D88A mutant of cytochrome P450nor provides kinetic evidence for
direct complex formation with electron donor NADH
Mariko Umemura
1
, Fei Su
1
, Naoki Takaya
1
, Yoshitsugu Shiro
2
and Hirofumi Shoun
3
1
Institute of Applied Biochemistry, University of Tsukuba, Japan;
2
The Institute of Physical and Chemical Research Institute, RIKEN
Harima Institute, Mikazuki-Cho, Sayo, Japan;
3
Department of Biotechnology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Japan
The haem-distal pocket of nitric oxide reductase cyto-
chrome P450 contains many Arg and Lys residues that are
clustered to form a putative access channel for NADH.
Asp88 is the sole negatively charged amino acid in this
positive charge cluster, and thus it would b e interesting to
know its functional r ole. Here we found the intriguing
phenomenon that mutation at this site of P450nor (D88A
or D88V) c onsiderably d ecreased t he overall nitric oxide
reductase activity without blocking the reducing half reac-
tion in which the ferric enzyme–NO complex is reduced
with NADH to yield a specific intermediate (I). The results

such diverse P450 species and is involved in fungal
denitrification [4–6]. I t functions as a n itric oxide ( NO)
reductase (NOR) t o reduce NO t o nitrous oxide (N
2
O),
with NADH or NADPH (NAD(P)H) as the electron
donor [4]. P450nor can complete this reaction without
the aid o f other p roteinaceous components such a s P450
reductase and thus receives electrons directly from
NAD(P)H (Eqn 1).
2NO þ NAD(P)H þ H
þ
! N
2
O þ NAD(P)
þ
þ H
2
O
ð1Þ
Fe
3
þ
þ NO ! Fe
3
þ
ÀNO ð2Þ
Fe
3
þ

O(Eqn4).We
assume I to be the two-electron reduced product of Fe
3
+
–
NO, formally the (Fe
3
+
–NO)
2–
state [7]. Several lines of
evidence support hydride (H
–
) t ransfer from N AD(P)H to
the Fe
3
+
–NO complex to form I in Eqn 3. For example, I is
formed upon reduction of the Fe
3
+
–NO complex with
sodium borohydride, and a kinetic isotope effect has been
observed in the reduction step for the proR hydrogen of
NADH [8]. This mean s that the eq uivalent of two e lectrons
and one proton is provided by NADH. The chemical form o f
I thus depends on when the second proton is provided. I
wouldbeinthe(Fe
3
+

+
)
2–
state.
The three-dimensional structure of P450nor [9] exhibits
unique features, although overall structural similarity to
other P450s is conserved. That is, there is an open space in
the haem-distal pocket that has a hydrophilic environment
including many hydrophilic amino acid residues and water
molecules [10]. This unique feature suggests that the
P450nor molecule has become d iversifiedsoastointeract
with the hydrophilic electron donor NAD(P)H, which is
distinct from the molecular evolution of u sual monooxy-
genase P450s that generally employ hydr ophobic organic
substances as substrates. We thus expect that this big haem-
distal pocket forms an access c hannel for NAD(P)H.
We are c urrently characterizing the distal-haem pocket o f
P450nor by means of s ite-directed mutagenesis s tudies in
order t o prove the working hypothesis that t he pocket forms
an access channel for NAD(P)H. There are many Arg and
Lys residues i nside and outside the haem-pocket of P450nor.
We have shown t hat this positive charge cluster plays a
crucial role i n a ttracting a nd binding to the n egatively
charged NAD(P)H molecule [11]. We have shown also that
the specificity of P 450nor for electron donors (NADH and
NADPH) is mainly determined by a few amino a cid residues
in the B¢-helix [12]. We have further shown that s ome
NADH analogues cause a specific spectral c hange of t he
bound haem upon mixing with P450nor, indicating that
these NADH analogues can bind to P450nor [12].

