Tài liệu Báo cáo khoa học: Critical roles of Asp40 at the haem proximal side of haem-regulated phosphodiesterase from Escherichia coli in redox potential, auto-oxidation and catalytic control doc

Critical roles of Asp40 at the haem proximal side of haem-regulated
phosphodiesterase from
Escherichia coli
in redox potential,
auto-oxidation and catalytic control
Miki Watanabe, Hirofumi Kurokawa, Tokiko Yoshimura-Suzuki, Ikuko Sagami and Toru Shimizu
Institute of Multidisciplinary Research for Advanced Materials Tohoku University, Sendai, Japan
In haem-regulated phosphodiesterase (PDE) from Escheri-
chia coli (Ec DOS), haem is bound to the PAS domain, and
the redox state of the haem iron regulates catalysis by the
PDE d omain. We generated mutants of Asp40, which forms
a hydrogen bond with His77 (a proximal haem axial ligand)
via two water molecules, and a salt bridge wit h Arg85 at the
protein surface. The redox potential of haem was marked ly
increased from 67 mV vs. the stan dard hydrogen e lectrode in
the wild-type enzyme t o 95 mV and 114 mV in the Ala and
Asn mutants, r espectively. Additionally, the auto-oxidation
rate of Ec DOS PAS was significantly increased from
0.0053 to 0.051 and 0.033 min
)1
, respectively. Interestingly,
the catalytic activities of the A sp40 mut ants were a bolished
completely. T hus, Asp40 appears to play a critical role in the
electronic structure of the h aem iron and redox-dependent
catalytic control of the PDE domain. In this report, we
discuss the mechanism of catalytic control o f Ec DOS,
based on the physico-chemical characteristics of the Asp40
mutants.
Keywords: auto-oxidation; h aem axial ligand; haem sensor;
phosphodiesterase; redox potential.
Haem-regulated phosphodiesterase (PDE) from Escheri-

chia coli (Ec DOS) is a haem sensor enzyme composed
of two functional domains: an N-terminal haem-bound
sensor domain and a C-terminal PDE catalytic domain
[1,2]. Catalysis by this enzyme is regulated by the haem
redox state in that PDE is functional in the Fe(II) haem-
bound enzyme, but not the F e(III) haem-bound enzyme
[2,3]. The crystal structure of the haem-bound domain
revealed a typical PAS structure [4,5]. PAS proteins
display a characteristic three-dimensional structure with a
glove-like fold consisting of five j uxtaposed b-sheets and
flanking a-helices [6–9]. The characteristic three-dimen-
sional structure of the PAS domain is commonly used
for discussing the signal transduction mechanism of
numerical signal transducing enzymes [6–9]. The crystal
structures of both the Fe(II) and Fe(III) forms of the
isolated haem-bound PAS domain (Ec DOS PAS)
disclose that haem axial ligand switching from His77/
hydroxide anion to the His77/Met95 ligand pair occurs
upon haem reduction. Moreover, haem ligand switching
induces conformational changes in the FG loop region
and movement of t wo subunits. These structural changes
may play critical roles in c atalytic regulation of the PD E
domain [4].
Structures of the haem-bound PAS domain have been
reported under various conditions, and structure–function
relationships are well documented [8–14]. FixL is an oxygen
sensor enzyme with a haem-bound PAS domain. Specific-
ally, O
2
association/dissociation to/from the haem switches

