From the Department of Medicine, University of
Freiburg, D-79106 Freiburg, Germany, § Department of
Chemistry and Biochemistry and
Department of Medicine,
University of California at San Diego, La Jolla, California 92093-0358, and ¶ Department of Biochemistry, University of Texas Health
Science Center, San Antonio, Texas 78284-7760
Received for publication, August 16, 2000, and in revised form, October 13, 2000
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ABSTRACT |
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Nitric-oxide synthases (NOS) catalyze the
conversion of L-arginine to NO, which then stimulates
many physiological processes. In the active form, each NOS is a dimer;
each strand has both a heme-binding oxygenase domain and a reductase
domain. In neuronal NOS (nNOS), there is a conserved cysteine motif
(CX4C) that participates in a ZnS4
center, which stabilizes the dimer interface and/or the
flavoprotein-heme domain interface. Previously, the Cys331
Nitric-oxide synthases
(NOS)1 constitute a family of
heme-thiolate-liganded proteins that catalyze the conversion of
L-Arg to NO and L-citrulline, requiring NADPH
and molecular O2 (1). Three isoforms of mammalian NOS are
known. Neuronal nitric-oxide synthase (nNOS) and endothelial
nitric-oxide synthase (eNOS) are constitutively expressed and are
Ca2+/calmodulin-dependent; inducible nitric-oxide synthase
(iNOS) is immunostimulated and Ca2+-independent. All three
are homodimers in their physiologic states; each monomer has a molar
mass between about 130 and 160 kDa. In each monomer, an oxygenase
domain is recognized that contains the heme (2-5) and requires
tetrahydrobiopterin (BH4) (6-8). All isoforms also have a
reductase domain, which is not of direct interest here.
A recent study (9) prepared nNOS (rat brain) protein with the point
mutation C331A expressed in Escherichia coli. The mutant was
characterized with respect to catalytic activity, the binding of
BH4 and substrates, the state of the iron as deduced from
EPR, and UV-visible spectral features. Because E. coli produces neither BH4 nor calmodulin, the
preparation is valuable for investigating reconstitution with those
factors. That report should be consulted for the several hypotheses
that motivated the investigation and for an extensive list of
citations. In this study, we added a brief account of the
kinetics of ligand binding. The major effect of the mutation was that
C331A failed to bind either BH4 or L-Arg. Arginine affinity was suppressed at least 100-fold, and the binding of
BH4 was undetectable at least for some time after mixing.
However, extended incubation (~12 h) with L-Arg restored
essentially normal behavior, including binding of BH4 and
almost full catalytic activity. Because L-Arg substrate and
BH4 bind near the heme, the findings (9) suggested that
"the primary dysfunction ... is a distorted or floppy heme
pocket." As to the reason for that dysfunction, the suggestion was
made that Cys331 is required for binding zinc at a
tetrathiolate site (10) (discovered just as the work with the mutant
was being completed) and that loss of zinc perturbs the structure,
especially near the heme site.
Because conformational defects (as might be introduced by mutations) on
either the proximal or distal side of the heme can have dramatic
effects on ligand binding, it seemed useful to us to characterize the
kinetics of heme ligation for CO and NO. That would test the conclusion
that the pocket is initially distorted and would provide a very
sensitive test of the ability of incubation with L-Arg to
restore normal functionality.
Carbon monoxide (99.8%), nitric oxide (99.9%), high purity
argon, and mixtures of CO (1-100%) and NO (0.1-10%) diluted
in argon were from Matheson. Nitric oxide was further purified
through a column of fresh KOH. Buffers (20 mM Tris-HCl, pH
7.8, 100 µM EDTA, 100 mM NaCl) were prepared
in a gas-tight syringe and deoxygenated by bubbling with argon for 40 min. When indicated, buffers contained 250 µM
BH4 and/or L-Arg at concentrations of 0.1-10
mM.
The point mutation C331A in rat nNOS was generated as reported
previously (9). The proteins were >90% homogeneous as determined by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein
solutions were degassed and then added to the buffer with the desired
ligand concentration. As needed, the protein solutions were
reduced using one part in 50 of 1% sodium dithionite solution. Overnight incubations of protein with L-Arg were carried
out at 3 °C. Spectra were consistent with previously published data
(9, 11-13).
Flash photolysis was carried out at 22 °C using procedures described
previously (14, 15). Kinetics were monitored at several wavelengths
showing both transient absorption and initial state bleaching.
Stopped-flow measurements were carried out at 20 °C following
protocols described earlier (16). Carbon monoxide dissociation rates
were determined by replacing CO with NO (17). After mixing, samples
contained 0.5 µM protein, 25 µM sodium
dithionite, 500 µM CO, and 1000 µM NO. (For
the ferrous hemes, which alone bind CO, affinity for NO is much larger
than for CO.) Absorption changes were monitored at 443 nm.
