From the Department of Biochemistry, School of Medicine, Keio University, Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
From the Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During the monooxygenase reaction catalyzed by
cytochrome P450cam (P450cam), a ternary
complex of P450cam, reduced putidaredoxin, and
d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron
of P450cam in the intermediate complex, the EPR spectrum of
reduced putidaredoxin with a characteristic signal at 346 millitesla at
77 K changed into a spectrum having a new signal at 348 millitesla. The
experiment with O2 was carried out by employing a mutant
P450cam with Asp251 Cytochrome P450cam
(P450cam)1
catalyzes the conversion of d-camphor to
5-exo-hydroxycamphor at the expense of 1 mol each of NADH
and dioxygen (1). In the reaction, two reducing equivalents from NADH
are sequentially transferred to P450cam via two
redox-linked proteins, putidaredoxin reductase (PdR), a FAD-containing
enzyme, and putidaredoxin (Pd), a 2Fe-2S protein, where Pd receives an electron from PdR and transfers it to P450cam. In the first
electron transfer, reduced Pd combines with ferric
d-camphor-bound P450cam and reduces it to the
ferrous form. In the second electron transfer, reduced Pd forms a
quaternary complex with the ferrous dioxygen complex of
d-camphor-bound P450cam to decompose it into the
reaction products, i.e. hydroxycamphor, water,
d-camphor-free ferric cytochrome P450, and oxidized Pd (2,
3).
The role of Pd described above is not replaceable by the other electron
donors. Low potential iron-sulfur protein such as spinach ferredoxin
and bovine adrenodoxin can donate the first electron to
P450cam but not the second electron, whereas reduced rubredoxin and cytochrome b5, which are
incapable of giving the first electron, can provide the second electron
yielding the reaction products (4). Much effort has been made therefore
to understand the interaction between P450cam and Pd;
results of UV-visible, electron paramagnetic resonance (EPR), and
recent resonance Raman studies indicated that high spin ferric
P450cam in the presence of d-camphor was
partially converted to a low spin form upon the binding of oxidized Pd
(5-7). Associated with this spin conversion, the heme axial ligand
stretching mode On the other hand, only a few reports have been found in the literature
on the structural changes of Pd upon binding to P450cam. Sligar and Gunsalus (10) reported an elevation of redox potential upon
binding to P450cam by 10 mV, whereas Pochapsky et
al. (11) noted two-dimensional NMR signal changes of some amino
acid residues in Pd upon P450cam binding. In this study, we
examined the effects of P450cam binding to the structure of
Pd with the aid of EPR spectroscopy. Results revealed that the binding
of reduced Pd with ferrous d-camphor-bound
P450cam induced an EPR-detectable conformational change in
the iron-sulfur cluster of reduced Pd. Furthermore, a change in the
spin state of heme iron by ligand binding to P450cam in the
same complex caused a distinct and even larger conformational change in
Pd than those that occurred in the complex formation. Thus, the
structural change in the active site of P450cam occurred as
the consequence of the altered spin state is transmitted to the redox
center of another component protein in the intermediate complex
presumably through pathway(s) including particular surface amino acid residues.
Growth of Bacteria--
Pseudomonas putida cells
(strain ATCC17453) were grown on d-camphor as a sole carbon
source as described elsewhere (12). The cells harvested by
centrifugation were washed twice with 50 mM potassium
phosphate, pH 7.4, containing 50 mM KCl and 1 mM d-camphor (buffer A), suspended in the same
buffer, and stored at Enzyme Preparations and Mutagenesis--
The P450cam
and Pd genes were mutated by employing an oligonucleotide-directed
mutagenesis system according to the manufacturer's protocol (Takara
Biomedical, Kyoto, Japan). The wild-type P450cam and its
mutants expressed in E. coli were purified with the
procedures described previously (13). Purified preparations with an
RZ value
(A392/A280) greater than
1.5 were employed in this study. Pd, its mutant protein, and PdR were
purified from E. coli to a homogeneity on SDS-polyacrylamide
gel electrophoresis according to the methods of Gunsalus and Wagner
(14). Chemicals used were of analytical reagent grade and were used
without further purification.
