(Received for publication, October 2, 1996, and in revised form, December 5, 1996)
From the Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 and the § Department of Molecular Biology & Biochemistry and Physiology & Biophysics, University of California, Irvine, California 92697-3900
Cytochrome P450BM-3 has the P450 heme domain and
FAD/FMN reductase domain linked together in a single polypeptide chain
arranged as heme-FMN-FAD. In the accompanying article (Govindaraj, S., and Poulos, T. L. (1997) J. Biol. Chem. 272, 7915-7921, we have described the preparation and characterization of
the various domains of cytochrome P450BM-3. One reason for undertaking
this study was to provide simpler systems for studying intramolecular electron transfer reactions. In particular, the heme-FMN version of
P450BM-3 that is missing the FAD domain should prove useful in studying
the FMN-to-heme electron transfer reaction. This version of P450BM-3
has been designated truncated P450BM-3 or BM3t. In this
study we have used laser flash photolysis techniques to generate the
reduced semiquinone of 5-deazariboflavin which in turn reduces the FMN
of BM3t to the semiquinone, FMN, at a rate constant
of 6600 s
1, whereas the heme is not reduced by the
5-deazariboflavin radical. The reduction of the heme by FMN
does not proceed in the absence of carbon monoxide (CO), whereas in the
presence of CO the FMN
to heme electron transfer rate constant
is 18 s
1. If a fatty acid substrate is present, this rate
constant increases to 250 s
1. Somewhat surprisingly, the
rate of heme reduction also is dependent on [CO] which indicates that
CO causes some change within the heme pocket and/or interaction between
the heme and FMN domains that is required for intramolecular electron
transfer.
Cytochrome P450BM-3 from Bacillus megaterium catalyzes
the NADPH-dependent monooxygenation of fatty acid
substrates (1). Unlike other P450s where the site of hydroxylation, the
heme center, and the site of NADPH oxidation, the FAD center, reside on
separate polypeptide chains, P450BM-3 has both activities on a single
polypeptide chain of 119 kDa (2). As with eukaryotic microsomal P450s, the electron transfer flow in P450BM-3 is NADPH FAD
FMN
heme. Unlike its eukaryotic counterparts, however, P450BM-3 provides an
excellent system for studying intramolecular electron transfer (ET)1 from the flavin domain to the heme
domain without the complications that arise from the protein-protein
recognition and binding steps required for the eukaryotic P450
systems.
Recombinant expression of heme and diflavin reductase domains has provided simplified systems for detailed biophysical and biochemical studies. Sevrioukova and Peterson (3) and Sevrioukova et al. (4) have investigated the ET rates within the FAD/FMN reductase domain using NADPH and dithionite as reductants. In addition, Sevrioukova et al. (4) have measured the rate of heme reduction in holo-P450BM-3 using the physiological reductant, NADPH. It appears that ET from FMN to the heme is most efficient when the FMN is in the one-electron reduced semiquinone form. In the accompanying article (5) and in previous work (6) it has been shown that it also is possible to express and purify the individual FAD and FMN domains. These studies demonstrate the modular architecture of P450BM-3 and also provide an opportunity to compare the properties of the domains to related proteins like flavodoxin (homologous to the FMN domain) and ferredoxin reductase (homologous to the FAD domain).
As described in the accompanying paper (5), we have prepared a version
of P450BM-3 consisting of residues 1-664 that contains both the heme
and the FMN domains (hereinafter referred to as a truncated BM3 or
BM3t). BM3t is a simplified system for
following the FMN heme ET reaction and provides the advantage of
removing the spectral and kinetic ambiguities introduced by the
presence of two flavin cofactors. In an attempt to directly measure the kinetics of intramolecular ET between the FMN and the heme, we have
utilized the ability of free flavin semiquinones, generated by laser
flash photolysis (7, 8), to act as in situ one-electron reductants. By this method, a brief laser pulse (<1 µs), in the presence of a sacrificial electron donor such as EDTA or semicarbazide, produces an exogenous flavin semiquinone which then acts as an efficient one-electron reductant of a redox protein. The optimal situation in this type of experiment can be achieved if initial reduction of BM3t occurs at the FMN center, rather than at
the heme, with a rate sufficiently faster than any subsequent
intramolecular ET reactions, since 1) the direction of electron flow is
from FMN to the heme in the presence of either O2 or CO, 2)
FMN is not readily reduced by NADPH (9), and 3) reduction of the FMN by
exogenous flavin semiquinone will generate a one-electron reduced FMN
species, which occurs in the holoenzyme during turnover prior to ET to
the heme (4).
