(Received for publication, August 4, 1994; and in revised form, November 9, 1994)
From the
Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of
nitric oxide to nitrous oxide with electrons directly transferred from
NADH (2NO + NADH + H
N
O
+ H
O + NAD
). We studied the
reaction of P450nor with NO and NADH using stopped-flow rapid scan and
low temperature spectroscopic methods. The NO ligand can bind to the
ferric enzyme to form the stable NO bound complex,
P450nor(Fe
NO). Reduction of
P450nor(Fe
NO) with NADH yielded an intermediate,
which transiently formed (
=
100 ms) and spontaneously
decomposed to the Fe
state. The optical absorption
spectrum of the intermediate was different from that of
P450nor(Fe
NO), which was formed by either a
one-electron reduction of P450nor(Fe
NO) with
Na
S
O
or NO binding to
P450nor(Fe
). On the basis of these observations, we
suggested that the intermediate is presumably a two-electron reduced
product of P450nor(Fe
NO) by NADH, formally the
(Fe
NO)
complex. We determined the
rate constants of these reactions at 10 °C for the NO binding to
P450nor(Fe
) (2.6
10
M
s
), the NADH reduction
of P450nor(Fe
NO) (0.9
10
M
s
), and the
spontaneous decomposition of the intermediate (0.027
s
). In these kinetic measurements, it was found that
the former two processes are fast enough, while the latter is extremely
slow, compared with the fast turnover of the catalytic reaction (1200
s
at 10 °C), which we measured by monitoring the
NADH consumption. Therefore, we suggested that in the catalytic cycle,
decomposition of the intermediate is fairly accelerated by free NO,
resulting in such a fast turnover. On the basis of several lines of the
spectroscopic and the kinetic evidence, we proposed a possible
mechanism of the NO reduction by P450nor.
The cytochrome P450nor ()is a heme enzyme found in
the denitrifying fungus Fusarium oxysporum(1, 2, 3) ; this fungus can reduce
NO
and NO
to N
O. Shoun and his co-workers (4) isolated
and purified this P450nor from F. oxysporum cultivated in the
presence of NO
and
NO
. As identified by its name, P450, this
enzyme contains protoheme in its active site as a prosthetic group, and
its ferrous (Fe
)CO complex exhibits a Soret
absorption at 447 nm in its optical absorption spectrum. Its ESR
spectrum in a ferric state also gives a typical feature of P450, in
which a high spin signal at g = 8 and a set of low spin
signals (g = 2.43, 2.25, and 1.91) are resolved at 4 K. (
)All of these spectroscopic data are indicative of ligation
of a thiolate anion from Cys as a fifth axial ligand of the heme iron.
Furthermore, its cDNA analysis suggested that this enzyme can be
involved in the superfamily of cytochrome P450s (5) (cytochrome
P450 55A1 is its systematic name given on the basis of its cDNA
sequence). Its amino acid sequence deduced from the cDNA analysis
showed about 25% identity with that of Pseudomonas putida P450cam and about 40% with that of Streptomyces P450
.
Despite these similarities in its structure
and properties, the biological function of P450nor is very unique
compared with other usual P450s. Although the usual P450 activates
O to catalyze a monooxygenation reaction, P450nor exhibits
nitric oxide reductase (nor) activity. This enzyme can reduce NO to
N
O but cannot catalyze the monooxygenation reaction. This
means that in the denitrifying process by the fungus F.
oxysporum, P450nor acts to rapidly diminish poisonous NO produced
in the cell by reducing it to N
O. In this P450nor reaction,
it was also noted that electrons required for the NO reduction are
directly transferred from NADH to P450nor, in sharp contrast with the
monooxygenase reaction by other usual P450s, where the electrons are
donated from NAD(P)H to the enzyme through a flavoprotein or an
iron-sulfur protein. No redox co-factor has been detectable in P450nor
except for protoheme, in contrast with some flavo-hemoproteins such as
P450
(6) , NO synthase(7, 8) ,
and so on.
Shoun and his co-workers (4) have defined the stoichiometry and the turnover number of the reaction catalyzed by P450nor from the product analysis with gas chromatography as follows.
