A Positively Charged Cluster Formed in the Heme-distal Pocket of
Cytochrome P450nor Is Essential for Interaction with NADH*
Takashi
Kudo,
Naoki
Takaya,
Sam-Yong
Park
,
Yoshitsugu
Shiro
, and
Hirofumi
Shoun§
From the Institute of Applied Biochemistry, University of Tsukuba,
Tsukuba, Ibaraki 305-8572, Japan and the
RIKEN Harima
Institute/SPring-8, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Received for publication, August 10, 2000, and in revised form, November 11, 2000
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ABSTRACT |
Arg and Lys residues are concentrated on the
distal side of cytochrome P450nor (P450nor) to form a positively
charged cluster facing from the outside to the inside of the distal
heme pocket. We constructed mutant proteins in which the Arg and Lys
residues were replaced with Glu, Gln, or Ala. The results showed that
this cluster plays crucial roles in NADH interaction. We also showed that some anions such as bromide (Br
) perturbed the
heme environment along with the reduction step in P450nor-catalyzed
reactions, which was similar to the effects caused by the mutations. We
determined by x-ray crystallography that a Br
, termed an
anion hole, occupies a key region neighboring heme, which is the
terminus of the positively charged cluster and the terminus of the
hydrogen bond network that acts as a proton delivery system. A
comparison of the predicted mechanisms between the perturbations caused
by Br
and the mutations suggested that
Arg174 and Arg64 play a crucial role in
binding NADH to the protein. These results indicated that the
positively charged cluster is the unique structure of P450nor that
responds to direct interaction with NADH.
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INTRODUCTION |
Cytochrome P450nor
(P450nor)1 is involved in
fungal denitrification as the nitric-oxide reductase (Nor) (1) that
catalyzes the following reaction (Scheme 1).
P450nor can complete this reaction without the aid of other
protein components such as P450 reductase and thus receives electrons directly from NADH. Furthermore, the high catalytic turnover (more than
1000 s
1 at 10 °C) (1, 2) is another
notable property that may arise from direct interaction with NADH. The
overall reaction can be divided into three partial reactions
(2).
First, the ferric resting form (Fe3+) of P450nor binds
the substrate NO to form a ferric iron-nitric oxide complex
(Fe3+-NO; Scheme 2). Thereafter, Fe3+-NO is
reduced with NADH to form a specific intermediate (I) with a
Soret absorption band at 444 nm (Scheme 3) and finally reacts with the
second NO to form the product, N2O (Scheme 4).
We isolated P450norA and P450norB from Fusarium
oxysporum (3) and P450nor1 and P450nor2 from
Cylindrocarpon tonkinense (4, 5). These isoforms differ in
terms of intracellular localization (mitochondria and cytosol). These
P450nor isoforms also have different levels of specificity against
electron donors. P450norA, P450norB, and P450nor1 prefer NADH as the
electron donor, whereas only P450nor2 prefers NADPH to NADH, indicating
that they can discriminate between NADH and NADPH and thus contain an
intramolecular site for binding a reduced pyridine nucleotide. We
showed from the characterization of chimeric P450norB and P450nor2
proteins that the N-terminal half is more important than the
C-terminal half for determining specificity against reduced pyridine
nucleotides (5).
In contrast to the usual monooxygenase P450s that receive electrons
from the heme-proximal side, we surmised that P450nor does so from the
distal side. This characteristic should be reflected in the structure,
although the crystal structure determined with respect to P450nor of
F. oxysporum was fundamentally identical to those of other
P450s (6). The most unusual structure outstanding in P450nor is that a
wide open space is formed in the heme-distal pocket. We assumed that
this space acts as the access channel for NADH (or NADPH). The crystal
structure also revealed that the heme-distal pocket is in a highly
hydrophilic environment compared with those of other P450s, which
contain many water molecules (6, 7). A specific hydrogen bond network
that comprises water-74, Ser286, water-33, and
Asp393 is formed when NO binds to the resting heme (Scheme
2). Water-74 is adjacent to the heme iron-bound NO, and thus the
network connects the active site to the bulk solvent and would act as a
proton delivery system when the Fe3+-NO complex is reduced
with NADH (Scheme 3). This heme-distal pocket abnormally
concentrates positively charged Lys and Arg residues, suggesting that
the positively charged cluster attracts the negatively charged NADH
molecule by ionic interaction. We constructed a series of P450nor
mutants by site-directed mutagenesis to examine the roles of the
positively charged cluster and showed that the unique structure is
probably responsible for the characteristic function of P450nor, namely
direct interaction with NADH.
