Báo cáo khoa học: Characterization of Met95 mutants of a heme-regulated phosphodiesterase from Escherichia coli ppt

Characterization of Met95 mutants of a heme-regulated
phosphodiesterase from
Escherichia coli
Optical absorption, magnetic circular dichroism, circular dichroism, and redox
potentials
Satoshi Hirata, Toshitaka Matsui, Yukie Sasakura, Shunpei Sugiyama, Tokiko Yoshimura,
Ikuko Sagami and Toru Shimizu
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
On the basis of amino acid sequences and crystal structures
of similar enzymes, it is proposed that Met95 of the heme-
regulated phosphodiesterase from Escherichia coli (Ec DOS)
acts as a heme axial ligand. In accordance with this proposal,
the Soret and visible optical absorption and magnetic
circular dichroism spectra of the Fe(II) complexes of the
Met95Ala and Met95Leu mutant proteins indicate that
these complexes are five-coordinated high-spin, suggesting
that Met95 is an axial ligand for the Fe(II) complex. How-
ever, the Fe(III) complexes of these mutants are six-coordi-
nated low-spin, like the wild-type enzyme. The latter spectral
findings are inconsistent with the proposal that the axial
ligand to the Fe(III) heme is Met95. To determine the
possibility of a redox-dependent ligand switch in Ec DOS,
we further analyzed Soret CD spectra and redox potentials,
which provide direct evidence on the environmental
structure of the heme protein. CD spectra of Fe(III) Met95
mutants were all different from those of the wild-type
protein, suggesting indirect coordination of Met95 to the
Fe(III) wild-type heme. The redox potentials of the
Met95Leu, Met95Ala and Met95His mutants were consid-
erably lower than that of the wild-type enzyme (+70 mV)
at )1, )26, and )122 mV vs. SHE, respectively. Thus, it is

reasonable to speculate that water (or hydroxy anion)
interacting with Met95, rather than Met95 itself, is the axial
ligand to the Fe(III) heme.
Keywords: heme sensor; magnetic circular dichroism; optical
absorption; phosphodiesterase; redox potential.
Heme-regulated phosphodiesterase from Escherichia coli
(Ec DOS) has been cloned, and its structure–function
relationships have been partially characterized by both
our group and the Kitagawa group [1,2]. Interestingly,
phosphodiesterase (PDE) is active only when the heme iron
is in the Fe(II) redox state. Consistent with this finding, the
PDE activity of Ec DOS was dramatically inhibited by
CO and NO, which display strong affinities for the Fe(II)
complex. Therefore, Ec DOS is likely to be a heme-based
sensor, in which the redox state of the heme iron appears to
control protein conformational changes, which in turn
transmit signals to other domains to initiate the PDE
catalytic function.
The heme-bound N-terminal portion of Ec DOS has
been identified as a PAS domain, based on the sequence and
tertiary structure. PAS is an acronym formed from the
names of proteins in which imperfect repeat sequences were
initially recognized specifically: Drosophila period clock
protein (PER) [3], vertebrate aryl hydrocarbon receptor
nuclear translocator (ARNT) [4], and Drosophila single-
minded protein (SIM) [5]. Site-directed mutagenesis studies
reveal that one of the two axial ligation sites of the heme
iron is occupied by His77 [1,2]. The Gilles-Gonzalez group
reported on the physicochemical properties of the isolated
N-terminal heme-bound PAS domain of Ec DOS and

suggested that the heme axial ligand trans to His77 is Met95
on the basis of sequence homology data and the crystal
structures of other heme-bound PAS proteins [6,7]. How-
ever, resonance Raman spectra of Met95Ala and Met95His
mutants of Ec DOS suggest that a different molecule
occupies this axial position of the Fe(III) complex [2].
Therefore, it is of interest to determine the characteristics of
Met95 mutants of Ec DOS using other spectroscopic
techniques and kinetic analyses.
Optical absorption [8–10], magnetic circular dichroism
(MCD) [10–12], and CD [13,14] spectroscopy are useful
tools for examining the heme coordination structure,
protein folding and the environment of aromatic amino
acid residues. Here, we present an initial report on the
optical absorption, MCD and CD spectra of Met95
mutants of both full-length Ec DOSandtheisolatedPAS
Correspondence to T. Shimizu, Institute of Multidisciplinary Research
for Advanced Materials, Tohoku University, 2-1-1 Katahira,
Aoba-ku, Sendai 980-8577, Japan.
Fax: + 81 22 217 5604, 5605, 5390, Tel.: + 81 22 217 5604, 5605,
E-mail:
Abbreviations: Ec DOS, heme-regulated phosphodiesterase obtained
from Escherichia coli; PDE, phosphodiesterase; PAS, acronym formed
from the names Drosophila period clock protein (PER), vertebrate aryl
hydrocarbon receptor nuclear translocator (ARNT), and Drosophila
single-minded protein (SIM); MCD, magnetic circular dichroism;
FixL, oxygen sensor heme protein from Rhizobium meliloti.
(Received 19 June 2003, revised 12 October 2003,
accepted 16 October 2003)
Eur. J. Biochem. 270, 4771–4779 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03879.x