off/on catalysis [8,9]. Global structural changes at the haem
distal side are i nduced upon O
2
binding to FixL, and these
changes contribute significantly to intramolecular s ignal
transduction [9–11]. For Ec DOS, site-directed mutations at
Met95, the axial ligand at the distal side in the Fe(II)
complex, induced significant changes i n t he redox potential
of the haem, rate constants o f CO, O
2
and CN b inding to
haem, and CD spectra in the Soret region [15–18],
suggesting that this residue is critical for maintaining the
electronic states of haem and ligand access channel.
However, the catalytic activities of Met95 mutants were
comparable to those of wild-type [14], implying no direct
involvement in the catalytic control of Ec DOS. A number
of site-directed mutagenesis [2,3,16] and crystallographic
[4,5] s tudies show that His77 is the proximal axial ligand of
haem. Therefore, a residue interacting with H is77 may p lay
an important role in regulation of catalysis. Ec DOS forms
hydrogen networks at the haem proximal side (Fig. 1)
including the Asp40 residue that interacts w ith His77 via
two water molecules and Arg85 at the protein surface [4].
In the present study we generated Asp40Ala and
Asp40Asn mutants and analysed their physico-chemical
Correspondence to T. Shimizu, Institute of M ultidisciplinary Research
for Advanced Materials, Tohoku University, 2-1-1 Katahira,
Aoba-ku, Sendai 980-8577, Japan. Fax: +81 22 217 5604/5390,
Tel.: +81 22 217 5604/5605. E-mail:

Abbreviations: PDE, phosphodiesterase; Ec DOS, full-length haem-
bound phosphodiesterase from Escherichia coli or a redox sensor from
Escherichia coli; PAS, an acronym formed from the following names:
Drosophila period clock protein (PER), vertebrate aryl hydrocarbon
receptor nuclear translocator (ARNT) and Drosophila single-minded
protein (SIM); FixL, an oxygen sensor haem p rotein from Rhizobia
melilori;ant-cAMP, 3¢,5¢-cyclic monophosphate, 2¢-O-anthraniloyl;
Ec, DOS PAS, isolated haem-bound PAS domain of Ec DOS;
SHE, standard hydrogen electrode.
(Received 20 June 2004, revised 9 Au gust 2004,
accepted 12 August 2004)
Eur. J. Biochem. 271, 3937–3942 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04331.x
characteristics, includ ing optical absorptio n spectra, cyanide
binding, redox potential and auto-oxidation rates of
Ec DOS PAS, and catalytic activities of full-length
enzymes. We found that the mutations at Asp40 markedly
altered t he redox potential and auto-oxidation rate. Fur-
thermore, PDE activity was completely abolished in the
Asp40 mutants. To our knowledge, this is the first report
showing that mutations in the haem environment substan-
tially influence the catalytic activity of haem-bound
Ec DOS.
Experimental procedures
Materials
The expression vector, pET28a(+) was from Novagen.
E. coli competent cells, XL1-blue (for cloning), and BL21
(for pr otein expression) were purchased from Stratagene.
Site-directed mutagenesis was performed using the
Quikchange Site-Directed Mutagenesis Kit
TM

(Stratagene)
with the following oligonucleotides: Asp40Ala:
5¢-TGTTAATTAACGAAAATGC TGAAGTGATGTTTT
TC-3¢ (forward); 3¢-GAAAAACATCACTTC AGCATTT
CGTTAATTAAC-5¢ (reverse); Asp40Asn: 5¢-GGTGTT
AATTAACGAAAATAAC GAAGTGATGTTTTTCA
AC-3¢ (forward); 3¢-GTTGAAAAACATCACTTCGTTA
TTTTCGTTAATTAACA-5¢ (reverse). Mutation sites are
shown in italics. Oligonucleotides were synthesized by the
Nihon Gene Research Laboratory (Sendai, Japan). Restric-
tion and m odification enzymes were from Takara Bio (Otsu,
Japan), Toyobo, New England Biolabs and Nippon Roche
K.K. The fluorescence substrate, adenosine 3 ¢,5¢-cyclic
monophosphate, 2 ¢-O-anthraniloyl (ant-cAMP) was from
Calbiochem. Calf intestine alkaline phosphatase was from
Takara Bio. DEAE Sephadex was from Amersham
Biosciences. Other chemicals were from Wako Pure
Chemicals.
Expression and purification of
Ec
DOS wild-type and
Asp40 mutant proteins
Expression and purification procedu res were performed as
described previously [2,3]. Purified proteins were more than
95% homogenous, as verified by SDS/PAGE. Yields of
Ec DOS and Ec DOS PAS from 1 L E. coli culture were
210 n mol and 610 nmol, respectively, in terms of haem
absorbance at 417 n m [2,3].
Optical absorption spectra
Spectral experiments were performed under aerobic condi-

