From the
The kinetics of CO geminate recombination in cytochrome
P450
Cytochromes P450 are globular heme protein enzymes that catalyze
the oxidation of various xenobiotics and endogenous compounds.
Cytochrome P450
Infrared experiments
(10, 11) reveal that
substrate-free P450
The reaction cycle
of P450
Although the bimolecular dissociation and association kinetics of CO
to P450 have been measured earlier
(9, 20, 21, 22, 23, 24, 25, 26, 27) ,
the nanosecond geminate recombination of CO to P450 has not been
reported previously. Such observations are crucial for a determination
of the fundamental kinetic rate constants of heme proteins
(18) ( e.g. ligand entry to the active site,
k
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
The fundamental rate constants can be calculated from the
experimentally determined geminate rebinding rate
( k
On-line formulae not verified for accuracy under the conditions that k
In this work, we present the CO geminate rebinding
kinetics of P450 and P420, both in the presence and absence of
substrate and in different glycerol/buffer solutions at different
temperatures.
P450m
The details of the flash photolysis experiment have been presented
elsewhere
(19) . The sample was photolyzed by the 532-nm line of
a frequency-doubled Nd:YAG laser (10-ns pulse width) and the kinetics
of the ligand rebinding was probed by an argon-pumped cw dye laser. The
absorption signal was monitored by a highly linear photomultiplier
circuit and averaged about 10
Since the P450m
The difference between the absorption spectra of the CO-bound
and unbound states of P450 (P420) can be utilized in flash photolysis
experiments to optically study the kinetics. The left panel of
Fig. 1
shows the absorption spectra of camphor-free P450 and P420
in both the CO-bound and unbound states. In the flash photolysis
experiments, CO is dissociated from the heme during the 10-ns
photolysis pulse. Some CO molecules remain in the heme pocket and
rebind to the heme, which gives rise to the geminate rebinding
kinetics. The remaining CO molecules diffuse out of the heme pocket
into the solvent. On longer time scales CO rebinds from the solvent
giving rise to the bimolecular rebinding kinetics. The time course of
CO binding measured at 447 nm was best resolved using two geminate (CO
concentration-independent) exponential phases and two bimolecular (CO
concentration-dependent) phases. In the right panel of Fig. 1,
we present the kinetics of P450mCOr in 50% glycerol solution at 275 K.
The solid line is a fit with two exponential geminate phases,
the dash-dot line is a fit with one exponential geminate
phase, and the dashed line is a fit with a
``stretched'' exponential geminate phase
(e
In
Fig. 2
we present the CO rebinding kinetics of P450mCOrs,
P450mCOr, and P420mCOr in pH 7 aqueous solution at 275 K. In the
left panel we show the kinetics of the substrate-bound form,
P450mCOrs and the substrate-free form P450mCOr monitored at 447 nm. The
P450mCOrs data ( trace A) show only a very small geminate yield
( I
In order to compare the kinetics of P420mCOr to
P450mCOr, trace D shows the N( t) data for
P420mCOr after scaling by their relative extinction coefficient changes
at 447 nm (see Fig. 1). In addition to having a relatively small
absorption change, the signal also undergoes a change in sign for t < 10
In Fig. 3, we present the kinetics of P450mCOr in pH
7.2 (75% glycerol) solutions as a function of temperature. When the
temperature decreases from 293 to 263 K, the amplitude of the fast
geminate phase increases by a factor of 1.23, but the rate remains the
same (see Table I). On the other hand, the corresponding changes of the
slow geminate rate k
The inset of Fig. 3shows the Arrhenius
fit to the fundamental rates for the fast rebinding conformation. The
ratio of the rates in the fast and slow states are kBAf/kBAs
The time course of CO rebinding to P450mCOr in aqueous solution at
different temperatures is shown in Fig. 4. The inset shows the expanded bimolecular process. When the temperature
increases from 273 to 293 K, the amplitude of the fast geminate phase
decreases
In summary, the CO
geminate rebinding kinetics of P450 and P420 were observed for the
first time and experiments as a function of temperature and
glycerol/water buffer have been carried out. P450mCOrs and P450mCOr
have drastically different geminate rebinding kinetics, which indicates
that substrate plays an very important role in the structure/function
relationships that govern the dynamics of diatomic ligand binding and
release. Such effects may occur via alterations of the heme pocket that
affect the stability of CO within the pocket (this might involve
displacement of water molecules, or direct steric interactions, for
example). The two geminate and two bimolecular CO kinetic phases of
P450mCOr suggest two conformational states of the protein, each one
with a different CO affinity. The specific structural changes
associated with the fast and slow states are not yet clear. One
possibility is that they correspond to conformational interconversions
of the protein that are linked to (camphor) substrate binding, which
also affect the diatomic ligand entry and exit rates in addition to the
rate of binding at the heme. In analogy to myoglobin
(34) ,
these states may correspond to slowly interconverting open and closed
substrate binding pockets. The open state has significantly faster
fundamental rates ( k
All the fundamental rates are calculated from Equation 1.