EPR Spectroscopy--
A 200-µl solution containing either Pd
or Pd plus P450cam in buffer A was transferred into a
screw-topped EPR tube. After screwing a cap with rubber septum into the
tube, the protein was degassed by repeating three cycles of evacuation
and subsequent flushing with oxygen-free N2 gas. Then
proteins were reduced with a trace amount of solid sodium dithionite,
which had been placed on the inside of the tube, and then allowed to
stand on ice for 5 min to complete the reduction. The mixture was
frozen by immersing the tube into liquid nitrogen, which usually took
15-30 s. When necessary, CO or NO gas was anaerobically introduced to
the tube containing reduced Pd and ferrous P450cam. When
O2 was introduced in place of CO and NO, the EPR tube was
rapidly immersed into liquid nitrogen to freeze the protein as quickly
as possible after the mixing of the protein and O2; the
procedure was completed within 3 s.
EPR measurements were carried out on a Varian E-12 EPR spectrometer
(San Fernando, CA) at 9.363 GHz of X-band microwave frequency under the
following instrumental parameters: microwave power, 5 milliwatts;
modulation frequency, 100 kHz; and modulation amplitude, 0.5 mT. An
immersion Dewar flask was used for the measurements at liquid nitrogen
temperature (77 K). The microwave frequency and magnetic field of the
instruments were calibrated by a microwave frequency counter (Takeda
Riken, model TR5212) using an Mn2+ signal in MgO as a
standard. Accuracy of the g values was approximately ±0.001. Other
details were described under appropriate figure legends.
Effects of Carbon Monoxide on the EPR Spectrum of the
P450cam-Putidaredoxin Complex--
EPR spectrum of reduced
Pd was measured in the presence and absence of camphor-bound ferrous
P450cam and CO at 77 K (Fig. 1). The spectrum of reduced Pd in its
free state has g
The addition of CO to the 1:1 complex resulted in further spectral
changes, particularly in the g
In Fig. 3, Pd was titrated with an
increasing amount of ferrous P450cam in the presence of
excess CO. With an increment of P450cam, the intensity of
difference spectra recorded against reduced Pd increased following a
set of isosbestic points. Further spectral change was not observed upon
addition of over an equimolar amount of P450cam, indicating
that the observed spectral changes are because of the formation of a
1:1 complex with CO-ferrous P450cam. The CO-induced
spectral changes of reduced Pd were found to be reversible; the
original spectrum was restored after an extensive evacuation of CO
under an illumination of light (data not shown). Thus, the binding of
CO to the heme in P450cam altered the EPR spectrum of
reduced Pd in its binary complex with camphor-bound ferrous
P450cam, suggesting that Pd senses the spin state changes of P450cam in the complex.
The effect of CO on the EPR spectrum was also observed in
situ in P. putida cells where PdR, Pd, and
P450cam were contained in a 1:8:8 ratio (16). The cells,
which were reduced with sodium dithionite, showed an EPR spectrum of
reduced Pd complexed with ferrous P450cam together with an
unidentified signal around g Effects of Nitric Oxide on the EPR Spectrum of the
P450cam-Putidaredoxin Complex--
Next we examined the
effects of NO binding to the heme of P450cam in the complex
of Pd with camphor-bound ferrous P450cam. In Fig.
5, EPR spectra of reduced Pd, NO-ferrous
P450cam, and reduced Pd in the presence of NO-ferrous
P450cam are compared. The g Effects of KCl and Putidaredoxin Reductase on the Ligand-induced
Spectral Change--
The present findings suggested that reduced Pd
sensed the binding of ligands such as CO and NO to the cytochrome P450
heme when Pd forms a 1:1 complex with d-camphor-bound
ferrous P450cam. The requirement for the complex formation
in this phenomenon was tested by the addition of reagents that promote
the dissociation of a protein-protein complex. The
Pd-P450cam complex is known to be stabilized by ion pairs
between acidic and basic residues of Pd and P450cam,
respectively (11, 18-20). When KCl concentration in the buffer (50 mM potassium phosphate, pH 7.4, supplemented with 50 mM KCl and 1 mM d-camphor) was
raised to 1 M, the ligand-induced spectral change was found
to decrease to approximately 40% of that in the original buffer (Fig.
6, B). In this experiment, the titration data shown in Fig. 3 were used as the calibration curve.
