position close to Glu183 of
CPO and mutation of Leu80 to His would potentially lead to have the
His80 (NE) residue at ~5.1 Å from the iron in CYP175A1 analogous to
that observed in case of Glu183 in CPO. Fig. 1 depicts the disposition of
the Leu80, His80 of CYP175A1 and Glu183 of CPO with respect to the
heme. The structural comparison of CYP175A1 and CPO also shows
that the proximal cysteine residue is almost equivalent in both of
these two enzymes. Based on the 3D superposition and analysis of the
structures of the two enzymes, mutations of the Leu80 residue in
CYP175A1 with a hydrogen bonding residue such as histidine,
2
.7. ABTS oxidation study
The peroxidase activity for wild type CYP175A1 and its variants
(
L80I, L80H & L80Q) were estimated from initial oxidation rate of the
+
•
4
−1
−1
ABTS. The product (ABTS , ε414 =3.6×10 M cm ) formation
was monitored at 414 nm. The steady-state kinetics was studied with
0
2
.2 μM enzyme in 50 mM potassium phosphate buffer (pH 7.4) at
5 °C. The concentration of H was 50 mM and the concentration of
2 2
O
ABTS was varied (0.5 μM–1 mM). The initial reaction rates obtained
were fitted to the Michaelis–Menten equation by non-linear least-
squares method by using ORIGIN software to determine the apparent
cat and K values. The control reactions were also conducted by
m
maintaining the same condition as above except the addition of
enzyme to the reaction mixture.
k

1188
R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
Fig. 1. The comparison of the crystal structures of the CYP175A1 (PDB: 1N97.pdb) and of the chloroperoxidase (CPO, PDB: 1CPO.pdb) depicting the dispositions of the Leu80 (A), that
is mutated to His80 (B) in CYP175A1 compared to the Glu183 (C) in CPO. The overlap of the hemes of the two enzymes is shown in D.
glutamine, arginine, lysine, aspartic acid or glutamic acid could thus
potentially mimic the active site of a peroxidase enzyme.
ferric P450 enzymes [38–40]. In contrast, the L80H and L80Q mutants
exhibit red shifted Soret absorption maxima at 424 and 420 nm
respectively. The two visible bands are also red shifted (by ~5 nm) for
both L80H and L80Q mutant compared to wild type CYP175A1
enzyme. The red shift in the Soret absorption maximum may arise
because of increase in the polarity of the heme environment [41].
Earlier studies [41,42] on A264E mutant of P450BM3 showed that
axial coordination of the glutamate causes analogous red shift of the
Soret absorption peak of the P450 enzyme. Thus, weak interaction of
histidine (in L80H) or glutamine (in L80Q) with the ferric heme in the
mutant of CYP175A1 might also be responsible for the red shift of the
Soret absorption maxima observed in the present case. P450
inhibitors such as imidazole and its analogues have earlier been
shown to cause red shift of the Soret band maximum to 425 nm [43].
Titration of the wild-type ferric CYP175A1 with imidazole (Fig. S1 in
Supporting information) also shows red shift in the UV–visible
spectrum of the protein supporting that binding of histidine may
also cause red shift in the Soret peak as observed in the L80H mutant
of the enzyme. Lowering of the pH of the L80H mutant to pH=4.5 was
found to cause blue shift of the Soret peak to 421 nm. These results
indicate that the histidine in L80H mutant may have weak interaction
with the ferric heme that decreases with decrease in pH. Considering
the structure of the heme cavity in the enzyme, direct coordination of
the histidine or glutamine to the ferric ion in the heme however seems
unlikely but these residues in the mutant enzyme (L80H or L80Q) may
act as hydrogen bond accepters and interact with the heme through
the axial water coordinated to the metal center in the substrate-free
enzyme. This may increase the electron density at the axial site
causing red shift in the Soret band of the mutant enzyme. It has earlier
been shown that on application of high pressure to CYP119 leads to a
red shift of the Soret band to 425 nm with concomitant changes in the
3
.2. Site-specific mutation of CYP175A1
Mutation of Leu80 to a strongly basic or acidic residues (i.e., Lys,
Arg, Asp or Glu) did not give the folded protein on expression possibly
because of destabilization of the predominantly hydrophobic distal
pocket of CYP175A1 leading to difficulty of folding of those mutants.
