Substrates modulate the rate-determining step for CO bindingin cytochrome P450cam CYP101 A high-pressure stopped-flow study 1 Max-Delbru¨ck-Center for Molecular Medicine, Protein Dynamic
Trang 1Substrates modulate the rate-determining step for CO binding
in cytochrome P450cam (CYP101)
A high-pressure stopped-flow study
1 Max-Delbru¨ck-Center for Molecular Medicine, Protein Dynamics Laboratory, Berlin, Germany;
2
Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 128, IFR24, Montpellier, France
The high-pressure stopped-flow technique is applied to study
the CO binding in cytochrome P450cam (P450cam) bound
with homologous substrates (1R-camphor, camphane,
nor-camphor and norbornane) and in the substrate-free protein
P450cam bound with substrates that do not contain methyl
complexed with substrates carrying methyl groups show a
the compressibility and the influx rate of water for the heme pocket of the substrate complexes it is concluded that the positive activation volume is indicative for a loosely bound substrate that guarantees a high solvent accessibility for the heme pocket and a very compressible active site In addition, subconformers have been found for the substrate-free and camphane-bound protein which show different CO binding kinetics
Keywords: high-pressure stopped-flow; cytochrome P450;
CO ligand binding; protein dynamics
Cytochromes P450 represent a big superfamily of heme-type
monooxygenases that catalyze the conversion of diverse
substrates [1] Besides the main route of the reaction cycle
from the substrate to the product there are side reactions
which lead to the production of cytotoxic oxygen species
such as hydrogen peroxide or of water in the oxidase
reaction These so-called uncoupling processes have been
observed in many cytochrome P450 systems [2] However,
the structural parameters of the protein and the substrate
which are responsible for the uncoupling process are not
well understood Data are increasingly accumulated
indica-ting that the dynamics of the protein structure and in
particular the accessibility of the active site for water
molecules are very important [3] In the oxidized form of
P450 the high-spin/low-spin state equilibrium reflects a
time-averaged population of water molecules at the sixth
iron co-ordination site This equilibrium can be monitored
using the heme Soret band [4] However, for the
iron-reduced form there is no spectral signal that could be used
directly to monitor the water exchange An indirect method
is a water replacement technique using a probe molecule In
a large number of studies [4–9] using different approaches
we found that the CO iron ligand is a good probe for the
polarity and therefore for the presence of water molecules in the heme environment of cytochrome P450cam
To get a further insight into the dynamics of the water exchange process in different substrate P450 complexes we used the high-pressure stopped-flow technique [10,11] The activation volume as well as the rate constant for the CO on-reaction obtained from such studies should allow us to quantitate dynamic properties of the heme pocket when P450 complexed with homologous substrates is studied High-pressure flash photolysis studies on ferrous heme model complexes and heme proteins with imidazole as proximal ligand show that the sign of the activation volume for the overall on-reaction depends on the nature of the ligand indicating two main steps, the iron-ligand bond
was found that the overall activation volume for the CO ligand binding in heme proteins with histidine proximal ligand is always negative indicating that the bond formation
is the rate-limiting step Considering these results it was surprising that cytochromes P450 do not seem to show the same behaviour Lange et al [11] have determined the activation volumes for the CO binding in several cyto-chromes P450 in the absence of a substrate using the stopped-flow technique under high pressure It turned out that all the proteins which have a cysteine as proximal ligand have a small positive activation volume of (+1)–
in the sulfur ligand class proteins is structurally very close to the ground state and that the negatively charged sulfur from the cysteine ligand produces specific electronic properties which may be the origin for this behaviour However, flash photolysis studies under pressure for P450cam in the presence of various substrate analogues [13] indicate that even negative activation volumes are possible Due to the
