(Received for publication, December 29, 1995; and in revised form, January 26, 1996)
From the
Nitric oxide synthase (NOS) has a thiolate-coordinated heme
active site similar to that of cytochrome P450 (P450). Both NOS and
P450 form stable nitric oxide (NO)-ferric heme complexes, whereas an
NO-ferric heme complex of methemoglobin, that has an
imidazole-coordinated heme active site, is easily reduced. The NO
complex stability of the thiolate-coordinated hemoproteins, however,
appeared irreconcilable with the strong electron-donating capability of
the cysteine thiolate. In the present study, NO bindings to cytochrome
P450 1A2 (P450 1A2) distal mutants were studied in the presence of
various substrates. We found that a mutation at Glu-318 to Ala in the
putative distal site of P450 1A2, suggested to be important in the
O activation of P450 reactions, markedly facilitates the
reduction of the NO-ferric complex. Addition of
1,2:3,4-dibenzanthracene or phenanthrene almost abolished the mutation
effect on the NO complex. Based on these results, together with other
spectral and kinetic data, it is suggested that the NO-ferric complex
stability of P450, and perhaps of NOS, is largely ascribed to an ionic
bridge between NO and the distal carboxyl group.
The microsomal P450 ()class of hemoproteins catalyze
monooxidation reactions of a variety of external and endogenous organic
compounds with O
and electrons ( (1, 2, 3) and references therein). Likewise,
NOS produces physiologically important NO from L-Arg and N
-hydroxy-L-Arg in the presence of
O
and NADPH ( (4, 5, 6, 7) and references
therein). A hemoprotein domain of NOS has a monooxidation catalytic
site that is likely to have a thiolate-coordinated heme iron similar to
that of
P450(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
NO is a product of the NOS reaction, but is believed to tightly bind
to the heme iron(10, 12, 19) . The NO-ferric
NOS complex is stable in the absence of O, and is similar
to
P450(10, 12, 19, 20, 21, 22) .
On the other hand, an imidazole-coordinated hemoprotein, metHb, is
easily reduced by the binding of NO under anaerobic
conditions(23) . NO-ferric-porphyrin complexes are also easily
reduced in polar solvents (24) . The NO complex stability of
the thiolate-coordinated ferric hemoproteins, thus, appeared
irreconcilable with the strong electron-donating capability of the
cysteine thiolate.
Spectral studies of the NO-ferric hemoproteins or water-soluble ferric heme complexes have to date been extremely difficult because of their intrinsic instability toward autoreduction and of NO instability under aerobic conditions(20, 23, 24) . Thus, most of the NO binding studies to hemoproteins have been done for ferrous complexes under anaerobic conditions ( (25, 26, 27, 28, 29) and references therein). Recently, it became feasible to carry out NO-binding studies for ferric hemoproteins as well as ferrous hemoproteins, because of the current commercial availability of NO-releasing (donating) reagents such as NOR1. With the use of this NO donor, spectral titration experiments of NO and laser flash photolysis experiments for ferric P450 enzyme are possible.
In order to understand the interaction between NO and the P450 heme active site, we studied the NO binding to the wild-type and putative distal mutant P450 1A2 enzymes by taking advantage of the NO donor. We found that E318A mutation at the putative distal site of P450 1A2 markedly facilitates the iron reduction with NO. The reducing capability of NO in the E318A mutant was largely influenced by substrates. We discuss the Fe-N-O conformation in the P450 1A2 distal site in association with the NOS distal structure.
NO was made from NOR1 purchased from Dojindo Laboratories (Kumamoto, Japan). Careful experiments with NOR1 indicated that almost the same (experimental errors within 5%) concentration of NO is released from the equimolar NOR1 in 30 min after NOR1 is mixed with the buffer (pH 7.4) at 25 °C. It was also found the hexeneamide compound derived from NOR1 after the NO release does not bind to the heme iron of the wild-type and mutant P450 1A2 enzymes in terms of the Soret and visible absorption spectra. Namely, addition of a large excess of NOR1 up to 2.0 mM to the P450 1A2 solution did not alter the high-spin band to the nitrogen- or oxygen-coordinated low-spin band under aerobic conditions(30) . Thus the hexeneamide part of NOR1 should not disturb the NO binding to the P450 1A2 heme active site.