These mutants (L80K/R/D/E) were either formed in inclusion bodies
which could not be properly folded or the yield was extremely low
hence could not be easily purified for further studies. The L80H and
L80Q mutants were found to fold properly with well defined
spectroscopic signatures of the heme bound to the active site of the
enzyme. The Leu80 residue mutated to histidine and glutamine
residues could potentially provide suitable hydrogen bonding
interaction to the peroxide enhancing the heterolytic cleavage of
the O–O bond forming the compound I intermediate in the mutant
enzyme and hence these mutants (L80H and L80Q) were subjected to
further studies. Mutation of the Leu80 residue to isoleucine (L80I) was
also made as a control where the hydrophobic residue was replaced
with another hydrophobic residue, and no enhancement in the
peroxidase activity is expected on such mutation.
3
.3. UV–visible absorption, near-UV and Heme tertiary CD spectroscopies
The UV–visible absorption spectra of wild type and mutant
CYP175A1 enzymes are shown in Fig. 2(A). The substrate-free ferric
form of the wild type as well as the L80I mutant of CYP175A1 show
Soret band at 417 nm and two visible bands at 534 and 568 nm. These
spectral signatures are characteristic of typical low spin substrate-free

R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
1189
protein backbone [45]. The above tertiary CD result suggests slightly
different heme tertiary environment for mutants compared to the
wild type CYP175A1. But these mutations have very little effect on the
secondary structure of the CYP175A1 enzyme upon mutation and the
far-UV CD spectra of the wild type and the mutant enzyme were
identical to each other (data not shown) as reported earlier [46].
3.4. Resonance Raman spectroscopy
The heme CD studies indicated small changes in the tertiary
structure of the enzymes on the mutations. In order to determine
whether these changes could arise due to any alteration in the
coordination geometry and/or oxidation state of the heme, we carried
out resonance Raman studies on the wild type and mutant CYP175A1.
The resonance Raman (RR) spectra of the wild type and mutant
enzymes were studied to determine the environment of the heme
center [47,48]. Fig. 3 shows the RR spectra of wild type CYP175A1 and
its variants obtained by excitation of a heme electronic transition.
Because of the resonance enhancement mechanism, when excited in
the heme band, heme proteins give RR spectra displaying exclusively
bands originating from the vibrations of the chromophoric group,
heme, according to their symmetry properties. Based on extensive
amount of experimental data on heme model compounds, most bands
in the RR spectra of P450 enzymes can readily be assigned to the
corresponding vibrational modes [48–50]. Some of these bands are
regarded as the marker bands for the metal oxidation, spin and
coordination states [47]. In particular, the RR bands between 1340 and
−
1
1
375 cm
while above 1450 cm
coordination and spin states [47]. The oxidation state marker band ν
are marker bands for oxidation state of the metal ion,
−
1
are considered as marker bands for the
4
−
1
of wild-type enzyme at 1371 cm
heme core size marker bands ν , ν and ν10 at 1502 cm , 1586 cm
3 2
and 1638 cm
indicates a ferric state and the
−
1
−1
Fig. 2. (A) UV–visible absorption, (B) near-UV and heme CD spectra of the ferric wild
type (WT) (solid line), L80I (short dash), L80H (dash dot dot) and L80Q (dash)
CYP175A1 in 50 mM potassium phosphate buffer (pH 7.4).
−1
respectively are characteristic of a hexacoordinate
low-spin heme (6cLS) [47]. The frequencies of the Raman lines of wild
type CYP175A1 are very similar to the values reported for the other
P450 enzymes [48–50]. The frequencies of these bands are the
characteristics of a specific heme pocket, and can be used to
discriminate between P450 enzymes having different heme environ-
ments [47]. Comparing the resonance Raman spectra of wild type
visible bands that are analogous to those observed in the L80H mutant
compared with the wild type CYP175A1 [44]. The pressure induced
change in the spectrum of CYP119 was proposed to arise due to
formation of a ‘reversible P420 form’ of the enzyme. The L80I, L80H as
well as the L80Q mutants of CYP175A1 were however found to be in
P450 forms as they showed the Soret absorption band at 450 nm of
the reduced CO complex of the enzyme (Fig. S2 in Supporting
information). However, formation of a species that is electronically
analogous to the ‘reversible P420 form’ in the L80H and L80Q cannot
also be ruled out in the ferric form of the enzyme.