Correspondence to C Jung, Max-Delbru¨ck-Center for Molecular
Medicine, Protein Dynamics Laboratory, Robert-Ro¨ssle-Strasse 10,
13125 Berlin, Germany.
Fax: + 49 30 94063329, Tel.: + 49 30 94063370,
E-mail: cjung@mdc-berlin.de
Abbreviations: P450, cytochrome P450; P450cam,
1R-camphor-hydroxylating P450 from Pseudomonas putida (CYP101); P420,
denatured and nonactive form of P450; TMCH,
3,3,5,5-tetramethylcyclohexanone; FTIR, Fourier transform infrared
(Received 28 January 2002, revised 9 April 2002, accepted 2 May 2002)
Trang 2fact that in all these substrate complexes the cysteine ligand
is the same, the specific electronic structure of the proximal
ligand cannot be the origin for the positive activation
volume observed for some substrate complexes To be sure
that this result is not only specific for CO rebinding induced
by flash photolysis we extended the high-pressure
stopped-flow study on P450cam by a homologous series of camphor
analogues (1R-camphor, camphane, norcamphor and
nor-bonane) and substrate-free protein These camphor
ana-logues lack characteristic groups which are relevant for a fit
of the substrate into the heme pocket (Fig 1) It will be
shown that the activation volume of the CO on-rate is
positive for P450cam bound with substrates which lack
methyl groups, are loosely bound, have a higher water influx
rate [3] and form a more compressible active site [7] In
addition, subconformers have been found for the
substrate-free and camphane-bound protein which show different CO
binding kinetics
M A T E R I A L S A N D M E T H O D S
Cytochrome P450cam from Pseudomonas putida expressed
in Escherichia coli TB1 was isolated and purified as
purified protein was 1.3 Substrate removal was performed
Sephadex G-25 (medium) gel chromatography and final
The concentrated substrate-free P450cam stock solution
aliquots of the P450cam stock solution were added 1R-camphor was from Sigma Camphane, norcamphor and norbornane were from Aldrich
Substrate analogues were added to the substrate-free protein as few microliters aliquot of an ethanolic stock solution Because the substrates have different dissociation constants [3] the substrate concentration was chosen such that substrate complex was completely formed The amount
Soret band spectrum of the oxidized protein using the fit procedure described earlier [4] The P450cam concentration
mixed equal volumes of an enzyme solution with the CO solution The buffer and substrate composition was the same in both volumes Both solutions were carefully deoxy-genated by purging with argon before the experiment, and the same amount of sodium dithionite was added to each syringe to have always a constant final dithionite
that P450 remained reduced during the stopped-flow experiment The CO containing solution was prepared by adding an appropriate volume of a CO saturated buffer stock solution to the syringe The CO stock buffer solution
Because the binding kinetics strongly differ for the different substrate complexes the final CO concentration has to be varied to stay in a time window which can be resolved by the stopped-flow-spectrometer All stopped-flow experiments
stopped-flow experiment the recovered protein solution was checked for possible P420 formation using the CO differ-ence spectrum There was no spectral differdiffer-ence to the solution at the beginning indicating that P420 was not formed during the high-pressure stopped-flow experiment The high-pressure stopped-flow apparatus used is inter-faced with the Aminco DW2 spectrometer and is described
in [10,11] All kinetic traces were recorded in the dual-wavelength mode of the Aminco using the dual-wavelength of
406 nm
We have previously found for P450s that the observed rate constant for the CO binding is linearly related to the
CO concentration indicating bimolecular binding kinetics
absorbance difference DA(t) were fitted with bimolecular kinetics as described recently [5] (Eqn 1)
concentration, the final P450-CO concentration, the initial
CO concentration, the extinction coefficient at 446 nm, and
indicates the first or second phase in case of two-phase kinetics (see below)
Fig 1 Structure of the active site of cytochrome P450cam and of
camphor analogues Top, heme and amino acids contacting the
sub-strate 1R-camphor, PDB accession no 3cpp; bottom, subsub-strate
ana-logues used in the high-pressure stopped-flow study.