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EXPERIMENTAL PROCEDURES |
Mutagenesis and Expression Plasmids--
Plasmid preparation,
digestion by restriction and modifying enzymes, and electrophoresis
proceeded according to standard methods (8). An expression vector for
P450nor (pT7-nor) comprised pRSET C (Invitrogen) with the T7 promoter
in which the N-terminal His tag was replaced with the full
length of P450nor cDNA (9). Site-directed mutagenesis of the
P450nor cDNA was achieved using polymerase chain reaction (10) with
pfp450-20 (9) as a template. M13-47 and M13-RV
(Takara, Japan) were primers specific for the vector pUC18. The primers
used to construct each mutant protein were as follows (mutated sites
are underlined): K62A, CTGAAAAGCTCTCCGCGGTCCGCACTCG; R62E,
CTGAAAAGCTCTCCGAGGTCCGCACTCG; R64E,
CTCCAAGGTCGAGACTCGCCAAGGC; R64Q,
CTCCAAGGTCCAGACTCGCCAAGGC; K77E,
GCGCCAGTGGAGAGCAAGCAGCC; K81E,
GCAAGCAGCCGAGGCAAAGCC; R174E,
GAACGCCATTGAGACAAATGGTAG; R174Q,
GAACGCCATTCAGACAAATGGTAG; R182E,
GCTCCACTGCCGAAGAGGCCTCTG; K291E,
GCACTAGCTATCGAGCGTACTGCC; K292E,
CTAGCTATCAAGGAAACTGCCAAGG; R392E,
CGCCTCTGAACGAAGATGTCGGAATC.
The resulting polymerase chain reaction product was inserted into
pGEM-T (Promega), and the mutation was confirmed by sequencing the
inserted nucleotide fragment. All plasmids expressing mutant proteins
were constructed by replacing the BssHII-PstI
fragment containing P450nor cDNA in pT7-nor with the corresponding
portion of mutated cDNAs. The introduced mutations were again
confirmed by sequencing the full length of the exchanged cDNA.
Expression--
The pT7-nor plasmid and derivatives were
introduced into Escherichia coli JM109 (DE3). The
transformed cells were cultured overnight at 30 °C in LA broth (1%
tryptone, 0.5% yeast extract, 0.5% NaCl, 25 µg/ml ampicillin)
supplemented with 0.5% glucose (preculture). Precultures (20 ml) were
inoculated into 2 liters of LA broth in a 5-liter Erlenmeyer flask with
baffles and incubated at 30 °C with rotation at 120 rpm for 6-7 h.
Cells were induced by an overnight incubation with 1 mM
isopropyl 1-thio-
-D-galactoside (IPTG).
Purification of Mutated P450nor--
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 then centrifuged at 10,000 × g at
4 °C for 30 min, and then the supernatant was dialyzed against Tris
buffer and centrifuged at 10,000 × g for 30 min. The
supernatant was applied to a DEAE-cellulose (DE52, Whatman)
column (bed, 30 ml) equilibrated with Tris buffer, and eluted with
0-0.4 M KCl gradient. The P450nor fraction was condensed
by dehydration with polyethylene glycol and dialyzed against Tris
buffer. The dialysate was then applied to a Mono-Q HR 5/5 column
(Amersham Pharmacia Biotech) equilibrated with the same buffer and
eluted with a 0-0.4 M KCl gradient. The P450nor fraction
was condensed, dialyzed, and stored at 4 °C for further analyses.
Analytical Methods--
P450nor concentration was determined as
reported (3) using a Beckman DU-7500 spectrophotometer. P450nor
interaction with anions was observed by spectrophotometric titration.
P450nor (10 µM) in 50 mM TES buffer (pH 7.2)
was mixed with an equal volume (100 µl) of a KBr, KCl, or potassium
phosphate solution in the same buffer in a cuvette (200 µl volume),
and the spectrum was recorded.