There are two negatively charged amino acid r esidues,
Asp88 and Asp393, in the haem-distal pocket o f P450nor in
addition to the positive charge cluster. Here we c onstructed
mutant protein of P450nor of Fusarium oxysporum in which
thenegativechargeofeachoftheseresidueswascancelled
by replacing it with a neutral residue. Some of the mutant
proteins were shown to exhibit intriguing properties,
providing kinetic evidence for the direct complex formation
of P450nor with NADH.
Materials and methods
Mutagenesis and expression plasmids
The construction of each mutant of P450nor of F. oxyspo-
rum was carried out according to a standard method [13].
Each recombinant protein was produced using an expres-
sion vector for P450nor (pT7-nor) [6]. Site-directed muta-
genesis w as performed by m eans of PCR [ 14] u sing template
pfp(450)-20 [15], which consists of the P450nor cDNA of
F. oxysporum and the pUC18 vector. The p rimers used were
M13-47 and M13RV ( Takara, Otsu, Japan), which are
specific for vector pUC18. The primers used to construct the
mutant proteins were as follows (mutated sites are under-
lined): D88A, 5¢-ACATTTGTC
GCCATGGATCC-3¢;
D88V, 5¢-GCCGACATTTGTC
GTCATGGATCC-3¢;
D88N, 5¢-GCCGACATTTGTC
AACATGGATCC-3¢;
and D393A, 5¢-CTGAACCGA
GCTGTCGGAAT-3¢.
The resulting PCR product was inserted into pGEM-T

Tris/
HCl, pH 8.0, 0.1 m
M
dithiothreitol, 0.1 m
M
EDTA, 10%
(v/v) glycerol] and then sonicated (200 watts, 10 min). The
suspension was centrifuged at 10 000 g at 4 °Cfor
30 min. The supernatant was dialyzsd against Tris buffer
and centrifuged at 10 000 g for 30 min, and then applied
to a DEAE–cellulose (DE52, Whatman) column (bed
volume, 30 mL) equilibrated with Tris buffer and e luted
with a 0 –0.4
M
KCl gradient. The P450nor fraction was
concentrated by dehydration with polyethylene glycol and
then dialysed against Tris buffer. The dialysate was
applied to a Mono Q HR 5/5 column (Amersham
Pharmacia Biotech) equilibrated with the same buffer
andelutedwitha0–0.4
M
KCl gradient. The P450nor
fraction was concentrated, d ialysed, and s tored at 4 °C
until further analyses.
2888 M. Umemura et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Stopped-flow rapid scan analysis
We analysed the P450nor reducing half-reaction by follow-
ing the appearance of the intermediate (I) at 444 nm upon
reduction of the Fe
3

100 m
M
potassium p hosp hate buffer pH 7.2 was m ixed
with an equal v olume (100 lL) of each concentration of
potassium chloride and then the spectrum of the mixture
(200-lL volume) was recorded. The dissociation constant
(K
d
) was calculated from the plot of Cl
–
concentration vs.
the difference in absorbance (DA) at 413 nm from that at
395 nm caused by Cl
–
binding.
Analytical methods
The Nor activity of P450nor was assayed as reported [4].
P450nor (6 n
M
) was incubated anaerobically with NO
(55 l
M
) in t he presence of 1.0 m
M
NADH in 100 m
M
potassium phosphate buffer p H 7.2 at 30 °C. The activity
was determined by m easuring the i nitial rate of pr oduct
(N
2

across the haem. Here we constructed mutant protein of
P450nor of F. oxysporum in which either Asp88 or Asp393
was replaced with a neutral amino acid. Each mutant
protein was expressed i n E. coli and purified. All of the
purified proteins exhibited the same spectral properties as
those of the native protein. A representative spectrum
(D88A) is shown in Fig. 2. D88A exhibited the same
characteristics as those of the wild-type P450nor, i.e. its
ferric resting form (Fe
3+
) comprises a mixture of high- and
low-spin states, a nd the CO-bound form gives a Soret band
at 448 nm. These spectra show that the environment of the
haem was not modulated very much by the m utation,
Fig. 1. Stereoview of the haem-distal poc ket of P450nor. Negative charge residues (D88 and D393) are depicted in r ed, haem in magenta, h aem-iron
in grey, a nd positive charge residues (K62, R6 4, R174, and K291) in blue. Data from PDB 1 CL6 [9].
Ó FEBS 2004 Asp88 in haem-distal pocket of P450nor (Eur. J. Biochem. 271) 2889
indicating that each mutant protein was properly folded in
the heterologous host c ells.
Mutations at Asp88
All mutations at Asp88 decreased the overall NOR activity
of P450nor to a considerable extent, as shown i n Table 1.
We further examined the partial reaction (reduction of the
Fe
3
+
–NO complex with NADH t o y ield the intermediate I;
Eqn 3) of the mutant proteins. T his reducing half reaction
can be observed as an isolated process under specific
conditions by following the time-dependent accumulation

