D88A mutant of cytochrome P450nor provides kinetic evidence for direct complex formation with electron donor NADH Mariko Umemura1, Fei Su1, Naoki Takaya1, Yoshitsugu Shiro2and Hirofumi S
Trang 1D88A mutant of cytochrome P450nor provides kinetic evidence for direct complex formation with electron donor NADH
Mariko Umemura1, Fei Su1, Naoki Takaya1, Yoshitsugu Shiro2and Hirofumi Shoun3
1
Institute of Applied Biochemistry, University of Tsukuba, Japan;2The Institute of Physical and Chemical Research Institute, RIKEN Harima Institute, Mikazuki-Cho, Sayo, Japan;3Department 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 be interesting to
know its functional role Here we found the intriguing
phenomenon that mutation at this site of P450nor (D88A
or D88V) considerably decreased the overall nitric oxide
reductase activity without blocking the reducing half
reac-tion in which the ferric enzymeỜNO complex is reduced
with NADH to yield a specific intermediate (I) The results
indicate that the catalytic turnover subsequent to the I
formation was blocked by such mutation This property of
the mutants made it possible to perform kinetic analysis of
the reduction step, which is impossible with the wild-type
P450nor These results are the first kinetic evidence for direct complex formation between P450nor and an electron donor (NADH or NADPH) The kinetic analysis also showed that the inhibition by chloride ions (ClỜ) is com-petitive with respect to NAD(P)H, which highlights the importance of the binding site for ClỜ(the anion hole) in the interaction with NAD(P)H We also characterized another mutant (D393A) of P450nor The results demon-strated that both Asp residues play important roles in the interaction with NADH, whereas the role of Asp88 is unique in that it must be essential for the release of NAD+rather than binding to NADH
Keywords: cytochrome P450nor; P450nor; NADH
Cytochrome P450 is the term used for a group of haem
proteins that widely exist in life from bacteria to higher
organisms such as mammals [1,2] P450 usually catalyses
a monooxygenase reaction, whereas its molecular and
functional diversity is so remarkable that some P450
species catalyse dehydration, isomerization, reduction,
CỜC bond cleavage, and so on [3] P450nor is one of
such diverse P450 species and is involved in fungal
denitrification [4Ờ6] It functions as a nitric oxide (NO)
reductase (NOR) to reduce NO to nitrous oxide (N2O),
with NADH or NADPH (NAD(P)H) as the electron
donor [4] P450nor can complete this reaction without
the aid of other proteinaceous components such as P450
reductase and thus receives electrons directly from
NAD(P)H (Eqn 1)
2NOợ NAD(P)H ợ Hợ! N2Oợ NAD(P)ợợ H2O
đ1ỡ
Fe3ợợ NO ! Fe3 ợ
Fe3ợNO ợ NAD(P)H ! I ợ NAD(P)ợ đ3ỡ
Iợ NO đợ Hợỡ ! Fe3 ợ
ợ N2Oợ H2O đ4ỡ The overall NOR reaction can be divided into three partial reactions [7]: first, the resting enzyme with ferric haem (Fe3+) binds the substrate NO to form a ferric enzymeỜNO complex (Fe3+ỜNO) (Eqn 2) Fe3+ỜNO is then reduced by NAD(P)H to yield a specific intermediate (I) exhibiting a Soret absorption peak at 444 nm (Eqn 3), and finally I reacts with a second NO to form the product N2O (Eqn 4) We assume I to be the two-electron reduced product of Fe3+Ờ
NO, formally the (Fe3+ỜNO)2Ờ state [7] Several lines of evidence support hydride (HỜ) transfer from NAD(P)H to the Fe3+ỜNO complex to form I in Eqn 3 For example, I is formed upon reduction of the Fe3+ỜNO complex with sodium borohydride, and a kinetic isotope effect has been observed in the reduction step for the proR hydrogen of NADH [8] This means that the equivalent of two electrons and one proton is provided by NADH The chemical form of
I thus depends on when the second proton is provided I would be in the (Fe3+ỜNO)2H+)2Ờstate, which is equivalent
to a ferric-hydroxylamine radical complex, if the second proton is also provided in the reduction step (Eqn 3) [8] On
Correspondence to H Shoun, Department of Biotechnology,
Gradu-ate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan Tel.:/Fax: +81 35841 5148,
E-mail: ahshoun@mail.ecc.u-tokyo.ac.jp
Abbreviations: DA, difference in absorbance; k dec , rate constant of
decomposition; k obs , observed (apparent) first order rate constant;
k red , rate constant of reduction; NOR, nitric oxide reductase;
P450, cytochrome P450.