domain. Mutations at Met95 did not alter the spin state
from six-coordinated low spin to five-coordinated high spin
in the Fe(III) complex in Soret and visible absorption and
MCD spectra, but caused significant changes in the Soret
CD peak position, and dramatically decreased the redox
potentials. We discuss the heme coordination structure of
this redox-sensitive heme sensor, Ec DOS, in view of our
spectral findings.
Experimental procedures
Materials and proteins
The fluorescent substrate, 2¢-o-anthraniloyl adenosine 3¢,5¢-
cyclic monophosphate (ant-cAMP), was purchased from
Calbiochem (La Jolla, CA, USA). Calf intestine alkaline
phosphatase was purchased from Takara Syuzo Co (Otsu,
Japan). DEAE-Sephadex and Sephadex G25 were obtained
from Amersham Biosciences (Uppsala, Sweden). Other
chemicals were acquired from Wako Pure Chemicals
(Osaka, Japan).
Cloning, mutations, expression in E. coli and purification
of full-length Ec DOS (amino acids 1–807) and the isolated
heme-bound PAS domain (amino acids 1–133) were
performed as described previously [1,2]. Site-directed muta-
genesis was performed using the PCR-based strategy with
the ODA-LA PCR kit from Takara Shuzo. The sequence
was confirmed by Sanger’s method using a DSQ-2000 L
automatic sequencer (Shimadzu Co.). Ec DOS-PAS mutant
proteins were expressed in BL21 cells. Purified full-
length Ec DOS and isolated PAS domain were more
than 95% homogeneous, as confirmed by SDS/PAGE.
Molar absorption coefficients of the wild-type Fe(III) and

Fe(II) complexes were 129 cm
)1
Æm
M
)1
(at 416 nm) and
175 cm
)1
Æm
M
)1
(at 427 nm), respectively, as determined by
the pyridine–hemochromogen method [18,19]. The coeffi-
cients of the Met95Ala (at 414 nm), Met95Leu (at 414 nm)
and Met95His (at 415 nm) mutant Fe(III) complexes were
124, 124 and 126 cm
)1
Æm
M
)1
, respectively.
Optical absorption, MCD, CD and fluorescence spectra
Optical absorption experiments were performed on Shim-
adzu UV-1650, UV-2500 and Hitachi U-2010 spectropho-
tometers maintained at 25 °C by a temperature controller.
MCD spectra were obtained on a Jasco J-500 spectro-
polarimeter equipped with an electromagnet which produces
a longitudinal magnetic field in the sample (up to 1.53 T).
CD spectra were obtained with Jasco J-720 and Jasco
J-500 CD spectropolarimeters. Fluorescence spectra were

obtained using a Shimadzu RF-5300PC spectrofluorimeter.
To ensure the appropriate temperature of the solution, the
reaction mixture was equilibrated for 10 min before each
spectroscopic measurement.
Redox potentials
Anaerobic spectral experiments were performed on a
Shimadzu UV-160A spectrophotometer in a glove box.
Redox potentials were obtained on the same spectro-
meter in the glove box. 2,3,5,6-Tetramethylphenylenedi-
amine (10 l
M
), N-ethylphenazonium ethosulfate (10 l
M
)
and 2-hydroxy-1,4-naphthoquinone (10 l
M
) were added as
mediators to the wild-type protein solution before titration
[15]. For Met95Ala and Met95Leu mutants, the dye
concentrations were changed to 5 l
M
. For the Met95His
mutant, 5 l
M
anthraquinone-2,6-disulfonate was added to
the solution. The heme protein concentration used was
15 l
M
. Spectral changes in intensity at 563 nm for wild-
type, 560 nm for Met95Ala, 557 nm for Met95Leu and