−
1
−1
− 1
− 1
(ν
4
: 1371 cm , ν
3
: 1502 cm , ν
2
: 1586 cm and ν10: 1638 cm
)
and its various mutants (ν
4
: 1370–1371 cm−1, ν
3
: 1500–1502 cm−1,
The near UV and heme tertiary CD spectra of the wild type and the
mutants of CYP175A1 are shown in Fig. 2(B). The circular dichroism
spectra of the wild type as well as the mutant CYP175A1 show
characteristic spectra with the Soret CD band (around 400 nm) and
delta CD band (around 350 nm) as observed in the substrate-free
P450 enzymes. The heme CD spectrum of the L80I mutant of
CYP175A1 was very similar to that of the wild type enzyme, though
the relative intensity of the delta CD band was slightly less in the L80I
mutant compared to that in the wild type enzyme, indicating
negligibly small change in the heme cavity on the mutation of leucine
by isoleucine. However, the CD spectra of the L80H and L80Q mutants
of CYP175A1 showed a red shift (4–6 nm) compared to the respective
bands of wild type CYP175A1, which corresponds to the shifts in the
UV–visible absorption spectra of the enzymes. Although the intensi-
ties of the delta CD bands were not much affected, the corresponding
Soret CD bands were found to be decreased in the mutants (except in
L80I). The heme CD provides information about the integrity of heme
active site of the protein. The origin of the CD in this region are due to
the coupling of heme π–π* electric dipole transition moments with
those of nearby aromatic residues and with π–π* transition of the
Fig. 3. Resonance Raman spectra of the ferric wild type (WT), L80I, L80H and L80Q
CYP175A1 (600–800 μM) in 50 mM potassium phosphate buffer (pH 7.4). All
measurements were carried out using excitation at 488 nm and incident powers of
5
3
–6 mW at room temperature. All spectra were obtained with data accumulated over
00 s averaged over 5 scans. The positions of the marker bands (ν , ν , ν , and ν10) are
2
3
4
shown by vertical lines.

1190
R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
: 1583–1585 cm− and ν10: 1634–1638 cm− 1) showed that the
1
recently for CYP119 [51] and the peroxidase activity was enhanced in
case of the L80H and L80Q mutants of CYP175A1. The peroxidase
activity of the control mutant, L80I however was analogous or smaller
than that of the wild type enzyme. The increase in activity in case of
L80H and L80Q mutants arises due to the introduction of the acid/base
catalytic residue at the Leu80 position and it is not due to any other
effect of the mutation as evidenced from the results of the control
mutant L80I. The initial reaction rates (v) were used to determine the
ν
2
structure of the heme active site was almost unchanged after the
mutation of the Leu80 position as the positions of the various marker
bands were almost the same as those of the wild type species. Hence
the coordination geometry, spin state and oxidation state of iron in
the heme of the L80I, L80H and L80Q mutants of CYP175A1 are
similar to those of the wild type enzyme. The resonance Raman
results support that the iron in the heme is in the low-spin six-
coordinated ferric state in the wild type as well as in all the mutants.
It is important to note that the resonance Raman spectroscopy does
not provide information on the exact nature of the sixth axial ligand
m
apparent kcat and K values by analyses of the data using the Michaelis–
Menten equation. The apparent kcat values for guaiacol oxidation are
given in Table 1. The peroxidase activity constant (kcat) of the L80H and
L80Q mutants are found to be respectively 3 and 4 fold higher than that
of the wild type CYP175A1 enzyme. These apparent kinetic constants
are compared with the corresponding apparent constants reported for
myoglobin, cyt b562, HRP and H42A mutant of HRP in Table 1. The
[
42]. The results however support that the ferric ion in the heme of
L80H as well as of L80Q mutants is in six-coordinated low-spin state
analogous to the wild type enzyme as discussed above.
