From the Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
The role of two essential residues at the
N-terminal hook region of neuronal nitric-oxide synthase (nNOS) in
nitric-oxide synthase activity was investigated. Full-length mouse nNOS
proteins containing single-point mutations of Thr-315 and Asp-314 to
alanine were produced in the Escherichia coli and
baculovirus-insect cell expression systems. The molecular properties of
the mutant proteins were analyzed in detail by biochemical, optical,
and electron paramagnetic resonance spectroscopic techniques and
compared with those of the wild-type enzyme. Replacement of Asp-314 by
Ala altered the geometry around the heme site and the substrate-binding
pocket of the heme domain and abrogated the ability of nNOS to form
catalytically active dimers. Replacement of Thr-315 by Ala reduced the
protein stability and altered the geometry around the heme site,
especially in the absence of bound
(6R)-5,6,7,8-tetrahydro-L-biopterin cofactor. These results suggest that Asp-314 and Thr-315 both play critical structural roles in stabilizing the heme domain and subunit
interactions in mouse nNOS.
Nitric-oxide synthase
(NOS)1 is a complex
flavo-hemoprotein that catalyzes the conversion of L-Arg to
citrulline in the presence of oxygen, NADPH,
6(R)-5,6,7,8-tetrahydro-L-biopterin
(H4BP), and Ca2+-calmodulin complex with
concomitant production of nitric oxide in two stepwise monooxygenase
reactions involving
N Oxygen activation in the regular cytochrome P450-type monooxygenase
reaction requires a proton donor at the distal side of the heme center
(11, 12, 28-35). In many regular cytochrome P450s, this proton donor
is associated with a water molecule, which is usually hydrogen bonded
to the conserved distal threonine residue or the adjacent conserved
aspartate/glutamate residue located in the central helix I; whenever
these conserved residues exist, they are arranged in a motif,
(Asp/Glu)-Thr, positioned ~100 amino acid residues upstream of the
cysteine ligand to the heme center (11, 29, 32, 33, 36-41). It was
postulated, before the x-ray crystal structure determination of the
dimeric iNOS heme domain by Crane et al. (14), that an
analogous proton donor might exist in the NOS isoforms to facilitate
the cytochrome P450-type monooxygenation reaction (42, 43). Amino acid
sequence comparisons of NOS isoforms and cytochrome P450cam and
P450BM-3 were made, with particular attention to the sequence motif,
(Asp/Glu)-Thr, conserved in regular cytochrome P450s (Fig. 1). Among
the 10 strictly conserved threonine residues in the heme domain of all
NOS isoforms, a single site in the N-terminal region, positioned
approximately 100 amino acid residues upstream of the cysteine ligand
(Cys-415 in mouse nNOS), was found to have the conserved motif Asp-Thr (Asp-314-Thr-315 in mouse nNOS; see Fig. 1). Thus, Asp-314 and Thr-315
of mouse nNOS were considered as candidates for the putative distal
proton donor. Site-directed mutagenesis studies were conducted with a
heme domain fragment produced in an Escherichia coli
expression system by us (42) and with full-length recombinant nNOS
produced in a yeast expression system by others (43). Although the
replacement of Asp-314 of the mouse nNOS heme domain fragment by Ala
led to the formation of an inactive cytochrome P420-like species
(42),2 the results of the
latter studies with recombinant full-length nNOS led to the proposal
that Asp-314 might serve as the putative distal proton donor by analogy
with regular cytochrome P450s (43). This controversy remains to be resolved.
The recent x-ray crystal structural analysis of the
H4BP-bound dimeric iNOS heme domain fragment has shown
unambiguously that neither Thr-315 nor Asp-314 is located at the distal
substrate-binding pocket of iNOS, and that these residues are in fact
located at the N-terminal hook region, far from the distal heme site
(14). The N-terminal hook of the iNOS heme domain fragment is
critically involved in the dimer interface and the binding of the
pterin cofactor of the dimeric heme domain, in close proximity to the H4BP-binding site (14). In this study, we report a detailed biochemical and spectroscopic investigation of full-length recombinant Asp-314 Materials--
Synthetic DNA oligomers were purchased from
either SCI-MEDIA (Tokyo, Japan) or Nissinbo (Tokyo, Japan), and DNA
modification enzymes and restriction enzymes were from either New
England Biolabs or Takara Biomedicals (Otsu, Japan). 2',5'-ADP
Sepharose 4B, Sephacryl S-200HR, DEAE-Sepharose Fast Flow, and Ampure
SA were purchased from Amersham Pharmacia Biotech. Calmodulin, FAD,
FMN, L-Arg, and L-citrulline were from Sigma,
and 6(R)-5,6,7,8-tetrahydro-L-biopterin (H4BP) was from the Schircks Laboratories (Jona,
Switzerland). L-[14C-U]Arg was obtained from
NEN Life Science Products. Water was purified by a Milli-Q purification
system (Millipore). Other chemicals used in this study were of
analytical grade.