Uncertainties in the fundamental rates can be estimated as described in
Table I.
are studied at room temperature subsequent to laser
photolysis. The geminate rebinding kinetics of P450 are strongly
affected by the presence of the camphor substrate. We observe a
2%
geminate yield for substrate-bound P450 and a 90% geminate yield when
the substrate is absent. The drastic difference in the geminate
kinetics suggests that the presence of camphor significantly alters the
CO rebinding and escape rates by modifying the heme pocket environment.
Two geminate phases and two bimolecular rebinding phases in the
substrate free protein were observed, which could arise from slowly
interconverting protein conformations. When the temperature or the
viscosity of the solution is changed, the fast geminate rate remains
the same, whereas the slow geminate rate and the two bimolecular rates
change significantly. The geminate rebinding yield of substrate-free
P420 is smaller than that of substrate free P450, but its geminate
rebinding rate is faster. This demonstrates that in the absence of
substrate, CO escapes from the pocket of P420 much more rapidly than
from P450 and suggests that the distal pocket environment is altered in
the P420 form.
, which catalyzes the hydroxylation of
camphor, is obtained from Pseudomonas putida and has been
widely studied as a model for P450 monoxygenases using many techniques
(1, 2, 3, 4) . The three-dimensional
structure of P450
in both substrate-free and -bound forms
has been determined by x-ray studies
(5, 6) . One
important result is that the heme active site of P450
is
deeply buried inside the protein, and there is no obvious access
channel for substrate and diatomic ligand binding. This means that the
protein must undergo structural fluctuations or interconversions, which
allow the passage of small molecules to the active site. Recent
photoacoustic calorimetry studies
(7) show that the presence or
absence of the camphor substrate in P450
significantly
affects the dynamics of the protein. The camphor evidently leaves the
heme pocket when CO is dissociated
(7, 8) , which
suggests that important conformational changes within the protein take
place upon photolysis that may be linked to the opening of an access
channel. Camphor-free P450 has a high CO affinity, but when camphor
binds, the CO affinity is reduced by a factor of 10, and the
association rate is slowed by about 2 orders of magnitudes
(9) .
-CO has a broad and slightly
structured CO-stretching band. The multiple signals indicate that
P450
exists in a dynamic equilibrium involving several
conformational substates. Binding of camphor or camphor analogues
strongly influences this equilibrium
(11) and analogous
resonance Raman experiments have demonstrated significant differences
in the Fe-CO vibrational frequencies as a function of substrate
(8, 12, 13) . Resonance Raman investigations
(8) have also shown that the presence of camphor substrate in
P420 (the inactive form of P450) samples has little effect on the Raman
spectra in the oxidized, reduced, or CO-bound states. The P420 heme
appears to be in equilibrium between a high-spin, five-coordinate form
and a low-spin six-coordinate form in both the ferric and ferrous
oxidation states
(8) . In the ferric state of P420,
H
O remains as a heme ligand, whereas alterations of the
protein tertiary structure lead to a significant reduction in affinity
for Cys
thiolate binding to the heme iron. In ferrous
P420, H
O and histidine are the most likely axial ligands
(8) . This evidence indicates that the heme environment and the
camphor-binding site of P420 differ significantly from that of P450. It
is likely that the altered tertiary protein structure leads to changes
in key rate constants associated with diatomic ligand binding to the
heme as well as substrate binding to the protein.