3
.5. Peroxidase activity of wild type and its mutants CYP175A1
m
specificity constant (kcat/K ) that indicates efficiency of the catalyst,
was found to be 5 and 2 folds higher respectively for the L80H and the
L80Q mutants than that for the wild type CYP175A1 enzyme for
guaiacol oxidation.
The steady-state kinetics of guaiacol and ABTS peroxidation by wild
type CYP175A1 and its variants (L80I, L80H and L80Q) have been
examined to evaluate the effect of introduction of residues such as
isoleucine, histidine and glutamine at position 80 in the distal heme
pocket of the enzyme. Fig. 4(A) depicts the time course of the oxidation
of guaiacol (tetra-guaiacol formation) with hydrogen peroxide by wild
type CYP175A1 and its variants (L80I, L80H and L80Q) along with that
of the control reaction at pH 7.4. The results suggest that wild type
CYP175A1 has distinct peroxidase activity analogous to that reported
Fig. 4(B) depicts the time course of the oxidation of ABTS (ABTS+
formation) with hydrogen peroxide by wild type CYP175A1 and the
mutants (L80I, L80H and L80Q) along with the control reaction at pH
7.4. Analogous to that observed in case of guaiacol, the results from
ABTS oxidation studies also support the role of acid/base properties of
the mutated residue (in L80H and L80Q) in the distal pocket in
enhancing the peroxidase activity of the mutant. The apparent rate
constant, kcat values for ABTS oxidation obtained from Michaelis–
Menten analysis (Fig. S5 in Supporting information) of the initial
reaction rate catalysed by the wild type as well as L80I, L80H and L80Q
mutants of CYP175A1 are shown in Table 2. The kcat values of L80H
and L80Q mutant are respectively about 3 and 8 fold higher than that
of the wild type CYP175A1 enzyme. Table 2 shows that the kinetic
•
constants (kcat/K
myoglobin and comparable to that for H42A mutant of HRP.
2 2
The Michaelis constant, K for guaiacol reaction with H O in case
m
) for the CYP175A mutants are higher than that for
m
of the wild type and mutant CYP175A1 were about one order smaller
compared to those in case of wild type myoglobin, cyt b562 and HRP
(
Table 1). Similarly the ABTS affinity constant, K
type and mutant CYP175A1 were about three to four orders smaller
compared to those for wild type myoglobin, cyt b562 and HRP. The K
m
values for the wild
m
values for the CYP175A1 variants were however larger than that for
the H42A HRP mutant, where a vacant site was created to enhance the
binding of the reducing substrate and for promoting peroxygenase
activity [52] (Table 2). The most peroxidases as well as myoglobin and
cyt b562 are relatively non-selective for the reducing substrate as they
lack specific recognition sites for the substrate at the heme active site.
As the heme active centre of the wild type CYP175A1 is buried inside
the protein matrix, the size of the distal pocket above the heme as well
as the interactions of the active site with the substrates would
possibly be important in the substrate association to the enzyme. The
m
smaller values of K possibly indicate stronger association of the
substrate in case of the wild type CYP175A1 and its mutants to the
enzyme compared to that in case of HRP, myoglobin etc., which
Table 1
Kinetic parameters for guaiacol oxidation at 25 °C for different heme proteins.