563 nm for Met95His accompanying the redox change were
monitored, as dye absorption hampers the detection of
Soret spectral changes. To ensure that the appropriate
temperature of the solution was maintained, the reaction
mixture was incubated for 10 min before spectroscopic
measurements. For reduction titration, the sample was
initially fully oxidized with potassium ferricyanide. After
each addition of sodium dithionite and allowing equilibrium
and stabilization, spectra were recorded. Titration experi-
ments were repeated at least three times for each complex.
Enzymatic assays
PDE activities were measured using a fluorescence band
of the product, as described previously [1,19,20]. Briefly,
Ec DOSwasincubatedat37°Cwithant-cAMPina
500 lL reaction mixture comprising 50 m
M
Tris/HCl buffer
(pH 8.5), 2 m
M
MgCl
2
and 1 m
M
dithiothreitol. To
terminate the reaction, Ec DOS was removed using an
Ultrafree-MC centrifugal filter (Millipore Co., Bedford,
MA, USA). Then 2 U alkaline phosphatase was added to
the mixture and incubated for 1 h at 37 °C. Next, the
reaction mixture was applied to a DEAE-Sephadex column
and washed with water. Finally, the fluorescence intensity

(excitation at 330–350 nm, emission at 410 nm) of the
eluted fraction was measured to determine the amount of
product [1]. At least four experiments were conducted to
obtain each value. Experimental errors were less than 20%.
Theenzymewasreducedwith10m
M
sodium dithionite.
Excess sodium dithionite was removed using a gel-filtration
column (Sephadex G25) in a glove box under nitrogen
atmosphere with an O
2
concentration of less than 50 p.p.m.
[18,19]. The reaction of the Ec DOS Fe(II) complex was
performed under anaerobic conditions in the glove box.
Results
Optical absorption spectra
Optical absorption spectra (350–650 nm) of the Fe(III),
Fe(II), and Fe(II)–CO complexes of wild-type and Met95
mutants of the isolated PAS domain are shown in Fig. 1
(lower), and summarized in Table 1. The absorption
spectrum (peaks at 416, 530 and 564 nm) of the Fe(III)
complex of wild-type Ec DOSisthatofatypicalsix-
coordinated low-spin complex. This spectrum closely
resembles that of cytochrome b
562
(peaks at 418, 530 and
564 nm), but shows less similarity to the spectra of other six-
coordinated low-spin heme proteins, including cytochrome
c and cytochrome b
5

[20–24] (Table 1). To identify the axial
ligand trans to His77, we generated Met95Ala, Met95Leu
and Met95His mutants. Soret and visible absorption peaks
of the Fe(III) Met95 mutant proteins disclosed six-coordi-
nated low-spin complexes essentially similar to that of the
4772 S. Hirata et al.(Eur. J. Biochem. 270) Ó FEBS 2003
wild-type enzyme (Fig. 1, Table 1). Notably, denaturation
of the Met95 mutants occurred occasionally at high
temperatures during expression or as a consequence of
auto-oxidation of the O
2
-bound Fe(II) complexes during
purification, often resulting in five-coordinated Fe(III) high-
spin complexes.
The close similarities between the absorption spectra of
wild-type Ec DOS and cytochrome b
562
are also evident in
the Fe(II) complexes (Table 1). Absorption spectra of Fe(II)
complexes of the Met95Ala and Met95Leu mutants were
characteristic of five-coordinated high-spin complexes sim-
ilar to Fe(II) myoglobin, whereas the spectrum of the Fe(II)
complex of the Met95His mutant was characteristic of a
six-coordinated low-spin complex, analogous to the Fe(II)
complex of the wild-type enzyme. Absorption spectra of
Fe(II)–CO complexes of the Met95 mutants were compar-
able to that of the wild-type enzyme and very similar to the
spectrum of the corresponding Fe(II)–CO myoglobin
complex (Table 1). Spectra generated using mutants of
full-length Ec DOS were essentially identical with those of

the isolated PAS domain.
To identify axial ligand(s), the effect of modulating pH on
absorption bands was examined using the isolated PAS
domain, as holoenzymes are easily precipitated when pH is
varied. Between pH 3.5 and 9, no spectral changes were
observed for either the wild-type or Met95His mutant PAS
domain. However, Met95Ala and Met95Leu mutants form
six-coordinated high-spin complexes between pH 4 and 7.5.
The Met95Leu mutant is likely to have a pK
a
at  4.6 (data
not shown), but partial denaturation occurs concomitantly
with this spin-state shift in the acidic region. The Met95Ala
mutant was easily denatured when pH was varied below 6,
andnoclearpKa value was obtained.
MCD spectra
Figure 1 (upper) displays the MCD spectra of Fe(III),
Fe(II) and Fe(II)–CO complexes of wild-type (A),
Met95Ala (B), Met95Leu (C) and Met95His (D) mutants
of Ec DOS. MCD positions and intensities are summarized
in Table 2. The MCD spectrum of the Fe(III) complex of
the wild-type enzyme contained a peak at 412 nm and a
trough at 426 nm in the Soret region, and a small peak at
554 nm and a small trough at 569 nm in the visible
region (solid line in Fig. 1A). The MCD spectral contour of
the wild-type enzyme Fe(III) complex is typical of a
Fig. 1. Optical absorption (lower panel) Soret
and visible MCD (upper panel) spectra for the
Fe(III) (—), Fe(II) (- - -), and Fe(II)-CO (
…