CO Ligation--
UV-visible spectra for the C331A mutant itself
and its association with CO were reported previously (9). The kinetics
of CO binding for both the C331A mutant and WT nNOS display two phases for concentration-dependent bimolecular combination that
differ in rate by a factor of 100. A typical example of absorption
changes following flash photolysis is shown in Fig.
1. Mutant and WT nNOS are similar in that
fast and slow phases are not perfect single exponentials but require
two or more rate constants distributed over a range of a factor of 3 or
4, as may be seen in Fig. 1. This was discussed at some length for WT
(14). Once the heterogeneity is noted, it is sufficient for most
purposes to consider an effective mean rate.
Mutant and WT proteins are also similar but not identical with respect
to the mean value of the fast and slow rate constants for CO addition,
as shown in Table I. The slow phase has
a rate constant that is almost the same in all circumstances.
The fast phase, however, has a rate constant in the mutant that
is about twice as fast as in the WT holoenzyme and heme
domain.
It is, however, the relative proportion of the two phases that is of
the most interest. Whatever the interpretation of the two kinetic
phases may be, the changes in their relative proportions as conditions
are varied offer a critical test for comparing the behavior between WT
and mutant preparations. Fig. 2 shows the fraction of the slow phase in two circumstances for C331A. It also
includes, for comparison, data on the WT holoenzyme and data for a
heme-binding domain (residues 1-714), both of which were reported
previously (14). The fraction of the slow phase is at its
maximum when both L-Arg and BH4 are
bound. It is intermediate when just one or the other is present, and it
is at its minimum when both are absent. In the WT holoenzyme
(back row), the appearance of the slow phase requires much
less than 1 mM L-Arg, and the association of
L-Arg with protein occurs faster than we can carry out
flash photolysis measurements, which require several minutes of
signal averaging. In the C331A mutant, the effects associated with
binding L-Arg and BH4 appeared only gradually
after extended incubation. The mutant C331A could be forced to display
100% slow phase but only after more than 12 h of incubation with
both BH4 and a large, 10 mM concentration of
L-Arg (third row, left side). Lesser
concentrations of L-Arg were not as effective. Either
BH4 or L-Arg alone was not nearly as effective
as is either alone in the WT protein. A 2-h incubation
(second row) had little effect.
Fig. 2 (two front rows) shows that CO binding to C331A
initially has some similarity to that in the heme domain of WT nNOS. Both BH4 and L-Arg must be present to achieve
much slow phase. In both cases, the fraction of slow phase increases
for the first few hours (data not shown). Incubation of the heme domain
could not be extended to 12 h because of the excessive formation
of the "P-420 analogue" (13, 18). Some formation of the
P-420 feature also occurred in C331A, especially at low CO
concentration and in the absence of BH4, consistent with
previous reports of the factors involved in P-420 conversion (13,
18).
As with the WT holoenzyme and heme domain (14), flash photolysis of the
CO-ligated C331A mutant displays, in addition to bimolecular
association, nanosecond transient absorbance changes that are plausibly
attributed to geminate recombination. In all cases, these
nanosecond transients are larger whenever the fast phase is larger.
Ligand dissociation of CO was also characterized. Illustrative cases
are shown in Table I. Dissociation in extensively incubated C331A is
about a factor of 2 faster than in either the WT holoenzyme or heme
domain. For both the WT holoenzyme and C331A with BH4 but
without L-Arg, somewhat better fits were obtained by using two exponentials (for C331A + BH4, 33% at 0.92 s NO Ligation--
The association of nitric oxide with C331A was
investigated briefly. Unlike CO, which binds only to ferrous iron, NO
binds to both ferrous and ferric forms. Association of NO was virtually identical for ferric and ferrous C331A, as was true for WT protein (15). Fresh solutions of C331A displayed association rate constants of
1-2 × 107 M
Although it is unwise to overinterpret rate constants that vary by only
a factor of 2, the essential finding for NO association parallels what
was observed for CO ligation; incubation with L-Arg modifies behavior to make it more similar to WT holoenzyme and less
like the heme domain fragment.