tions on Shimadzu UV-1650, UV-2500 and Hitachi U-2010
spectrophotometers maintained at 25 °C using a tempera-
ture controller [2,3,15,17,18]. Anaerobic spectral experi-
ments were conducted in the glove box of a Shimadzu
UV-160 A spectrophotometer. Following reduction of the
haem b y sodium dithionite, excess dithionite was removed
in the glove box by using a Sephadex G25 column. To
ensure that the temperature of the solution was maintained
at 25 °C, the reaction mixture was i ncubated for 10 min
prior to spectroscopic measurements.
Cyanide binding
The association rate of cyanide to haem was observed by
monitoring changes at 417 nm in spectra of the haem
protein in 50 m
M
Tris/HCl pH 7.5, as described earlier [17].
Redox potential
Anaerobic spectral experiments on Ec DOS PAS proteins
were performed i n the glove box on a Shimadzu UV-160 A
spectrophotometer and a n O RION Research (Tokyo,
Japan) Model 701 digital p H meter equipp ed with a TOA
(Tokyo, Japan) ORP g old/calomel combination microelec-
trode. Redox potentials were measured using the same
apparatus. 2,3,5,6-Tetramethyl phenylenediamine (5 l
M
),
N-ethyl phenazonium ethosulfate (5 l
M
) a nd 2-hydroxy-
1,4-naphthaquinone (5 l

M
) were added as m ediators to the
wild-type protein solution before titration [2,15]. The
concentration o f h aem protein used was 15 l
M
. Spectral
changes in i ntensity at 563 nm accompanying redox changes
were monitored, as dye absorption hampers the d etection o f
Soret spectral changes. To ensure that the appropriate
temperature o f the solution was maintained, the r eaction
mixture was incubated for 10 min prior to spectroscopic
measurements. Titration experiments were re peated at least
three times for ea ch complex.
Auto-oxidation rate
To measure auto-oxidation rates, Ec DOS PAS proteins
were reduced with sodium dithionite in 50 m
M
Tris/HCl
pH 7.0 containing 1 m
M
EDTA. Excess dithionite was
removed in the glove box using a Sephadex G-25
column. After removal from the glove box, the mutant
wasdilutedto1mLwith50m
M
potassium phosphate
pH 7.0 containin g 1 m
M
EDTA. The auto-oxidation
rates of Asp40Ala and Asp40Asn mutants were meas-

ured at 25 °C. Oxidation of t he sample was observed b y
Fig. 1. Structure of ferric Ec DOS PAS (PDB code 1V9Y). Asp40
interacts with the imidazole r ing o f His77 via t wo wate r molecules (W3,
W4).Asp40additionallyformsasaltbridgewithArg85atthesurface
of the m olecu le. T he h ydrogen bonds and fl exible FG loop are depicted
by black dotte d and cyan broken lines, respectively. The figure was
obtained using
PYMOL
[20].
3938 M. Watanabe et al. (Eur. J. Biochem. 271) Ó FEBS 2004
recording entire visible spectra or monitoring the decrease
in absorbance at a single wavelength (567 nm) on a
Hitachi U-2010 spectrometer. All auto-oxidation reac-
tions were characterized by only two spectral species with
sharp i sosbestic points, and the time course patterns
revealed a simple first-order process under each set of
conditions.
Enzymatic assay
Full-length Ec DOS protein was incubated at 37 °Cwith
ant-cAMP in a reaction mixture of 500 lL containing
50 m
M
Tris/HCl pH 8.5 a nd 2 m
M
MgCl
2
in a glove box
under a nitrogen atmosphere with an O
2
concentration