In the present study we have utilized 5-deazariboflavin semiquinone
(dRFH·) as the exogenous reductant, which has a quite negative
midpoint potential (630 mV) (10). This ensures that the initial
bimolecular reduction of a redox protein is sufficiently fast
(108-109 M
1
s
1 (8)) that any subsequent intramolecular ET reaction
can be readily resolved both kinetically and spectrally. In view of the enzymatic function of P450BM-3, we have evaluated the effects of
substrate (myristate) and CO binding on both the initial reduction reaction and the subsequent intramolecular ET between FMN and heme.
BM3t was prepared and isolated as described in Govindaraj and Poulos (5). Steady-state photoreduction of BM3t (10 µM) was performed in a 100 mM phosphate buffer solution (pH 7.0) containing 5 µM deazariboflavin (dRF) and 5 mM semicarbazide as a sacrificial electron donor. The solution was bubbled with either oxygen-free argon or CO for at least 1 h prior to anaerobic addition of BM3t. Irradiation was carried out in 30-s illumination increments using a 30-watt tungsten illuminator. Difference spectra were collected with an OLIS-modified Cary 15 spectrophotometer using the fully oxidized BM3t as a reference spectrum.
Laser flash photolysis was carried out as described elsewhere (8). For
all reactions, a 100 mM phosphate buffer solution (pH 7.0)
containing 100 µM deazariboflavin and 2 mM
semicarbazide was used. The solution (minus enzyme) was made anaerobic
by bubbling for 1 h with either argon or CO-nitrogen mixtures
which had been passed through a 1.5 × 100-cm column containing an
oxygen removing catalyst (R3-11, Chemical Dynamics). The absence of
O2 was monitored by the amplitude and decay of the
5-dRFH· transient signal obtained at 500 nm upon laser
excitation of the reaction prior to addition of the enzyme. In order to
maintain anaerobiosis, all aliquots of added protein (5 µl) or
substrate (0.5 µl) were subjected to a flow of the
O2-sparged gas prior to mixing with the bulk reaction
solution (0.5 ml). The protein concentration was always larger than the
concentration of 5-dRFH· generated by the laser flash, so that
pseudo-first order conditions applied and no more than a single
electron could enter each protein molecule. Transient kinetic data were
collected using a Tektronix TDS 410A digitizing oscilloscope and
analyzed on a PC using KINFIT (OLIS). Kinetic traces corresponding to
FMN reduction or reoxidation and heme reduction could be well fit with
a single exponential equation.
In order to characterize the
spectral species generated in the laser flash photolysis experiments,
BM3t, in a solution containing dRF and the sacrificial
donor semicarbazide, was reduced by white light irradiation for a
series of fixed time intervals (~30 s) under either anaerobic or
CO-saturated conditions. In the absence of CO and substrate
(myristate), the reduced minus oxidized difference spectrum shown by
the solid line in Fig. 1 was obtained. This spectrum, with a minimum at ~470 nm, is consistent with the
conversion of the oxidized FMN prosthetic group of BM3t to
the fully reduced species. The absence of significant positive
absorption in the region from 580 to 610 nm indicates that there is
little or no neutral FMN semiquinone present in this solution. Further
irradiation of the sample resulted in the reduction of the oxidized
heme (data not shown), despite the absence of substrate and CO.
However, extensive illumination times were required to accomplish this, consistent with the expectation that the midpoint potential of the FMN
is more positive than that of the heme.