The principal changes in ligand binding kinetics brought
about by the mutation C331A in nNOS were variations in the
relative amount of fast and slow phases for CO binding. The rate of
binding must reflect conditions in the distal "pocket" and/or
tension exerted by the proximal ligand, which in nNOS happens to be a cysteine, but not Cys331. That the presence of bound
L-Arg and/or BH4 increases the fraction of slow
phase in the WT nNOS was well established by prior work. The first
observation was reported by Matsuoka et al. (19) as a small
part of a broad study. Our own more detailed study (14) confirmed the
two phases (albeit with a larger 100-fold ratio in rate constants) and
the effect of L-Arg on them but also added information
about the effect of BH4, demonstrated that the two phases
are each slightly heterogeneous, and showed that the oxygenase domain
alone also displayed dual behavior but was biased much more
toward the faster process. In addition, we used scavenging to measure
dissociation rate constants near 0.3 s Native WT protein exists largely in the fast-reacting form in the
absence of L-Arg and BH4, but it converts
rapidly to the slow form even at low concentrations of those agents.
Modifying the protein either by truncation to the oxygenase (heme)
domain or by mutating C331A renders it more difficult to achieve the slow-reacting active form, but sufficiently large concentrations of
L-Arg and extended incubation eventually accomplish much
the same change that occurs more readily in the native holoenzyme. In
the absence of BH4 and L-Arg, CO association
with C331A behaves much like that in the WT heme domain fragment.
Extended incubation restores almost normal behavior. The dissociation
of CO, however, follows a slightly different pattern. The dissociation
from the WT heme domain and WT holoenzyme is very similar, whereas CO
dissociation from C331A is faster by a factor of 2 and remains so even
after extended incubation with 10 mM L-Arg.
This suggests that the restoration of the normal structure by
incubation of C331A is not quite perfect.
Association with NO shows similar effects. There is only one
phase for NO association under any condition, but there is a slight
difference in the rate between the WT heme domain and WT holoenzyme.
That difference is replicated in C331A before and after incubation.
Complementing the kinetic studies discussed here may be resonance Raman
measurements of vibrational frequencies for ligands bound to heme.
Recent publications (22, 23), building on earlier investigations cited
therein, infer "open" and "closed" conformations for the
immediate environment of the iron binding site with the open form
correlating with the fast-reacting species observed in kinetic
measurements and the closed form correlating with the slow-reacting
form. A plausible inference is that both L-Arg and BH4 bind close to the iron, affecting both ligand access to
and bonding with the iron. It is conceivable that their effect on ligation is attributable to simple steric blockage. However, it is more
likely that the effects of L-Arg and BH4 are
better described as inducing conformational change either in the distal
pocket or at the proximal cysteine bond, or both. The Raman
investigation of the immediate neighborhood of the iron explores a very
different length scale than do the light-scattering studies that
inferred different hydrodynamic volumes. Kinetic data involve both
scales. There are presently too many uncertainties for us to propose a model that can explain the data from Raman spectroscopy,
light-scattering measurements, and ligation kinetics. (For example,
full understanding of kinetics requires data on geminate recombination
in the picosecond regime.) However, some ideas may be inspired by
crystal structures, which are starting to appear.
In rat brain nNOS, the C331A mutant involves substitution into the
generally conserved motif (CXXXXC) of the two (identical) subunits as follows.
Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of
L-Arg and tetrahydrobiopterin (BH4). Because
binding L-Arg and BH4 to wild type nNOS
profoundly affects CO binding with little effect on NO binding, ligand
binding to the mutant was characterized as follows. 1) The mutant
initially has behavior different from native protein but reminiscent of
isolated heme domain subchains. 2) Adding L-Arg and
BH4 has little effect immediately but substantial effect
after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS.
Such incubation was shown previously to restore most but not all
catalytic activity. These kinetic studies substantiate the hypothesis
that zinc content is related to a structural rather than a catalytic
role in maintaining active nNOS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Typical data for the rebinding of CO to C331A
nNOS. In this case, CO (at 1 atm) is combining, after photolysis,
with protein incubated overnight with 10 mM
L-Arg and no BH4. The data illustrate the
presence of two kinetic phases and show that the slow phase is not a
perfect single exponential. In practice, the fast phase, shown as a
steep drop in the main figure and expanded in the inset,
would be recorded at a much higher time resolution to prove that it
also is not a single exponential. In this figure, the fractional
amplitudes of the two phases are nearly equal.
Rate constants for ligand binding to wild type nNOS and to C331A
incubated 12 h
View larger version (129K):
[in a new window]
Fig. 2.
Percentage of amplitude in the slow phase for
CO association with various nNOS preparations. Front
row, results for the heme domain (HmDom) of the WT
enzyme, incubated approximately 2 h. Second row, C331A
incubated approximately 2 h. Third row, C331A incubated
approximately 12 h. Back row, data for the WT
holoenzyme, incubated approximately 2 h. Along each row, data
labeled with B have 250 µM BH4;
those labeled with A have 10, 1, or 0.1 mM
L-Arg as indicated.