(Received 10 November 2003, revised 6 April 2004,
accepted 6 May 2004)
Trang 2the other hand, however, if the second proton is provided in
the subsequent step, as indicated above (Eqn 4), I should be
in the (Fe3+–NOH+)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 diversified so as to interact
with the hydrophilic electron donor NAD(P)H, which is
distinct from the molecular evolution of usual
monooxy-genase P450s that generally employ hydrophobic organic
substances as substrates We thus expect that this big
haem-distal pocket forms an access channel for NAD(P)H
We are currently characterizing the distal-haem pocket of
P450nor by means of site-directed mutagenesis studies in
order to prove the working hypothesis that the pocket forms
an access channel for NAD(P)H There are many Arg and
Lys residues inside and outside the haem-pocket of P450nor
We have shown that this positive charge cluster plays a
crucial role in attracting and binding to the negatively
charged NAD(P)H molecule [11] We have shown also that
the specificity of P450nor for electron donors (NADH and
NADPH) is mainly determined by a few amino acid residues
in the B¢-helix [12] We have further shown that some
NADH analogues cause a specific spectral change of the
bound haem upon mixing with P450nor, indicating that
these NADH analogues can bind to P450nor [12]
The above results are highly indicative that the reduction
step (Eqn 3) comprises two steps obeying a normal
enzymatic reaction, i.e reversible complex (Michaelis
inter-mediate) formation between P450nor and NADH, and a
subsequent catalytic (electron transfer) reaction (Eqn 5) On
the other hand, the possibility cannot be ruled out yet that
the electron transfer from NADH to P450nor is carried out
in a manner like in a chemical reaction (one-step reaction;
Eqn 6), as the reaction of P450nor is too rapid (more than
1000 s)1at 10C) [7] for an enzymatic reaction in which
electron transfer from or to NAD is involved To
discrim-inate enzymatic and chemical reactions, classical kinetic
analysis is still effective for determining whether or not the
rate of an enzymatic reaction is saturated as to the substrate
concentration However, the electron transfer from NADH
to P450nor (Eqn 3) is too rapid to be followed with a high
NADH concentration, even with a rapid reaction analyser
Thus, such kinetic analysis has not been performed yet
Eþ NADH $ E-NADH ! I þ NADþðE : Fe3 þ
NOÞ ð5Þ
There are two negatively charged amino acid residues,
Asp88 and Asp393, in the haem-distal pocket of P450nor in
addition to the positive charge cluster Here we constructed
mutant protein of P450nor of Fusarium oxysporum in which
the negative charge of each of these residues was cancelled
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-rumwas 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 was performed by means of PCR [14] using template pfp(450)-20 [15], which consists of the P450nor cDNA of
F oxysporumand the pUC18 vector The primers 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
The resulting PCR product was inserted into pGEM-T (Promega), and then the mutation was confirmed by sequencing of the inserted nucleotide fragment All plasmids expressing mutant proteins were constructed by replacing the BssHII–PstIfragment containing P450nor cDNA [15] in pT7-nor with the corresponding portion of the mutant cDNA The introduced mutations were again confirmed by sequencing of the full-length exchanged cDNA
Expression The pT7-nor plasmid and derivatives of it were introduced into Escherichia coli JM109 (DE3) The transformed cells were cultured overnight at 30C in
