From the CCLRC Daresbury Laboratory, Warrington,
Cheshire, WA4 4AD, United Kingdom and the ¶ Department of
Biological Chemistry, John Innes Centre, Colney,
Norwich, NR4 7UH, United Kingdom
Received for publication, June 20, 2000, and in revised form, November 15, 2000
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ABSTRACT |
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We have collected synchrotron x-ray
solution scattering data for the MoFe protein of Klebsiella
pneumoniae nitrogenase and show that the molecular conformation
of the protein that contains only one molybdenum per
Biological nitrogen fixation is catalyzed by nitrogenase, a
two-component metalloenzyme system that couples the hydrolysis of MgATP
to the reduction of dinitrogen in the reaction,
2
2 tetramer is different from that of the
protein that has full occupancy i.e. two molybdenums per
molecule. This structural finding is consistent with the existence of
MoFe protein molecules that contain only one FeMo cofactor site
occupied and provides a rationale for the 50% loss of the specific
activity of such preparations. A stable inactive transition state
complex has been shown to form in the presence of MgADP and
AlF
-subunit of the MoFe protein. This observation demonstrates that the
conformation of the
-subunit or the
subunit pair that lacks
the FeMo cofactor is altered and that the change is recognized by the
Fe protein. The structure of the 1:1 complex reveals a similar change
in the conformation of the Fe protein as has been observed in the low resolution scattering mask and the high resolution crystallographic study of the 1:2 complex where both cofactors are occupied and with the
Fe protein bound to both subunits. This extensive conformational change
observed for the Fe protein in the complexes is, however, not observed
when MgATP or MgADP binds to the isolated Fe protein. Thus, the large
scale conformational change of the Fe protein is associated with the
complex formation of the two proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUSION
REFERENCES
Molybdenum-containing nitrogenases are made up of a
molybdenum-containing (MoFe protein or component 1; ~230 kDa) and an iron-containing protein (Fe protein or component 2; ~60
kDa).1 During enzyme turnover
the Fe protein functions as a specific MgATP-dependent
electron donor to the MoFe protein (1-4). The x-ray crystal structures
of both individual proteins isolated from Azotobacter
vinelandii (Av proteins) and Clostridium pasteurianum (Cp proteins) have been determined (5-9). The x-ray structure of MoFe
protein from K. pneumoniae (Kp protein) has also been determined (10). The MoFe proteins have an
2
2 subunit structure in which each
subunit pair binds a unique Fe8S7 cluster (P
cluster) positioned at the subunit interface and the active site of the enzyme, a Fe7S9 molybdenum homocitrate cluster
(FeMo cofactor), within the
-subunit (11). The Fe protein is a
2 dimer that has a single
[Fe4S4] cluster at the subunit interface and
two nucleotide binding sites, one on each subunit (6, 9). The binding
of MgADP or MgATP to the isolated Fe protein results in an altered
reactivity and spectroscopic properties of the Fe-S cluster, which have
been well documented (see Ref. 4).
The crystal structures of Av2 and Cp2 (6, 9) display a peptide folding
pattern similar to other nucleotide-binding proteins, including the
ras and G-protein family, and myosin, where transient protein complexes couple nucleotide hydrolysis to signal and energy transduction processes (1). MgATP hydrolysis by nitrogenase requires
the presence of both the Fe protein and the MoFe protein, and recently
several groups have exploited these similarities to form stable but
inactive nitrogenase complexes of A. vinelandii (12, 13) and
K. pneumoniae (14, 15) using
AlF
Extensive kinetic and modeling work has shown that following each
electron transfer, the Fe protein and the MoFe protein complex dissociates in what is the rate-limiting reaction of nitrogenase turnover (see Ref. 4). It has been demonstrated that this transient complex can be stabilized in the presence of ADP and
AlF
We have recently reported that the kinetics of the formation of
the K. pneumoniae transition state complex are consistent with MoFe protein lacking one
FeMoco2 center having an
altered molecular conformation, which the Fe protein can recognize
(15). To test this proposal experimentally, we have obtained solution
x-ray scattering data of Kp1 containing one or two FeMoco centers per
molecule and generated their molecular shapes at ~20-Å resolution
for both forms. In addition, the 1:1 complex MoFe protein-(Fe
protein·ADP·AlF
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EXPERIMENTAL PROCEDURES |
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Sample Preparation
Nitrogenase Component Proteins-- All manipulation of the air-sensitive nitrogenase components was done under an atmosphere of nitrogen. The nitrogenase proteins Kp1 and Kp2 were purified from K. pneumoniae to homogeneity as described previously (27, 28). In the case of Kp1, this method (27) resolved species containing 1.1 and 1.9 molybdenum atoms per molecule and were shown to be free of any contaminating proteins by SDS gel electrophoresis. The specific activities expressed as nmol of hydrogen evolved/min/mg of protein were as follows: Kp1(1.9 Mo), 2100; Kp1(1.1 Mo), 1100; Kp2, 1399.