This reaction was hardly inhibited by CO, implying that a
ligand-free Fe state is not involved in the P450nor
catalytic cycle. It must also be noted that the ligand-free
Fe
form of P450nor cannot react with NADH, but its
ferric NO-bound form can. On the basis of these observations, Shoun et al.(4) proposed a molecular mechanism for the NO
reduction by P450nor as is shown in Fig. SI. In this
hypothetical mechanism, NO binds to the Fe
state of
the enzyme to yield the Fe
NO complex. It is then
reduced with ``one electron'' from NADH to be converted to
the Fe
NO form. In this stage, the electron moves from
the iron to the ligand NO, and subsequently the resultant
Fe
NO
complex spontaneously
decomposes to the Fe
state, releasing
NO
. The NO
produced is
non-enzymatically coupled with another NO
to form
N
O(9) .
Scheme I:
In this hypothetical mechanism, however,
we have two problems. First, NADH acts as a one-electron reductant,
although it generally donates two electrons to an acceptor in the form
of a hydride anion. Second, we have never identified the
FeNO complex of P450nor because it is too unstable at
room temperature to be detected spectrophotometrically. In the present
study, we examined the following three reactions using stopped-flow
rapid scan, flash-photolysis, and low temperature spectroscopic
methods.
On the basis of the spectroscopic and kinetic data, we propose a
possible mechanism for the NO reduction to NO catalyzed by
P450nor.
Figure 1:
Time course of the absorption change
of the reduced form of NADH at 340 nm in the NO reduction reaction
catalyzed by P450nor. The optical cell containing 2.5 ml of the NADH
solution (about 0.16 mM (A =
1)) was treated with bubbling N
/NO mixed gas for 15
min. After incubation at 10 °C for 5 min, recording of the
absorption change was then started. Upon adding 30 µl of P450nor
solution (0.45 µM), the reaction was started (t = 0), as shown by the arrow in the figure. In the
catalytic reaction, the reduced form of NADH was consumed, resulting in
a decrease in the absorption at 340 nm. The initial rate (v)
was obtained from the slope at t = 0. Inset shows the double-reciprocal plot (e/v versus 1/[NO]). From the y and the x intercepts in this plot, the turnover and K
values were estimated,
respectively.
The turnover (1200
s) at 10 °C we obtained here is seemingly larger
than that (500 s
) at 30 °C previously reported
by Nakahara et al.(4) , which was determined by the
product analysis with gas chromatography. However, because such a fast
reaction is over the limitation of measurement with their
method(4, 10) , they suggested that the real value
might be much larger than 500 s
at 30 °C.
Figure 2:
Absorption spectra of P450nor in various
oxidation/ligation states measured at 10 °C with the stopped-flow
rapid scan method. A, a ligand-free Fe, a
NO-bound Fe
, and a ligand-free Fe
; B, a NO-bound Fe
, a NO-bound
Fe
, and its NADH-reduced (I) states. The
spectra of the ligand-free Fe
, the
Fe
NO, and the ligand-free Fe
enzymes are the same as the corresponding ones measured by
conventional spectroscopy. The spectrum of the Fe
NO
complex was obtained about 50 ms after mixing of the Fe
enzyme with the NO-dissolving buffer. It is indistinguishable
from that obtained upon mixing the Fe
NO complex with
Na
S
O
solution.
P450nor(Fe
NO) was stable for about 100 ms after the
formation but was gradually converted to the Fe
state. Formation of the NADH-reduced product of
P450nor(Fe
NO) (I) is described in the legend
of Fig. 4and Fig. 6.
Figure 4:
A,
absorption spectral change of P450nor(FeNO) 8
36
ms after mixing the enzyme solution with NADH (100 µM) in
the stopped-flow experiment at 10 °C. The spectra measured at 8,
13, 16, 21, 26, and 36 ms after the NADH mixing are shown. The Soret
band for the Fe
NO complex at 431 nm decreases in
intensity, while that for the intermediate, I, at 444 nm
concomitantly increases in intensity. An isosbestic point is located at
440 nm. The gate time for the measurement was 1 ms. B, its
difference spectral change based on the spectrum at 8 ms after the
mixing. C, absorption spectral change 0.4
204.9 s after
the NADH mixing. A new isosbestic point is observed at 427 nm. The
Soret absorption for I at 444 nm decreases in intensity, while
that for the Fe
enzyme at 413 nm concomitantly
increases in intensity. D, its difference spectral change
based on the spectrum at 0.4 s. In these measurements, the NO
concentration was set to be a small excess amount of the enzyme
concentration (
5
µM).