LA broth [1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 25 lgÆmL)1 ampicillin] supplemented with 0.5% (w/v) glucose (preculture) Precultures (20 mL) were inoculated into 2 L LA broth in 5 L Erlenmeyer flasks with baffles, and incubated at 30C with rotation at 120 r.p.m for 6–7 h Cells were induced
by overnight incubation with 1 mM isopropyl thio-b-D -galactoside
Purification of mutant P450nor Each mutant P450nor protein was purified from a cell extract as reported [12] The transformed cells were harvested and suspended in Tris buffer [20 mM Tris/ HCl, pH 8.0, 0.1 mM dithiothreitol, 0.1 mM EDTA, 10% (v/v) glycerol] and then sonicated (200 watts, 10 min) The suspension was centrifuged at 10 000 g at 4C for
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 eluted with a 0–0.4M 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 and eluted with a 0–0.4M KCl gradient The P450nor fraction was concentrated, dialysed, and stored at 4C until further analyses
Trang 3Stopped-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 Fe3–NO complex with NAD(P)H (Eqn 3)
at 10C using a Unisoku rapid scan analyser (Osaka,
Japan) in 100 mMpotassium phosphate buffer pH 7.2 as
reported [7,11] The P450nor enzyme (final concentration
5 lM) in the Fe3–NO complex was mixed with an equal
volume of NADH or NADPH (final concentration 0.1 mM
unless otherwise stated) anaerobically, and then the
spec-trum of the mixture (200 lL volume) was recorded The
concentration of NO was kept almost equal to that of
P450nor to avoid catalytic turnover [7] The gate time was
set at 1 ms, and the rate of I formation (kobs) was calculated
as described [7,11]
Titration of chloride ions (Cl–)
We investigated Cl– binding to P450nor by means of
spectrophotometiric titration [11,12] P450nor (10 lM) in
100 mM potassium phosphate buffer pH 7.2 was mixed
with an equal volume (100 lL) of each concentration of
potassium chloride and then the spectrum of the mixture
(200-lL volume) was recorded The dissociation constant
(Kd) 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 nM) was incubated anaerobically with NO
(55 lM) in the presence of 1.0 mM NADH in 100 mM
potassium phosphate buffer pH 7.2 at 30C The activity
was determined by measuring the initial rate of product
(N2O) formation N2O was determined by gas
chromato-graphy [4] Spectrophotometric analyses were carried out with a Beckman DU-7500 spectrophotometer The amount
of P450nor was determined from the CO-difference spec-trum using the value of 86.3 mM )1Æcm)1 [16] for the molecular extinction coefficient of the difference between
at 448 nm (CO-bound form) and 490 nm (dithionite-reduced form) [17]
Results
Negative charges in the haem-distal pocket The location of the negatively charged amino acid residues
in the haem-distal pocket of P450nor is shown in Fig 1 The B¢-helix and F,G-loop form the entrance of the pocket There is an intriguing tendency in the distribution of charged amino acid residues that form the putative access channel for NADH Most of the charged residues (Arg64, Asp88, Lys62, Lys291, and Arg292; from the top to the bottom of the pocket) are located on one side of the pocket beneath the B¢-helix, whereas only one charged residue (Arg174) is present on the other side beneath the F,G-loop (Fig 1) [9] Asp88 is located in the middle of the access channel, between the positive charges of Arg64, Lys291, and Lys62 Asp393 occupies a site away from the access channel 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 in 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 (Fe3+) comprises a mixture of high- and low-spin states, and 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 mutation,
Fig 1 Stereoview of the haem-distal pocket of P450nor Negative charge residues (D88 and D393) are depicted in red, haem in magenta, haem-iron
in grey, and positive charge residues (K62, R64, R174, and K291) in blue Data from PDB 1 CL6 [9].