The process by which NO binds to the resting ferric form of P450nor was
observed by flash photolysis as reported (2). The reaction of P450nor
with NADH was analyzed by following the appearance of the specific
intermediate (Soret peak at 444 nm) upon reduction of the
Fe3+-NO complex with NADH at 10 °C using a Unisoku rapid
scan analyzer as described (2), except 50 mM TES buffer
(pH7.2) was used instead of phosphate buffer.
The Nor activity of P450nor was assayed as reported (1). P450nor (4 nM) was incubated anaerobically with NO (55 µM) in the presence of 0.5 mM NADH, and the
amount of N2O formed was determined by gas chromatography.
X-ray Crystallography--
Br
derivatives of P450nor were searched by co-crystallization
(6). Co-crystals were grown in 10 mM KBr, 100 mM MES buffer at pH 5.6 using polyethylene glycol (PEG
4000) as a precipitant. The initial drops were composed of 2 ml of
protein (45 mg ml
1) and 2 ml of precipitant
solution that were equilibrated with precipitant in a 1-ml reservoir.
Crystals obtained after 1 week at 293 K were suspended on a loop in a
thin liquid film of stabilizing solution and frozen at 100 K directly
in a cold nitrogen gas stream in a Cryostream Cooler (Rigaku
Cryosystems). Data were obtained at the synchrotron radiation source of
the BL44B2 (RIKEN Beam Line 2) station in SPring-8, Harima, Japan (11).
All data sets were detected on R-AXIS IV imaging plates. Measurements
were performed at 100 K and a wavelength of 1.0 Å. Diffraction data
were integrated and scaled with DENZO (12) and SCALEPACK (12). The
derivative crystal (P212121, unit
cell parameters: a = 54.32, b = 78.05, c = 86.6 Å) was isomorphous to the native
crystal. Data collection statistics are summarized in Table I.
Difference Patterson synthesis at 2.0 Å resolution calculated using
the CCP4 package indicated two major binding sites in the asymmetric
unit. The map shows Br
self-vectors on the Harker
sections with more than 4 root-mean-square deviations of the map. The
position of Br
was refined as (0.175 0.060 0.240 and
0.804 0.076 0.228) by vector space refinement (13).
The atomic model was built using the program TURBO-FRODO (14), and
improved with SigmaA-weighted |2Fo
Fc| and |Fo
Fc| maps iteratively with X-PLOR 3.851 (15).The
structures of the native P450nor enzyme in the orthorhombic space group
(Protein Data Bank identification number 1ROM), without the coordinates
of the water molecules, were used as an initial model for refinement of
the derivative structures. After a first round of rigid body, simulated
annealing and B-factor refinements, a |2Fo
Fc| map was calculated. After manually rebuilding
the water molecules, other rounds of refinement were completed, which
included positional, individual B factors and bulk solvent correction
of the protein model. Water molecules were placed at positions where
spherical densities were above 1.5 s in the
|2Fo
Fc| map and above 3.0 s in the |2Fo
Fc|
map and where stereochemically reasonable hydrogen bonds were allowed.
Structural evaluations of the final models of the derivative enzymes
using PROCHECK (16) indicated that 90.8% of the residues were in the
most favorable regions of the Ramachandran plot and that no residues
were in disallowed regions. The refined model included residues in each chain and 116 water molecules, yielding an R-factor of
21.0% and an Rfree-factor of 27.8%. The
root-mean-square deviations in bond lengths and angles were 0.007 Å and 1.2°, respectively. The refinement statistics are summarized in
Table I. Coordinates of the derivative P450nor have been deposited at the Protein Data Bank (identification code 1GED).
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Table I
Crystal parameters, data collection, and structure refinement
Values in the outer shell are for the highest shell with a resolution
of 2.09-2.0 Å.
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RESULTS |
Positively Charged Cluster on the Distal Side of P450nor--
The
crystal structure revealed many Lys and Arg residues around or inside
the heme pocket on the distal side of P450nor (6), as shown in Fig.
1. Among these positively charged
residues, Lys77, Lys81, Arg182, and
Arg392 are located on the surface of the entrance of the
distal-heme pocket, and Lys62, Arg64,
Arg174, Lys291, and Arg292 are
buried inside the pocket. We constructed a series of mutant proteins by
site-directed mutagenesis in which these five Arg and four Lys residues
were replaced, respectively, with a negative charge (Glu) or a neutral
(Gln or Ala) amino acid residue. We then investigated their properties
to examine our hypothesis that this unique structure (positive charge
cluster) is responsible for the direct interaction of P450nor with
NADH. The results are summarized in Table
II. On the other hand, the negatively
charged groups, Asp88 and Asp393, are also
located inside the pocket. The presence of so many charged groups along
with many water molecules (7) makes the environment of the distal
pocket highly positive and hydrophilic unlike those of other P450s.