For data collection, Kp1 was in 25 mM HEPES buffer, pH 7.5, containing 250 mM NaCl and 2 mM Na2S2O4. Kp2 was in the same buffer except at 50 mM, and in some experiments 10 mM MgCl2 and 5 mM ATP were present. After exposure to the x-ray beam and after the scattering data had been collected, samples of Kp2 were removed from the cell, and the specific activity was remeasured; typically, the recovery was 80% or higher.
Preparation of the 1:1
ADP·AlF2
2 tetramer. The complex was separated
from the excess Kp2 and reaction mixture components by anaerobic gel
filtration using an Amersham Pharmacia Biotech FPLC system and a 26/60
SuperdexTM 200 column equilibrated with 25 mM HEPES buffer,
pH 7.5, 2 mM Na2S2O4, 5 mM AlF3, and 50 mM KF. Further
purification was carried out using an anion exchange Mono Q 5/5 column
developed with a linear gradient of NaCl from 0.1 to 1 M in
50 mM Tris buffer, pH 7.5, containing 5 mM
AlF3 and 2 mM
Na2S2O4. This procedure resulted in
the separation of three species, which subsequent analytical gel
filtration showed to be homogeneous species with retention volumes
corresponding to Kp1 and the 1:1 and 1:2 complexes. The work described
in this paper utilized the purified 1:1 complex.
X-ray Scattering Experiments and Data Analysis
Due to the extreme sensitivity to oxygen, all manipulations of
nitrogenase proteins were conducted in an anaerobic chamber (glove box)
in an atmosphere of nitrogen containing a very low concentration of
oxygen (<5 ppm of O2). All samples were filtered (0.2-µm
pore size) and loaded in the glove box into a brass cell (containing a
Teflon ring sandwiched by two mica windows that defines the sample
volume of 120 µl and a thickness of 2.5 mm). The cell was sealed with
plastocene and then transferred immediately to the x-ray station.
Scattering data were collected on beamline 8.2 at the Synchrotron
Radiation Source (Daresbury, UK) (29) at an electron energy of 2 GeV
and with beam currents between 150 and 250 mA. At the
sample-to-detector distance of 3.3 m (2.5 m) and the x-ray
wavelength of =1.54 Å, a momentum transfer interval of 0.002 (0.004) Å
1
s
0.030 (0.035) Å
1 was covered on a
position-sensitive quadrant multiwire proportional counter (30). Values
in parentheses refer to the measurements for the Fe protein only. The
modulus of the momentum transfer is defined as s = (2sin
)/
, where 2
is the scattering angle. The scattering
pattern from an oriented specimen of wet rat tail collagen was used to
calibrate the detector. Samples were measured at room temperature
(~20 °C) at concentrations between 0.5 and 5 mg/ml. To minimize
systematic errors, each data set consisted of buffer followed by
protein data collection. The experimental data were recorded in frames
of 100 s allowing on-line checks for changes in the scattering
profiles and corrected for background scattering (subtraction of the
scattering from the camera and a cell filled with buffer), sample
transmission and concentration, and positional nonlinearities of the
detector. Off-line data reduction was done with the OTOKO software
package (31). Maximum particle dimensions Dmax,
the radius of gyration Rg, the distance distribution function p(r), and the extrapolated
forward scattering value I(0) were evaluated with the
program GNOM (32). The latter allows the estimation of molecular mass
when calibrated against the scattering from proteins with known
molecular mass (apart from fully loaded Kp1, 225 kDa, as standard for
an anaerobic protein sample, nitrous oxide reductase, 134 kDa, was
used). The volume V of the particle can be calculated from
the Porod invariant (33), including the outer part of the scattering
profile. A correction factor is applied to alleviate the difficulties
of the limited range of scattering data (described in Ref. 34). More
details concerning data collection and reduction are given elsewhere
(35).