"
P450
1
½P4500
½CO0
#
ð1Þ
Trang 3Three shots at each pressure were taken and fitted The
averaged values were used for further analysis to get
@P
T
#
@P
T
For the camphane complex and the substrate-free
complex two bimolecular processes were required to get a
reasonable fit indicating subconformer equilibrium We
used a linear combination as the simplest approximation
(Eqn 3) The fraction w for one phase is used to estimate the
equilibrium The reaction volume DV between the
subcon-former is calculated according to Eqn 2
R E S U L T S
Figure 2 shows the typical time traces obtained from the
stopped-flow measurements There is a delay time of 0.622 s
after the trigger signal was initiated and before the two
volumes with the enzyme and the CO are mixed At the
lowest absorbance the time is set to zero for fitting The data
for the different substrate complexes show that the binding
rate can decrease or increase with increasing pressure As an
example, Fig 2 demonstrates the results for 1R-camphor,
where the rate increases with pressure, and for norbornane,
where the rate decreases with pressure The bimolecular rate
constants given in Fig 2 are obtained by nonlinear
least-square fitting the time curves using a single bimolecular
process according to Eqn (1) Figure 3 shows the plot of the
logarithm of the rate constant vs the pressure which is linear
The activation volume, obtained from the slope of this linear
the CO binding in the 1R-camphor-bound P450cam In
contrast, the activation volumes are positive for the
norcam-phor-bound as well as for the norbornane-bound proteins
In contrast to P450cam bound with 1R-camphor, nor-camphor and norbornane, the time curves for substrate-free P450cam and P450cam bound with camphane could not be fitted satisfactorily with only one bimolecular process Figure 4 shows the data for the camphane complex as an example As the simplest approximation we used a linear combination of two bimolecular processes to fit the curves
At 1 bar the fractions of the slow and the fast phases are approximately equal For the camphane complex the fraction of the slow phase is almost constant up to 1150 bar (52–55%) but increases to 75% at further pressure elevation up to 1380 bar (Fig 5, Table 1) The activation
than 1150 bar, the activation volumes become even more negative (Table 1)
For substrate-free P450cam the fraction w of the fast phase
logarithm of the equilibrium constant between the fast phase conformer to the slow-phase conformer, vs the pressure,
the slow phase; Table 1, Fig 6) The binding rate constants for both phases in substrate-free P450cam are significantly
(kon,slow 1.6 · 104
M )1Æs)1)
com-parison to the other substrate complexes which also have positive activation volumes (norcamphor, norbornane), the rate constant for the fast phase in substrate-free P450cam is similar or slightly lower, while for the slow phase it is approximately 10 times smaller (Table 1)
D I S C U S S I O N
The high-pressure stopped-flow study on the CO binding in cytochrome P450cam revealed two important results: (a) The substrate complexes studied can be divided into two
Fig 2 Time-dependent absorbance change at
446 nm recorded at low and high pressure in the
stopped-flow experiment on cytochrome
P450cam bound with two different substrates.
Bottom, 1R-camphor; top, norbornane The
curves for norbornane were offset for better
view The rate constants k on are obtained by
fitting the curves with a bimolecular kinetics as
described in Materials and methods The k on
mean values are given with their ± SD.
Experimental conditions are summarized in
Table 1.
Trang 4groups The one group is characterized by a positive
activation volume and a fast CO binding (substrate-free,
norcamphor and norbornane) The other group shows a
negative activation volume and slow CO binding kinetics
(1R-camphor and camphane) (b) There are two complexes
which show two-phase CO binding kinetics (substrate-free,
camphane) In the following both these findings will be
discussed
The presence of methyl groups in the substrate changes
the rate-determining step for CO binding
Unno et al [13] reported CO flash photolysis experiments
under high pressure on cytochrome P450cam bound with
various camphor analogues and on the substrate-free
protein They found that 1R-camphor, fenchone,
show negative activation volumes and slow rebinding
kinetics while the substrates norcamphor and adamantane
and the substrate-free protein have positive activation
volumes and fast rebinding kinetics Stopped-flow and flash
photolysis studies should give comparable results at normal
qualita-tively the finding by Unno et al although other camphor
analogues except norcamphor have been used Combining
the data from the flash photolysis and the stopped-flow
studies, we sort the substrate analogues into two classes:
fenchone, 3-endo-bromocamphor and
norcamphor, norbornane and adamantane and the
sub-strate-free protein) All class I substrates possess methyl
groups while class II substrates do not We conclude that the
methyl groups present in the substrate are the relevant
structural entities which modulate significantly the CO
binding properties of P450cam The crystal structure for
1R-camphor-bound protein [17] shows that 1R-camphor is
# and
kon
4 M
1 Æs
1 )
1 Æs
1 )
3 Æmol
1 )
3 Æmol
1 )
14–1515 4–1311
kon
Fig 3 Plot of lnk on against the pressure for cytochrome P450cam
bound with different substrates The experimental conditions are given in
Table 1 r 2 is the regression coefficient for the linear regression
ana-lysis: 1R-camphor (0.97); norcamphor (0.71) and norbornane (0.93).