basically includes four stable intermediates
(1, 14) . The initial state is the substrate-free
cytochrome, P450m
(ferric iron, spin 1/2). Upon binding the
camphor substrate, a heme-ligated H
O molecule is expelled
to form the high-spin state, P450m
(ferric iron, spin
5/2). When the heme iron is reduced to P450m
(ferrous
iron, spin 2), dioxygen can bind to the heme forming
P450mO
rs. Subsequent to the input of a second electron, the
oxygenated intermediate decays with rapid product formation. To better
understand the catalytic process, we have studied the kinetics of
diatomic ligand binding to P450
in the presence and
absence of the camphor substrate. Such studies are sensitive to the
heme environment as well as the protein structure and dynamics. In this
investigation we explore the ligand binding kinetics of CO to P450. We
utilize CO because it serves as a stable ``reporter group''
that allows easy comparison to other heme protein systems (MbCO, HbCO)
that have been extensively studied
(15, 16, 17, 18, 19) .
; ligand escape to the solvent,
k
; and ligand binding to the heme,
k
) as shown below for the three-state model.
), geminate amplitude ( I
),
and bimolecular rate ( k
),
,
k
k
, k
= k`
[ CO] with
[ CO]
[ P450], and the
photolysis/off rate, k
0 when t > 0.
was generated and purified from P. putida as described previously
(28) . High purity P420m
was prepared by exposure of P450m
to a pressure of 2
kbar at room temperature for 1-2 h
(29, 30) .
Substrate-bound samples contained 0.4-1.0 m
M camphor and
100 m
M KCl. Concentrated frozen samples were thawed and
diluted in 0.1
M sodium phosphate buffer at pH 7.0-7.2.
The substrate-free protein P450m
was obtained by passing a
concentrated P450m
sample through a small column of
Sephadex G-25 which contained the same buffer. A cold centrifuge was
used to reconcentrate the sample. The purity of the P450m
preparation was determined by checking the absorbance ratio at
416.5 nm (the Soret peak of P450m
) relative to 390 nm (the
Soret peak of P450m
). In our experiments, the ratio was
2.7 which indicates the purification of P450m
was
better than 97%. P450m
was reduced to P450m
by
anaerobically adding a small amount of concentrated sodium dithionite
solution (
20:1 with respect to protein concentration). P450mCOr
was obtained by bubbling CO gas slowly through the P450m
sample for 20 min. When preparing glycerol/buffer mixed samples,
a longer gas equilibration time (
1 h) was used. All the
preparation procedures were carried out at 0-2 °C, in order
to maintain the sample
(8) . CO solubility changes as a function
of temperature
(31) and glycerol concentration
(32) .
The changes of the concentration of CO as a function of the temperature
in 75% glycerol were treated as proportional to the change in water.
The CO concentrations used for the samples at different temperatures
and solutions are listed in . These concentrations are used
to find the bimolecular CO association rates, k
.
times using a 350-MHz digital
oscilloscope. The voltage signal was subsequently converted to a
differential absorbance between the bound and unbound states, using
A( t) =
log( V( t)/ V(0) ), where V(0) and V( t) are proportional to sample
transmittance before and after photolysis. The quantity
A is renormalized to give the surviving unbound population,
N( t) =
A( t)/
A(0) , where
A(0) is the absorbance difference at t = 0 as determined from equilibrium measurements on bound
and unbound samples (or from their relative extinction coefficients).
Fitting functions are allowed to have a variable normalization,
N
, and the deviation of N
from unity, which is a measure of the goodness of fit at t = 0, is considered in the assessment of the fitting
function's validity. If N
lies outside the
range 1.0 ± 0.2, the fit is considered unreliable, since
non-equilibrium relaxation processes are unlikely to alter the t = 0 absorbance changes by more than ± 20%
(19) .
sample is less stable at
high temperature, most experiments were carried out at 275 K. Special
anaerobic cells were used to maintain CO gas pressure, and the sample
temperature was stabilized with a circulating bath (±1 K). The
optical spectra of the samples were taken before and after addition of
CO and at the conclusion of the experiment to check the quality of the
sample and to determine the absolute difference between the P450mCOr
and P450m
absorbance (
A(0) ).