as compared with 45 s
)1
for the wild-type
enzyme) agreed well with that in the overall N OR activity
(36.5%) of the D88N mutant, showing that the inactivation
caused by the mutation arose from blocking of the
reduction step.
P450nor of F. oxysporum shows electron donor specific-
ity towards NADH [4,5,12]. The accumulation of I is not
observed when the wild-type enzyme is reduced by
NADPH, a less effective electron donor (Fig. 3A). This
suggests that a higher formation rate is required for the
accumulation of I. I must be highly re active with free NO to
complete the overall reaction (Eqn 4), and Fe
3
+
–NO
complex formation (Eqn 2) should be in rapid equilibrium
between association and dissociation [7]. Thus, during the
I-forming process, previously formed I has a chance t o
further react with NO even under the conditions used (with
no excess NO) by taking it from the remaining Fe
3
+
–NO
complex i f the I formationrateismuchslowerthanthe
following step (Eqn 4), which results in n o accumulation of
I. This should b e the case for the reduction of wild-type
P450nor by NADPH (Fig. 3A and Table 1). The I forma-
tion step, the rate-limiting step of the overall reaction [7],

Table 1. Kinetic parameters for the reduction step f or P4 50nor w ild-type an d mutant enzymes. k
obs
, Ob served fi rst-order rate constant for reduction
(I formation ); k
dec
, first-order rate constant for spontaneous decomposition of I; ND, not determined.
P450nor
Overall
activity
(%)
NADH (m
M
) NADPH (m
M
)
0.1 0.5 0.1 1.0
k
obs
(s
)1
) k
dec
(s
)1
) k
obs
(s
)1
) k
obs

obs
is not sufficiently high for conversion of
all of the 431 nm (Fe
3
+
–NO) species to I. This situation is
intermediate between the results in Fig. 3A and B. Thus, if
the r eduction of the D393A mutant is performed with
NADH at a higher con centration, the formation of I should
be more complete, affording a clearer isosbestic point.
Saturation kinetics of the intermediate formation
by the D88A or D88V mutant
The D 88A or D88V mutation e nables the intermediate I to
accumulate even after slow reduction by NADPH. This
suggests that the k
obs
can be d etermined in a wide range of
NADPH concentrations for kinetic analysis when these
mutant proteins are utilized, which is impossible with the
wild-type P450nor, a s noted above. As shown in Fig. 5, the
k
obs
for the reduction step for the D88A mutant showed
saturation kinetics in terms o f the NADPH c oncentration,
affording V
max
(k
red
; first order reduction rate) and K
m

The k
obs
for I formation is usually obtained from the time-dependent
decrease in the absorbance at 427 nm, at which the isosbestic point
between the spectra of I (444 nm species) and the Fe
3
+
state (413 nm
species) exists (cf. Fig. 6). Thus, a time-dep endent trace of I formation
can avoid the interf erence due to the s pontan eous dec omposition of I
that follows its formation, although the rate of decomposition is m uch
slower than that of I formation (cf. Figure 6).
Fig. 5. Saturation kinetics observed o n the reduction (I formation) of th e
Fe
3
+
–NO complex of the D 88A m utant with N ADPH. The k
obs
for the
reductionwasobtainedateachNADPHconcentrationasdescribedin
the l egend to F ig. 3. The mean value fo r two to four experiments w ith
each NADPH concen tratio n was used for each plot. The data we re
fitted with
KALEIDAGRAPH
(Abelbeck Software), w hich gave V
max
and
K
m
values of 12.7 ± 0.55 s

proceeds in an enzymatic man ner (Eqn 5) and not in a
chemical reaction manner (Eqn 6), and thus the ternary
complex of P450nor, NO, and NADPH should be formed
prior to the electron transfer from NADPH to form I.This
mechanism should b e ascribed to the reduction step due to
NADH (Eqn 3).
Spontaneous decomposition of intermediate
I
The intermediate I is so stable that its a ccumulation can be
observedwitharapidscanapparatus,whereasI slowly
decomposes after completion of the reduction ( I formation)
to give th e resting form ( Fe
3
+
) (Fig. 6). The decomposition
process comprises single exponential decay, and t he rate
constant for the decomposition (k
dec
) can be obtained by
following the c hange in a bsorbance at 440 nm, which is the
isosbestic point between the Fe
3
+
–NO state and I [7]. We
observed this process in addition to the I-forming process
with each mutant protein, and the obtained k
dec
(I decomposition) values a re listed i n Table 1. The k
dec
value f or each mutant (0.10–0.19 s