)
complexes of wild-type (A), Met95Ala (B),
Met95Leu (C) and Met95His (D) mutants of
isolated heme-bound PAS domain. MCD
spectra of the wild-type and Met95 mutants of
the full-length enzyme were similar to those of
the isolated heme-bound PAS domain. Spec-
tra were obtained in 0.1
M
Tris/HCl (pH 8.0)
buffer. Molar absorption coefficients deter-
mined are described in Experimental pro-
cedures.
Ó FEBS 2003 Met95 mutants of heme-regulated phosphodiesterase (Eur. J. Biochem. 270) 4773
six-coordinated low-spin complex. The intensity (De/T)
of the Soret trough band  80
M
)1
Æcm
)1
ÆT
)1
is also typical
of a low-spin complex [11]. Interestingly, the Met95Ala,
Met95Leu and Met95His mutants of Ec DOS displayed
similar MCD spectra (solid lines in Fig. 1B–D) to the wild-
type enzyme, although a specific decrease in Soret MCD
intensity was observed for the Met95Ala and Met95Leu
mutants (Table 2). Note that denaturation occurring during
the purification procedures often generated five-coordinated

high-spin Fe(III) complexes for the Met95 mutants, inclu-
ding Met95Phe (not shown).
The Fe(II) complex of the wild-type enzyme had a peak at
 430 nm in the Soret region at a comparable intensity to
the Fe(III) complex (Fig. 1A, broken line). MCD intensities
recorded in the visible region for the wild-type enzyme are
more than twofold higher than those in the Soret region,
unlike those of the Fe(III) complex. A similar MCD pattern
was observed for the Met95His mutant (Fig. 1D). However,
MCD band intensities in the visible spectra of the Met95Ala
and Met95Leu mutant proteins were much lower than those
in the Soret region. The position of the MCD peak (437 nm)
in the Soret region of Met95Ala, Met95Leu and Met95His
mutants was higher than that (430 nm) of the wild-type
protein. Moreover, the Soret MCD spectra of the wild-type
enzyme and the Met95His mutant started on the plus side
and switched to the minus side (from lower to higher
wavelengths), which was reversed for Met95Ala and
Met95Leu mutants. The MCD data indicate that the Fe(II)
complexes of the wild-type and Met95His mutant are
characteristic of spectra of the low-spin complexes in that
intensities of the visible MCD bands are much higher than
those of the Soret MCD bands [10,11]. On the other hand,
the Met95Ala and Met95Leu mutant complexes are high-
spin, as the MCD intensities on the plus side in the Soret
region are much higher than those in the visible region
[10,11].
The Soret MCD peaks of the Fe(II)–CO complexes of the
wild-type and Met95 mutants were located at  420 or
421 nm, with troughs at  430 or 431 nm (Fig. 1 and

Table 2, dotted lines). The MCD peaks and troughs in the
visible region of these proteins were observed at  562–
564 nm and 579–580 nm, respectively. However, the MCD
spectra of the Fe(II)–CO complexes of the wild-type and
Met95His mutant proteins differed from those of the
Met95Ala and Met95Leu mutants. Specifically, the Soret
MCD intensities of the Fe(II)–CO complexes of the
Met95Ala and Met95Leu mutants were 20–30% higher
than those of the Fe(II)–CO complexes of the wild-type
enzyme and Met95His mutant (Table 2).
Soret CD spectra
The a-helix and b-sheet contents of the full-length enzyme
andisolatedPASdomainweredeterminedfromtheUV
region of the CD spectrum in a previous study by our group
[1]. Neither reduction of the heme iron nor Met95 mutations
altered the CD spectrum in this region (data not shown).
Soret CD bands of the proteins under various conditions are
shown in Fig. 2 and summarized in Table 3. The Soret CD
band of the Fe(III) wild-type complex was presented at
 421 nm on the plus side with De ¼ 26.2
M
)1
Æcm
)1
(solid
line in Fig. 2A). Reduction with sodium dithionite led to a
shift in the band position to 431 nm and a slight increase in
CD intensity (up to De ¼ 32.3
M
)1

