Substrates modulate the rate-determining step for CO binding
in cytochrome P450cam (CYP101)
A high-pressure stopped-flow study
Christiane Jung
1
, Nicole Bec
2
and Reinhard Lange
2
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.
The activation volume DV
#
of the CO on-rate is positive for
P450cam bound with substrates that do not contain methyl
groups. The k
on

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

)1
. It was concluded that the transition state
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:
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)
Eur. J. Biochem. 269, 2989–2996 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02980.x
fact 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-

M
. To have comparable conditions to previous
other experiments we used 100 m
M
potassium phosphate
buffer, pH 7.3 (20 °C), 10% (w/w) glycerol to which
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
of high-spin state content at 20 °Cwasestimatedfromthe
Soret band spectrum of the oxidized protein using the fit
procedure described earlier [4]. The P450cam concentration
before mixing was 5–6 l
M
in all experiments. We always
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 concen-
tration of 1.7 m
M
. This dithionite concentration guaranteed
that P450 remained reduced during the stopped-flow

[11,16]. To get the k
on
rate constants the time curves for the
absorbance difference DA(t) were fitted with bimolecular
kinetics as described recently [5] (Eqn 1).
[P450]
0
, [P450CO]
1
,[CO]
0
, e,andl are the initial P450
concentration, the final P450-CO concentration, the initial
CO concentration, the extinction coefficient at 446 nm, and
the optical pathlength, respectively. eÆl, [P450CO]
1
,and
k
on,i
were used as fit parameters. The subscript letter i
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, substrate ana-
logues used in the high-pressure stopped-flow study.
DA
i
ðtÞ¼e Á ‘ Á
"

k
on
values for the same pressure were averaged. The
averaged values were used for further analysis to get
the activation volumes DV
#
according to Eqn. 2.
@ ln k
on
@P








T
¼À
DV
#
RT
;
@ ln K
@P





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
dependence, is strongly negative by % )19.6 cm
3
Æmol
)1
for
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
(% +8 cm
3
Æmol
)1
for both, Table 1). While the CO binding
in 1R-camphor-bound P450cam is very slow (k
on
% 3 ·
10
4
M
)1
Æs
)1

elevation up to 1380 bar (Fig. 5, Table 1). The activation
volumes DV
#
for both binding phases are negative. The
absolute value of DV
#
for the fast phase is approxi-
mately twice that of the slow phase ()18.2 cm
3
Æmol
)1
vs.
)10.6 cm
3
Æmol
)1
, Table 1). In the pressure range higher
than 1150 bar, the activation volumes become even more
negative (Table 1).
For substrate-free P450cam the fraction w of the fast phase
gradually increases from % 54% at 1 bar to %65% at 1000
bar. The plot of ln(w/(1 ) w)), which corresponds to the
logarithm of the equilibrium constant between the fast phase
conformer to the slow-phase conformer, vs. the pressure,
allows the estimation of the reaction volume DV ¼ V
fast
)
V
slow
to be approximately +11 cm

4
M
)1
Æs
)1
) compared to the respective values for camphane
(k
on,slow
% 1.6 · 10
4
M
)1
Æs
)1
and k
on,fast
% 7.8 · 10
4
M
)1
Æs
)1
)
andalsofor1R-camphor (k
on
$ 3 · 10
4
M
)1
Æs

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, 3-endo-
bromocamphor and 3,3,5,5-tetramethylcyclohexanone
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
temperatures (> 5 °C). Indeed, our data confirm 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:
classI(negativeDV
#
,smallk
on
:1R-camphor, camphane,
fenchone, 3-endo-bromocamphor and 3,3,5,5-tetramethyl-
cyclohexanone) and class II (positive DV
#

)
Substrate
(l
M
)
CO
(l
M
)
T
°C Pressure(bar)
Slow phase
(10
4
M
)1
Æs
)1
)
Fast phase
(10
4
M
)1
Æs
)1
)
Pressure range
(bar)
Slow phase

20.0
1
1
3.00 ± 0.03 (100%)
2.95 ± 0.04 (100%)
–
–
14–1515
4–1311
)19.6 ± 0.9
)13.2 ± 0.8
–
–
a
100m
M
potassium phosphate buffer, pH 7.3, 10% (w/w) glycerol, substrate dissociation constants [3]: norcamphor (345 l
M
), norbornane (47 l
M
), camphane (1.1 l
M
), 1R-camphor (0.8 l
M
), 1.7 m
M
sodium dithionite, values for concentrations correspond to the mixture.
b
The mean values for k
on

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 cm
3
Æmol
)1

and activation entropy DS
on
#
of CO binding in substrate-free
and 1R-camphor-bound P450cam determined from flash
photolysis studies by Kato et al.[25].DH
on
#
is 31.8 kJÆmol
)1
for camphor-bound P450cam. This value is increased to
61.9 kJÆmol
)1
in substrate-free protein. The activation
enthalpy may be written as DH
on
#
¼ DE
on
#
+ PÆDV
#
[15]
where DE
on
#
is the internal energy of activation which may be
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

)1
for
camphor-bound and +98.7 JÆK
)1
Æmol
)1
for substrate-free
P450cam. The energetic contribution of the entropic
term (–TÆDS)at5°C to the free enthalpy of activation DG
on
#
is 12.13 kJÆmol
)1
and )27.45 kJÆmol
)1
for camphor-bound
and substrate-free P450cam, respectively. Therefore, DG
on
#
for substrate-free P450cam is lower (34.44 kJÆmol
)1
)than
DG
on
#
for camphor-bound P450cam (43.93 kJÆmol
)1
)mean-
ing that 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.
Substrate Method
T
(°C)
k
on
(10
4
M
)1
Æs
)1
)
DV
#
(cm
3
Æmol
)1
)
Water influx
rate DHS%/
(KÆ10 min) [3]
b
a
(GPa
)1

)
& 132.5 (8%; 1951.9 cm
)1
)
& 381.3 (31%; 1960.1 cm
)1
)
–
Norbornane SF 3.8 332 8.4 0.538 –
F-FTIR
b
26.8 343.8 (1953.3 cm
)1
)–
Norcamphor SF 4.5 381 7.6 0.571 0.01445
F 20.0 1000 3.0
F-FTIR
b
26.8 340.8 (1946.1 cm
)1
)–
Adamantane F 20.0 1300 7.0 – 0.0113
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 ¼À
1
V

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 Ôhypothet-
icalÕ 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.
Subconformers of P450cam have different
k
on
and D
V
#
for CO binding
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
activation volume for the pressure range lower than % 1100

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 fre-
quency increases by % 11% with increasing pressure (from
% 62% at 1 bar to % 73% at 1600 bar) and the reaction
volume is in the order of 9 cm
3
Æmol
)1
. In the present
stopped-flow experiment we found that the fraction w of the

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
than % 1100 bar (Fig. 5) might indicate that the volume
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
Ó FEBS 2002 High-pressure stopped-flow for P450 CO binding (Eur. J. Biochem. 269) 2995
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