It has been suggested by Holden et al. (19) that the binding
site of P450cam in Pd is overlapped with that of PdR based on the observation that mutations of some amino acid residues in Pd
altered both interactions of Pd with P450cam and PdR. Thus, the Pd-P450cam complex is expected to dissociate by an
excess amount of PdR. Addition of a 5 M excess of PdR over
the complex made the CO-induced spectral change decrease by about 60%
(Fig. 6, C), giving additional evidence for a competitive
binding of PdR to Pd with P450cam as described previously
(19). It is of note that the addition of PdR to reduced Pd did not show
a detectable spectral change of the latter. The significance of a small
peak around the g Effects of Oxygen Binding on the P450cam-Putidaredoxin
Complex--
A complex of reduced Pd with oxyferrous
P450cam is not stable and hence does not allow its EPR
measurements under ordinary conditions; oxygen is a substrate for the
P450cam-catalyzed reaction. One way to see the effects of
dioxygen coordination to ferrous P450cam on the spectra of
reduced Pd is to employ a mutant P450cam, which forms a
stable complex. A mutant of P450cam with Asn, Ala, or Gly
substituted for Asp251 may be such a mutant; the oxyferrous
form of these mutants readily forms the complex with reduced Pd, but
its decay into reaction products is 500-1000-fold smaller than that of
the wild-type P450cam (21, 22). Then the effects of
dioxygen on Pd were tested in the complex of Pd and the mutant
P450cam. Fig. 7 shows the EPR spectra of reduced Pd in its complex with the two mutants of
P450cam before and after oxygenation. The complex was
frozen immediately after exposure to O2 around 0 °C. As
seen, oxygenation changed the spectrum of reduced Pd in the
g Essential Amino Acid Residues for the Ligand-induced Spectral
Changes--
To elucidate the mechanism(s) by which CO, NO, and
O2 bind to the heme in P450cam and to induce
the structural changes of Pd in the P450cam-Pd complex, we
tested the effects of mutations of Arg79,
Arg109, and Arg112 as well as those of
Asp251 and Thr252 in P450cam on the
EPR spectral changes. The former 3 amino acid residues are in the
putative binding site of P450cam for Pd (11), whereas the
latter two locate in the distal region of the active center of
P450cam (23) and are essential for the monooxygenation reaction (13, 21, 24). A mutation of Ala252,
Asn251, or Gln79 gave similar results with
those obtained with the wild-type P450cam (data not shown),
whereas the mutant at positions 109 and 112 did not. Reduced Pd
complexed with the mutant with Gln109, Lys109,
Met112, or Lys112 did not show spectral changes
observed in the complex formation with the wild-type
P450cam (Fig. 8, dotted
line in spectrum A, B, C, or
D, respectively). Furthermore, upon the addition of CO to
the complex, a spectral change, which occurred with the wild-type P450cam, was not observed except in the Lys mutants; a
subtle change (indicated by arrows) was observed. In the
measurements with the mutants at positions 109 and 112, the
concentrations of P450cam were raised from 200 µM to 2 mM against 200 µM Pd. We did so because a mutation at a surface amino acid residue such as
Arg112 Substitution of Amino Acid Residues Around the 2Fe-2S Cluster of
Putidaredoxin--
In the next series of experiments, the structure of
Pd was perturbed by changing the amino acid residues that surrounded
the 2Fe-2S cluster using site-directed mutagenesis; Asp38,
Ser44, Thr47, and Cys85 were
replaced by Gly, Ala, or Val. These amino acids sit next to
Cys39, Cys45, Cys48, and
Cys86, which coordinate directly to Fe3+ atoms
in the iron-sulfur center of Pd. As seen in Fig.