DNA Manipulations--
The baculovirus-Spodoptera
frugiperda (Sf9) insect cell expression system
(44) and E. coli pCWori+ expression system (45-47) were
employed for the expression of mouse full-length wild-type nNOS and the
single-point mutant enzymes T315A, T315S, and D314A. Unless otherwise
stated, vectors were constructed by utilizing E. coli HB101
as the host strain. The site-directed mutagenesis was done by using a
Muta-Gene Phagemid in vitro mutagenesis kit (Bio-Rad). All
of the altered DNA sequences were analyzed by using a Sequenase version
2.0 DNA sequencing kit (United States Biochemical Co).
Introduction of Amino Acid Substitutions--
E. coli strain
CJ236 (Bio-Rad) was transformed with pTZ19RNOS1, and this transformant
in the mid-log phase was infected with helper phage M13KO7 (Bio-Rad) in
the presence of ampicillin, chloramphenicol, and kanamycin to prepare
single-stranded DNA. The phage particles were collected by polyethylene
glycol precipitation, and the single-stranded DNA was extracted with
phenol-chloroform and recovered by ethanol precipitation. The following
5'-phosphorylated DNA oligomers were utilized to construct the nNOS
variants: 5'-GAT GTG GTC CTC ACT GAT GCA TTG CAC CTG AAG AGC ACG-3' for
T315A, 5'-GAT GTG GTC CTC ACC GCG ACC CTG CAC CTG AAG AGC ACG-3' for
D314A, and 5'-GAT GTG GTC CTC ACT GAT TCC CTG CAC CTG AAG AGC ACG-3'
for T315S.
Expression of the Full-length Wild-type nNOS and the Variants
Using the E. coli Expression System--
A heterologous expression
system for the full-length wild-type nNOS and the variants in E. coli was constructed with pCWori+ vector (45-47) and the
chaperonin expression vector pKY206 (pACYC184 (Nippon Gene, Toyama,
Japan) carrying the E. coli chaperonin groELS genes (kindly provided by Dr. K. Ito, Kyoto University) as reported (46, 47), with the following minor modifications. Firstly, a
NdeI site was newly generated to include the first Met codon of the cDNA for the wild-type nNOS in pTZ19RNOS1 by using the mutation primer (NOSNde I: 5'-GAC GGC CAG TGA GAA CAT ATG GAA GAG CAC
ACG-3'). Because the resultant plasmid
(pTZ19RNOS1/NdeI) has two NdeI sites, it
was partially digested with NdeI, followed by complete
digestion with XbaI. The NdeI-XbaI
fragment containing the full-length nNOS1 cDNA was then inserted
between the NdeI and XbaI sites of the
multicloning linker of pCWori+. The pCWori vectors for the variants
(pCWoriNOS1/D314A, pCWoriNOS1/T314A, and pCWoriNOS1/T315S) were
individually prepared by exchanges of the
AflII-XbaI fragment encoding the D314-T315
region. The resultant pCWori+ vectors and pKY206 were introduced by
repeated transformation into the host strain, E. coli strain
BL21 (Takara Biomedicals), which lacks the two proteases lon
and ompT. The recombinant enzymes were expressed as
described in the literature (46, 47).
Expression of Full-length Wild-type nNOS and the Variants Using
the Baculovirus-Sf9 Insect Cell System--
The plasmid vector
pTZ19RNOS1, carrying the full-length cDNA for mouse nNOS reported
previously (48), and the baculovirus transfer vector pJVP10Z (49)
kindly provided by Dr. S. Kawamoto (Yokohama City University) were
used. The transfer vectors carrying the cDNA for the wild-type nNOS
and the variants were individually constructed for recombination of
AcNPV as follows: an NheI site was newly generated upstream
of the first Met codon of the cDNA for the wild-type nNOS in
pTZ19RNOS1 with a phosphorylated DNA oligomer (NOSNhe I: 5'-GTA AAA CGA
CGG CCA GTG AGC TAG CAT GGA AGA GCA CAC G-3'). The DNA fragment coding
the altered 5'-leader sequence and the N-terminal region of nNOS was
exchanged for the corresponding region of the variants by utilizing two
unique restriction enzyme sites, the ScaI and
AflII sites. The obtained plasmids (pTZ19RNOS1/NheI, pTZ19RNOS1/NheI/D314A,
pTZ19RNOS1/NheI/T315A, and pTZ19RNOS1/NheI/T315S)
were excised by NheI and XbaI digestion, and the
DNA fragment encoding the full-length nNOS gene was ligated into the
NheI site of pJVP10Z utilizing the compatibility between XbaI and NheI sites. The direction of the
cDNA was identified by DNA sequencing.