The computation of the molecular envelopes was based on the ab
initio shape determination procedure of Svergun and Stuhrmann (36). If we assume that the scattering is caused by a globular, homogeneous molecule, one can define its molecular shape by the angular
envelope function F(,
) such that the particle density
(r) is unity inside the molecular boundary and vanishes
elsewhere. F(
,
) can be expanded into a series of
spherical harmonics Ylm(
,
) according to Refs.
37 and 38,
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(Eq. 1) |
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RESULTS AND DISCUSSION |
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Formation of a 1:1 Complex--
In a previous study, the rate of
formation of the transition state complex
Kp1-(Kp2·ADP·AlF
To detect complexes formed by Kp1 preparations that are partially
active due to incomplete occupancy of the FeMoco binding sites, a
method was developed to monitor complex formation that was independent
of activity measurements. The extent of formation of the transition
state complexes was determined using gel permeation chromatography of
reaction mixtures containing a range of molar ratios of Kp1 and Kp2
from 0.25 to 5 Kp2/Kp1 in the presence of MgADP and
AlF
dimer. At the lowest Kp2/Kp1 ratio tested, the profile is dominated by
free Kp1, which has a retention volume of 10.3 ml (bottom
trace in Fig. 1). At this ratio, no peak corresponding to
free Kp2 is evident, but a shoulder on the elution profile of Kp1
arising from a higher molecular weight species with a retention volume
of 9.8 ml has been formed. As the Kp2/Kp1 ratio was increased to 0.5 Kp2/Kp1, this species became the dominant feature, but at higher ratios
it was replaced by a peak with a retention volume of 8.9 ml. A peak
corresponding to free Kp2 with a retention volume of 12.35 ml was also
detectable under these conditions. The Kp2 band was first evident at a
ratio of 1:1 Kp2/Kp1 and continued to grow as the ratio was increased
(Fig. 1). These data are consistent with the formation of two types of
stable complexes by Kp1 lacking a full complement of cofactor centers,
as the Kp2/Kp1 ratio is varied. We propose that initially a 1:1 complex
is formed as an intermediate on its way to the 1:2 complex, which
predominates at high Kp2/Kp1 ratios. The kinetic data of Yousafzai and
Eady (15) are consistent with Kp2 in this complex binding to the
2
2 subunit pair, which contain the metal
redox centers. When similar experiments were carried out with Kp1
containing 1.9 molybdenum atoms per
2
2
tetramer, a similar behavior was observed, but the formation of the 1:2
complex occurred at a lower ratio of Kp2/Kp1 (data not presented),
consistent with a difference in the stability of the 2:1 complexes
formed by the two species of Kp1.
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To isolate the 1:1 complex in sufficient quantity to allow structural studies, it was purified from the components formed in an incubation mixture containing Kp1 lacking a full complement of cofactor centers as described under "Experimental Procedures." This procedure resulted in the separation of three species, which subsequent analytical gel filtration showed to have retention volumes corresponding to Kp1 and the 1:1 and 1:2 complexes.
Solution Structure of Fully Loaded and Half-loaded MoFe
Protein--
Fig. 2 compares the x-ray
scattering patterns (with error bars) for the fully loaded Kp1
(i.e. with the full complement of the cofactor) and Kp1 with
only half the cofactor centers present (from now on denoted as
Kp11/2). The two scattering profiles are distinct, crossing
each other at an intermediate s, suggesting that a
significant structural difference exists. As shown in Table
I, the geometrical parameters increase in
the absence of a full metal cofactor complement, indicating an
expansion of the overall conformation. The main differences in the
p(r) curves (Fig. 2, inset) occur for
longer distances and in the maximum of p(r),
which is shifted for Kp11/2 to larger distances (by
approximately 5 Å) to 47 Å. To evaluate possible protein aggregation,
scattering patterns have been obtained in the concentration range from
0.5 to 5 mg/ml. The concentration-dependent values for
radii of gyration are revealed in Fig.