Æcm
)1
: broken line in
Table 1. Optical absorption maxima (nm) of the wild-type and Met95 mutants of isolated Ec DOS PAS domain. Putative coordination structures are
specified in parentheses. Optical absorption wavelengths of full-length enzymes were similar to those of the isolated PAS domain. SwMb, Sperm
whale myoglobin.
Proteins Fe(III) Fe(II) Fe(II)–CO Reference
Wild-type 416, 530, 564
(6c-LS)
427, 532, 563
(6c-LS)
423, 540, 570
(6c-LS)
This work
Met95Ala 414, 534, 563
(6c-LS)
432, 560
(5c-HS)
423, 541, 571
(6c-LS)
This work
Met95Leu 414, 533, 561
(6c-LS)
434, 558
(5c-HS)
423, 541, 570
(6c-LS)
This work
Met95His 415, 533, 564
(6c-LS)

427, 532, 561
(6c-LS)
423, 541, 570
(6c-LS)
This work
His, Met: axial ligands (6c-LS)
Cyt c 408, 530, 695 413, 521, 550 [20]
Cyt c
2
416, 550 [21]
Cyt c
551
410, 520, 551 416, 520, 551 [22]
Cyt b
562
418, 530, 564 427, 531, 562 [23]
His, His: axial ligands (6c-LS)
Cyt c
7
408 418, 522, 552 [24]
Cyt b
5
413, 540 (br) 423, 526, 556 [23]
His: axial ligand
SwMb
(H
2
O) 410, 505, 635
(6c-HS)
434, 556

(5c-HS)
423, 542, 579
(6c-LS)
[8]
(OH
–
) 414, 542, 582
(6c-LS)
[8]
4774 S. Hirata et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 2A). The Fe(II)–CO complex displayed a Soret band
at  425 nm on the plus side with high intensity
(De ¼ 58.5
M
)1
Æcm
)1
: dotted line in Fig. 2A). Soret CD
bands of the Fe(III) complexes of all the Met95 mutants
were observed at  414 nm, in contrast with that of
the wild-type enzyme complex, which was at  421 nm.
The distinct Soret CD positions of the Fe(III) complexes of
the wild-type enzyme and the Met95 mutants is important,
in view of the fact that the optical absorption, MCD, and
resonance Raman spectra of the wild-type and mutant
proteins are essentially similar [1,2]. The Soret CD bands of
the Fe(II) Met95Ala and Met95Leu mutants were presented
at  435–437 nm, at a slightly higher position than those of
the wild-type enzyme and Met95His mutant (430–431 nm).
Redox potentials

Electrochemical titrations were conducted for the Met95
mutants of the isolated PAS domain, as full-length enzymes
precipitate in the presence of mediators. The one-electron
midpoint potential of the Ec DOS heme was determined
from the absorbance change at 560 nm (Fig. 3). Redox
potentials obtained for the Met95 mutants ()1to)122 mV
vs. SHE) summarized in Table 4 are much lower than that
of the wild-type enzyme (+70 mV). Oxidative titrations
were also conducted for the Met95 mutant proteins to
determine whether a redox-dependent ligand switch occurs.
No significant differences were observed between the mid-
point potentials of reductive and oxidative titrations.
Interestingly, wild-type Ec DOS has a positive potential,
similartohemeproteinsinvolvedinelectrontransferorO
2
storage, while Met95 mutants displayed lower, negative
potentials similar to the enzymes involved in O
2
or H
2
O
2
activation, such as cytochromes P450 and peroxidases
(Table 4).
Table 2. MCD spectra of wild-type and Met95 mutants of isolated
Ec DOS PAS domain. Intensities (indicated in parentheses) are
expressed as De/H (
M
)1
Æcm

)1
ÆT
)1
). MCD peaks and intensities of full-
length enzymes were similar to those of the isolated PAS domain.
Soret Visible
Peak Crossover Trough Peak Crossover Trough
Fe(III)
Wild-type 412
(78.9)
420 426
()61.4)
554
(12.3)
563 569
()9.8)
Met95Ala 410
(68.1)
418 425
()55.2)
556
(8.7)
567 580
()7.5)
Met95Leu 410
(65.0)
418 425
()48.9)
556
(8.9)