Coinfection with AcNPV DNA and constructed transfer vectors was
conducted by using a Linear Transfection Module (Invitrogen), and the
screening of the recombinant virus was carried out according to the
manufacturer's manual, Max Bac Baculovirus Expression System Manual
(Invitrogen), and the literature (44). The recombinant enzymes were
produced in the presence of riboflavin, hemin, and sepiapterin as
described in the literature (20, 50, 51).
Activity Measurement--
NOS activity was measured by
monitoring the conversion of L-[14C-U]Arg to
L-[14C-U]citrulline as described previously
(52). The standard assay was performed at 25 °C in assay mixture
containing 16.7 mM HEPES-NaOH buffer, pH 7.4, 4.2 mM Tris-HCl buffer, pH 7.4, 667 µM EDTA, 167 µM EGTA, 667 µM dithiothreitol, 16.7 µM L-[14C-U]Arg, 667 µM NADPH, 1.2 mM CaCl2, 6.7 µg
of calmodulin, 1.25 µM FAD, 1.25 µM FMN,
2.5 µM H4BP, and the enzyme, in a total
volume of 30 µl. The specific activity of
L-[14C-U]Arg used in the assays was 11.84 GBq/mmol.
Purification of Recombinant nNOS--
Purification of
recombinant enzymes produced in E. coli strain BL21 was
performed on ice or at 4 °C essentially as described in the
literature (47), except that purification was conducted using a
2',5'-ADP Sepharose 4B column chromatography (Amersham Pharmacia
Biotech), followed by Sephacryl S200HR and DEAE-Sepharose Fast Flow
column chromatography (Amersham Pharmacia Biotech), and that the
overnight dialysis step (47) was omitted. H4BP (10 µM) was supplied in the ultrasonification step, unless
otherwise stated. The catalytic activity and the purity of the purified wild-type enzyme, nNOS1, were comparable to those previously reported for recombinant nNOS1 by others (47).
Recombinant enzymes produced using the baculovirus-insect cell
(Sf9) expression system were partially purified on a
2',5'-ADP Sepharose 4B column (Amersham Pharmacia Biotech), followed by a fast desalting column, Ampure SA (Amersham Pharmacia Biotech).
Analytical Procedures--
Absorption spectra were recorded
using a Hitachi U3210 spectrophotometer or a Beckman DU-7400
spectrophotometer. EPR measurements were carried out using a JEOL
JEX-RE1X spectrometer equipped with an Air Products model LTR-3
Heli-Tran cryostat system, in which the temperature was monitored with
a Scientific Instruments series 5500 temperature indicator/controller
as reported previously (53). EPR spectra of several different batches
of recombinant nNOS samples were also measured at JEOL Ltd. (Tokyo,
Japan), using a JEOL JES-TE200 spectrometer equipped with an ES-CT470
Heli-Tran cryostat system, in which the temperature was monitored with
a Scientific Instruments digital temperature indicator/controller model
9650, and the magnetic field was monitored with a JEOL NMR field meter
ES-FC5. All spectral data were processed using KaleidaGraph software,
version 3.05 (Abelbeck Software).
Purified nNOS was estimated by using the Coomassie protein assay
reagent (Pierce) with bovine serum albumin as a standard. The multiple
sequence alignments of the NOS isoforms and cytochrome P450s were
performed using the CLUSTAL X graphical interface (54) with small
manual adjustments.
Among 10 conserved threonine residues in the heme domain of all
NOS isoforms, only Thr-315 in the N-terminal region of mouse nNOS lies
in an Asp-Thr motif (Asp-314-Thr-315 in mouse nNOS) (42, 43),
resembling that found at the helix I region of many regular cytochrome
P450s (29, 32) (Fig. 1). To investigate the possible function of these conserved residues, Asp-314 and Thr-315,
at the N-terminal hook of mouse nNOS, each of the two residues was
subjected to single-point mutagenesis. The resultant full-length mutant
enzymes were heterologously produced in E. coli and
baculovirus-insect cell (Sf9) expression systems and characterized biochemically and spectroscopically as described below.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-hydroxy-L-arginine as an
intermediate (1-6). Studies on the heme centers of the NOS isoforms by
absorption, electron paramagnetic resonance (EPR), resonance Raman, and
magnetic circular dichroism spectroscopy, together with mutational
analysis, have strongly suggested that an endogenous thiolate sulfur
donor ligand is coordinated to the central heme iron, as in the cases
of cytochrome P450 and chloroperoxidase (7-12). This was confirmed by
the x-ray diffraction analysis of inducible NOS (iNOS) heme domain
fragments (13, 14). A unique feature of the NOS isoforms is the
requirement of dimerization, which is facilitated by binding of the
pterin cofactor (5, 6, 15-27), for the citrulline- and NO-forming activity.