Enzyme
pH
k
(
cat
K
m
(M)
k
M
cat/K
m
Reference
min−1)
3.3± 0.2 (3.1 ± 0.3)×10−
−1
min−1)
4
4
WT CYP175A1
L80I CYP175A1
L80H CYP175A1 7.4 12.4± 0.5 (2.5± 0.2)×10
7.4
7.4
1.1×10
This work
This work
This work
This work
[7]
[9]
[17,52]
[17,52]
1.2± 0.5 (1.5± 0.3)×10−
4
0.83×10
5.0×10
2.4×10
1.2×10
0.4×10
4.3×10
23×10
4
4
4
3
3
6
4
Fig. 4. The time profile for (A) guaiacol oxidation (monitored at 470 nm) and (B) ABTS
oxidation (monitored at 414 nm) due to the peroxidase activity of the wild type (WT)
−4
−4
L80Q CYP175A1 7.4 14.6± 0.7 (6.1± 0.6)×10
(solid line), L80I (short dash), L80H (dash dot dot) and L80Q (dash) and control, i.e., no
−3
Myoglobin
WT Cyt b562
HRP
7
6
6
6
1.7
0.5
2.5×10
0.9
1.5×10
1.2×10
5.8×10
4×10
enzyme (short dot) experiment. The enzyme (0.2 μM) and H (50 mM) were used for
2
O
2
−3
−3
−6
the oxidation of guaiacol (750 μM) and ABTS (75 μM) in 50 mM potassium phosphate
buffer (pH 7.4) at 25 °C. The initial slope of the absorbance was used to calculate the
peroxidase activity.
4
H42A HRP

R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
1191
Table 2
bonding to the axially coordinated water as indicated from the
spectroscopic studies of the mutants would retard the replacement of
the axial water (or hydroxide) molecule by hydrogen peroxide and
Kinetic parameters for ABTS oxidation at 25 °C for different heme proteins.
Enzyme
pH
k
(
cat
min−1)
K
m
(M)
k
cat/K
m
Reference
(M min−1
−1
)
2 2
thus may lead to an increased activation barrier for binding of H O to
7.4 6.1± 0.3 (6.4 ± 0.6)×10−6
9.5×105
the iron. This effect would thus try to decrease the reactivity, while the
acid/base property of these residues (His and Gln) would favor
increase in the peroxidase activity of the mutant and the overall kcat
would be a resultant of these two opposing factors.
WT CYP175A1
This work
This work
This work
This work
[7]
−
6
3.4×105
14.0×105
6.6×105
1.1×104
L80I CYP175A1 7.4 3.5± 0.4 (10.0 ± 1.0)×10
L80H CYP175A1 7.4 15.4± 0.2 (11.0± 1.0)×10−
6
−
6
L80Q CYP175A1 7.4 46.3± 0.5 (70.0± 2.0)×10
−
3
Myoglobin
HRP
H42A HRP
7
11
1×10
4.6 2.5×105
4.6 24.6
0.8×10
−3
6
3.1×10
8
[17,52]
[17,52]
−
4.1×106
6×10
3.6. Temperature dependence of the peroxidase activity
The rate of an enzymatic reaction generally increases by increasing
temperature up to a certain temperature and then starts decreasing
due to the thermal denaturation at elevated temperatures. Hence the
enhancement of the enzymatic turnover with increasing temperature
is compromised by the competing effects of protein denaturation at
high temperatures. It is thus very important to measure the activity at
different temperatures to determine the optimum temperature that
supports high enzymatic activity without undergoing significant
protein denaturation. The thermophilic nature of the CYP175A1
enzyme makes it a better suited enzyme at elevated temperatures
compared to its mesophilic homologue. The thermal and conforma-
tional stabilities of the native enzyme have been studied and found to
consist of the active site partially exposed to the surface. Tables 1 and
also show that the K values for ABTS are much smaller compared to
those for guaiacol in case of wild type CYP175A1 albeit guaiacol being
smaller in size than ABTS. This suggests that not only the size of the
substrate but also the interactions of the substrate with the protein
2
m
need to be considered for rationalizing the variations in the K
m
values.
3
The molecular size of Leu (166.7 Å ) is larger than that of His
3
3
(
153.2 Å ) or that of Gln (143.8 Å ) [53], but the replacement of Leu80
by His or Gln causes increase in the value of K for ABTS (Table 2)
m
while this effect is much less prominent for guaiacol (Table 1). These
results possibly indicate that the effects of conformation of the side
chains of the mutated amino acids as well as their polarity might have
important roles in the formation of the enzyme–substrate complex in
the present case.
m
have the melting temperature (T ) of 87 °C [33,46]. It is well known
that the enzymes from thermophilic sources show higher activity at
elevated temperatures especially at their dwelling temperature
[8,31,51]. The optimum temperature required for the growth of T.