Trang 5between its keto group and the hydroxyl group of the
amino-acid residue Tyr-96, and (b) by hydrophobic contacts
of its methyl groups C-8, C-9 to Val295 and Asp297 in the
I helix and Thr185 in the F helix (Fig 1) Disturbing these
interactions leads to a higher substrate mobility and
acces-sibility of the heme pocket for water molecules [5–9,18,19
We have recently found for the same homologous series
of camphor analogues used for this stopped-flow study that
the amount of high-spin state content which can be trapped
by a negative temperature jump (fast freezing) from 297 K
to 77 K depends strongly on the presence of substrate
methyl groups and correlates with the initial high-spin state
content at 297 K [3] The slope of the loss of the
high-spin-state content DHS with the temperature change DT (from
297 K to 77 K within 10 min) represents a water influx rate
for the heme pocket The inverse value of the water influx
rate has been defined in [3] as rigidity factor As seen in
Table 2 the water influx rate is clearly smaller for substrate complexes with negative activation volume for CO binding (camphor and camphane) compared to those substrate complexes with positive activation volumes (norcamphor, norbornane) In addition, the resulting CO complex has a smaller compressibility for substrates causing a negative activation volume compared to those with positive activa-tion volume (Table 2)
It has been discussed in various papers [12,13,23] that a positive activation volume indicates that the entry of CO into the protein is the rate-limiting step of CO binding In contrast, a negative activation volume points to the Fe-CO bond formation as the rate-limiting step However, the Fe-CO bond formation step itself (geminate binding) is very fast and independent of CO concentration [24] if the CO molecule has found the optimal place close to the iron It is
Fig 6 Plot of lnk on against the pressure for substrate-free cytochrome P450cam Inset: logarithm of the equilibrium constant K ¼ w/(1 ) w) with w being the fraction of the fast phase The activation volume DV# (10.9 ± 0.8 cm3Æmol)1) is obtained from the slope of the linear fit r2is the regression coefficient for the linear regression analysis: slow phase (0.37, this slow regression coefficient is caused essentially by the extreme points around 35 bar and 300 bar); fast phase (0.71).
Fig 5 Plot of lnk on against the pressure for cytochrome P450cam
bound with camphane Inset: fraction of the slow phase r2 is the
regression coefficient for the linear regression analysis: slow phase (0.92
for P < 1200 bar, 0.75 for P > 1200 bar); fast phase (0.72 for
P < 1000 bar, 0.93 for P > 1000 bar).
Fig 4 Time-dependent absorbance change
at 446 nm recorded in the stopped-flow
experi-ment on cytochrome P450cam bound with
camphane The experimental curve is fitted
with a single and with two bimolecular
bind-ing processes accordbind-ing to Eqns (1) and (2).
Only two processes fit the experimental curve
well Experimental conditions are summarized
in Table 1.
Trang 6the probability of finding this optimal place which causes
the rate limitation This suggestion can be explained for
and 1R-camphor-bound P450cam determined from flash
for camphor-bound P450cam This value is increased to
assigned to the energy needed to break bonds or other
contacts (e.g hydrogen bonds) or to induce a conformational
change accompanied with forming the transition state for
applied to the system for CO binding The energetic
substrate-free P450cam at 1 bar using the activation volumes from
P450cam indicates therefore that stronger bonds or more
bonds have to be broken during CO binding which let
one expect a slower binding rate compared to
P450cam The energetic contribution of the entropic
major contribution which makes CO binding in substrate-free P450cam faster than in the presence of camphor [23] The large positive activation entropy in substrate-free P450cam may indicate that the CO molecule travels along many pathways to the heme iron Along each pathway, however, many contacts (e.g contacts to many water molecules) have
to be broken, reflected in the large positive activation enthalpy The higher flexibility, respective stronger compres-sibility (Table 2) of the structure in substrate-free P450cam is
in agreement with this view
In contrast, in the presence of camphor the activation state
is highly ordered as seen by the negative activation entropy Camphor makes the protein and the heme pocket more rigid (smaller compressibility, Table 2) and the CO molecule has few or even only one pathway to approach the heme iron where it immediately sticks in the right position for bond formation leading to volume contraction (negative activa-tion volume) Along each of these few pathways obviously only a small number of contacts are necessary to cleave (low
present) In conclusion, CO binding in camphor-bound P450cam is statistically disfavoured and therefore slow
Table 2 Comparison of k on and DV # for the CO binding in cytochrome P450cam bound with class I and II substrates obtained from stopped-flow (SF, Table 1), flash photolysis (F [13]), and FTIR-flash photolysis (F-FTIR [5]), studies.