1 plus 67% at 0.21 s
1, and for WT + BH4, 17% at 15 s
1 plus 83% at 0.16 s
1).
1
s
1 with the faster rate in the absence of
L-Arg and BH4 and the slower rate in the
presence of both. This is identical to values reported previously for
the WT heme domain (15). Table I shows the results for C331A samples
incubated overnight with L-Arg. After incubation, the NO
association constants match values measured previously for the WT
holoenzyme (15), which are reduced by about a factor of 2 from the
value for fresh C331A solutions and the heme domain. (These rate
constants for NO are all mean rates; detailed fits showed
heterogeneity, requiring comparable amplitudes of at least two
exponentials differing by a small factor, around 5.) Photolysis of
NO-ligated nNOS has only a small amplitude. Presumably much of the
photolyzed NO undergoes geminate recombination before it can escape to
the solvent. Because no geminate recombination was measured with the
nanosecond spectrometer, it was determined that most geminate
recombination must occur during time scales under a few nanoseconds.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1, and we detected
substantial absorbance changes on nanosecond time scales, attributed to
geminate recombination. Subsequently, Sato et al. (20)
studied CO association with nNOS (rat). In the course of a study of a
variety of substrates and inhibitors, they confirmed again that with
both L-Arg and BH4 present, one observes
essentially a single phase with the slow rate constant found earlier.
They also reported careful measurements of equilibrium constants from
which one may deduce a CO dissociation rate constant equal to 0.3 s
1, which is in agreement with our kinetic determination.
Recently, Tetreau et al. (21) confirmed the 100-fold factor
in rates between the two phases, the need for a distribution of
rate constants within each phase, and the presence of nanosecond
absorbance changes. They added information about CO association
kinetics at cryogenic temperatures. In an effort to explain the
differences between phases, they measured the hydrodynamic volume of
the protein under different conditions. When the slow phase dominated,
the volumes were reasonable for a globular protein of the mass of NOS.
When the fast phase dominated, the hydrodynamic volume was 10-fold larger. They inferred that the fast-reacting form of nNOS involves either substantial unfolding of the protein or aggregation or both.
A crystal structure of nNOS is not yet reported, but that of eNOS
has been (24). In bovine eNOS the analogous position is
Cys101, whereas in human eNOS it is Cys99. The
four cysteines coordinate to a zinc in both eNOS (24) and
nNOS.2 This ZnS4
center stabilizes the dimer interface and/or the flavoprotein-heme domain interface. (We have argued elsewhere (24) that iNOS also requires a similar, tetrahedrally coordinated zinc.) In the C331A mutant of nNOS, there are structural perturbations that make binding both BH4 and L-Arg more difficult, but
sufficiently long incubation at large concentrations of
L-Arg allows binding to occur. Once binding has
occurred, a structure is stabilized that is similar to the native
conformation and is kinetically active. Still, the incubated C331A is
probably not quite identical to WT. The enzymatic activity of the
incubated C331A is 84% of that in WT nNOS, and CO dissociation
is twice as fast.
Cysteine 331 is not the heme-binding amino acid, nor is it involved
directly in the binding of either BH4 or L-Arg.
The question then is how the mutation C331A affects their binding. An
explanation may be offered on the basis of the crystal structure data
(24). Those data indicate that L-Arg is a structural mimic
of BH4 and that L-Arg and BH cross-talk
with each other through heme propionate. In eNOS, the BH4
hydrogen bonds to Ser104, which is a part of the chain that
is connected to the zinc atom, which is tetrahedrally coordinated to
the cysteine residues on the two chains of the dimer. Zinc is one of
the few metals capable of readily forming tetrahedral (most often),
octahedral, and square planar (occasionally) structures. Thus the
structure around the zinc should be able to switch among multiple
conformations. Any change in the stereochemistry around the zinc will
be transmitted to the binding of BH4 (and in turn to the
L-Arg through the heme-proprionate bridge) via
Ser104 or its analog in nNOS. Thus the data from ligand
binding kinetics and enzyme reactivity bring out rather dramatically
the importance of the tetrahedrally coordinated zinc for functional
integrity in the enzyme.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL30050 and GM52419 (to B. S. S. M. ) and Grant AQ-1192 from the Robert A. Welch Foundation (to B. S. S. M. ).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.: 858-534-3199; Fax: 858-534-0130; E-mail: dmagde@ucsd.edu.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M007461200
2 C. S. Raman, H. Li, T. L. Poulos, P. Martásek, and B. S. S. Masters, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: NOS, nitric-oxide synthase(s); NO, nitric oxide; CO, carbon monoxide; nNOS, neuronal nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; BH4, tetrahydrobiopterin; WT, wild type.
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