Trang 4indicating that each mutant protein was properly folded in
the heterologous host cells
Mutations at Asp88
All mutations at Asp88 decreased the overall NOR activity
of P450nor to a considerable extent, as shown in Table 1
We further examined the partial reaction (reduction of the
Fe3+–NO complex with NADH to yield the intermediate I;
Eqn 3) of the mutant proteins This reducing half reaction
can be observed as an isolated process under specific
conditions by following the time-dependent accumulation
of I (444 nm species; Fig 3B and Fig 4) with a rapid scan
apparatus [7] Such conditions can be attained by adding a
similar amount of NO to that of the enzyme (P450nor), with
which I cannot react further with a second NO because no
free NO remains; thus I could be in a quasi-stable state
suitable for accumulation (cf Figs 3 and 4) The apparent
first-order rate constant (kobs) for the reduction (I
forma-tion) can be obtained from the time-dependent decrease
during the process in the absorbance at 427 nm, which is the
isosbestic point of the spectrum of I and the resting enzyme
Fe3+(cf legend to Fig 3) [7] The kobsvalue was obtained
for each mutant protein and is presented in Table 1 It is
intriguing that the kobsdue to NADH did not change or was even enhanced by the mutation replacing Asp88 with a hydrophobic amino acid residue (D88A or D88V), whereas the mutation replacing Asp88 with a hydrophilic residue (D88N) decreased the kobs The extent of the decrease in kobs (36%; 16 s)1 as compared with 45 s)1 for the wild-type enzyme) agreed well with that in the overall NOR 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 reactive with free NO to complete the overall reaction (Eqn 4), and Fe3+–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 to further react with NO even under the conditions used (with
no excess NO) by taking it from the remaining Fe3+–NO complex if the I formation rate is much slower than the following step (Eqn 4), which results in no accumulation of
I This should be 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 reacforma-tion [7], must not be much slower than the subsequent steps for I to accumulate Therefore, the accumulation of I even after slow reduction of the D88A or D88V mutant with NADPH (Fig 3B and Table 1) means that the mutation decelerated the subsequent steps, so that the reaction rate became comparable to or even lower than the slow reduction Mutation at Asp393
We previously showed that a hydrogen bond network including Asp393, Ser286, and a few water molecules is formed upon Fe3+–NO complex formation, and that the network should play a key role in providing a proton that is required for intermediate formation [9,10] As shown previously, mutation at Asp393 (D393A) greatly blocked the reduction step as well as the overall activity (Table 1) However, when the reduction was examined with a higher NADH concentration (0.5 mM), we could observe the I formation of the mutant protein (Fig 4), suggesting that the
Fig 2 Absorption spectra of the D88A mutant of P450nor The spectra
are for the ferric resting (solid line), dithionite-reduced (dotted line),
and CO-bound (broken line) forms, respectively, of P450nor (5.0 l M )
in 10 m M potassium phosphate buffer pH 7.2.
Table 1 Kinetic parameters for the reduction step for P450nor wild-type and mutant enzymes k obs , Observed first-order rate constant for reduction (I formation); k dec , first-order rate constant for spontaneous decomposition of I; ND, not determined.
P450nor
Overall
activity
(%)
k obs (s)1) k dec (s)1) k obs (s)1) k obs (s)1) k dec (s)1) k obs (s)1) k dec (s)1)
D88A 25.5 ± 1.5 41 ± 4 0.10 1.6 ± 0.2 0.10 7.6 ± 0.8 0.10 D88V 22.5 ± 0.5 99 ± 6 0.089 7.3 ± 0.5 0.19 22 ± 2 0.11
Trang 5hydrogen bond network containing Asp393 is essential for
the binding of NADH rather than the electron transfer to
form I Confusion regarding the isosbestic point (at 440 nm)
in the latter stage of the process (Fig 4) is due to the
appearance of the 413 nm species (resting Fe3+) This
means that the kobsis not sufficiently high for conversion of
all of the 431 nm (Fe3+–NO) species to I This situation is
intermediate between the results in Fig 3A and B Thus, if
the reduction of the D393A mutant is performed with
NADH at a higher concentration, 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 D88A or D88V mutation enables the intermediate I to accumulate even after slow reduction by NADPH This suggests that the kobscan be determined in a wide range of NADPH concentrations for kinetic analysis when these mutant proteins are utilized, which is impossible with the wild-type P450nor, as noted above As shown in Fig 5, the
kobs for the reduction step for the D88A mutant showed saturation kinetics in terms of the NADPH concentration, affording Vmax (kred; first order reduction rate) and Km values (10C) for NADPH of 12.7 s)1 and 0.64 mM,
Fig 3 Spectral changes during reduction with NADPH of the Fe3+–
NO complex of the wild-type (A) and D88A mutant (B) of P450nor.