< 50 p.p.m., as described previously [2,3]. At least five
experiments were conducted to obtain e ach value.
Results
Optical absorption spectra
Figure 2 depicts the optical absorption spectra of Fe(III),
Fe(II) and Fe(II)-CO complexes of the Asp40Ala and
Asp40Asn mutants. Table 1 summarizes the spectral
absorption maxima of these complexes. Despite subtle
differences in the spectral maxima in the visible region of
the Fe(III) species, spectra of the Asp40 mutants were
essentially similar to that of the wild-type protein.
Additionally, spin states of the Fe(III), Fe(II) and
Fe(II)-CO complexes of Asp40 mutants were similar to
those of the wild-type enzyme in that all mutant proteins
were in the six-coordinated low-spin state. T herefore, we
propose that Asp40 mutations do not significantly affect
the h aem e nvironment or coordination structures. More-
over, these mutants are suitable for further spectral and
catalytic characterization.
Cyanide binding
Kinetic and e quilibrium studies on cyanide binding provide
valuable inform ation on t he haem distal structure o f
Ec DOS PAS [17]. As the structure of the cyanide-bound
complex of FixL is similar to that of its oxygen-bound
complex [10], a cyanide binding study should facilitate
elucidation of the structure and catalytic mechanism of
Ec DOS. Cyanide-bound Fe(III) complexes of t he Asp40
mutants displayed optical absorption spectra containing a
sharp Soret peak at 421 nm and a broad visible band
around 540 nm (data not shown), analogous to that of

the wild-type enzyme [17]. Optical absorption changes
observed for both the Asp40 mutants upon cyanide
binding were composed of only one phase. The first-order
rate co nstants f or cyanide binding to Asp40 mutants were
dependent on the cyanide concentration. The rates of
cyanide association to t he Asp40Ala and Asp40Asn
mutants were 0.065 and 0.073 m
M
)1
Æs
)1
, respectively,
which are only slightly higher than that (0.045 m
M
)1
Æs
)1
)
of the wild-type protein (Table 2). The data i ndicate that
mutation of Asp40 a t the haem proximal side alters the
cyanide b inding property only slightly, a nd may not
significantly affect the exogenous ligand binding access
channel a t t he haem distal side and/or structure in t erms of
cyanide binding in the Fe(III) complex.
Redox potentials
The redox state of haem is related to the PDE activity of
Ec DOS, since it is a haem redox-sensing enzyme [2,3].
Thus, it is important to determine the redox potentials of the
Asp40 mutants of Ec DOS. Asp40 m utant proteins w ere
converted from the Fe(III) complex to the Fe(II) complex

by reductive titration, accompanied by clear isosbestic
points, similar to previo usly documented data for the wild-
type and Met95 mutant proteins [2,15]. Electrochemical
reductive titration of the mutant protein is depicted in
Fig. 3. The redox potential values of the m utants as well as
wild-type protein are summarized in Table 2. Marked
increases in the redox potential value from 67 m V vs. the
standard hydrogen electrode (SHE) (wild-type) up to 95 and
114 mV were observed f or the Asp40Ala and Asp40Asn
mutants, respectively. This tendency is opposite to that
observed on mutating Met95 at the haem distal site.
Specifically, Met95 mutations led to significant decreases in
redox potential [15]. O xidative titration experiments were
additionally conducted. No remarkable differences were
detected in the potentials between reductive and oxidative
titration. Accordingly, we propose that Asp40 is located
close enough to the haem iron or axial ligand to influence
the haem electronic state of Ec DOS.
Fig. 2. Soret and visible optical absorption spectra of Fe(III) (solid line),
Fe(II) (broken line) and Fe(II)-CO (dotted line) complexes of the
Asp40Ala (upper) and Asp40Asn (lower) mutants of Ec DOS P AS. The
small peak seen around 670 nm is occasionally appears when we
purified mutant enzymes. It is likely to be a minor component of a
denatured form.
Ó FEBS 2004 Haem proximal side of a haem-regulated phosphodiesterase (Eur. J. Biochem. 271) 3939
Auto-oxidation rates
As Ec DOS was initially identified as an O
2
sensor enzyme
[1], we examined the auto-oxidation rates of the Asp40