Ala and Thr-315
Ala mutants of mouse nNOS produced using E. coli and baculovirus-insect cell
(Sf9) expression systems either in the absence or
presence of added H4BP. Our results strongly suggest that
Asp-314 and Thr-315 both play critical structural roles in maintaining
protein stability and the geometry around the heme site. These
conclusions are consistent with inferences based on the recent x-ray
structural analysis of the dimeric iNOS heme domain (14) and provide
complementary evidence for the structural importance of the hairpin
loop of the N-terminal hook of mouse nNOS. A part of the present work
has been presented elsewhere (42).
EXPERIMENTAL PROCEDURES
RESULTS
View larger version (39K):
[in a new window]
Fig. 1.
Multiple amino acid sequence alignments of
the putative N-terminal hook region of selected NOS isoforms and the
central helix I region of cytochromes P450cam and P450BM-3. The
sequence data were retrieved from data bases using the BEAUTY and BLAST
network service (59), and the multiple sequence alignments of the
N-terminal region of the NOS isoforms and cytochrome P450s
(shaded) were performed using a CLUSTAL X graphical
interface (54) with minor manual adjustments. The secondary structural
elements, N-terminal hook, N-terminal pterin-binding segment, and the
heme-binding site defined by the x-ray crystal structure of the dimeric
iNOS heme domain fragment (14) are indicated. The positions of
the two strictly conserved residues, Asp-314 and Thr-315 of mouse nNOS,
are highlighted, together with Cys-415, which serves as a
thiolate ligand to the central heme iron. It should be noted that
Asp-314 and Thr-315 of mouse nNOS are located at the hairpin turn of
the putative N-terminal hook region, whereas Asp-251 and Thr-252 of
cytochrome P450cam are located at the central helix I (29).
Expression and Biochemical Characterization of the Wild-type and Mutant Enzymes Produced Using the E. coli Expression System-- The recombinant full-length wild-type and mutant enzymes, T315A, T315S, and D314A, were produced in an E. coli expression system co-producing E. coli chaperonin GroELS (46, 47). Fig. 2 shows the CO-reduced minus reduced difference spectra of the crude wild-type and mutant enzymes in the E. coli lysate prepared in the presence of added H4BP (10 µM). A cytochrome P450-like heme center was apparent in the difference spectra when the recombinant wild-type enzyme, T315A, or T315S was produced in E. coli but was virtually absent when D314A was produced (see Fig. 2).2 T315S was constructed because the serine residue has a hydroxyl group, as does the threonine residue, although essentially the same results were obtained for T315A and T315S. Parallel measurements of the citrulline-forming activity of the crude enzymes in the presence of 10 µM H4BP suggested that the recombinant wild-type enzyme and T315A were active, T315S was less active, and D314A was completely inactive (data not shown; see below).
|
The wild-type and T315A enzymes were purified to near electrophoretic homogeneity in the presence of added H4BP (10 µM) (see under "Experimental Procedures"). The purified T315A showed a single 160-kDa band in SDS-polyacrylamide gel electrophoresis (data not shown). The as-isolated mutant enzyme showed citrulline-forming activity of ~80 nmol/mg/min at 25 °C, which was lower than that of the wild-type enzyme (~220-280 nmol/mg/min at 25 °C; see Table I). This activity was greatly enhanced after preincubation of the recombinant enzyme in the presence of L-Arg and H4BP, when the purified T315A showed activity as high as ~390 nmol/mg/min at 25 °C with an apparent Km for L-Arg of 1.1 µM, which is comparable to the values of the wild-type enzyme (~360-450 nmol/mg/min at 25 °C with an apparent Km for L-Arg of 1.3 µM), as summarized in Table I.
|
Spectroscopic Characterization of the Wild-type and T315A Enzymes Purified in the Presence of H4BP-- Fig. 3 shows the visible absorption spectra of the wild-type and T315A enzymes purified in the presence of 10 µM H4BP (solid traces). The optical properties of the two are essentially identical in that the resting enzymes were predominantly high spin as isolated, exhibiting a broad Soret band at around 395-400 nm (Fig. 3, A and B, solid traces), and they showed a sharp peak at 444 nm in the CO-reduced minus reduced difference spectra (Fig. 3C, solid traces).
|
The properties of the ferriheme center of the purified enzymes were further investigated by EPR spectroscopy, which is a sensitive technique to detect minor changes at the ferriheme site (Fig. 4). The EPR spectra at 16 K of the ferriheme center of the resting wild-type enzyme showed a high spin component at g = 7.68, 4.07, and ~1.8 as the predominant species (Fig. 4A). The rhombicity (defined as the ratio of the rhombic and axial zero field splitting parameters, E/D) of this high spin ferriheme species is 0.075, which is lower that of the hydroxyarginine-bound high spin ferriheme species (E/D = 0.077; data not shown). Incubation of the enzyme with 10 µM H4BP in the absence of added L-Arg for 30 min did not cause any change of the apparent g values of the high spin ferriheme; instead, a decrease of the relative intensity of the g = 7.68 signal was observed (Fig. 4B). This implies that the H4BP cofactor may bind to substrate-free ferric nNOS with minimal disruption of the ligation geometry of the high spin heme site and/or may stabilize the ferrous state, although the details remain to be investigated.