Each spectrum was recorded with a rapid scan apparatus at the
indi-cated time after mixing the Fe 3 +
–NO complex solution with NADPH solution (final concentration, 0.1 m M ), as described in Materials and
methods In (A) the Fe3+–NO complex (431 nm species) was
con-verted to the resting (Fe 3 +
) state (413 nm species) due to catalytic turnover without accumulation of the intermediate I In (B) Fe 3 +
–NO was converted to I (444 nm species) upon reduction with NADPH.
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 Fe3+state (413 nm
species) exists (cf Fig 6) Thus, a time-dependent trace of I formation
can avoid the interference due to the spontaneous decomposition of I
that follows its formation, although the rate of decomposition is much
slower than that of I formation (cf Figure 6).
Fig 5 Saturation kinetics observed on the reduction (I formation) of the
Fe 3 + –NO complex of the D88A mutant with NADPH The k obs for the reduction was obtained at each NADPH concentration as described in the legend to Fig 3 The mean value for two to four experiments with each NADPH concentration was used for each plot The data were fitted with KALEIDAGRAPH (Abelbeck Software), which gave V max and
K m values of 12.7 ± 0.55 s)1and 0.64 ± 0.077 m M , respectively.
Fig 4 Formation of the spectral intermediate (I ) observed upon reduction of the Fe3+–NO complex of the D393A mutant with a higher concentration of NADH Each spectrum was obtained as in Fig 3 after mixing the Fe3+–NO complex of the D393 mutant with NADH (final, 0.5 m M ).
Trang 6respectively Similarly, the D88V mutant exhibited
satura-tion kinetics (data not shown) with kredand Kmvalues of
29.4 s)1and 0.29 mM, respectively The results demonstrate
that reduction of the P450nor–NO complex by NADPH
proceeds in an enzymatic manner (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 be ascribed to the reduction step due to
NADH (Eqn 3)
Spontaneous decomposition of intermediateI
The intermediate I is so stable that its accumulation can be
observed with a rapid scan apparatus, whereas I slowly
decomposes after completion of the reduction (I formation)
to give the resting form (Fe3+) (Fig 6) The decomposition
process comprises single exponential decay, and the rate
constant for the decomposition (kdec) can be obtained by
following the change in absorbance at 440 nm, which is the
isosbestic point between the Fe3+–NO state and I [7] We
observed this process in addition to the I-forming process
with each mutant protein, and the obtained kdec
(I decomposition) values are listed in Table 1 The kdec
value for each mutant (0.10–0.19 s)1) was increased by
several fold as compared with that (0.027 s)1) for the
wild-type enzyme, indicating that I became more unstable with
mutation The kdecvalue was also shown to be independent
of the NAD(P)H concentration used for the I-forming
process, as previously observed for the wild-type enzyme [7]
Competitive inhibition by chloride ions
Halogen ions such as chloride and bromide are reverse type
I ligands for P450nor, and inhibit its enzymatic reaction
[4,11] Two binding sites for bromide were revealed by
X-ray crystallography [11], one of which was located near
the haem and termed the anion hole Chloride ions (Cl–)
caused spectral perturbation of reverse type I in the bound
haem of the D88A mutant (Fig 7A), which is similar to that
observed on its binding to wild-type P450nor [11,18] The
Kdfor the complex was determined by spectrophotometric titration (Fig 7B) to be 0.69Mfor the mutant, which was almost equal to the value for the wild-type enzyme (data not shown) The results indicated that the environment around the anion hole was not modulated by the mutation It has now become possible to kinetically 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 with NADPH, the Kibeing 0.70M(Fig 8) The excellent agreement of the Kd (Ki) values obtained with the different methods (Figs 7 and 8) strongly supports the conclusion above that the ternary complex between the Fe3–
NO binary complex and NADPH is formed prior to the electron transfer from NADPH to the binary 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 NOR activity was decreased to a
Fig 6 Spontaneous decomposition of intermediate I The Fe3+–NO
complex of the D88A mutant was reduced with 0.1 m M NADH as in
Fig 3, and each spectrum was recorded at the indicated time (after
mixing), it being shown that I is in a quasi-stable state, i.e it is
decomposing slowly.