7-Ethoxycoumarin, anthracene, phenanthrene, and dibenzanthracene were obtained from Aldrich. All other chemicals of the highest guaranteed grade available were purchased from Wako Pure Chemical Industry (Osaka, Japan) and were used without further purification.
Laser flash photolysis
experiments were carried out in the 10 1-mm cell at 25 °C
with the second harmonic (532 nm) of a Nd:YAG laser (Quanta-Ray,
GCR-130) producing an excitation flash of 125 millijoules with a pulse
width of 6 ns. Details of this machine are described
elsewhere(34, 35) . Kinetic data was analyzed on a
Power Macintosh 6100/60AV personal computer using DeltaGraph
software. Experimental errors were within 20%.
Figure 1:
Optical absorption spectra of
Fe and NO-Fe
wild-type P450 1A2
complexes (A), Soret absorption spectral changes of
Fe
wild-type P450 1A2 caused by the addition of NO (B), and double-reciprocal plots of the absorbance change at
431 nm versus the concentration of free NO in the presence of
7-ethoxycoumarin (0.3 mM) (R
= 0.755)
(
) and dibenzanthracene (0.02 mM) (R
=
0.885) (
) (C). NO titration experiments were repeated 3
times and linear least-square fittings were performed on a Power
Macintosh 6100/60AV personal computer with DeltaGraph
software as described
previously(33) .
Glu-318 in P450 1A2 is conserved as Glu/Asp
throughout most P450s and will be important in activating O for catalytic function of
P450s(36, 37, 38) . The conserved Glu/Asp is
located at the distal site in the heme active site cavity based on the
crystal structure of P450cam(39) , P450BM3(40) ,
P450terp(41) , and P450eryF(42) . An E318D mutation at
the putative distal site of P450 1A2 did not change the high-spin type
spectrum, but increased the K
values of NO by
1.8-fold (Table 2). All substrates except for L-Arg and N
-hydroxy-L-Arg largely increased the K
values for this mutant (Table 2).
Especially dibenzanthracene increased 4.7-fold the K
values of NO for the E318D mutant. In the presence of L-Arg and N
-hydroxy-L-Arg,
spectral changes of the mutant with NO binding were not linearly
increased.
An E318A mutant at the putative distal site of P450 1A2
without NO was almost in the low-spin state(30, 37) .
By adding NO to this mutant, a peak at 431 nm ascribed to the NO-ferric
low-spin complex was formed. But later, a new peak at 400 nm appeared
concomitant with the disappearance of the 431 nm peak (Fig. 2).
This new peak is ascribed to a 5-coordinated NO-ferrous
complex(20, 21, 22) . The spectral change
rate from the NO-bound ferric low-spin complex to the NO-bound ferrous
complex of the mutant was 1.2 10
min
(Table 3) in terms of the spectral
change monitored at 431 nm. Interestingly, in the presence of
dibenzanthracene, the spin state of the mutant was a high-spin state
and essentially no redox change from ferric to ferrous state was
observed (Table 3). Phenanthrene also markedly disturbed the
redox change by 10-fold. On the other hand, the presence of other
substrates did not essentially alter the spectral change rate from
NO-bound ferric to ferrous complex (Table 3).
Figure 2:
Absorption spectral changes of the E318A
mutant (2.0 µM) by adding 0.1 mM NO (A)
and time course curves monitored at 431 nm () ascribed to
NO-Fe
and at 400 nm (
) ascribed to
NO-Fe
after the addition of NO (B).
Note that the 5-coordinated NO-ferrous complex with the 400-nm peak is not denatured. When CO gas was bubbled to the NO-ferrous wild-type complex with the 400-nm peak, only a 450-nm peak appeared, but no peak around 420 nm appeared (Fig. 3). When CO gas was bubbled to the NO-ferrous E318A mutant complex with the 400 nm peak, almost no spectral change was observed, probably because NO affinity to the ferrous mutant will be much higher than CO affinity.