571 580
()10.2)
Met95His 410
(71.5)
417 425
()67.7)
5.54
(9.9)
563 571
()8.3)
Fe(II)
Wild-type 430
(70.9)
558
(176)
561 565
()179)
Met95Ala 437
(94.5)
Met95Leu 437
(109)
Met95His 436
(36.1)
555
(95.9)
559 563
()97.7)
Fe(II)–CO
Wild-type 420
(68.9)

427 431
()26.9)
564
(18.4)
571 580
()25.1)
Met95Ala 421
(88.6)
427 431
()31.7)
563
(21.8)
571 579
()24.9)
Met95Leu 420
(92.1)
426 430
()36.2)
563
(25.0)
570 579
()28.1)
Met95His 420
(73.6)
427 431
()27.4)
562
(19.2)
570 579
()22.6)

Fig. 2. Soret CD spectra of the Fe(III) (—),
Fe(II) (- - -), and Fe(II)-CO (
…
) complexes of
wild-type (A), Met95Ala (B), Met95Leu (C)
and Met95His (D) mutants of isolated heme-
bound PAS domain. CD spectra of wild-type
and Met95 mutants of the full-length enzyme
were similar to those of the isolated heme-bound
PAS domain. Concentrations are 7.6 l
M
for
wild-type, 5.8 l
M
for the Met95Ala mutant,
4.5 l
M
for the Met95Leu mutant, and 5.6 l
M
for the Met95His mutant in 0.1
M
Tris/HCl
(pH 8.0) buffer.
Ó FEBS 2003 Met95 mutants of heme-regulated phosphodiesterase (Eur. J. Biochem. 270) 4775
cAMP PDE activities
Wild-type Ec DOS displays PDE activity only in the Fe(II)
form [1]. PDE activities of Fe(II) Met95 mutants of the
holoenzyme were obtained under anaerobic conditions.
Interestingly, all Met95 mutant proteins displayed PDE
activities that were comparable to that of the wild-type

enzyme. In addition, the Fe(III) Met95 mutants exhibited
no PDE activity, similar to the wild-type enzyme. Our data
clearly demonstrate that Met95 is not essential for PDE
activity with cAMP.
Discussion
Optical absorption and MCD spectra
A comparison of the absorption spectra of Fe(III) and
Fe(II) Ec DOS proteins with those of the corresponding six-
coordinated low-spin complexes of other heme proteins
reveals very similar spectral patterns between Ec DOS
proteins and cytochrome b
562
(Table 1). Although cyto-
chrome b
562
contains His/Met axial ligands, the spectra are
distinct from those of other cytochromes with His/Met or
His/His ligation. Therefore, the coordination structure
and/or heme environment of Ec DOS appears similar to
that of cytochrome b
562
, if the axial ligands of the two heme
proteins are the same.
Our previous data indicate that His77 is one of the axial
ligands of the heme in Ec DOS [1,2]. Amino-acid sequences
and crystal structures of PAS proteins indicate that the axial
ligand trans to His77 is possibly Met95 [6,7,31]. Construc-
tion of the distal heme site of Ec DOS by replacing the
Ec DOS sequences on the FixL backbone indicates that
Met95 is possibly the axial ligand trans to His77, based on

the finding that there are no other amino acid(s) in a suitable
position to fulfil this role, except for coordination of a water
molecule or a main chain amide. However, there remains
another possibility, that the water molecule or hydroxy
anionthatinteractswithMet95isanaxialligandtothe
Fe(III) heme.
The Fe(III) complexes of all three Met95 mutants
displayed the same coordination structure as the wild-type
enzyme (six-coordinated low-spin in terms of the Soret and
visible optical absorption and MCD spectra) (Tables 1 and
2). Resonance Raman spectra of the Met95Ala mutant in
addition disclosed that the Fe(III) form of this mutant is six-
coordinated low-spin [2]. If Met95 is an axial ligand to the
heme, the Fe(III) forms of the Met95Ala and Met95Leu
mutants should display five-coordinated high-spin states,
because Ala and Leu have nonpolar aliphatic side chains
and cannot coordinate to the heme. Changes dependent on
pH were observed in the absorption spectra of Met95Ala
and Met95Leu mutants, although the Met95Ala mutant
was denatured to some extent at acidic pH. It is possible that
ahydroxyaniontrans to His77 in the six-coordinated low-
spin complex is protonated, resulting in a five-coordinated
high-spin complex bound by His77 with the release of water.
Table 3. Soret CD spectra of wild-type and Met95 mutants of the iso-
lated Ec DOS PAS domain. Intensities are expressed as De (
M
)1
Æcm
)1
).