Figure 6:
A, time course of the absorption change at
427 nm after mixing P450nor(FeNO) with NADH (from the
Fe
NO to I) measured at 10 °C by the
stopped-flow apparatus. In this measurement, the NO concentration was
set to be almost equal to the enzyme concentration (
5
µM). Under this condition, we could obtain the single
exponential kinetic trace shown here. We estimated the apparent rate
constant (k
) at ambient NADH concentration by
analyzing this kinetic trace with the Gugenheim curve-fitting method. Inset shows a plot of the k
value
against the NADH concentration employed. By analyzing the NADH
concentration dependence of k
, we estimated the
bimolecular reaction rate constant (k
) at 0.9
10
M
s
. B, time course of the absorption
change at 440 nm from I to the Fe
state at
10 °C. The kinetic trace is a single exponential. The apparent rate
constant, k
, in this reaction is independent of
the NADH concentration, as shown in the inset, and we then
estimated the unimolecular reaction rate constant (k
) at 0.027s
. C, time
course of the absorption change at 427 nm in the presence of a large
amount of NO at 10 °C. Tracea was recorded at 1
mM of the NO concentration. As NO was purged with bubbling
N
, tracesb-e were recorded.
Finally, when the NO concentration became to be almost equal to the
enzyme concentration (
3 µM), the trace in the single
exponential feature was recorded (see A).
Next, we tried to obtain the spectrum of the
NO-bound ferrous form (P450nor(FeNO)) by the same
method. Due to its instability at room temperature, the spectrum of
P450nor(Fe
NO) has never been observable by the
conventional spectrophotometric method. Using the stopped-flow
technique, we succeeded in measuring the spectrum of
P450nor(Fe
NO), which was formed by binding NO to the
Na
S
O
reduced enzyme
(P450nor(Fe
)) or by reduction of
P450nor(Fe
NO) with
Na
S
O
. In the spectrum of
P450nor(Fe
NO) (Fig. 2B), we observed
the diminished Soret absorption at 434 nm and the single broad visible
band at 558 nm. The same spectrum was obtained with the low temperature
(-10 °C) spectral measurement (data not shown). The spectral
features resemble those of the ferrous NO complex of P. putida P450cam (P450cam(Fe
NO)), in which the Soret and
the visible absorption are at 438 and 557 nm,
respectively(11, 12) .
The NO binding to
P450nor(Fe) was so fast even at low NO concentration
(
5 µM) and at 10 °C that we could not follow it
by the stopped-flow measurements. The rate constant (k
in ) was estimated to be larger than 10
M
s
.
Figure 3:
A, spectral change of
P450nor(FeNO) after the laser irradiation in the
flash photolysis experiment. The spectra measured at 1, 5, 10, 15, and
30 µs after the flash was shown. The gate time was set to be 1.5
µs for the measurement. B, time course of the absorption
change at 434 nm in the flash photolysis experiment. In this
measurement, the NO concentration was 2.5 mM at 10
°C.
Using the time course of
the absorption change at 434 nm (Fig. 3B), which was a
single exponential decay, we estimated the rate constant for the NO
binding to the Fe state (k
) to
be 2.6
10
M
s
at 10 °C.
The intermediate, I, was
also detectable with a low temperature spectroscopic method in the
reaction of P450nor(FeNO) with NADH, as shown in Fig. 5. Although an absorption for the Fe
enzyme is concomitantly observed at 413 nm, the spectral features
are basically the same as those measured by the stopped-flow rapid scan
method. When we compared the spectra of I and
P450nor(Fe
NO) in Fig. 2B, we noted
that the Soret (445 nm) and visible (544 nm) absorption positions of I are substantially different from those of
P450nor(Fe
NO) (434 and 558 nm); I is formed
on reduction of P450(Fe
NO) with NADH, while
P450nor(Fe
NO) is formed on reduction with
Na
S
O
.
Figure 5:
Absorption spectra of
P450nor(FeNO) and its NADH-reduced product, I, at -10 °C measured by Hitachi U3000. In the
spectrum of I, the absorption for the Fe
form concomitantly appeared at 413 nm. In this measurement, the
NADH and NO concentrations were about 100 and 40 µM,
respectively. The spectral features were exactly the same as those
measured by the stopped-flow rapid scan method (see the spectrum at 0.4
s in Fig. 4C). Even at -10 °C, the spectrum
of I gradually converted to that of the Fe
form.