Therefore, this environment is very likely the structure that attracts
negatively charged, hydrophilic NAD(P)H.

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Fig. 1.
Stereo view of the heme-distal pocket
and the positively charged cluster of P450nor. Positively and
negatively charged amino acid residues are shown in blue and
red, respectively. Heme (violet) and charged
residues are represented by the stick model. Other residues
are illustrated by the ribbon model. Amino acid residues are
represented by one-letter abbreviations.
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Table II
Kinetic parameters of the P450nor mutants
Averaged values of experiments repeated at least three times are
presented. Experimental error was below 20% with the exception of the
spin state ratio (less than 10%). ND, not detected; , not
determined.
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Effects of Mutations on Overall Nor Activity--
All of
these mutations, except that at Lys81, modulated overall
Nor activity to various extents (Table II). Mutations at the residues
inside the distal pocket (Lys62, Arg64,
Arg174, Lys291, and Arg292)
decreased the activity more than those outside the pocket
(Lys77, Lys81, Arg182, and
Arg392), and mutation to a negative charge (K62E,
R64E) decreased the activity more than mutation to a neutral amino acid
at the same position (K62A, R64Q). R174E, R174Q, R64E, and R292E
completely lost overall activity, and mutations at Lys62
also considerably decreased the activity. All of these mutant proteins
except R292E seemed to be folded correctly in heterologous E. coli cells because they formed complexes with carbon monoxide (data not shown). By contrast, R292E was formed as an unfolded protein
without heme. This finding is reasonable because Arg292
corresponds to the conserved Arg residue (for example
Arg299 in P450cam) that is essential for binding heme to
the protein moiety (17). Arg292 of P450nor also interacts
with one of the propionate side chains via a water
molecule.2 Large amounts of
these mutant proteins were expressed and purified for further characterization.
Spectral Perturbation by Mutations--
Fig.
2A shows that the ferric
resting heme of native P450nor is in a mixture of high and low spin
states (1). All of the mutations inside the pocket caused a reverse
type-1 spectral change in the bound heme of each resulting mutant
protein, that is, an increase in the ratio of the low spin state (Fig.
2B, Table II), whereas those at Lys77,
Lys81, and Arg182, which are located outside
the pocket, did not. The extent of the spectral perturbation did not
correlate with the remaining overall activity but seemed to reflect the
distance between the mutation site and heme. The mutations at
Lys62 and Lys291, which are located
close to heme, induced the largest perturbation, whereas mutations at
Arg174, which is located further away from
heme, caused a complete loss of the activity but only a weak
perturbation. However, mutation at the side chain of
Arg392, which is located on the entrance of the pocket and
oriented toward the outside, caused moderate inactivation and spectral perturbation. Its neighbor, Asp393, is involved in the
proton delivery network (6, 7). Therefore, the mutation of
Arg392 may have perturbed this network, which in turn
resulted in spectral changes in heme and partial
inactivation of the enzyme.

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Fig. 2.
Optical absorption spectra of native
(A) and R174E mutant (B) P450nor in a
ferric resting state in the presence (solid line) or
absence (dashed line) of 1.0 M KBr.
Purified protein was dissolved in 50 mM TES buffer (pH
7.2).
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Effects of Mutations on the Binding of NO--
Each mutant protein
was further characterized by analyzing its partial reactions (Schemes
2-S4). During the first step of the Fe3+-NO complex
formation (Scheme 2), all mutant proteins except R292E bound NO (data
not shown). Their Fe3+-NO form afforded essentially the
same spectra as native P450nor with a Soret peak at 431 nm
(cf. Figs. 3 and 4). This
binding process was observed by flash photolysis (2). This step is very
rapid, and thus the minor modulation of the apparent rate constant
(kobs) observed with each mutant (R64E, R64Q,
and R174E; Table II) cannot be used to explain such a large decrease in
the overall activity.

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Fig. 3.