9, every Pd mutant with a substitution at
Ser44 (spectra A and C) or
Thr47 (spectra E, F, and
G) showed a different EPR spectrum from that of the wild
type except for the Ala44 mutant (spectrum B),
the latter of which was indistinguishable from that of the wild type
(spectra D and H). Among them, the EPR spectrum
that mimicked well the spectrum of reduced Pd upon the ligand binding
to the heme of P450cam in the P450cam-Pd
complex (Fig. 1, C) was that of the Gly44
mutant; upon CO binding, the trough in the g It has been known that a ternary complex of ferrous
P450cam with d-camphor and reduced Pd is formed
as an obligatory intermediate of the reaction during the catalysis of
d-camphor monooxygenase (2, 3). With the aid of EPR
spectroscopy, we have shown in this study that a change in the spectrum
of reduced Pd occurs upon its binding to ferrous P450cam in
the presence of d-camphor, indicating that the formation of
the intermediate complex accompanies a structural change in the
iron-sulfur center of Pd. More interestingly, a further structural
change was observed in the reduced Pd upon binding of O2,
CO, or NO to the ferrous heme of P450cam in the intermediate complex; it was distinct from that induced by the binding
to P450cam. When O2 is the ligand, the
resulting complex of ferrous P450cam with
d-camphor, O2, and reduced Pd is another obligatory intermediate of the reaction, the degradation of which leads
to the formation of the final reaction products,
5-exo-hydroxycamphor, H2O, ferric
P450cam, and oxidized Pd. Such a structural change in Pd
induced by the heme ligand is unlikely to be an artificial observation
in vitro because we observed a similar spectral change upon
the binding of CO and NO to P450cam in situ in
P. putida (Fig. 4).
The structural changes in Pd induced by the binding of O2,
CO, and NO were indistinguishable from each other as judged from their
EPR spectra, and all these ligands are known to convert the spin state
of the heme iron from a high to a low spin state. Accordingly, the
change in the spin state of heme iron in the intermediate complex
appears to trigger a series of structural changes, which are
transmitted to Pd within the complex. Then questions arise as to the
nature of the structural change in Pd as well as to the mechanism(s)
for the signal transduction from the heme to the iron-sulfur redox center.
The EPR spectrum of Pd at g As to the altered structure of Pd, effects of amino acid substitution
in Pd on the EPR spectrum give us further information about it. As
described above, the trough position of the composite g The side chain of Arg112 of P450cam at the
putative binding site for Pd is oriented toward the inside of the
protein and forms a hydrogen bond with the propionyl side chain at the
heme periphery. The same propionyl is also hydrogen-bonded to
His355, the second amino acid residue from the endogenous
heme ligand Cys357. Accordingly, it is not surprising that
the Arg112 residue is involved in the sensing of a
structural change occurring at the heme and its vicinity. As stated
above, ligand binding to the ferrous camphor-bound P450cam
alters the spin state of a heme iron from high to low spin. This spin
change conceivably accompanies structural alteration of
P450cam at least in its active site, involving the movement
of ferrous iron from the out of plane position to the in plane position
as observed in myoglobin and hemoglobin (29, 30). Therefore we propose
that a structural change occurred at the heme and its vicinity in
P450cam, which upon liganding is transmitted to reduced Pd
via Arg112 as illustrated schematically in Fig.
10. On the other hand,
Arg109, which was also essential to the ligand-induced
structural change of Pd, has no direct bonding interaction with the
heme and hence may not have a primary role for the sensing. Rather,
this residue would be essential for a proper docking of Pd, thus
assisting a signal transduction via Arg112. It is possible
that the signaling pathway in the binary complex of Pd and
P450cam proposed here is also responsible for the electron transfer from the iron-sulfur cluster of Pd to the heme iron of P450cam. In any event, modulation of the Pd structure by
the conformational change in the P450cam active site
associated with the altered spin state of heme iron could be an
important factor in the electron and proton transfers in the reaction
intermediate and hence in the d-camphor monooxygenation
catalyzed by P450cam.
Asn or Gly where the
rate of electron transfer from putidaredoxin to oxyferrous
P450cam is considerably reduced. Such a ligand-induced EPR
spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the
neighborhood of the iron-sulfur cluster of putidaredoxin revealed that
a Ser44
Gly mutation mimicked the ligand-induced
spectral change of putidaredoxin. Arg109 and
Arg112, which are in the putative putidaredoxin binding
site of P450cam, were essential for the spectral changes of
putidaredoxin in the complex. These results indicate that a change in
the P450cam active site that is the consequence of an
altered spin state is transmitted to putidaredoxin within the ternary
complex and produces a conformational change of the 2Fe-2S active center.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Fe-S upshifts by ~3
cm
1 from 350.5 cm
1 (7). It was also found
that the 15N NMR resonance signal from the
15N-cyanide complex of ferric P450cam shifted
from 500 to 477 ppm upon association with oxidized Pd (8). With the
ferrous d-camphor-bound P450cam, the binding of
reduced Pd to P450cam produced a shift in
CO of the heme-bound CO from 1940 to 1932 cm
1 (9). However, the significance of such structural
change evoked by the binding of Pd in the reaction catalyzed by
P450cam is unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C until use. For the expression of
P450cam, Pd, and PdR, Escherichia coli strain
JM109 was grown in LB medium supplemented with ampicillin and
isopropylthio-
-D-galactoside as described previously
(13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
signal at 2.02 (331 mT) and
g
signal at 1.94 (345 mT) (Fig. 1, A) as
originally reported by Tsibris et al. (15). Upon the
addition of camphor-bound ferrous P450cam, Pd changed its
spectrum in the g
region (Fig. 1, B). The
trough of the derivative type signal was broadened with an appearance
of a shoulder around 348 mT. The spectral changes were more evident in
a difference spectrum presented in Fig.