|
The resting wild-type enzyme also contained at least two overlapping
low spin ferriheme species at g = 2.45-2.41, 2.28, and 1.91-1.90, which are more clearly observed at ~20-25 K (data not shown), in addition to a sharp flavin semiquinone radical at
g = 2.0. The calculated crystal field parameters of
tetragonality (/
) and rhombicity
(V/
) of the predominant low spin ferriheme species at g = 2.41 are consistent with the axial
coordination of an oxygen ligand trans to the proximal
cysteine thiolate ligand (56). Addition of 0.1 mM
L-Arg to the full-length wild-type enzyme resulted in
disappearance of the remaining g = 2.41 low spin
ferriheme and the formation of a new high spin species at g = 7.59, 4.08, and 1.81 (Fig. 4C), and a
decrease of the rhombicity E/D of the high spin ferriheme
(E/D = 0.073) was observed. This suggests that
L-Arg binds to the wild-type enzyme without direct coordination to the ferriheme center.
The EPR spectrum of the ferriheme center of the resting mutant enzyme T315A purified in the presence of 10 µM H4BP (Fig. 4D) showed a high spin species, but with slightly different g values (g = 7.62, ~4.07, and ~1.82); its rhombicity E/D of 0.074 is similar to that of the L-Arg-bound high spin ferriheme species at g = 7.59, 4.08, and 1.81 (E/D = 0.073). Its high spin ferriheme content was typically slightly lower by 10-20% than that of the wild-type enzyme, which may contribute to the lower citrulline-forming activity of T315A as isolated (prior to preincubation, see Table I). Nevertheless, addition of 0.1 mM L-Arg resulted in the conversion of the remaining low spin ferriheme of T315A to the high spin species (Fig. 4E), the apparent g values and rhombicity of which (E/D = 0.073) were essentially identical to those of the L-Arg-bound wild-type enzyme (Fig. 4C). These data suggest that the binding of L-Arg causes a local conformational change in both the wild-type and T315A enzymes that results in a substantial change in the geometry and spin-state of the ferriheme site. Thus, the EPR results confirm that the mutant enzyme T315A purified in the presence of H4BP is capable of binding L-Arg without direct coordination to the central heme iron and that the resultant H4BP- and L-Arg-bound high spin ferriheme is virtually identical to that of the wild-type enzyme.
Spectroscopic Characterization of D314A Purified in the Presence of H4BP-- D314A partially purified in the presence of H4BP was very unstable even in the presence of added L-Arg and H4BP and exhibited no citrulline-forming activity (data not shown). Gel filtration analysis with a calibrated Tosoh G-3000SWXL column connected to a high performance liquid chromatography system suggested that purified D314A is predominantly multioligomeric, and not dimeric (Fig. 5, bottom trace). It has been reported previously that the multioligomeric form of NOS is inactive and exhibits only a monomer band (but no SDS-resistant dimer band) in low temperature SDS-polyacrylamide gel electrophoresis, indicating that it probably represents the multioligomeric state of monomer molecules (see Refs. 19, 20, and 57). Thus, replacement of Asp-314 by Ala affected the ability of mouse nNOS to form catalytically active dimers even in the presence of added pterin cofactor.
|
The visible absorption spectrum of D314A as isolated showed a Soret band at 418 nm, and split peaks at 421 and ~446 nm were seen in the ferrous-CO state, indicating the heterogeneity of the ferrous-CO heme site (Fig. 6A). The CO-reduced minus reduced difference spectrum showed that the P450-like form of the ferrous-CO complex in the presence of L-Arg has a peak at 447 nm (Fig. 6B), which is red-shifted by 3 nm as compared with that of the wild-type enzyme (444 nm) (Fig. 3C).
|
The EPR spectrum at 7 K of D314A prepared in the presence of
L-Arg and H4BP showed a high spin ferriheme
component at g = 6.02 at 7 K (Fig.