thermophilus is reported to be in the range of 65–70 °C [58]. The
The activity constant, kcat values were found to increase by
introducing a residue, which can act as acid–base catalyst (such as His
or Gln) at the distal side of the heme in the wild type CYP175A1
enzyme (Tables 1 and 2). The mutation of the Leu80 residue with
isoleucine did not increase the activity constant, rather the L80I
mutant showed a small decrease in the kcat compared to the wild type
enzyme, indicating that the effect of the His and Gln as acid–base
catalyst was indeed important for enhancement of the catalytic
activity of the enzyme. The values of the kcat for the mutants of
CYP175A1 were also found to be higher with ABTS than with guaiacol
as reported in case of HRP. The kcat values for L80H as well as for L80Q
mutant were found to be higher compared to those for myoglobin and
for cyt b562 but was much lower compared to that for HRP at 25 °C.
The mechanism of the peroxidase activity involves formation of two
ferryl heme redox intermediates viz., compound I and compound II of
m
melting temperatures (T ) of 84 °C, 82 °C, 82 °C and 86 °C for the
heme tertiary structure of the wild type [46], L80I, L80H and L80Q
mutants of CYP175A1 respectively were determined from the
temperature dependent heme CD data (Fig. S3 in Supporting
information). Unlike ABTS and guaiacol, Amplex Red and its oxidation
product (resorufin) are stable at temperatures up to 90 °C. Fig. 5(A)
shows the temperature dependence of the relative peroxidase
activities of the wild type, L80H and L80Q mutant CYP175A1 enzyme
determined using Amplex Red as the reducing substrate. Since the
control mutant, L80I showed even lower peroxidase activity than that
of the wild type enzyme at room temperature, the temperature
dependence of activity of L80I was not performed.
The peroxidase activities of the wild type as well as of the mutants
of CYP175A1 were found to increase up to ~70 °C (Fig. 5(A)) and then
sharply decreased with further increase in temperature. The L80H and
L80Q mutant had higher peroxidase activity compared to wild type
enzyme at all temperatures in the range of the present study. Fig. 5A
shows that the peroxidase activity of HRP is much higher compared to
wild type CYP175A1 at temperatures up to 45 °C, and the activity of
HRP decreased drastically with increase in temperature above 45 °C
because of denaturation of the enzyme. The L80Q mutant of
CYP175A1, although had much lower activity compared to HRP at
room temperature, showed higher activity than HRP above ~70 °C.
These results suggest that the higher peroxidase activity of the L80Q
mutant of CYP175A1 at high temperature arises because of high
thermostability of the enzyme compared to HRP.
m
the enzyme [12]. The values of kcat (as well as K ) thus give the
apparent catalytic parameters only. The present observation that
incorporation of the hydrogen bonding residues enhances the rates of
2 2
the catalysis, possibly indicate that the H O bound heme is indeed
involved in the process. Heterolytic cleavage of the peroxide complex
by the ‘Push–Pull’ mechanism is the accepted pathway of formation of
the ferryl heme species of the compound I of the peroxidases, which
seems to be obeyed in the present case also [54,55]. The results in the
present studies suggest that the change in the values of the kcat on
introduction of hydrogen bonding residues (histidine or glutamine) at
Leu80 site possibly arises due to facilitation of heterolytic cleavage of
the O–O bond in the peroxide complex of the iron of heme.