When this procedure was performed in a CO-saturated buffer (~960 µM), the difference spectrum (dashed line) shown in Fig. 1 was obtained. This spectrum corresponds to CO-bound reduced heme (HCO) minus oxidized heme. Note that the contribution of the FMN to this difference spectrum is minimal, the major characteristic being the large increase in absorbance at 450 nm. Further irradiation of the sample resulted in a loss of absorbance in the region from 450 to 500 nm, indicating reduction of the oxidized FMN (not shown). It was also determined that exposure of the oxidized heme to CO, in the absence of any irradiation, did not alter the heme spectrum, indicating that on the time scale of this experiment there was no CO-induced autoreduction of the BM3t heme.
Time-resolved difference spectra, resulting from laser flash induced
one-electron reduction of the enzyme, are also shown in Fig. 1. When a
CO-saturated solution containing 5 µM BM3t, myristic acid (40 µM), dRF, and semicarbazide was
irradiated using laser flash excitation at a wavelength equivalent to
the absorption maximum of dRF (~395 nm), the semiquinone of the free
flavin, dRFH·, was rapidly (<1 µs) generated, and subsequent
one-electron reduction of BM3t occurred via a bimolecular
reaction (cf. Fig. 2A). It should
be stressed that under the conditions of these experiments, [dRFH·] [BM3t]ox, and, in as much as
dRFH· is a one-electron reductant, any given enzyme molecule was
reduced by only 1 eq (7, 8). The initial spectral species generated by
this process is shown by the filled circles in Fig. 1. These data were obtained from the magnitude of the transient signals obtained
at 0.5 ms after the laser flash. The rationale for this is as follows.
Upon flash irradiation of the reaction solution, a very rapid (<1
µs) increase in absorbance occurs, consistent with the formation of
the deazariboflavin semiquinone (cf. Fig. 2A).
This is followed by a slower change in absorbance consistent with
reduction of the enzyme and concomitant oxidation of the deazariboflavin semiquinone. At the concentration of enzyme used to
obtain the transient redox difference spectra in Fig. 1, the reaction
is complete in <0.5 ms. Thus, the signal amplitude at this point
represents the initially reduced enzyme species. In the absence of CO,
regardless of the presence or absence of substrate, this initial
reduction product is stable for at least 300 ms after laser excitation
(data not shown.) The initial reduction species has spectral properties
similar to that obtained by steady-state white light irradiation in the
absence of CO (solid line in Fig. 1). However, there is a
significant difference, mainly in the region from 470 to 525 nm, where
the laser flash difference spectrum is more positive. The spectrum
obtained by subtracting the data obtained by steady-state illumination
of BM3t (solid curve) from the initial
time-resolved difference spectrum (closed circles) is also
shown in Fig. 1 (open diamonds). This corresponds to a spectral species having an absorbance maximum at ~490 nm, which is
consistent with the anionic semiquinone form of FMN (11), and not the
neutral semiquinone species which typically has an absorption band at
580 nm (11). We thus conclude that ET from 5-dRFH· to
BM3t results in the conversion of FMN to FMN
.
Sevrioukova et al. (6, 12) have shown that two-electron
reduction of the holoenzyme by NADPH occurs at the FAD site forming the
hydroquinone species. Subsequent to this bimolecular reduction, a
slower intramolecular ET reaction results in the formation of the
neutral semiquinone of FAD (FADH·) and the anion semiquinone of
FMN (FMN
). Thus, despite the fact that the BM3t is
lacking the FAD domain, the anion semiquinone form of FMN is apparently
the preferred one-electron reduced species in this construct as well as
in the holoenzyme. Also shown in Fig. 1 is the initial flash-induced
reduction difference spectrum obtained in the absence of substrate
(open circles). There is no significant difference in either
the magnitude or the shape of the spectra obtained in the absence or
presence of substrate.
Following the initial reduction of BM3t at the FMN site, a second spectral species (filled triangles in Fig. 1) is produced on a longer time scale (20 ms after the laser flash; cf. Fig. 2C). This spectrum, which is superimposable on that obtained upon steady-state photoreduction of BM3t in a CO-saturated buffer (dashed curve in Fig. 1), represents the conversion of the oxidized heme to HCO, with a strong absorbance maximum at 450 nm and a weaker band at 550 nm. The magnitude and shape of this flash-induced difference spectrum is also independent of substrate (data not shown). It should also be noted that if flash photolysis experiments are conducted in the absence of CO, only FMN reduction is observed on time scales up to 2 s after the laser flash, irrespective of the presence or absence of substrate (not shown).