T (C)
k on
(104M )1 Æs)1)
DV #
(cm3Æmol)1)
Water influx rate DHS%/
(KÆ10 min) [3]
b a
(GPa)1) [7]
Class I
F-FTIR b 26.8 9.8 (1939.1 cm)1) – Camphane SF 4.8 1.6 (52%) & 7.8 (48%) )10.6 & )18.2 0.302 0.00638
F-FTIR b 26.8 10.4 (61%; 1939.4 cm)1)
& 49.7 (25%; 1949.2 cm)1)
–
Class II
Substrate-free SF 5.0 29.5 (46%) & 297 (54%) 4.6 & 10.2 – 0.01228
F-FTIR b 26.8 158.1 (54%; 1941.1 cm)1)
& 132.5 (8%; 1951.9 cm)1)
& 381.3 (31%; 1960.1 cm)1)
–
F-FTIR b 26.8 343.8 (1953.3 cm)1) –
F-FTIR b 26.8 340.8 (1946.1 cm)1) –
a
b is the isothermal compressibility determined from the following equation using the absolute value for the slope of the linear pressure-induced red-shift of the Soret band maximum m in P450cam-CO m 0 is the Soret band maximum extrapolated to 1 bar using the regression parameters for the particular substrate complex given in [7] const has been assumed to be equal to 1 b ¼ 1
V ½ @V T const: 1
o ½ @
@P T ;bThe values in parantheses give the percentage population and the CO stretch mode frequency of the substate The FTIR data are obtained for a
D 2 O buffer solution [5].
Trang 7Extending this conclusion to all the other substrate
complexes of P450cam studied, we note that for the class I
substrates CO binding is disfavoured because of a rigid
heme pocket and the search for the optimal place near the
heme for CO-iron bond formation appears to be
rate-limiting In contrast, the CO entry into the protein and the
CO migration through the protein to the heme iron favours
statistically CO binding for class II substrates The lack of
methyl groups in the substrate and the higher substrate
mobility and water accessibility are the relevant structural
parameters which allow that another step besides diffusion
becomes rate-determining when going from the
hypothet-ical protein-free heme to the protein This step is purely
entropically driven The positive activation volume in P450
is therefore indicative rather for a high solvent accessibility
of the heme pocket than for a diffusion limited process
The CO binding time traces for substrate-free and
cam-phane-bound P450cam had to be fitted with two processes
Biphasic kinetics were also observed for substrate-free
P450cam in the flash photolysis study under pressure by
Unno et al [13] At a first glance one could suppose that
cytochrome P420 was formed during the experiment as
discussed by Unno et al However, in our studies the
spectral analysis before and after the stopped-flow
experi-ments as well as a spectral comparison with the substrate
complexes with mono-phase behaviour clearly excludes this
possibility (data not shown) Because biphasic kinetics are
observed already at ambient pressure we conclude that
rather an equilibrium of subconformers with different CO
binding behaviour exists than a pressure dependence of the
bar Indeed, conformational substates in P450cam have
been observed and extensively studied by FTIR using the
CO stretch vibration mode as spectroscopic probe [6,8,9,
14,26] Many of the substrate complexes of P450cam-CO
studied reveal conformational substates at low temperatures
(< 160 K) At room temperature however, the transitions
between substates become rather fast resulting in an
averaged CO stretch infrared band or in shift of the
equilibrium to only one substate Many of the substrate
complexes appear therefore as a single substate at room
temperature [9] (e.g 1R-camphor, norcamphor,
norborn-ane) In contrast, the infrared spectra of substrate-free and
camphane-bound P450cam-CO are an overlap of several
subconformer bands even at room temperature which can
be merged into two main subconformer ensembles
beha-viour could explain the biphasic CO binding kinetics
observed in our stopped-flow studies The fractions of slow
and fast phases match approximately the population of the
main subconformer ensembles in both P450 complexes
Because the activation volumes for both phases in
substrate-free, respective camphane-bound, P450cam are qualitatively
similar (positive for substrate-free and negative for
cam-phane) we exclude that one of the two phases in the
camphane complex is caused by a fraction of P450 that has
not bound camphane Recently, we have found by CO flash