The peroxidase activity constants (kcat) of L80H and L80Q mutants
of CYP175A1 were lower than those expected for a peroxidase (e.g.,
HRP) that consists of a five-coordinated heme at the active site. Earlier
reports [8,56,57] showed that the formation of a six-coordinated
heme indeed decreases the peroxidase activity. The M69A/V49D
mutant cyt c552 from T. thermophilus was shown to lose its peroxidase
activity above 70 °C owing to the formation of a six coordinated heme
species [8]. Similarly in HRP, the mutation of distal Arg by Lys results
in the formation of a six coordinated system showing low enzymatic
activity [56,57]. The UV–visible spectra as well as resonance Raman
results suggest that the wild type as well as the mutants of CYP175A1
consist of six coordinated ferric heme species. Interaction of histidine
3.7. pH dependence of the peroxidase activity
The understanding of the pH dependence of the activity of an
enzyme is very important in optimizing the assay conditions. The
tertiary structure of the enzyme, which is responsible for its activity, is
usually sensitive to pH, and denaturation of the enzyme occurs
generally at extremely low and high pH values. Fig. 5B shows that the
peroxidase activity of wild type as well as mutant CYP175A1 depends
strongly on pH. The optimum pH for the peroxidase activity of the
wild type enzyme was found to be at pH ~5 and the activity becomes
insignificant at pH above 7.5. On the other hand, the peroxidase
(
in L80H) or glutamine (in L80Q) with the heme through hydrogen

1192
R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
H O
2 2
bound to the iron of the heme [59]. Similar effects have been
observed in case of the H42A [17] and H42E [59] mutants of HRP,
indicating that the apparent pKa of H bound to the iron in heme
2 2
O
might have important contribution to the stabilization of the redox
intermediate. The decrease in the activity at high pH (above pH ~9) for
the mutants of CYP175A1 could arise due to decrease in the stability of
+
•
the oxidized product (e.g., ABTS radical ion [60]) at high pH.
.8. Hydrogen peroxide dependent guaiacol oxidation
The H dependence of the peroxidase activities (guaiacol
3
2 2
O
oxidation) was studied and the apparent kinetic constants for wild
type CYP175A1 and its mutants were determined. The concentration of
the H O was varied in the range 5 mM–450 mM and the enzyme was
2 2
not denatured in this concentration range of the peroxide in the
experimental time range [61]. The initial rate of guaiacol oxidation by
H O
2 2
catalysed by the wild type and mutants of CYP175A1 showed
typical Michaelis–Menten kinetics (Fig. S6 in Supporting information).
The apparent kinetic parameters for H were determined by fitting
initial oxidation rate of guaiacol oxidation to Michaelis–Menten
equation (Table 3) at different H concentrations. The kcat values
determined from the H concentration dependence kinetics of
2 2
O
2 2
O
2 2
O
guaiacol reaction for wild type and mutant CYP175A1 (Table 3) were
similar in magnitude to the corresponding kcat values for the enzyme
variants obtained for the guaiacol dependent peroxidase activity
(
Table 1). This indicates that the value of the kcat corresponds to the
reaction of guaiacol with the redox intermediates of the enzyme. The K
values (Table 3) for the H dependence kinetics however correspond
m
2 2
O
to interaction of the peroxide with the enzyme and were different from
those determined for the guaiacol interaction (Table 1). The apparent
substrate (H O ) affinity constant (K ) for H O reaction with wild type
2 2 m 2 2
Fig. 5. (A). Temperature dependence initial rate profile of the oxidation of Amplex Red by
peroxidase reaction of the wild type (WT) (■), L80H (●), L80Q (▲) mutants of CYP175A1
and HRP (☆).The enzyme (0.2 μM) and H O (4 mM) were used for the oxidation of
2 2
Amplex Red (10 μM) in 50 mM potassium phosphate buffer (pH 7.4). The initial oxidation
rate of Amplex Red by wild type CYP175A1 enzyme at 70 °C was set as 100%, and the rates
for other enzymes are expressed with respect to (w.r.t.) that of the wild type enzyme.
CYP175A1 enzyme (65 mM) was higher than that reported for HRP
(22 μM) and other peroxidases (Table 3). The P450s generally have low
affinity towards H O compared to HRP and peroxygenases (such as
2 2
P450SPα and P450BSβ) possibly due to the presence of hydrophobic
environment around the distal site of the heme [4] in the P450 enzymes.