Photoexcitation of each heme in the sample volume is assured by use of
a polarization scrambler, counter propagating beam geometry, and
sufficient pumping energy (10 mJ/pulse). Due to the limited time
resolution of the instruments, kinetics faster than 10 ns are not
observed. This leads to 10-20% ``missing'' amplitude,
as determined by the expected equilibrium absorption change
(
A(0) ) between P450mCOr and P450m
at
the probe wavelength. It should be noted that this methodology does not
account for the possibility of spectral evolution caused by
nonequilibrium protein relaxation
(19) ; however, such effects
are expected to be relatively small (5%). Another source of uncertainty
in the geminate kinetics arises from radiofrequency noise from the
Nd:YAG laser. This effect has been limited by shielding and grounding
the instruments and by subtracting the background response, with the
probe light blocked, from the signal. The remaining noise still affects
the accuracy of the measurement at the level of
1% near
10
s.
Figure 1:
The left panel shows the Soret
band absorption spectra of camphor-free P450 in the unbound
P450m ( trace A) and CO-bound P450mCOr ( trace
B) forms, along with camphor-free P420 in the unbound
P420m
( trace C) and CO-bound P420mCOr ( trace
D) forms. The right panel shows a comparison of fits to
the P450mCOr kinetics in 50% glycerol at 275 K. The solid line utilizes two exponential geminate phases and two bimolecular
phases (Equation 2). The dash-dot line uses a single geminate
exponential phase and two bimolecular phases, whereas the dashed and dotted lines each uses a stretched exponential (
= 0.5 and 0.25, respectively) for the geminate phase and two
bimolecular exponential phases. The inset is the expansion of
these fits.
One simple explanation for the
two geminate phases and two bimolecular rebinding rates observed for
P450mCOr is that there are two conformations having different CO
affinity.(
)
The differences in affinity could be
due to a variety of conformationally controlled factors, ranging from
distal pocket steric effects to heme geometry and iron spin state.
Analogous kinetic
(19) and resonance Raman investigations
(33) , as well as double-pulse flash photolysis experiments
(34) , have revealed the presence of pH-dependent
``open'' and ``closed'' distal pocket protein
conformations in myoglobin. The specific conformational changes have
recently been confirmed by x-ray crystallography studies
(35) .
The double-pulse protocol
(34) kinetically selects the most
rapidly rebinding (open pocket) fraction of the ensemble and determines
the time scale for averaging between the open and closed states. The
interconversion time scale (1-10 µs) for myoglobin is fast
compared with the rate of ligand migration from the solution to the
heme pocket (
10
s) so that a time-averaged,
single exponential population analysis, rather than a superposition of
the open and closed states describes the ligand association and
dissociation kinetics of myoglobin. The ligand association kinetics of
P450mCOr may also involve different protein conformations (to
acknowledge that a variety of factors might affect the kinetics, we
refer to them as ``fast'' and ``slow'' rebinding
conformations), but the interconversion rate may be slower than the CO
entry and exit rates. Under this circumstance, two bimolecular rates
should be observed. As mentioned above, recent infrared
(11) and Raman
(12, 13) experiments report
multiple FeCO-stretching bands in substrate-free P450mCOr, supporting
the view that several conformational substates exist.
= 2.5%, k
=
3.6
10
s
) with 97% of the
amplitude associated with the two bimolecular phases
( I
= 11%,
k
= 240
s
, I
= 86%, k
= 26 s
). On the other hand, the
P450mCOr data ( trace B) reveal a significant geminate yield
( I
= 91%, k
=
4.8
10
s
; I
= 3.6%, k
= 6.9
10
s
) along with two bimolecular phases
(see ). The bimolecular rates of P450mCOrs and P450mCOr
agree reasonably well with other literature values
(9, 27) . The significant difference in the geminate
kinetics between P450mCOrs and P450mCOr indicates that the presence or
absence of substrate not only affects the coordination and spin state
of the ferric heme iron, but also significantly changes the heme
environment in the reduced state. The observation of significant
geminate rebinding in the absence of substrate explains the early work
of Shimada et al. (22) which found that the quantum
yield of CO photodissociation was only
6% for P450mCOr and
100% for P450mCOrs. It also supports the observation that the CO
affinity of camphor-bound P450mCOrs is 10 times weaker than
camphor-free P450mCOr
(9) , primarily because of a decreased
heme binding rate (
factor 7), k
, and an
increased CO escape rate (
factor 20) in P450mCOrs that reduces the
geminate amplitude ( I
=
k
/( k
+
k
)) and, thus, the probability of ligand binding
when CO enters the heme pocket.