Absorption spectral change during reduction
(I formation, Scheme 3) observed with native P450nor
by the rapid scan (A) or the stopped-flow
(B) method. P450nor in
Fe3+-NO (final, 5 µM) was mixed with NADH
(final, 20 µM) anaerobically at 10 °C in 50 mM TES buffer. The gate time was set to 1 ms. The single
exponential kinetic trace in panel B analyzed using the
Guggenheim curve fitting estimated the value of
kobs.
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Effects of Mutations on the Interaction with NADH--
Rapid scan
analysis (2) detected a spectral intermediate l, with a
Soret peak at 444 nm, when the Fe3+-NO complex was mixed
anaerobically with NADH, which is assumed to have arisen from the
2-electron reduction of Fe3+-NO with NADH (Scheme 3) (2,
18). Fig. 3 shows a spectral change typical of this process with native
P450nor. Although the reduction step (Scheme 3) would be rate-limiting
in the overall reaction (2), I can accumulate when the
amount of NO added is similar to that of the enzyme (P450nor). Under
such conditions I cannot react further with the second NO
because no free NO remains, and thus I can be in a
quasi-stable state suitable for accumulation (Fig. 3A) (2).
Fig. 3B shows the value of kobs for
the reduction (I formation) obtained from the
time-dependent decrease in the absorbance at 427 nm, which
is the isosbestic point of the spectrum of I and the resting
enzyme Fe3+ (with 413 nm peak) (2). We found that
kobs (reduction) for each mutant protein was
decreased in parallel with the decrease in the overall activity (Table
II). A typical spectral change during the reduction of a mutant protein
(R64E) is shown in Fig. 4. When
Fe3+-NO was reduced with a low concentration of NADH (Fig.
4A), I was undetectable. However, when the
concentration of NADH was increased, a small amount of l was
detected (Fig. 4B). These results indicate that since the
formation rate of I of the mutant is very slow, it is
comparable with the decomposition rate, resulting in no accumulation
of I and only Fe3+ species formation (Fig.
4A).

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Fig. 4.
Spectral change during reduction of R64E
mutant. Experiments were performed in the same manner as
described in the legend to Fig. 3. NADH concentrations: A,
20 µM; B, 0.5 mM.
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The results with other mutants were similar, showing that the decline
in the overall Nor activity in each mutant arose from interruption of
the reduction step because of the mutations. R174E and R174Q did not
form I at all even after reduction with higher concentrations of NADH, which is consistent with their complete loss of
Nor activity and highlights the crucial role of Arg174 in
the interaction with NADH.
Effects of Mutations on the Interaction of I with the Second
NO--
This process (Scheme 4) cannot be observed as an isolated
reaction by rapid reaction analyses, unlike the former two steps. However, this step apparently was not modulated so much by each mutation. This was suggested from the finding that the amount of
accumulated I as a result of the reduction of each mutant
decreased in parallel with the decrease in kobs
(reduction) (only the result obtained with R64E is shown in Fig. 4),
which showed that the next step (Scheme 4) was much faster than the
lowered reduction step. This conclusion is also consistent with
the finding that the overall activity of each mutant decreased
approximately in parallel with kobs (reduction)
(Table II).
Interactions with Anions--
Halogen anions (19) or phosphate
(present results) cause a reverse type-1 spectral change in the bound
heme (Fig. 2A) and inhibit the overall reaction of P450nor
(1). The Kd values obtained by spectrophotometric
titration in TES buffer for bromide (Br
), chloride
(Cl
), and phosphate were 100, 100, and 30 mM,
respectively (data not shown). These anions inhibited the reduction
step (Scheme 3). Fig. 5 shows the results
obtained with only Br
. The half-inhibitory concentrations
for Br
and Cl
were 50 mM (Fig.
5) and 120 mM (data not shown), respectively, which
essentially agreed with the respective Kd values. These results demonstrated that the reduction step of P450nor was
inhibited by the binding of these anions, which caused a decline in the
overall Nor activity. Thus, the introduction of a negative charge(s)
into the heme pocket by either mutation or anion binding exerted
similar effects, that is, a reverse type-1 spectral change and
inhibition of the reduction step.

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Fig. 5.
Inhibition of the reduction step (Scheme 3)
of native P450nor by bromide. The kobs
value was determined as described for Fig. 3B in the
presence of the indicated concentration of KBr.