(
B). Profile of initial reaction rate versus pH for the oxidation of ABTS in the peroxidase
reaction of wild type (WT) (■), L80H (●) and L80Q (▲) mutant CYP175A1enzyme along
with that of HRP (☆). The enzyme (0.2 μM) and H (50 mM) were used for the oxidation
The K
reported to be 15 mM [24], and that with liver microsomal P450 was
50 mM [4,23], which are in the similar range as that observed in the
present study with CYP175A1 (Table 3). The K values were found to be
80 mM and 277 mM respectively for L80H and L80Q mutants, which
m 2 2
value for the H O reaction with cytochrome P450BM3 was
2 2
O
2
ofABTS(1 mM)in50 mM universalbufferat 25 °C. Theinitial oxidationrateofABTSbyWT
CYP175A1 enzyme at pH 5 was set as 100%, and the rates for other mutants of CYP175A1
are expressed with respect to (w.r.t.) that of the wild type enzyme. The results of HRP has
been taken from earlier report by Savenkova et al. [17].
m
3
are respectively about six and four fold higher than that of the wild type
enzyme. The hydropathy index for leucine is 3.8 while those for
histidine and glutamine are −3.2 and −3.5 respectively. Thus, both
histidine and glutamine provide hydrophilic environment around the
activity of the L80H and L80Q mutants of CYP175A1 was increased up
to pH ~9, indicating that the mutation of the Leu80 by His and Gln
drastically enhance the peroxidases activity of the enzyme at high pH.
The peroxidase activity of HRP has earlier been shown to depend on
pH [17] (Fig. 5B). The role of distal histidine on the pH dependence of
the activity of HRP is well recognized, though the H42A mutant of HRP
also showed linear increase in activity with pH [17]. Careful inspection
of the pH dependence of the activity of the mutants of CYP175A1
showed that the peroxidase activity was initially increased with pH up
to pH 5 similar to that in case of the wild type CYP175A1 enzyme.
However, the activity of the mutants (L80H and L80Q) reached a
plateau in the range pH 5 to pH ~7 and subsequently increased steeply
up to pH ~9. The tertiary structure of the enzyme was found to remain
unaffected in the pH range 3.7 to 9.0 (Fig. S4 in Supporting
information), indicating that the effects of pH on the activity stems
mainly from the mutation of the Leu80 residue by His or Gln in the
present case. The steep rise of the peroxidase activity of the mutant
enzymes may thus also arise due to increase in basicity of the histidine
3
distal site of the heme. Moreover, the volume of Leu (166.7 Å ) is larger
3
3
than that of His (153.2 Å ) as well as that of Gln (143.8 Å ) [53]. On the
basis of hydropathy index and size of amino acids, K values for L80H
and L80Q were expected to be less than that for wild type, but actually
an increase in K values was observed in both these mutants of
CYP175A1. The present results thus indicate that thehigher values of the
for these mutants of CYP175A1 compared to the wild type enzyme
m
m
K
m
might arise due to the weak interaction of the His80 and Gln80 to the
ferric ion in L80H and L80Q mutants as proposed above. The interaction
Table 3
2 2
Kinetic parameters for guaiacol oxidation at different concentration of H O in 50 mM
potassium phosphate buffer (pH 7.4) at 25 °C for the wild type and its mutant
CYP175A1 enzyme.
Enzyme
k
cat
K
m
(M)
k
cat/K
m
References
(min−1)
(M min−1
−1
)
(
in L80H) or of the glutamine (in L80Q) at higher pH that facilitate the
−3
WT CYP175A1
L80H CYP175A1
L80Q CYP175A1
HRP-C at pH 5.0
2.7± 0.5
(65.0± 0.8)×10
42.0
18.0
37.0
This work
This work
This work
[62]
6.8± 0.7 (380.0± 5.0)×10−
3
formation of the ferryl intermediate by forming hydrogen bond with
the peroxo complex. One other possible origin of the observed
increase in the peroxidase activity of the mutants of CYP175A1 at
higher pH might be the increased efficiency of proton abstraction from
−
3
10.3± 0.3 (277.0± 4.0)×10
a
−6
6
32,200
22×10
145.5×10
a
ABTS was used as the substrate.

R.K. Behera et al. / Journal of Inorganic Biochemistry 104 (2010) 1185–1194
1193
of the histidine (in L80H) or glutamine (in L80Q) to the heme through
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3] S.-i. Ozaki, T. Matsui, M.P. Roach, Y. Watanabe, Coord. Chem. Rev. 198 (2000)
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(
2 2 m
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4
. Conclusions
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