7D), which is similar to the high spin
ferriheme of horse metmyoglobin at g = 5.89 with axial
coordination of water trans to the proximal histidine ligand
(Fig. 7E). This signal is probably attributed to the
cytochrome P420-like ferriheme species (see Fig. 6), which has axial
coordination of a non-thiolate ligand (presumably histidine). It should
be noted that no g = 7.59 EPR signal could be detected
with D314A even in the presence of excess L-Arg (400 µM) (as opposed to the case of the wild-type enzyme; see
Fig. 7B), indicating that the pentacoordinated ferric form is not formed at the substrate-binding pocket of the mutant enzyme. Instead, the EPR spectrum at 16 K of D314A showed a weak low spin ferriheme component at g = 2.46, 2.27, and ~1.85
(Fig. 4F), which is different from the low spin ferriheme
species of the resting wild-type enzyme at g = 2.41 and
may indicate axial coordination of an unknown nitrogen donor ligand
trans to the proximal cysteine thiolate ligand based on
comparisons with calculated crystal field parameters of tetragonality
(/
) and rhombicity
(V/
) (56). No strong radical feature at
g = 2.0 could be detected. In conjunction with the
absorption spectra of the ferrous-CO complex of D314A, showing the
presence of both P450- and P420-like forms (Fig. 6), these data
strongly suggest that the distal substrate-binding pocket of D314A is
significantly modified in such a way that substrate binding does not
induce an appropriate conformational change to form the catalytically
active pentacoordinated ferriheme center having a proximal cysteine
thiolate ligand. Taken together, the spectroscopic data suggest that
replacement of Asp-314 of mouse nNOS with Ala alters the geometry of
the heme site and the distal substrate-binding pocket.
|
Spectroscopic Characterization of T315A and D314A Purified in the Absence of H4BP-- The x-ray crystal structure of the dimeric iNOS heme domain fragment suggested that the N-terminal hook, which contains residues corresponding to Asp-314 and Thr-315 of nNOS, is critically involved in the dimerization and the pterin cofactor binding (14) (see Fig. 1). Therefore, the spectroscopic properties of the mutant enzymes T315A and D314A purified in the absence of H4BP throughout the purification were also investigated.
Gel filtration analysis suggested that T315A purified in the absence of H4BP was predominantly multioligomeric and monomeric, but could in part be converted to the dimeric form in the presence of added H4BP (30 µM) (Fig. 5, top). The resting form of the H4BP-free T315A enzyme was predominantly low spin, exhibiting a Soret band at around 414 nm (Fig. 3B, dashed trace) and showing split peaks at 444 and 421 nm in the CO-reduced minus reduced difference spectrum (Fig. 4C, dashed trace), implying the presence of both P450- and P420-like forms. The EPR spectrum at 7 K of such preparations typically showed two distinctive, weak high spin ferriheme signals at g = 7.62 and 6.02 in the resting form (Fig. 7C), indicating that partial replacement of the proximal thiolate ligand coordinated to the central ferriheme iron by certain other amino acid residue. This was not observed with the wild-type enzyme purified under the same conditions (Figs. 3A, 3C, and 7A).
The partially purified, H4BP-free mutant enzyme D314A showed a single peak at 421 nm in the CO-reduced minus reduced difference spectrum, as observed with the E. coli lysate containing crude D314A mutant enzyme (Fig. 2). The EPR analysis confirmed the absence of any g = ~7.6 species (data not shown), suggesting that H4BP-free D314A exists predominantly in a cytochrome P420-like form.
Expression and Characterization of the Wild-type and Mutant Enzymes
Using the Baculovirus-Insect Cell Expression System--
Because of
the absence of a biosynthetic pathway for the H4BP cofactor
in the E. coli system (46, 47), we attempted to confirm the
above results using the mutant proteins expressed in a
baculovirus-insect cell (Sf9) expression system.
Thus, recombinant full-length wild-type nNOS and the mutant enzymes
T315A and D314A, were produced in the insect cells supplied with
riboflavin, sepiapterin, and hemin as reported (20, 50, 51) and
purified as described under "Experimental Procedures." Although the
wild-type enzyme showed an NADPH-, Ca2+-calmodulin-, and
L-Arg-dependent citrulline-forming activity, the mutant enzymes T315A and D314A showed no enzymatic activity (summarized in Table I). Western blot analysis of the wild-type enzyme
after SDS-polyacrylamide gel electrophoresis (using commercially available anti-nNOS antibody raised against the C-terminal part of the
P450 reductase domain of nNOS) suggested that the protein retained its
mature size of 160 kDa. On the other hand, the mutant enzymes were
extensively proteolyzed to five major polypeptide fragments with
apparent sizes of 105, 90, 80, 60, and ~40 kDa (data not shown); they
probably correspond to domains and subdomain fragments containing the
C-terminal part of mouse nNOS, as judged from the proteolytic cleavage
studies on nNOS by Lowe et al. (55). These results suggest
that replacement of Thr-315 or Asp-314 by Ala modified the
domain-domain and/or subunit-subunit interactions of recombinant nNOS,
resulting in loss of the citrulline-forming activity and increased
sensitivity to proteolysis (see Table I).