Figure 2:
The CO rebinding kinetic responses of
P450mCOrs, P450mCOr, and P420mCOr in pH 7 aqueous solution at 275 K. In
the left panel, traces A and B represent the
kinetics of P450mCOrs and P450mCOr, both monitored at 447 nm. The
solid lines are the fit using Equation 2. In the right
panel, trace C is P420mCOr monitored at 420 nm, and the solid
line is a fit using two geminate exponential phases plus a
stretched exponential bimolecular phase with = 0.5;
( trace D) is
A( t) of the same P420mCOr
sample monitored at 447 nm and scaled down by its extinction
coefficient changes at 447 nm relative to P450mCOr for comparison;
trace E is a mixture of 35% P450mCOr with 65% P420mCOr
monitored at 447 nm. The
A( t) of traces A,
B, C, and E were normalized using the values of
A expected at t = 0 for a fully
photolyzed sample from equilibrium measurements of the bound and
unbound material.
The right panel of
Fig. 2
shows the kinetics of P420mCOr (the pressure inactivated
form of P450), which was monitored at both 420 nm ( C) and 447
nm ( D). At 420 nm (the peak absorption of P420mCOr), the
kinetics shows a geminate yield of about 60% ( I= 53%, I
= 11%) with geminate
rates that are significantly faster than for P450mCOr
( k
= 1.5
10
s
, k
= 2.7
10
s
). This indicates that CO
escapes from the pocket of P420
(
)
much more
rapidly than from P450 (see ) and suggests that the distal
pocket environment is altered in the P420 form. The bimolecular phase
of P420mCOr could not be fit with only two exponentials, so a stretched
exponential ( I
e(k
t))
was used. The fitting result, with
fixed at 0.5, is
I
= 36%, k
= 1.8
10
s
. The
nonexponential behavior may be due to the fact that ``P420''
is actually composed of a heterogeneous mixture of slowly
interconverting conformational states. Raman spectra support this
suggestion and indicate that the P420 heme is in equilibrium between a
high-spin five-coordinate form and low-spin six-coordinate form
(8) .
s. This latter observation suggests
that protein relaxation takes place in P420 following photolysis
( i.e. in analogy to myoglobin
(19, 36) , at
short times following photolysis, the Soret band of P420m
is red shifted or broadened relative to its equilibrium value.)
More importantly, for a sample that is a mixture of P450mCOr (35%) and
P420mCOr (65%), the kinetics ( trace E) can be seen to be a
combination of the kinetic traces ( traces B and D),
with proper weighting. These results demonstrate that the two geminate
phases and two bimolecular phases of P450mCOr monitored at 447 nm
( trace B) are not the result of P420mCOr contamination of the
sample.
and two bimolecular rates
( k
and
k
) are much larger over the
same temperature range (). All the least squares fitting
parameters from Equation 2 are listed in .
Figure 3:
The CO rebinding kinetics of P450mCOr in
pH 7 75% glycerol solutions at 263, 273, 283, and 293 K. The probe
light is at 447 nm, and the data are normalized by A(0)
from equilibrium absorption measurements. The solid lines are
the fits using Equation 2. The fitting parameters are listed in Table
I. The inset is the Arrhenius fit to the fundamental rates of
the fast state (Table II). Traces A, B, and C represent the fit of kBAf, koutf, and
kinf.