2, a (spectrum B
minus A of Fig. 1). Titration of reduced Pd with ferrous
P450cam in the presence of d-camphor indicated
that changes in the spectrum of Pd were caused by the formation of a
1:1 complex of the two proteins (data not shown).
View larger version (15K):
[in a new window]
Fig. 1.
EPR spectra of reduced putidaredoxin in the
absence and presence of P450cam and CO. EPR spectra of
200 µM Pd were taken at 77 K in 50 mM
potassium phosphate, pH 7.4, containing 50 mM KCl and 1 mM d-camphor in its free state (A),
in the presence of 200 µM P450cam without CO
(B), and with excess CO (C).
View larger version (13K):
[in a new window]
Fig. 2.
EPR spectral changes of reduced putidaredoxin
by the addition of P450cam and P450cam plus
CO. Spectrum a (dotted line), spectrum
B minus A of Fig. 1; Spectrum b
(solid line), spectrum C minus B of
Fig. 1.
region as seen in Fig. 1,
C; the position of the trough at 346 mT moved to 348 mT with
a shoulder at 346 mT. The CO-induced spectral changes were also
demonstrated in a difference spectrum (spectrum C minus
B of Fig. 1) in Fig. 2, b, showing clearly that
the CO addition caused larger spectral changes in Pd than those that
occurred upon binding to P450cam. The two difference
spectra in Fig. 2 also indicated that spectral changes of Pd induced by
cytochrome P450 binding to Pd and those of CO binding are different
from each other in that the positions of the major trough and peak were
clearly distinct. As seen in Fig. 1, C, a minor change was also observed in the g
region by the addition of CO; a
small increase in the g value was observed. In the absence of
P450cam, exposure of reduced Pd to CO did not alter the
spectrum (data not shown).
View larger version (17K):
[in a new window]
Fig. 3.
EPR spectral changes of reduced putidaredoxin
by the addition of an increasing amount of ferrous P450cam
in the presence of excess CO. A, spectral changes of Pd
were presented as differences against the spectrum of reduced Pd
recorded in the absence of P450cam. 100, 150, 300, and 400 µM P450cam were added to 200 µM
Pd. B, the signal intensities of the difference spectra
(peaks at 346 mT minus troughs at 348 mT) were plotted against the
ratios of P450cam to Pd. Other details are in Fig. 1.
(333 mT) (Fig.
4, A). The g
signal arising from Pd changed on exposure of the cells to CO to an
almost identical one that is shown in Fig. 1, C (Fig. 4,
B).
View larger version (16K):
[in a new window]
Fig. 4.
EPR spectra of dithionite reduced P. putida cells in the absence (A) or presence
(B) of CO. The cells contained PdR, Pd, and
P450cam in a 1:8:8 ratio (16).
signal of Pd in
the complex with NO-ferrous P450cam (Fig. 5, C)
was indistinguishable from that of the CO-induced signal in Fig. 1,
C. Other signals, except for the g
of reduced Pd, were derived from NO-ferrous P450cam, whose spectrum is
presented in Fig. 5, B for comparison. Inspection of the
spectra of NO-ferrous P450cam and its complex with reduced
Pd also leads to the finding that the g = 2.075 signal of
NO-ferrous P450cam shifted to g = 2.070 upon complex
formation with Pd. Such a spectral change suggests an alteration of the
NO-heme geometry as had been observed for the structure of heme-bound
CO in CO-ferrous P450cam (9). NO is known to react with an
iron-sulfur protein yielding an EPR-active nitrosyl Fe3+
(17), but it was not the case here; reduced Pd did not exhibit any
spectral change upon exposure to NO, although an addition of NO to
oxidized Pd, which is EPR silent, was found to produce EPR signals
indicative of a nitrosyl Fe3+ formation (data not shown).