![]() |
DISCUSSION |
---|
The present biochemical and spectroscopic analyses of two single-point mutant enzymes of mouse nNOS, T315A and D314A, produced in an E. coli expression system, were designed to explore the role of the strictly conserved Asp-314-Thr-315 array in the mouse nNOS heme domain (Fig. 1). Our results can be summarized as follows. (i) The Asp-314-Thr-315 array of mouse nNOS plays a critical structural role in stabilizing the domain-domain and/or subunit-subunit interaction (Table I). (ii) Replacement of Asp-314 by Ala abrogated the ability of nNOS to form catalytically active dimers and altered the geometry around the heme site and the substrate-binding pocket of the heme domain, resulting in a formation of an inactive P420-like species as the predominant species (Figs. 2, 5-7). (iii) Replacement of Thr-315 by Ala had little effect on the heme site or citrulline-forming activity of the H4BP-bound T315A mutant enzyme produced in E. coli (Figs. 2-4) but impaired the protein stability and altered the heme site geometry of the recombinant mutant enzyme purified in the absence of H4BP (Figs. 4, 5, and 7). These results could not be confirmed with mutant proteins produced in a baculovirus-insect cell (Sf9) expression system because of extensive proteolytic cleavage of the recombinant proteins. Nevertheless, the overall results strongly indicate a critical structural role for both Asp-314 and Thr-315 in stabilizing the heme domain and subunit interactions in mouse nNOS. The individual contributions of these residues are discussed below.
Critical Structural Role of Asp-314 of Mouse nNOS Heme Domain-- The importance of the dimerization of NOS isoforms for citrulline and NO-forming activity is well known (5, 6, 15-27). The replacement of Asp-314 by Ala abrogated the ability of mouse nNOS to form catalytically active dimers and resulted in the formation of a multioligomeric form of the subunit as the predominant species, with loss of the citrulline-forming activity (Table I). A further consequence was distortion of the remote heme site geometry, leading to the formation of a P420-like species, although this change could in part be prevented by addition of H4BP. Interestingly, resonance Raman analysis of the cytochrome P420-like form of the H4BP-free nNOS and iNOS isoforms suggested partial conversion of the proximal thiolate ligand to a nitrogen-donor ligand (presumably histidine) in the ferrous-CO form (58). The present EPR analysis of the P420-like form of the D314A mutant enzyme also suggested that the P420-like form of ferric nNOS is predominantly high spin, having an axial ligand replaced by an unidentified non-thiolate ligand (presumably histidine, based on the spectral similarity to horse metmyoglobin; see Fig. 7). Moreover, the ferriheme center of the P450-like form of H4BP-supplemented D314A most likely has a hexa-coordinated low spin structure, even in the presence of excess L-Arg, which indicates that the substrate-binding pocket of this mutant enzyme is structurally modified.
On the basis of these results, we suggest that Asp-314 of mouse nNOS is
critically involved in the dimerization of the enzyme, and its
replacement with Ala affects the orientation and/or structure of other
element(s) in the vicinity, leading to loss of citrulline-forming activity due to multiple structural distortions of the molecule. Indeed, the x-ray crystal structure of dimeric iNOS heme domain fragment 65 (residues 66-498) (14) has shown that Asp-92
and Thr-93 of mouse iNOS (corresponding to Asp-314 and Thr-315,
respectively, of mouse nNOS; see Fig. 1) reside near the C-terminal end
of the
2' strand at the hairpin loop of the N-terminal hook
(schematically illustrated in Fig. 8),
which binds to the other
subunit.3 The N-terminal hook
not only constitutes a part of the dimer interface of the dimeric iNOS
heme domain fragment but also interacts with the N-terminal
pterin-binding loop (residues 108-114), which is critically involved
in binding of the H4BP cofactor (14) (see also Figs. 1 and
8). Thus, our results obtained with the D314A mutant enzyme of mouse
nNOS are in line with the x-ray structural analysis of the iNOS heme
domain fragment (14). On the other hand, they do not support the
earlier hypothesis by Sagami and Shimizu (43) that Asp-314 might serve
as a distal proton donor of the NOS isoform on the basis of activity
measurements and the analogy with the regular cytochrome P450s (29,
32).
|
Cross-talk between the N-terminal Dimer-linking Region and the Remote Heme Site of nNOS-- Thr-315 of mouse nNOS is also located at the N-terminal hook region (Figs. 1 and 8), although the effect of its replacement by Ala (or Ser) was substantially different from that in the case of the adjacent residue, Asp-314. Thus, although the H4BP-free T315A is a mixture of monomeric and multioligomeric forms and shows a tendency to form the inactive P420-like species, the H4BP-bound mutant enzyme is predominantly dimeric and active and has a heme site geometry that is indistinguishable from that of the wild-type enzyme, especially in the presence of bound L-Arg. A plausible interpretation of these results is that a structural bias at the remote heme site caused by replacement of Thr-315 by Ala is annulled upon binding of H4BP, which also facilitates dimerization and prevents the partial replacement of the axial ligand to the central heme iron.