The
fundamental rates for ligand entry, exit, and binding to the heme can
be calculated using Equation 1. The rates associated with the fast
bimolecular and geminate rebinding kinetics are used to calculate the
fundamental rates associated with the fast protein structure, whereas
the slower pair of geminate and bimolecular parameters are used to find
the rates associated with the slow structure. presents
the fundamental rates calculated for both fast (f) and slow (s) states
of P450mCOr in 75% glycerol at different temperatures.(
)
6-30, koutf/kouts
6-8, kinf/kins
16-20
over the temperature range 293-263 K. Surprisingly, the value of
kBAf for the fast state does not appear to change significantly with
temperature. This suggests a very small barrier for ligand binding at
the heme in comparison with the slow state. Compared with the weak
temperature dependence of kBAf, koutf, and kinf are much more sensitive
to temperature. The Arrhenius barriers are found to be: HBAf
0
kJ/mol, Houtf
27kJ/mol, and Hinf
33kJ/mol. We note that,
due to experimental error and the small amplitudes associated with the
kinetic response of the slow state, only contains
estimates for the fast state barriers. The significant increase of the
rates in the fast state indicates the possibility of protein
conformational control of diatomic ligand binding in cytochrome P450.
4%, the rate increases
5%, and the slow geminate
rate increases
20% (see ). On the other hand, the
bimolecular rates ( k
and
k
) increase significantly
(factors of 2.5 and 3) over the same temperature range. All the least
squares fitting parameters from Equation 2 are listed in .
The fundamental rates, analyzed using Equation 1, are listed in
and follow a trend that is similar to the kinetics of
P450mCOr in 75% glycerol solution ( i.e. kBAf is nearly
independent of temperature, whereas koutf and kinf change
significantly).
Figure 4:
The CO
rebinding kinetics of P450mCOr in pH 7 aqueous solutions at different
temperature. The probe light is at 447 nm, and the data are normalized
by A(0) from equilibrium absorption measurements. The
solid lines are the fits using Equation 2. The fitting
parameters are listed in Table I. The inset is the expansion
of the bimolecular phase. Traces A, B, and C represent the kinetics of P450mCOr at 293, 283, and 273
K.
A systematic difference in the rebinding kinetics of
P450mCOr in aqueous solution and glycerol/buffer mixtures is observed
by comparing Figs. 3 and 4. As the solution changes from aqueous to 75%
glycerol at 273 K, the fast geminate amplitudes decrease from 92 to
77%, whereas the slow geminate amplitude increases from 3 to
10%.(
)
On the other hand, the geminate rates do
not change significantly and the bimolecular rates change by factors
2 (see ). The glycerol-dependent results show that the
kinetics are not dramatically sensitive to solvent environment. This
indicates that, unlike the camphor substrate, glycerol has no major
effect on the structural features of the heme pocket that determine the
geminate amplitude and rebinding kinetics.
, k
,
k
) than the closed state, which indicates that
the protein conformation can control ligand binding to the heme as well
as its motion between the solvent and the heme pocket. The geminate
kinetics of P450mCOr were affected by changes in the temperature and
viscosity, but major structural changes were not apparent. This
indicates that the structure of the P450mCOr heme pocket is stable to
such perturbations. In comparison with P450, the P420mCOr data show
faster geminate rebinding rates and a nonexponential bimolecular rate,
suggesting that multiple conformations and an altered heme pocket are
associated with the P420 form of the enzyme.
Table: 0p4in
The P420mCOr data were fit with two
exponential geminate phases and a stretched exponential bimolecular
phase with
= 0.5.
Table:
The fundamental rate constants of P450 and P420
is reduced to 0.25 ( dotted
line) the fit improves between 10
to
10
s, but the absorbance normalization factor,
N
, deviated significantly from unity
( N
= 6.69). This signals that the fitting
function is overshooting the zero time point determined by
A(0) and N(0) = 1. Such effects lead to a
gross overestimate of the geminate amplitude at the expense of the
bimolecular amplitude. The chi-square for the
= 0.25 fit
is a factor of 3 better than for
= 0.5, but still a factor
of 5 worse than the bi-exponential fits. The bi-exponential also gives
a superior fit at the zero time point ( N
=
1.18) compared with the
= 0.5 case ( N
= 1.7).
observed for P450mCOrs. The level of P450mCOrs
contamination would need to scale roughly with the amplitude of the
slow bimolecular phase of P450mCOr in order to account for this effect.
However, contamination by P450mCOrs cannot explain the two phases
associated with the P450mCOr geminate kinetics, since its small
geminate amplitude would preclude its observation. The two geminate
phases could also arise from protein relaxation processes, and further
studies, involving isobestic monitoring and double pulse protocols,
will be needed to assess this possibility.
, associated with the slow
geminate phase of P450mCOr, is much smaller than I
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.