Fig 7 Spectral perturbation upon binding of Cl – to the D88A mutant (A) Absolute spectra of the ferric resting (solid line) and Cl––bound (broken line) forms of the mutant protein KCl, 1.0 M (B) Titration with KCl The difference between the spectra with and without Cl–was recorded at each KCl concentration, in 10 m M potassium phosphate buffer pH 7.2.
Trang 7considerable extent (Table 1) Accumulation of I could be
observed even after slow reduction of the mutants by
NADPH (Fig 3B and Table 1) in contrast to the little
accumulation on the reduction of wild-type P450nor under
the same conditions (Fig 3A) It is evident that this
accumulation of I did not arise from the stabilization of I
as regards spontaneous decomposition, as the decomposition
rate (kdec) 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 turnover,
i.e dissociation of NAD(P)+from the protein and
subse-quent reaction of I with the second NO (Eqn 4) Because
Asp88 is 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 cause of
the inactivation of P450nor by the mutation
It is also intriguing that a mutation to also replace Asp88
with a hydrophilic amino acid (D88N) had an inhibitory
effect on I formation, in contrast with other mutations
(D88A and D88V) On the other hand, the accumulation of
Iwas still observed even in the case of slow reduction of the
D88N mutant with a higher concentration of NADPH
(1.0 mM; Table 1), suggesting that the subsequent process
was also blocked in this mutant It therefore seems that the
replacement of Asp88 with a hydrophilic residue (D88N)
blocked both I formation and the subsequent step The
opposite effects on the I formation of these mutations are
rather difficult to explain, while it would appear that the
hydropathy of the amino acid residue at the 88th site would
affect the hydrogen bond network containing many water
molecules, which would be important for the reduction step
[10]
Determination of the properties of the D88A mutant made it possible for the first time to perform kinetic analyses
of the reduction step and the inhibition by Cl– The kinetic analysis of the competitive inhibition (Fig 8) was based on the assumption that enzyme–substrate complex (Michaelis complex) and enzyme–inhibitor complex formation are both in rapid equilibrium as compared with the following catalytic process (electron transfer from NADPH in this case) The excellent agreement between the Ki andKdvalues, respectively, obtained by kinetic (Fig 8) and spectrophoto-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 to the Fe3–NO complex 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 (Figs 7 and 8) also provided the first evidence that Cl–binds to P450nor in a manner competitive in terms of NADH (or NADPH) The 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 acid residues located inside the haem-distal pocket have been identified as being important for the interaction with NAD(P)H They are Lys62, Arg64, Arg174, Lys291, Arg292 [11], Ser286 [9,10], Thr243 [19], Asp393 [9,10] (present study), and Asp88 (present study) All of these charged or hydrophilic amino acid residues are conserved among P450nor isozymes [6,20] It is noteworthy that many of these charged amino acid residues (Lys62, Arg64, Asp88, Arg174, Lys291, and Arg292) are concen-trated in a rather narrow area in the pocket (Fig 1), suggesting that these charged residues form an access channel for NADH It is also noteworthy that Asp88 is exceptional among these amino acid residues in that its mutation (D88A or D88V) decreased the overall NOR activity without blocking the I formation step This phenomenon could be attributed to blocking by the mutation of the steps subsequent to I formation, as noted above On the other hand, immediate dissociation of NAD+ is also essential for attaining the extremely high catalytic turnover of the P450nor reaction It would therefore appear that the inclusion of a negative charge (Asp88) in the positive charge cluster is important for releasing NAD+, leading to a charge balance in the access channel This charge balance would be important for both binding to NADH and release of NAD+
It is evident from our present and previous results that P450nor has evolved so as to interact directly with NAD(P)H by having many charged and hydrophilic amino acid residues in its distal pocket This unique molecular evolution of P450nor is in sharp contrast with that of other members of the P450 superfamily that have evolved a 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 the
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 ).
Trang 81 Nelson, D.R (1999) Cytochrome P450 and the individuality of
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