CD peaks and intensities of full-length enzymes were similar to those
of the isolated PAS domain. Note that CD intensities and band
positions for the Fe(II) and Fe(II)–CO complexes in [1] are incorrect.
Peak (nm) Intensity
Fe(III)
Wild-type 421 26.2
Met95Ala 414 28.3
Met95Leu 414 27.0
Met95His 414 27.6
Fe(II)
Wild-type 431 32.3
Met95Ala 437 30.1
Met95Leu 435 37.6
Met95His 430 28.8
Fe(II)–CO
Wild-type 425 58.5
Met95Ala 423 61.7
Met95Leu 423 70.7
Met95His 423 56.9
Fig. 3. Reductive titrations of the isolated Ec DOS PAS domain wild-
type (d), the Met95Leu (n), Met95Ala (h)andMet95His(s)mutants
from right to left. The fraction of the ferrous form was plotted as a
function of the potential. Solid lines represent theoretical Nernst curves
for the reduction of wild-type and Met95 mutants. Oxidative titrations
did not lead to any significant shifts.
Table 4. Redox potentials of wild-type and Met95 mutants of the
isolated Ec DOS PAS domain.
Protein mV vs. SHE n Reference
Wild-type +70 or +67 0.95 This work, [1]
Met95Leu )1 0.98 This work

Met95Ala )26 0.98 This work
Met95His )122 0.96 This work
Cytochrome c +260 [16]
Cytochrome b
562
+140 (pH 8.5) [23]
Myoglobin +59 or +46 [25–27]
Cytochrome b
5
+3 or +20 [23,28]
Cytochrome
P450cam
)170 (+ substrate) [27,29]
303 (– substrate) [27,29]
Horseradish
peroxidase
)220 or – 270 [26,27]
Microsomal
cytochrome P450
)310 [30]
4776 S. Hirata et al.(Eur. J. Biochem. 270) Ó FEBS 2003
However, the apparent pK
a
of 4.6 is unusually low for a
transition between an hydroxy anion and water. This
suggests significant structural differences in the heme
environments of the wild-type enzyme and the Met95Ala
and Met95Leu mutants. It is possible that an unknown
ligand, such as water/hydroxylate anion (or an amino-acid
main chain), coordinates to the Fe(III) in Met95Ala and

Met95Leu mutants to produce low-spin complexes. This
type of coordination may occur because of close proximity
of the water/hydroxyl to Met95 or flexibility of the
environment on the heme distal side.
For the Fe(II) complexes, optical absorption spectra of
the Met95 mutants are consistent with predictions from
the amino-acid sequences and crystal structures of the
PAS proteins. Specifically, the Met95Ala and Met95Leu
mutants form five-coordinated high-spin complexes,
whereas the complex of the Met95His mutant is six-
coordinated low-spin, similar to that of the wild-type
enzyme, indicating that His is the sixth axial ligand in the
Fe(II) Met95His complex.
Optical absorption spectra of the Fe(II)–CO complexes
of the wild-type and Met95 mutants are similar, indicating
the presence a common internal axial ligand, His77, as
expected from the resonance Raman spectra of the proteins
[2].
The spin and electronic states determined from the MCD
spectra of the Met95 mutants analyzed in this study are
consistent with those obtained from the optical absorption
spectra. The MCD spectra of the Met95 mutants indicate
that substitution with Ala, Leu and His did not alter the
spin state of the Fe(III) complex, whereas Met95Ala and
Met95Leu mutations changed the spin state of the Fe(II)
complex from low spin to high spin. However, the Soret
MCD intensities of the Fe(II)–CO complexes of the
Met95Ala and Met95Leu mutants were higher than those
of the wild-type enzyme and Met95His mutant. It appears
that ligand displacement accompanies the Met95 mutations,

andMet95maybethesixthaxialligandintheFe(II)
complex. Therefore, it suggests that redox-dependent axial
ligand exchange occurs, as indicated by resonance Raman
spectroscopy [2].
Soret CD spectra
The Soret CD band of the Fe(III) form of Ec DOS is
located exclusively on the plus side. This is distinct from
cytochrome b
562
[32] and cytochrome c [33] complexes,
which display bands on the minus side and both sides,
respectively. The Soret CD band of Ec DOS shifted from
421 nm to 425 nm and the intensity increased on reduction,
suggesting a change in the heme environment. The origin of
the Soret CD band of the heme protein may be due to
interactions of the p to p* transition of nearby aromatic side
chains with the delocalized p-electron system of the heme
prosthetic group and/or the unique conformation of the
polypeptide surrounding the heme [13,33–35]. In fact, Phe
mutations markedly altered the Soret CD band shape of
cytochrome c [33]. Importantly, the Soret CD positions of
all the Fe(III) Met95 mutants are 7 nm lower in wavelength
than that of the wild-type enzyme. The Soret CD spectra of
the Fe(III) Met95 mutants thus indicate that substitutions at
this position result in similar and profound conformation
changes near the heme prosthetic group in this particular
oxidation state. Therefore, it is reasonable to speculate that
the Met95 mutations affect the 6th ligand (water/hydroxy
ion) of the Fe(III) complex at the heme distal side.
From the Soret CD bands of the Fe(II) complexes, it