Kinetics of FMN and Heme ReductionThe kinetics of the change
in absorbance at 480 nm, corresponding to reduction of the
BM3t FMN by dRFH·, in a CO-saturated buffer and in
the absence of substrate, is illustrated in Fig. 2A. The
smooth line through the data represents the fit to a single exponential
equation, giving a rate constant of 6600 s1. Fig.
3 shows a plot of the values of kobs
versus [BM3t]ox for the
initial FMN reduction step for experiments conducted in both CO-free
and CO-saturated buffers. A second order rate constant of 7.8 × 108 M
1 s
1 is
obtained which, within experimental error, is independent of the
presence or absence of CO. It is interesting to compare these results
with those obtained previously in this laboratory for the reduction of
microsomal P450 reductase (13, 14). One-electron reduction of the
microsomal reductase was shown to occur at the FAD cofactor with a
second order rate constant of 8 × 107
M
1 s
1, followed by a slow
intramolecular ET (70 s
1) to the oxidized FMN, forming
the neutral semiquinone of FMN. Thus, the second order rate constant
for reduction of the FMN in BM3t is an order of magnitude
greater than that for FAD reduction in the microsomal reductase,
suggesting a much larger degree of steric exposure of the FMN in the
BM3t construct than either the FAD or the FMN cofactors in
the microsomal reductase.
As noted above, in a flash experiment in the absence of CO we observed
no heme reduction on time scales 2 s, whereas in a CO-saturated
buffer formation of the CO-bound reduced heme was readily observed.
This is shown in the kinetic traces at 460 nm obtained in the absence
of substrate in Fig. 2B and in the presence of substrate in
Fig. 2C. Under these conditions, there is no evidence for
the formation of CO-free reduced heme. In the absence of myristate and
in CO-saturated buffer, the observed rate constant determined from the
single exponential fit shown in Fig. 2B, corresponding to
the coupled ET/CO binding step, has a value of ~20 s
1.
This same rate constant value was also obtained at wavelengths corresponding to oxidation of the reduced FMN (cf. Figs.
4B and 5B; also see below).
Furthermore, the rate constants at either wavelength are independent of
enzyme concentration up to 40 µM (not shown). These
results demonstrate that initial reduction of the enzyme, even in the
presence of CO and/or substrate, occurs at the FMN site and that direct
reduction of the heme group by dRFH· is not a significant
occurrence. In addition, in the absence of CO the reduced FMN is unable
to transfer an electron to the heme on a time scale of
2 s, whereas
the presence of CO allows the FMN-to-heme ET reaction to occur.
The Effect of Substrate on Heme Reduction
If a substrate-free
CO-saturated solution is titrated with myristate, there is a marked
increase in the value of kobs for heme
reduction, as shown in the kinetic transient obtained at 460 nm in Fig.
2C (compare with Fig. 2B). At [myristate] = 35 µM, the rate constant obtained from the single
exponential fit to the data in Fig. 2C has a value of 250 s1. The dependence of kobs on
[myristate] is shown in Fig. 4A. The data clearly follow a
hyperbolic curve, with a limiting value of 350 s
1 for the
rate constant for formation of the reduced heme-CO complex, obtained
from a fit of the data to the equation for a two-step mechanism
involving binding followed by electron transfer. From the plot we
determined an apparent dissociation constant for substrate binding
(Kd) as 6.8 µM which is greater than
0.5 µM estimated using spectral titration methods (15).
This difference is probably due to differences in experimental
conditions. Substrate binding is strongly favored at pH 8.0 and high
ionic strength and the Kd = 0.5 µM
spectral titration was carried out at pH 8.0 in 0.5 M
buffer (15) while in the present study we used a 0.1 M pH 7 buffer. However, if we repeat the spectral titration experiment as in
Ref. 15 except using the same buffer used for the flash photolysis
experiment, a Kd
5 µM is obtained, in excellent agreement with 6.8 µM estimated in the
present study.