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The effects of Br
were also examined with the mutant
proteins with respect to the spectral perturbation. The spectrum of
each of K77E, K81E, and R182E did not alter by the mutations. These mutant proteins were perturbed spectrally upon mixing with
Br
to almost the same extent as the native protein (data
not shown). By contrast, K62A, K62E, and K291E, which were already
perturbed greatly by the mutations, were not further perturbed at all
by Br
(data not shown). The mutations at
Arg64 and Arg174 caused moderate spectral
changes. Mixing these mutant proteins with Br
caused
further reverse type-1 spectral change, as shown in Fig. 2B
with the results on R174E. These results demonstrated that the
mutations and the binding of Br
perturbed the heme
environment in the same manner.
Identification of Br
Binding Sites--
Fig.
6 shows the Br
binding sites identified by x-ray crystallography. The most
ordered Br
(Br1), located under the F-helix, was
surrounded by the side chains of Asn241 and
Ser150 and the backbone carbonyl of Thr168.
This site is situated rather far from heme (Fig. 6A), beyond the middle of the I-helix where the helix is interrupted (6). Another
ordered Br
(Br2) was buried deep inside the pocket in a
space between heme and the polypeptide backbone near
Ala289. The polypeptide backbone is bent upward at this
position to create a space for Br
(anion hole).

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Fig. 6.
Stereo view of the heme-distal pocket
of P450nor focused on Br binding sites.
A, ribbon diagram of heme-distal pocket of
P450nor focused on Br binding sites (red
spheres). -Helices are shown as blue coils and loops
as yellow tubes. The temperature factors of the bromide ions
were 29 and 30 Å2, respectively. Heme is represented by
the ball-and-stick mode. This figure was produced using
MOLSCRIPT (25). B, stereo view of the final electron density
map (|2Fo Fc|) for
Br . The map was calculated from reflections in the
resolution range of 30.0 to 2.0 Å and is contoured at 1.5 s,
where s is the S.D. of the electron density map. Atomic positions are
indicated by specific colors: yellow, carbon; sky
blue, nitrogen; and red, oxygen. The position of Br2 is
represented by a red sphere. This figure was produced using
TURBO-FRODO (14).
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 |
DISCUSSION |
Halogen (Br
) ions exerted effects that were
apparently similar to those caused by mutations at Arg or Lys residues
inside the heme pocket, that is, a reverse type-1 spectral change and inhibition of the reduction step. However, the extent of spectral perturbation by mutations was not proportional to that of concomitant inactivation (Table II) but seemed to depend on the distance of the
mutation site from heme. Thus, introducing a negative charge into the
neighborhood of heme would cause a larger spectral perturbation. Based on this assumption, the spectral change along with the inhibition of the reduction caused by Br
arose mainly from the
binding of Br
to the site near heme (Br2 in
Fig. 6; anion hole). The preference of anions as the binding
site for this hole to the positive charge cluster composed of Arg and
Lys residues suggests that the anion hole can attract a negative charge
or an electron-rich moiety. This anion hole occupies a key spot in the
pocket, neighboring heme, the terminus of the positively charged
cluster (Lys62), and the terminus of the proton delivery
network (Ser286). These results suggest that this anion
hole is the location of electron and proton transfer to the
Fe3+-NO complex (Scheme 3). If so, electron transfer from
NADH should be interrupted if Br
occupies this hole. On
the other hand, the spectral perturbation suggests that the hydrogen
bond network around heme, which contains many water molecules (7), was
altered or disrupted upon the binding of Br
or mutations.
Such a misalignment of the network induced in the Fe3+-NO
complex would also interrupt the electron transfer that must accompany
the concomitant transfer of proton(s) supported by the hydrogen-bond
network (7).
The discussion above implies that Br
bound at Br2
interrupts the electron transfer from NADH to heme rather than the
binding of NADH. Conversely, the findings that mutations at
Arg174 completely disrupted the reducing step but only
slightly perturbed the heme environment (Table
II) along with the results that the R174E mutant was further perturbed by Br
(Fig.
2B) are highly indicative that the mutant proteins (R174E, R174Q) were inactivated because their NADH binding ability was lost.