The spectral change
observed in the stopped-flow rapid scan measurements indicated that at
least three states, i.e. the FeNO, I, and the Fe
states of P450nor, are
involved in its catalytic reaction and the enzyme changes during the
catalytic cycle as shown in Fig. SII. To gain further insight
into the P450nor catalytic mechanism, we then tried to obtain the
reaction rate constant of each step under a single turnover condition,
where the NO concentration was limited to be almost equal to the enzyme
concentration.
Scheme II:
Under this condition, after a single reaction cycle from the
FeNO to the Fe
states through I, the reaction is supposed to stop because no more NO is
present in the system, and the NO-free Fe
state of
P450nor can never be reduced with NADH.
We spectrophotometrically
monitored the FeNO
I reaction at 427
nm, which is the isosbestic point between I and the
Fe
states, and obtained the single exponential decay
of the absorption (Fig. 6A). Because the decay was
dependent on the NADH concentration, as shown in inset of Fig. 6A, we estimated the second order rate constant (k
in ) at 0.9
10
M
s
at 10 °C.
On the other hand, the rate constant of the spontaneous decomposition
of I to the Fe
states (k
in ) was estimated from the time course of the
absorption change at 440 nm, which is the isosbestic point between the
Fe
NO state and I. As shown in Fig. 6B, the change is also a single exponential decay
but independent of the NADH concentration, so that we obtained the
unimolecular rate constant (k
) at 0.027
s
at 10 °C.
When we measured the spectral
change (Fig. 4) and the kinetics (Fig. 6, A and B) of P450nor(FeNO) in the reaction with
NADH, we set the NO concentration to be almost equal to the enzyme
concentration (a stoichiometric reaction condition). It is because the
effect of the very fast catalytic turnover on the spectral and the
kinetic measurements must be excluded. Indeed, when we observed the
NADH reduction of P450nor(Fe
NO) at 427 nm (Fig. 6C) in the presence of a large amount of NO
(
1 mM), the kinetic trace did not exhibit a normal single
exponential feature and changed depending on the NO concentration. When
the spectral change was followed under the same condition (data not
shown), we observed the change of the spectrum of the
Fe
NO state to that of the Fe
states
without detecting the spectrum of I, indicating that the
kinetic trace in Fig. 6C corresponds to the direct
conversion of P450nor(Fe
NO) to
P450nor(Fe
). These observations will be discussed in
more detail in the ``Discussion'' section.
In the present study, we spectroscopically and kinetically
examined the reactions of P450nor with NO and NADH and proposed a
possible reaction mechanism for the NO reduction to NO
catalyzed by P450nor, as shown in Fig. SII. The catalytic cycle
consists of three elementary steps: NO binding to the Fe
enzyme (step 1), reduction of the Fe
NO enzyme
with NADH (step 2), and conversion of the NADH-reduced product (I) to the Fe
enzyme (step 3). In this
proposed mechanism, we obtained two interesting findings with respect
to the intermediate I, its absorption spectral property, and
its spontaneous decomposition rate.
In general, NADH
donates two electrons to the acceptor, while
NaS
O
acts as a one-electron donor.
On the basis of this biochemical knowledge, we could suggest that I is possibly the two-electron reduced product of
P450nor(Fe
NO), formally the
(Fe
NO)
state. This electronic
structure seems consistent with the absorption spectral feature of I; its Soret band (444 nm) is located at a longer wavelength
than those of the Fe
NO state (434 nm) and of the
Fe
NO state (431 nm), likely showing that a more
negative charge resides on the FeNO moiety in I than in the
Fe
NO and the Fe
NO states.
We
also noted that the spontaneous decomposition of I to the
Fe state is surprisingly slow. In the kinetic
results, it was found that steps 1 (k
= 2.6
10
M
s
) and 2 (k
= 0.9
10
M
s
) are fast enough, in good consistency with
the fast catalytic reaction by P450nor. However, the k
value (0.027 s
) is too small to account for
the large turnover number (1200 s
). Therefore, some
other factor must affect conversion of I to the Fe
state, eventually resulting in the fast turnover.
As stated
above, we measured the k value under the condition
of a stoichiometric reaction, where the NO concentration is almost
equal to the enzyme concentration (about 5 µM). On the
other hand, the catalytic turnover was measured in the presence of a
large excess amount of NO (2.5
1.0 mM) relative to the
enzyme concentration (5.4 nM). Because the k
value was independent of the NADH concentration (see Fig. 6B), the difference in the NO concentration is the
only difference in the reaction condition affecting the k
and the turnover values. This fact suggests that
excess NO significantly influences the decomposition rate of I to the Fe
state.