mutants. As shown in Fig. 4, s emi-logarithmic t ime-
dependent changes in optical absorption spectra were linear,
and composed of only one phase. Auto-oxidation rates and
half-lives of the O
2
-bound Fe(II) complexes of Asp40
mutants are summarized in Table 2 . Marked increases in
the auto-oxidation rate (from 0 .0053 min
)1
for the wild-
type enzyme up to 0.033 and 0.051 min
)1
) were observed i n
the Asp40 mutants.
PDE activity
Electronic states of haem, such as redox potential and
autoxidation rate, largely regulate the PDE activity of full-
length Ec DOS enzyme. Interestingly, no PDE activity was
detected for the two Asp40 mutants analysed in this study.
This finding is in contrast with data on mutants of Met95 at
the haem distal side, which disclosed no effect on PDE
activity [15].
Discussion
The present study reveals an interesting aspect of the
structural and functional relationships of Ec DOS. Muta-
tions at Asp40 did not essentially affect the optical
absorption spectra of this enzyme. Howeve r, Asp40 muta-
tions at the haem proxima l side significantly altered the
redox potential values, auto-oxidation rates, and PDE
activities. These results aid in elucidating the transduction

mechanism of this enzyme.
Recent c rystal structure analyses of t he isolated haem-
bound PAS domain indicate that in the Fe(III) complex, a
hydroxide anion (or w ater molecule) is an axial ligand tra ns
to His77, the e ndogenous (proximal) axial ligand [4]. U pon
haem reduction, ligand switching from the hydroxide anion
to the side chain of Met95 i s evident at the haem distal s ide.
Met95, in turn, becomes the direct axial ligand f or the F e(II)
complex trans to His77 [4,5]. The haem redox potential
value was decreased from +67 mV vs. SHE (wild-type) to
)26, )1, and )122 mV in t he Met95Ala, M et95Leu and
Met95His mutants, respectively [15]. The auto-oxidation
rate was altered from 0.0058 to 0.0013, 0.0017 and
0.018 min
)1
in the Met95Ala, Met95Leu and Met95His
mutants, respectively [18]. Mutations at Met95 also signi-
ficantly affect the binding of exogenous ligands, such as the
cyanide anion, to the Fe(III) complex [17], and O
2
and CO
to the Fe(II) complex [18]. H owever, the PDE activities of
these mutants are comparable to that of the wild-type
enzyme. B ased on the data, we propose that Met95
Table 2. Cyanide binding rates, r edox potentials and auto-oxidation
rates of the Asp40Ala and Asp40Asn mutants of Ec DOS PAS. Cyanide
binding rate, experimental errors are within 20%; k
ox
, autoxidation
rate; t

1/2
, Half life.
Ec DOS PAS
CN
(m
M
–
1Æs
)1
)
Redox potential
(mV vs. SHE)
k
ox
(min
)1
)
t
1/2
(min)
Wild-type 0.045 67 (n ¼ 0.93) 0.0053 132
Asp40Ala 0.065 95 ( n ¼ 0.92) 0.051 14
Asp40Asn 0.073 114 (n ¼ 0.96) 0.033 21
Table 1. Optical absorption spectral maxima (nm) of the Asp40Asn and As p40Ala mutants of Ec DOS PAS.
Fe(III) Fe(II) Fe(II)-CO
Soret baSoret baSoret ba
Wild-type 416 530 564 427 532 563 423 540 570
Asp40Ala 415 535 566 428 533 563 424 539 565
Asp40Asn 417 535 565 427 532 563 423 541 572
Fig. 3. Electrochemical reductive titrations of the Asp40Asn m utant of