the anion hole was not modulated by the mutation . It has
now become possible to k inetically analyse inhibition by Cl
–
utilizing the I-forming process due to NADPH of the D88A
mutant. As expected, Cl
–
inhibited the process in a manner
competitive w ith N ADPH, the K
i
being 0 .70
M
(Fig. 8 ). The
excellent agreement of the K
d
(K
i
) values obtained with
the different methods (Figs 7 and 8) strongly supports the
conclusion above that the ternary complex between the Fe
3
–
NO binary complex and NADPH is formed prior to the
electron transfer from NADPH to the b inary complex.
Discussion
Here we found an intriguing phenomenon concerning the
properties of mutants D88A and D88V. The reducing half
reaction of these mutants yielding I was not blocked
although the overall N OR activity was decreased to a
Fig. 6. Spontaneous decomposition of intermediate I. The Fe
3

accumulation of I did not arise from the stabilization of I
as r egards spontaneous decomposition, as the decomposition
rate (k
dec
) increased with the mutation (Table 1). As noted
above, the I formation (Eqn 3) and the following reaction
of I with the second NO (Eqn 4) compete with each other
for free NO under the conditions used, and the
accumulation of I means that the former reaction
(I formation) overcomes the competition. Acquisition of
the ability by the mutant proteins to accumulate I after
slow reduction indicates that the rate-limiting step in the
NADPH-dependent overall reaction changes with the
mutation, and that the new rate-limiting step should be
the process subsequent to the formation of I. Two events
must occur following I formation during catalytic t urnover,
i.e. dissociation of NAD(P)
+
from the p rotein and subse-
quent reaction of I with the s econd NO (Eqn 4). Because
Asp88 i s located rather far away from the bound haem,
blocking of the release of NAD(P)
+
is more probable than
that of Eqn 4 (which must involve the haem) as the cau se of
the inactivation o f P450nor by the mutation.
It is also intriguing that a m utation to also replace Asp88
with a hydrophilic amino a cid (D88N) had an inhibitory
effect on I formation, in contrast with other m utations
(D88A and D88V). On t he other h and, the accumulation of

metric (Fig. 7 ) analyses means that this assumption is valid.
It can thus be concluded that the reduction step (Eqn 3)
progresses in an enzymatic manner (Eqn 5), that is,
reversible complex formation between P450nor and
NADPH (or NADH) precedes the electron transfer from
NAD(P)H t o the Fe
3
–NO c omplex to yield I. Thus, the
present results are the first kinetic evidence supporting our
assumption that P450nor directly binds to NAD(P)H
[11,12], although such direct binding of NADH is unpre-
cedented for a P450. The kinetic analyses (Fig s 7 and 8 ) a lso
provided the first evidence that C l
–
binds to P450nor in a
manner c ompetitive in terms of NADH (or N ADPH). T he
competitive inhibition by Cl
–
highlights the key role of
the anion hole (the Br
–
binding site near haem) [ 11] in the
interaction with NAD(P)H.
Now, many amino a cid residues located inside the h aem-
distal pocket h ave b een identified a s b eing important for the
interaction with NAD(P)H. They a re Lys62, Arg64,
Arg174, Lys291, Arg292 [11], S er286 [9,10], Thr243 [ 19],
Asp393 [9,10] (present study), and Asp88 (present study).
All o f t hese charged or hydrophilic amino acid residues are
conserved among P450nor isozymes [6,20]. It i s noteworthy

hydrophobic haem-distal pocket.
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science 14104005 (to H.S.).
Fig. 8. Inhibition by Cl
–
of the NADPH-dependent I formation of t he
D88A mutant. The k
obs
was obtained as in Fig. 5 at each NADPH
concentration in the presence of the indicated amount of KCl (0, 0.1 or
0.5
M
).
Ó FEBS 2004 Asp88 in haem-distal pocket of P450nor (Eur. J. Biochem. 271) 2893
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