Our above suggestion can be
indirectly but reasonably supported by comparison of the kinetic and
spectroscopic results in the reaction of
P450nor(FeNO) with NADH under various amounts of NO.
The kinetic traces in Fig. 6C are substantially
different from those in the stoichiometric reaction (Fig. 6A), and their features depend on the NO
concentration, qualitatively indicative of the effect of NO on the
overall catalytic reaction. The kinetic feature in Fig. 6C could be explained as follows. The catalytic reaction turns over
during the kinetic measurement, the Fe
NO state is
constantly accumulated in the system, and amount of NO in the system
governs the features of the overall reaction. Steps 1 and 3 are fast
enough relative to step 2 during the catalytic turnover, so that the
spectrum of I was undetectable under this reaction condition.
Namely, the reaction in step 3 in the presence of large amounts of NO, i.e. the conversion of I to the Fe
state in the catalytic cycle, is much faster than that in the
spontaneous decomposition of I. It is likely to suggest that
the decomposition of I ((Fe
NO)
to the Fe
state) is highly accelerated by
another NO.
Although this reaction mechanism is speculative, the charge balance is chemically allowed in this reaction, and acceleration of the decomposition reaction of I by bulk NO can be qualitatively explained. In comparison, the usual P450 reaction can be represented as follows:
where the two-electron reduced O on the iron site
reacts with a substrate (RH), as we know. The P450nor and the
usual P450 reactions are considered to be basically similar.
It is well known that the P450 monooxygenation reaction is supported by an acid catalyst located at the heme distal pocket, a hydroxyl group of the Thr residue, e.g. Thr-252 for P450cam(13, 14) . This Thr residue is highly conserved in all P450, and P450nor also has Thr at the 243 position in the heme pocket(5) . Therefore, Thr-243 is one possible candidate for the proton donor in the P450nor reaction. Furthermore, we found in the previous study that the channel to the heme pocket of P450nor is widely opened (15) for a second NO to have easier access to its heme pocket. All information on the structure and kinetic properties in the P450nor reaction appears to be consistently explained.
Other NO
reducing heme enzymes have been found in other microorganisms such as Paracoccus denitrificans and Pseudomonas
stutzeri(9, 10, 16) . For example, the
NO reductase isolated from P. denitrificans by Hollocher and
his co-workers (9) consists of one cytochrome b and
two cytochrome c components (a cytochrome bc complex). For the NO reduction mechanism by this enzyme, they
suggested a one-electron reduction of the FeNO moiety
and NO
release from the resultant ferrous complex,
followed by dimerization of NO
, protonation,
dehydration, and N
O formation. The former process, i.e. the one-electron reduction of the Fe
NO moiety,
is basically the same as the two-electron reduction of the
Fe
NO complex proposed by us for the P450nor reaction.
However, the NO
release from the Fe
state is not acceptable for our mechanism because the ligand-free
Fe
state is not involved in the P450nor reaction.
Because the turnover by the NO reductase of P. denitrificans (27 s
at 30 °C using phenazine
methosulfate/ascorbate as an electron donor) is much slower than that
of P450nor, acceleration of the reaction by bulk NO may not be
necessary for the reaction by the P. denitrificans NO
reductase.
In summary, we proposed the mechanism for the NO
reduction to NO catalyzed by P450nor, where the
two-electron reduced complex of the Fe
NO reacts with
another NO in the heme pocket to form N
O and H
O
(see Fig. SII). This mechanism is novel and unprecedented, in
comparison with the mechanism proposed so far for other copper- or
iron-containing NO reductases. For example, in the NO reduction
mechanism proposed for denitrifying enzymes by Averill and his
co-workers(17, 18) , the iron- or copper-bound NO is
converted to N
O via a metal-nitrosyl
(M-NO
) intermediate. However, this is not the case for
P450nor because a nucleophile such as N
does not seriously affect the P450nor reaction(4) , indicating
no involvement of the electrophile NO
in the reaction.
The electronic structure of the intermediate I observed in
the P450nor catalytic reaction is a clue to more clearly demonstrate
the reaction mechanism. The (FeNO)
state we proposed here has been rarely reported so far in
hemoprotein chemistry, so we need to explore the reactivity, structure
and electronic state of I in more detail. Because the life
time of I is about 100 ms, it must be rapidly quenched after
the reduction of P450nor(Fe
NO) with NADH for further
spectroscopic studies such as ESR and resonance Raman measurements.
These studies are now in progress in our laboratory.