Figure 3:
Optical absorption spectra of
Fe, dithionite-reduced Fe
,
NO-Fe
, and CO-Fe
complexes of the
wild-type P450 1A2. The NO-Fe
complex was formed by
adding NO to the dithionite-reduced Fe
complex. The
CO-Fe
complex was formed by bubbling CO for 2 min to
the NO-Fe
complex. Due to high affinity of NO to
Fe
complex, fully CO-bound Fe
complex was not formed but apparently mixed with
NO-Fe
complex in terms of the Soret intensity.
Denatured P420 with absorption peak at 420 nm was less than 10%
estimated from the absorption intensity.
Thr-319 of P450 1A2 is also very
conserved at the putative distal site of P450s and is known to be
involved in the O activation associated with the catalytic
function of P450s(43, 44, 45) . The K
value of an NO-ferric T319A mutant complex was
28 µM, and this is higher than the corresponding values of
the wild-type and the E318D mutant (Table 2). However, in
contrast to the E318A mutant, no change of the redox state from ferric
to ferrous low-spin complex was observed in the NO binding to the T319A
mutant.
Figure 4:
Laser flash photolysis of the NO-wild type
complex. A, laser flash photolysis time course curve at 431 nm
for the NO rebinding to the Fe wild-type (8
µM) in the presence of 2 mM NO. Other
experimental conditions are described under ``Experimental
Procedures.'' B, dependence of the first-order rate
constants (R
= 0.876) of the NO binding to the
Fe
wild-type (5 µM) on the NO
concentration.
The k value (3.2
10
M
s
) of NO to the
E318D mutant was about 2-fold higher than the wild-type (1.7
10
M
s
) (Table 2). 7-Ethoxycoumarin, anthracene, and phenanthrene
markedly increased the k
value of the E318D
mutant without substrate up to 6.2-fold. Dibenzanthracene decreased the k
value of the mutant by 4.5-fold (Table 2).
The k value of NO for the
T319A mutant was 3.8
10
M
s
. This is similar to that of the E318D
mutant.
Calculated k values of NO from the
wild-type P450 1A2 in the presence of phenanthrene or dibenzanthracene
were much larger than in the absence of the substrates. Calculated k
value from the E318D mutant was 3.6-fold
higher than the wild-type. Addition of 7-ethoxycoumarin, anthracene,
and phenanthrene increased the k
value by 5.2
8.9-fold, whereas dibenzanthracene did not change the k
value (Table 2).
Quantum yield for the laser-induced photodissociation of NO from P450 1A2 enzymes was about 0.01 under present conditions. We only compared relative quantum yield under specific conditions (Table 4). Relative quantum yields of the wild-type and the E318D mutant enzymes with dibenzanthracene were higher than those observed for other enzyme solutions by 7.2- and 3.5-fold, respectively (Table 4).
The K value of the axial ligands to the
hemoprotein will reflect the structure of the final bound
state(30, 31) . 7-Ethoxycoumarin and anthracene
markedly decreased the K
value by 3.4-fold and by
more than 37-fold, respectively. Phenanthrene and dibenzanthracene
markedly increased the K
value by more than 8-fold (Table 2). Perhaps the Fe-N-O bond angle in P450 1A2 may not be
180°, and is slightly bent due to distal amino acid constraint,
even without any substrates. It seems that 7-ethoxycoumarin and
anthracene bindings at or near the distal site of heme may partially
release this constraint, leading to a more linear Fe-N-O bond. In
contrast, bindings of other larger hydrocarbons such as phenanthrene
and dibenzanthracene, that have an extra benzene ring(s) on the side of
the long axis, may further distort the less linearity of the Fe-N-O
bond. L-Arg and N
-hydroxy-L-Arg
are rather flexible and have an ionic character in contrast to other
hydrocarbon substrates studied here. Thus, effects of these flexible
and ionic substrates on the K
value will be less
pronounced than other aromatic hydrocarbon substrates (Table 2).
Distal site structure of P450 1A2 is altered by the E318D mutation,
and thus effects of those substrates on the K value were less marked in the mutant than the wild-type.
Nevertheless, dibenzanthracene most increased the K
value among substrates studied here (Table 2).
The
NO-E318A mutant complex was easily reduced. NO-metHb complex and
NO-hemin complex in polar solvents are also easily
reduced(23, 24) . NO has a strong electron donating
character known to be a 3-electron donor (46) . In the NO-metHb
complex, presumably an ionic bridge consisting of distal His, NO, and
heme iron is formed (Fig. 5A)(23, 24, 25, 26, 27, 28, 29) .