photolysis time-resolved FTIR studies [5] that the subcon-formers have different CO rebinding rate constants This finding agrees with the observation in the present stopped-flow study Within the same P450 complex the subcon-formers with the higher CO stretching mode frequency generally rebind faster (Table 2)
In addition, in substrate-free P450cam-CO the popula-tion and the CO stretching mode frequency shift of the subconformers with higher CO stretch frequencies show an inverse behaviour on changes of hydrostatic and osmotic pressure [6] This indicates that the CO ligand in these subconformers is more influenced by the solvent, which is in line with the higher positive activation volume for the fast phase compared to the slow phase of the CO binding curves obtained in the stopped-flow experiments (Table 1) In the static pressure dependence study [6] the population of the subconformer with the higher CO stretching mode
62% at 1 bar to 73% at 1600 bar) and the reaction
stopped-flow experiment we found that the fraction w of the
65% at 1000 bar) which may reflect a pressure-induced shift of the subconformer equilibrium to a higher-frequency (faster CO binding) subconformer The reaction volume
be in reasonable agreement with the value of the static high-pressure study
In contrast to substrate-free protein, the fast phase in stopped-flow CO binding kinetics of the camphane com-plex, which we assign to the fast rebinding in the FTIR flash photolysis experiment and to the higher-frequency CO stretching mode, shows a more negative activation volume
This behaviour is different to substrate-free P450cam This might indicate that the subconformers in the camphane complex do not originate from different solvent accessibility but for example from different orientations of the substrate itself within the heme pocket The strong increase of the negative value of the activation volume at pressures higher
is actually pressure dependent or the compressibility is changed, for example, due to substrate rearrangement in the heme pocket
Summarizing the outcome of the present high-pressure stopped-flow study under consideration of the different flash photolysis studies and diverse other studies on P450cam we suggest that the accessibility of the protein for water molecules is a relevant property which is modulated by substrate binding The positive sign of the activation volume for CO binding is rather indicative for solvent accessibility and flexibility of the protein than for diffusion-controlled CO binding or for a specific electronic structure of the thiolate proximal ligand compared to the imidazole proximal ligand as earlier assumed [11] Con-cerning the functional significance one may conclude at least for the camphor-hydroxylating cytochrome P450cam sys-tem that a suboptimal fit of the substrate in the heme pocket increases the mobility of the substrate, facilitates the access for water molecules and makes the heme pocket more compressible Under these conditions the tight structural coupling for a specific proton transfer is disturbed which
Trang 8may favour the formation of hydrogen peroxide or of water
in the oxidase reaction over the substrate hydroxylation
consumed dioxygen is released as hydrogen peroxide while
substrate P450cam complexes show monophasic CO
bind-ing but with different sign of the activation volume (negative
for camphor and positive for norcamphor)
A C K N O W L E D G E M E N T S
We thank Dieter Schwarz for critical reading of the manuscript.
Financial support from the Deutsche Forschungsgemeinschaft (Sk35/
3–1,2,4), the Institut National de la Sante´ et de la Recherche Me´dicale
and the Deutscher Akademischer Austauschdienst in the frame of the
PROCOPE programme (312/pro-ms) is acknowledged.
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... ligand compared to the imidazole proximal ligand as earlier assumed [11] Con-cerning the functional significance one may conclude at least for the camphor-hydroxylating cytochrome P450cam sys-tem... pocket and the search for the optimal place near theheme for CO- iron bond formation appears to be
rate-limiting In contrast, the CO entry into the protein and the
CO migration... the value of the static high-pressure study
In contrast to substrate-free protein, the fast phase in stopped-flow CO binding kinetics of the camphane com-plex, which we assign to the fast