Close inspection of the dimeric structure of the iNOS heme domain
fragment (14) suggests that the N-terminal hook makes direct contact
with the N-terminal pterin-binding loop and the substrate-binding helix
7a (residue nos. 370-378 in mouse iNOS) within the same subunit
(Fig. 8), thereby constituting the dimer interface together with the
adjacent helices
8,
9,
10,
11a, and
11b, and the
-strand
12a in the presence of bound H4BP. The
substrate-binding helix
7a of the dimeric iNOS heme domain fragment
contains several key residues involved in the binding of both the
substrate and pterin cofactor, and directly spans above the
substrate-binding distal heme pocket (14). These findings indicate a
close structure-function relation among H4BP binding, dimerization, the shape of the substrate-binding distal heme pocket, and the citrulline-forming activity of the dimeric NOS isoforms.
Unfortunately, the three-dimensional structure of the H4BP-free, monomeric NOS heme domain with the N-terminal hook region has not yet been determined, and it is currently not possible to predict the structural changes of the N-terminal hook and the neighboring regions associated with the monomer-dimer conversion of the NOS isoforms. Nevertheless, the replacement of either Asp-314 or Thr-315 at the N-terminal hook region of the H4BP-free mouse nNOS by Ala resulted in marked modification of the geometry of the remote heme site and alteration of the oligomeric state of the mutant enzymes, which would not be expected if no structural interaction between the N-terminal hook and the remote heme site existed in H4BP-free nNOS. Thus, appropriate orientation and/or structure of the hairpin loop of the N-terminal hook are also required in H4BP-free mouse nNOS to prevent exchange of the proximal thiolate ligand and to maintain the geometry of the remote heme site.
In conclusion, our results provide evidence for a crucial structural
role of the conserved Asp-314-Thr-315 array at the N-terminal hook
region of mouse nNOS, in accordance with recent x-ray structural studies on iNOS heme domain fragments (14). In particular, our biochemical and spectroscopic analyses of the mutant enzymes T315A and
D314A demonstrate that appropriate orientation and/or structure of the
hairpin turn of the N-terminal hook are essential to maintain the
integrity of the substrate-binding pocket at the distal side of the
heme center and the geometry of the remote heme site (presumably via
the substrate-binding helix 7a; see Ref. 14 and Fig. 8), as well as
to facilitate formation of the active dimer. Thus, appropriate
cross-talk between the N-terminal hook region and the substrate-binding
distal pocket of nNOS is essential to facilitate the proper activation
and regulation of the citrulline- and NO-forming activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. T. Ogura and H. Esumi (National Cancer Center Research Institute, East) for their kind gift of cDNA encoding the mouse wild-type nNOS gene, Dr. Dr. S. Kawamoto (Yokohama City University) for his kind gift of the baculovirus transfection vector, and Dr. K. Ito (Kyoto University) and Drs. H. Taguchi and M. Yoshida (Tokyo Institute of Technology) for their kind gift of a plasmid vector harboring E. coli groELS genes. We also thank Dr. D. J. Stuehr (Cleveland Clinic) for providing us unpublished structural information about the dimeric iNOS heme domain, Drs. K. Tamura and T. Iizuka (The Institute of Physical and Chemical Research) for allowing us to utilize the X-band EPR facility, Dr. T. Oshima (Tokyo University of Pharmacy and Life Science) for allowing us access to the large-scale fermenter facility, and Y. Kurahashi and J. Mizushima (Nippon Medical School) for their excellent technical assistance at the initial stage of this work.
![]() |
FOOTNOTES |
---|
* This investigation was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas "Biometallics" (08249104) (to T. N.) and by Grants-in-aid 09480167 (to T. N.) and 8780599 (to T. I.) from the Ministry of Education, Science, Sports and Culture of 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.
To whom correspondence should be addressed. Tel.: +81-3-3822-2131,
ext. 5216; Fax: +81-3-5685-3054.
2 The glutathione S-transferase-heme domain fusion proteins of these mutant enzymes all contained a bound heme center as judged from the visible absorption spectra, indicating their ability to incorporate heme into the corresponding heme domain (T. Iwasaki, H. Hori, and T. Nishino, unpublished results).
3 Dr. D. J. Stuehr (Cleveland Clinic), personal communication. The location of the corresponding residues in the dimeric iNOS heme domain fragment can be viewed in the stereo figures at the web site of Dr. Tainer's laboratory at the Scripps Research Institute (14).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: iNOS, inducible nitric-oxide synthase; NO, nitric oxide; NOS, nitric-oxide synthase; nNOS, neuronal nitric- oxide synthase; H4BP, (6R)-5,6,7,8-tetrahydro-L-biopterin; EPR, electron paramagnetic resonance; MOPS, 4-morpholinepropanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|