Thus, the aforementioned effects of NO on Pd associated with
P450cam suggested that NO binding to the heme iron of the
cytochrome was sensed by Pd as was found in CO binding. A similar
spectral change of Pd was obtained by in situ EPR
measurements of P. putida cells with NO.
View larger version (23K):
[in a new window]
Fig. 5.
EPR spectra of reduced putidaredoxin
(A), NO-ferrous P450cam
(B), and reduced putidaredoxin with NO-ferrous
P450cam (C). 200 µM
each of Pd and P450cam were used for the measurements.
Other details are in Fig. 1.
View larger version (13K):
[in a new window]
Fig. 6.
Effects of KCl and putidaredoxin reductase on
the CO-induced EPR spectral changes of putidaredoxin. Spectra of
200 µM each of Pd and P450cam were measured
without any additive (A), at 1 M KCl
(B), and in the presence of 5 M excess of PdR
(C). Other details are in Fig. 1.
signal at 333 mT (Fig. 6,
C) is unknown at present.
signal region; a new signal at 348 mT appeared to be
accompanied by the reduction of a trough at 346 mT to a shoulder of the
new signal. It also accompanied a slight shift in the g
signal to a lower magnetic field (Fig. 7, A and
B). These spectral characteristics were very similar to the
spectral changes of Pd on exposing the Pd-P450cam complex to CO or NO. As will be described below, the mutation at
Asp251 (per se) did not affect the CO- and
NO-induced EPR spectral changes of Pd (data not shown).
View larger version (14K):
[in a new window]
Fig. 7.
Effects of dioxygen on EPR spectra of reduced
putidaredoxin complexed with the Asn251
(A) and Gly251
mutants (B) of
P450cam. Spectra of dotted and solid
lines are before and after exposure to dioxygen, respectively.
Other details are in Fig. 1.
Lys has been known to lower the binding affinity
of P450cam to reduced Pd (20). The subtle changes observed
were not intensified by a further addition of the Lys109
and Lys112 mutant P450cam. The significance of
the subtle changes that occurred with the Lys mutants is unknown at
present, but these changes proved that a complex of P450cam
and Pd was in fact formed under the experimental conditions; otherwise
no effect of CO exposure on Pd spectrum could be observed. Thus the
lack of change in the EPR spectrum of Pd with the 112 and 109 mutants
is not because of their incapability to form the Pd-P450cam
complex but is because of the uncoupling of ligand binding to the
structural change in Pd.
View larger version (14K):
[in a new window]
Fig. 8.
EPR spectra of reduced putidaredoxin in the
presence of ferrous mutant P450cam before (dotted
line) and after (solid line) exposure to
CO. 2 mM Arg109 Gln (A),
Arg109
Lys (B), Arg112
Met
(C), and Arg112
Lys (D) mutants
of P450cam were added to 200 µM Pd. Other
details are in Fig. 1.
signal
shifted to a higher magnetic field, although essentially no change was observed in the g
signal region. Replacement of
Thr47 with Val also produced a similar spectrum to that
obtained upon the ligand binding to the heme in the
P450cam-Pd complex in the g
region, although
the g
signal moved to a higher frequency by 1 mT. On the
other hand, mutations at Asp38 or Cys85 showed
no change in the EPR spectrum (data not shown). It is of note that the
trough of the g
signal was sensitive to the size of the
amino acid residues incorporated into the position; the trough shifted
from a higher to a lower magnetic field as the volume of side chain
increased, Gly < Ala
Ser < Val (Fig. 9,
A-C). The EPR spectrum of reduced Pd was also
sensitive to the mutation at Thr47, but the changes were
apparently random as seen in Fig. 9, E-G.
View larger version (17K):
[in a new window]
Fig. 9.
EPR spectra of reduced putidaredoxin and its
mutants. The purified preparation of the wild-type Pd and its
mutants were reduced with sodium dithionite under anaerobic conditions.