Figs. 4B and 5 illustrate that the kinetics corresponding to
heme reduction and CO binding are identical to those corresponding to
FMN oxidation. Fig. 5 shows the absorbance changes at
455 nm (heme) and 480 nm (FMN) on a 20-ms time scale, following
reduction of the enzyme by dRFH· in a solution containing 45 µM myristate (~7-fold larger than the
Kd value given above) and 0.8 mM CO
(85% saturation). The rate constant obtained from both data sets has a
value of 275 s1. Fig. 4B shows a plot of
kobs versus [CO] for the formation
of the HCO complex, in the presence of 45 µM substrate.
The close agreement between the rate constants obtained at the two
wavelengths clearly shows that the reduction of the heme and CO binding
is concomitant with FMN reoxidation over the accessible range of [CO]. Furthermore, there is a linear dependence on [CO], indicating that the reaction is strictly bimolecular, with an apparent second order rate constant, k2 = 3.1 × 105 M
1 s
1. These
data suggest that the rate-limiting reaction in the overall mechanism
is CO binding.
In this study we have found that dRFH·
rapidly reduces the FMN in BM3t to the anionic semiquinone,
which is thought to be the active species in the steady-state reaction
of the holoenzyme of P450BM-3 (4), without significant dRFH·
reduction of the heme center. In the presence of CO, this is followed
by a slower ET reaction from FMN to heme, the reaction of most
interest in the present study. Other key observations made in this work
are as follows: 1) FMN
reduction of the heme does not occur in
the absence of CO; 2) the kobs for FMN
to heme ET is dependent on [CO]; 3) the presence of substrate
increases the rate constant still further, and 4) there is no build up
of reduced heme and oxidized FMN as an intermediate on the way to forming the reduced heme-CO complex. We consider these observations in
light of the following two schemes, where H = heme and S = substrate.
![]() |
![]() |
![]() |
![]() |
Alternatively, we consider Scheme 2 in which there is a
pre-equilibration step between CO and (FMN Hox)S.
As noted above, in the absence of both substrate and CO,
Hox is not reduced by FMN
. However, when CO is
added, ET to Hox occurs. This effect of CO is likely due to
an increase in the heme redox potential (16). This implies the presence
of CO leads to some structural perturbation within the heme pocket.
Presumably, this does not involve formation of an Fe-CO bond, since the
iron is still oxidized (Fe3+) and CO does not affect the
spectral properties of BM3t in the ferric heme state. To
account for the [CO] dependence and the lack of (FMN
Hred)S build-up according to Scheme 2, k2
k1 and the
rate-limiting step is conversion of the heme environment by CO to a
form suitable for accepting an electron from FMN
. Precisely what the nature of this perturbation might be remains unknown. However,
we do know that the P450BM-3 heme domain exists in at least two
different conformational states. The substrate-free crystal structure
shows that the substrate access channel adopts an open conformation,
whereas the substrate-bound crystal structure (17) shows that the
access channel closes. This requires substantial movement of several
elements of secondary structure. In addition, Phe-87, which is directly
adjacent to the O2 binding site, is perpendicular to the
heme without substrate, but parallel to the heme when substrate binds.
Consistent with recent NMR studies (18), the crystal structure also
shows that the substrate is too far from the iron,
7.8 Å, for
hydroxylation. Additional NMR studies have shown that the substrate
moves much closer to the heme iron upon reduction of the iron from
Fe3+ to Fe2+. Therefore, P450BM-3 appears to
exist in multiple conformational states, some requiring rather large
global changes and others requiring alterations within the heme pocket.
As a result, it is not unreasonable to consider that a relatively
hydrophobic molecule like CO can enter the large substrate pocket, even
when the heme is in the ferric state, and induce changes that favor reduction of the heme. Precisely how remains an interesting question but could involve local perturbations within the heme pocket or, possibly, adjustments between the FMN and heme domains. The methods employed in this study together with mutagenesis and additional crystal
structure work should help to answer some of these questions.