Also, a polar solvent will form an ionic bridge with NO and heme iron.
This special ionic bridge consisting of the Fe-N-O-H bond may
facilitate the iron reduction in these NO-Fe complexes by forming a proper Fe-N-O orientation to push
electrons. On the other hand, the carboxyl group of Glu-318 may make a
different ionic bridge consisting of Fe-N-O-H at P450 1A2 in the way of
forming an improper Fe-N-O orientation to reduce the heme iron (Fig. 5B). This postulate bridge is possible, although
it does require postulation of an effectively low pK
of Glu-318. The cysteine thiolate of the P450 1A2 proximal site
should push electrons to the heme iron and facilitate the iron
reduction. In the NO-wild-type P450 1A2 complex, however, the iron
reduction may be in part canceled out by electrons from both proximal
and distal (even slightly) sites. In the NO-E318A mutant complex, there
is no more ionic bridge in the distal site and thus a strong electron
donating character of cysteine thiolate or NO may be manifested. It is
also possible that the distal site conformational change caused by the
mutation alters the polarity of the heme environment and/or redox
potential of the heme iron.
Figure 5: Hypothetical heme active sites of Hb (A) and P450 1A2 (B) in the presence of NO.
NO-heme (Fe) reduction
of Hb was explained by a mechanism (47) as: Fe
+ NO
(Fe NO)
; (Fe NO)
+ 2OH
(Fe
NO
)
+ H
O; (Fe
NO
)
+ NO
(Fe NO)
+ NO
. H
O or
alcohol is possible to participate in the Fe
-NO
reduction with such a mechanism (48) as shown in the following
(B, base),
The E318A mutant is the low-spin state and is likely that
HO or OH
is located near the distal site (30, 49) . Since the redox change of the NO-P4501A2
complex appears to be associated with the spin state of the mutant,
accessibility of hydroxide ion to NO may also contribute to the redox
stability of the NO-Fe
complex.
Larger hydrocarbons
such as phenanthrene and dibenzanthracene may largely distort the
Fe-N-O conformation in the wild-type in terms of the K value (Table 2). Similar marked distortion caused by these
hydrocarbons must also take place at the distal site in the E318A
mutant and disturb the Fe-N-O reduction in the mutant (Table 3).
The NO-ferrous forms of the wild-type and E318A
mutant enzymes have a Soret absorption at 400 nm (Fig. 2A and Fig. 3), suggesting that 5-coordinated complexes with
only NO as an axial ligand are formed (20, 21, 22, 50, 51) . NO
is tightly bound to Fe-heme, and thus the
Fe
-NO bond has a covalent character(46) . As
a result, the trans thiolate bond with Fe
will be dissociated to compensate the electron density at
Fe
.
The change of the k value
will reflect the change of the access channel of NO in P450 1A2(31).
All substrates studied here may release steric constraint at the NO
access channel and/or enlarge the NO access channel. In accordance with
this, relative quantum yield of the photoinduced dissociation of the
NO-P450 1A2 complex was increased by the addition of the substrates (Table 4). Dibenzanthracene increased the quantum yield mostly
among the substrates studied. Dibenzanthracene may distort the
approximate linearity (but not an angle of exactly 180°) of the
Fe-N-O bond and simultaneously the NO access channel by limiting the
distal space for the ligand approach to the heme iron. Thus, the
quantum yield of the NO complex in the presence of dibenzanthracene is
the highest among those in the absence and presence of other
substrates.
In conclusion, 1) conserved Glu/Asp in the distal site of P450 1A2 (and presumably of NOS) must be crucial in the redox stability of the NO-ferric complex; 2) non-linear aromatic hydrocarbons, such as phenanthrene and dibenzanthracene, markedly change the kinetic values associated with the NO binding; 3) phenanthrene and dibenzanthracene also markedly disturbed the reduction of the NO-E318A mutant complex. The ionic bridge in the NO-P450 1A2 complex hypothesized here will require further studies to clarify this finding by comparing with P450nor (52) and metHb distal structures.