Spectra A, B, and C are of Pd
substituted for Ser44 with Gly, Ala, and Val, respectively.
Spectra E, F, and G are of Pd
substituted for Thr47 with Gly, Ala, and Val, respectively.
Spectra D and H are of the wild-type Pd
illustrated as comparison. Other details are in Fig. 1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 1.94 is a composite of the
gx and gy signals (25, 26). The gy
signal has a derivative-type line shape and that of gx
appears as a trough. These two signals were observed very closely to
each other, thus being distinct from those of other 2Fe-2S proteins
such as spinach and parsley ferredoxins (25); in the latter case, the
derivative type gy signal at g = 1.96 is well
separated from the gx appearing as a trough at g = 1.89. A separation of the two signals in 2Fe-2S proteins becomes wider
as the rhombic character at ferrous iron is intensified, and Pd is
reported to be less rhombic than spinach and parsley ferredoxins (26).
If the ligand-induced EPR spectral change of reduced Pd in the
Pd-P450cam complex resulted from a splitting of the
gx signal from the composite, the structural change of Pd
described here is interpretable as an intensification of the distortion
at the ferrous iron. However, it is also of note that, by an increase
in line width of the gx signal without any change in the g
value, a trough can appear at a higher magnetic field in the
g
region as observed here without shifting the
derivative type signal centered at g = 1.94.
signal of reduced Pd is sensitive to a side chain volume of the 44th
residue of Pd; Gly with the smallest side chain made the signal shift
to a position similar to the ligand-induced signal. In the tertiary
structure of Pd elucidated by using NMR, the iron-sulfur redox center
locates closely to a protein surface (27) where Ser44, a
surface residue of Pd, was located at the boundary separating the redox
center from an aqueous phase. Accordingly, it has been considered that
the substitution for Ser with smaller Gly increases solvent access to
the cluster, because they are located at a position that can affect the
solvent access to the iron-sulfur cluster. Consideration of the effects
of the mutation on the EPR spectrum of reduced Pd leads to a hypothesis
that an association of reduced Pd with liganded camphor-bound ferrous
P450cam induces the structural alteration of Pd in a way to
increase the solvent accessibility of the iron-sulfur cluster.
Moreover, because Ser44 is in the vicinity of the ferrous
iron of reduced Pd (28), a change occurred at Ser44, which
altered the access of solvent to the iron-sulfur cluster, induces
distortion around the ferrous iron simultaneously.
View larger version (44K):
[in a new window]
Fig. 10.
Possible pathway(s) for transduction of a
ligand binding signal from the heme of P450cam to
putidaredoxin in their binary complex. Arg109 and
Arg112, which are in the putative Pd binding site of
P450cam, were essential for the signal transduction.
Arg112, which is hydrogen-bonded to the propionyl side
chain of the heme, senses the structural changes that occurred in the
redox center upon a change in spin state of heme iron via the heme
propionyl and/or via the peptide chain consisting of the heme ligand
Cys355, Leu356, and His357, the
last of which is hydrogen-bonded to the same propionyl side chain of
the heme as Arg112. Arg109 could not be a
primary site for the sensing, but rather it would assist a proper
docking with Pd. C39,
C45, C48, and
C86 denote the cysteine residues of Pd described
with one-letter symbols; they are the ligands for iron atoms in the
iron-sulfur center of Pd. For other details, see
"Discussion."
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Yoko Minowa for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japanese Government, by the Special Coordination Funds of the Science and Technology Agency of Japanese Government, and by grants from Keio University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Dept. of Biochemistry,
School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku,
Tokyo 160-8582, Japan. Tel.: 81-3-3353-1997; Fax: 81-3-3358-8138;
E-mail: shimada{at}med.keio.ac.jp.
§ Present address: Protein Research Inst., Osaka University, Yamadaoka, Suita, Osaka 565-8611, Japan.
¶ Present address: Inst. for Chemical Reaction Science, Tohoku University, Sendai 980-8557, Tohoku, Japan.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: P450cam, cytochrome P450 (CYP101) isolated from P. putida, which catalyzes the conversion of d-camphor to 5-exo-hydroxycamphor; Pd, putidaredoxin; PdR, putidaredoxin reductase; mT, millitesla.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|