Ec DOS PAS. Changes in a bsorption intensity were monitored at
563 nm.
Fig. 4. Time-dependent optical absorption changes of the Fe(II)-O
2
complexe s o f Ec DOS PAS wild-type (solid line, ·), Asp40Ala (broken
line, 4) and Asp40Asn (dotted line, s) mutants monitored at 578 nm.
3940 M. Watanabe et al. (Eur. J. Biochem. 271) Ó FEBS 2004
modulates the redox potential to relatively high values
(+61 mV), but is not directly involved in catalytic control
and/or interactio ns with the PDE domain, which are critical
in catalysis.
In contrast to data obt ained with Met95Ala and
Met95Leu mutants [15–18], Asp40 mutations increased
the redox poten tials and auto-oxidation rates. The effects of
Asp40 mutations are opposite to those of Met95 mutations.
Asp40 mutants are more easily reduced than the wild-type
enzyme, and favour the Fe(II) s tate to the Fe(III) state.
Accordingly, we suggest that Asp mutations alter the haem
environment to a more cationic state, and the F e(II) state is
more stabilized.
The crystal structur e of the PAS domain d iscloses two
water molecules between His77 and Asp40 (Fig. 1). There-
fore, the hydrogen bond ing network involving His77, two
water molecules and Asp40 should function in regulating
the electronic state affecting the redox potential and auto-
oxidation rate.
A signal transduction mechanism for the PAS domain
has b een proposed by Cr osson et al. [19] to explain
catalytic c ontrol. The investigators suggested that a well-
conserved salt bridge on the surface of PAS proteins is

important in the signal transduction mechanism.
Although a conserved salt bridge exists between Glu59
and Ly s104 in Ec DOS [ 4], the roles of these amino acids
remain to be elucidated. In Ec DOS, Asp40–Arg85 salt
bridges (Fig. 1) appear to be important for catalytic
control. Breakage of th e salt b ridge by m utations at
Asp40 abolishes PDE activity.
In a previous report, we proposed that movement of
the FG loop in Ec DOS accompanying haem reduction
regulates the catalytic switch [4]. The FG loop is rigidified
in the Fe(II) haem protein, but very flexible in t he Fe(III)
haem protein. Ec DOS is active only in the Fe(II) h aem
form. Notably, Asp40 forms a salt bridge with Arg85,
which is located near the FG loop (Fig. 1) [4]. Mutations
at Asp40 should break the salt bridge w ith Arg85. It is
possible that these mutations maintain flexibility of the
FG loop, even when the haem is the Fe(II) state, and thus
lead to inactive enzyme ( Fig. 1). Thus, switching the FG
loop from the ÔorderedÕ to ÔdisorderedÕ form by substitu-
tions at Asp40 may explain the loss of catalytic activity of
these mutant p roteins. Our recent study showed that
mutations at Trp residues markedly changed fluorescence
intensities, but did not significantly alter the environment
of the haem [21]. A single-Trp containing mutant, in
which only one Trp residue is located near or a t the FG
loop region, may be u seful to analyse flexibility of the FG
loop in Asp40 mutants. Our proposal could also be
substantiated by limited proteolysis t o see a change in
signals upon haem r eduction. These studies remain to be
carried out.

In summary, mutations at Asp40 markedly alter the
redox pot entials, auto-oxidation ra tes and cataly tic
activities of Ec DOS. Breakage of the salt bridge between
Asp40 and Arg85, and t he hydrogen bond network
consisting of Asp40, two water molecules and His77,
appear to be critical for these significant changes. In the
mutants, the FG loop region may become disordered,
even in the Fe(II) state, resulting in a marked decrease in
catalysis.
Acknowledgements
This work was supported in part by a Grand-in-Aid from the Ministry
of Culture, Education, Science, Sp orts and Technology of Japan to
H.K.
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