appears that the heme environment structures of the Fe(II)
wild-type enzyme and the Met95His mutant are distinct
from those of the Fe(II) Met95Ala and Met95Leu mutants.
These observations appear consistent with the optical
absorption, MCD and resonance Raman spectra, which
suggest that the Fe(II) wild-type and Met95His mutant are
in the low-spin state, whereas the Fe(II) Met95Ala and
Met95Leu mutants are high-spin [1,2]. The Soret CD band
positions and intensities of the Fe(II)–CO complexes of the
wild-type and Met95 mutants are similar.
Redox potentials
If a redox-dependent ligand switch occurs, the value for the
oxidative titration would be expected to be different from
that for the reductive titration. However, no such difference
was observed in the two directions. However, if a redox-
dependent ligand switch occurs and one of the axial ligands
in either redox state is a water molecule or hydroxy anion, it
is possible that the values for the two directions are similar,
because the ligand switching does not need high energy.
The redox potential of the wild-type protein
was +70 mV vs. SHE, which is within the range of
electron-transfer hemoproteins, cytochromes, and myoglo-
bin (Table 4). On mutation of Met95 to Ala, Leu and His,
the redox potential was markedly decreased in all the
mutants studied (Table 4), suggesting that the mutations
stabilize the Fe(III) form rather than the Fe(II) form.
Optical absorption and MCD spectral data are consistent
with the theory that Met coordination favours the Fe(II)
form, whereas His coordination favors the Fe(III) form.
Again, it is implied that Met95 is not an axial ligand in the

Fe(III) complex. The redox potentials of the Met95 mutants
are negative, similar to cytochrome P450 enzymes and
peroxidases. On the other hand, redox potentials of
cytochromes and myoglobin are positive (Table 4). There-
fore, Met95 of Ec DOS appears to be an important redox
potential control residue, preventing the enzyme from
exhibiting peroxidase-like and/or P450-like behavior and
avoiding activation of molecular oxygen or H
2
O
2
under
aerobic conditions.
Catalytic activities
All Met95 mutants are active only when the heme iron is in
the Fe(II) state, similar to the wild-type Ec DOS enzyme [1].
The Fe(II) complexes of Met95Ala and Met95Leu mutants
are five-coordinated high-spin, in contrast with the Fe(II)
form of the wild-type enzyme, which is six-coordinated low-
spin. From the spectra, it appears that the axial ligand trans
to His77 is Met95 in the Fe(II) form [2]. Therefore, the
changes in the Fe(II) heme coordination structure and
electronic state caused by the Met95 mutations did not
greatly influence the PDE activity of Ec DOS. These
structural changes are induced on the heme distal side,
where CO and O
2
are expected to bind. Ec DOS activity is
sensitive to the heme oxidation state. Accordingly, we
Ó FEBS 2003 Met95 mutants of heme-regulated phosphodiesterase (Eur. J. Biochem. 270) 4777

speculate that the heme proximal site structure (including
the Fe-His N distance and/or conformation), which alters
depending on the oxidation state, is critical for regulating
the PDE activity of Ec DOS. Detailed structural studies on
the environment near the heme are required to explain the
redox-sensitive PDE activity of this enzyme.
Crystal structure
After we submitted this manuscript, crystal structures of the
isolated heme-bound PAS domain of the wild-type Ec DOS
protein (resolution 1.3 A
˚
, R ¼ 0.16) were determined in this
laboratory (H. Kurokawa, unpublished results). The struc-
tures indicate that the axial ligands of the Fe(III) complex
are His77 and water, whereas those of the Fe(II) complex
are His77 and Met95. The redox-dependent ligand switch
suggested from the solution and crystallographic studies of
the enzyme agree well with each other, and this fact
increases the value of both approaches. Thus, artifacts
resulting from amino-acid substitution, experimental con-
ditions, data misinterpretation or the crystallization process
can be ruled out in the 3D structure determination work.
Acknowledgements
We thank Dr Hirofumi Kurokawa for valuable suggestions.
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