Whether the binding or the electron transfer was disrupted could be
confirmed if the Km for NADH and the maximum reduction rate could be compared between native and mutant proteins. However, we could not determine these kinetic constants with native P450nor because the reduction rate was too high even at 10 °C and
was therefore beyond the capability of the instrument to analyze the rapid reaction.
The Lys and Arg residues examined in this study were distributed from
outside the entrance of the heme-distal pocket to the vicinities of
heme (Fig. 1). The present results showed that these positively charged
amino acid residues are critically important for interaction with NADH,
although the extent of their contribution to the interaction would vary
depending on the role of each residue. All positive charges located
outside the entrance would introduce NADH to the entrance. Four of the
positive charges inside the pocket (Lys62,
Arg64, Arg174, and Lys291) have
side chains that are oriented toward the space of the pocket. The
B'-helix and the loop structure between the F- and G-helices (F-, G-loop) forms the entrance gate of the pocket, and
Arg64 and Arg174 are located just beneath
B'-helix and the F-, G-loop, respectively. Their side chains are
opposite each other (Fig. 1). The side chain of Lys291
extends upward from the bottom of the pocket, and its top reaches the
vicinity of the side chain of Arg64. Thus, these residues
form a positively charged barrier just beneath the entrance gate, which
should immobilize the NADH molecule by placing between these
residues the negatively charged pyrophosphate moiety of NAD(P)H.
In addition, Lys62 should also be important for the
interaction with NADH. Mutations at this site resulted in both an
enormous decline of kobs (reduction) and a large
spectral perturbation. Therefore, to identify whether electron transfer
or binding was disturbed is difficult. Lys62 is located in
the vicinity of heme within the positive cluster, and its side chain is
oriented toward heme (Fig. 1). This side chain might orient an NADH
molecule toward the correct direction when NADH accesses heme.
The positively charged cluster should be the novel motif for
interacting with NADH that is responsible for the unique catalytic property of P450nor, namely direct interaction with NAD(P)H. The motif
is in sharp contrast to the Rossman fold structure (20, 21).
Comparisons of several crystal structures of P450 species indicate that
substrate recognition site 1 (SRS-1) (according to Gotoh) (22), which
contains a B'-helix, is the most variable structure among P450
molecular species (23, 24). This finding also appears to hold true for
P450nor, as shown in Fig. 7. SRS-1 of
P450nor contains 6 positive amino acid residues including 4 (Lys62, Arg64, Lys77, and
Lys81) that we investigated in the present study and 3 negative groups (Glu71, Asp88, and
Asp90); this is in contrast to the corresponding region of
P450cam, which contains only 1 positive and 5 negative groups. The
other essential residues, Arg174 and
Lys291, are located in SRS-2 and SRS-5,
respectively. Furthermore, Ser286 and Asp393,
which are involved in the proton delivery system (6, 7) are also
involved, respectively, in SRS-5 and SRS-6. The present results
indicate that P450nor has evolved to fit an interaction with NAD(P)H
rather than with an organic substrate to be hydroxylated by
changing mainly the amino acid residues in SRS regions. The results of
the present study and others indicate that P450nor receives electrons
from NADH on the distal side in contrast to other monooxygenase P450s
that receive electrons on the proximal side.

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Fig. 7.
Alignment of the primary structures
of SRS-1 containing the B'-helix of cytochromes P450cam and
P450nor. Positively and negatively charged residues are shown in
blue and red, respectively. The extent of the
B'-helix is indicated only for P450nor.
|
|
 |
FOOTNOTES |
*
This study was supported by the Program for Promotion of
Basic Research Activities for Innovative Bioscience (PROBRAIN),
Grant-in-aid 11116205 for Scientific Research from the Ministry of
Education, Science, Culture and Sports of Japan, and the Sakabe Project
of the High Energy Accelerator Research Organization (KEK), Tsukuba, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1ROM and 1GED) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§
To whom correspondence should be addressed. Tel.:
+81-298-53-4603; Fax: +81-298-53-4605; E-mail:
p450nor@sakura.cc.tsukuba.ac.jp.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M007244200
2
S.-Y. Park, H. Shoun, and Y. Shiro, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
P450nor, cytochrome
P450nor;
NO, nitric oxide;
Nor, nitric-oxide reductase;
TES, N-tris(hydroxymethyl)methyl-2-aminoethane-sulfonic acid;
MES, 2-morpholinoethanesulfonic acid;
Br
, bromide.
 |
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