3a, highlighting the
difference between the two protein samples. r
itinf;g values (extrapolated to infinite dilution) differ by as
much as 1.5 Å, and the Rg versus
concentration curve shows very different slopes for Kp1 and
Kp11/2. This may reflect a change in the electrostatic
properties of the Kp1 and Kp11/2 surface. The difference in scattering behavior is further illustrated in Fig. 3b, where
the ratio of the scattering curves for Kp1 and Kp11/2 are
plotted (upper trace). It is clear that
scattering data for the two protein samples differ over much of the
range, and only beyond s 0.02 Å
1, the ratio hovers around unity. As a
control, the lower trace in Fig. 3b
shows the intensity ratio of scattering profiles from half-loaded Kp1
recorded at two different concentrations. It is clear that in this case
the ratio is unity over almost the whole data range. Moreover, a
careful analysis of I(0) did not reveal changes in molecular
mass between Kp1 and Kp11/2 (the mass of the metal cofactor is
small compared with that of the protein molecule).
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Previously, we have reported the molecular shape of the fully loaded
Kp1 (41) and shown this to be in good agreement with the overlaid
crystal structure of the MoFe protein; i.e. essentially flexible polypeptide segments appear outside the molecular envelope (see also Fig. 4a). In these
calculations, a 2-fold symmetry was assumed for the
2
2 tetramer; thus, shape restoration up
to harmonics L = 6 was justified. To assess if there
are differences in the molecular structure due to the absence of one of
the cofactors, we have undertaken shape restoration without assuming a
2-fold symmetry (Fig. 4b). As a control, a shape restoration
of Kp1 containing both cofactors was attempted, where no symmetry is
assumed. In this case, spherical harmonics of only up to
L = 4 are permissible (19 free parameters). Fig.
4b (left panel, yellow
envelope) shows two views of the molecular shape of Kp1
restored with L = 4. A comparison with Fig.
4a shows that the two shapes resemble each other
closely, demonstrating that shape restoration with L = 4 is sufficient to recognize the characteristic features of the molecule such as the presence of a 2-fold symmetry. Fig. 4b
(right panel, pink
envelope) provides two views of the Kp11/2 shape at
L = 4, where again no symmetry was assumed (fits to the experimental data with final residual R = 2.1 and 2.4%
for Kp1 and Kp11/2, respectively, are shown in Fig. 2). The
shape for Kp11/2 differs significantly from Kp1; an extension
or bulge appears that breaks the familiar view of 2-fold symmetry. An
assessment of the differences of both molecular envelopes (Kp1
versus Kp11/2 at L = 4) demonstrates that the
left half of the molecule remains essentially the same in the two cases
(see Fig. 4b), but significant expansion is observed on the
right half of the molecule in the absence of the cofactor. These shapes
indicate that the missing FeMo cofactor in Kp1 results in a
significantly less compact structure, which is underlined by the
geometrical parameters given in Table I. This structural expansion may
also explain the slightly larger volume of Kp11/2 as a result
of water filling the created cavities and clefts. Although the
molecular shape for both Kp1 and Kp11/2 (represented by an
average envelope deduced from several shape reconstruction runs using
different starting conditions) offers a qualitative insight into the
structural change as a result of FeMoco absence, the use of available
crystallographic information allows us to assess the experimental
findings more accurately. For that reason, we attempted to model the
structure of Kp11/2 based on the following rationale. The FeMo
cofactor is entirely contained in the
-subunit, at the boundary
between three domains. Inspection of the protein environment around the
FeMo cofactor shows primarily hydrophilic residues forming a shallow
cavity for metal cofactor anchoring. This interdomain location and the distinctive collection of polar and charged groups are likely to play
an important role in stabilizing the protein's native conformation.
Significant conformational rearrangements and destabilization would be
expected in the absence of the metal cluster. Consequently, as a result
of the missing FeMoco center, an opened
-subunit is conceivable in
which domain III (domains are labeled according to Ref. 5) is rotated
against domains I and II around a hinge comprising the residues
54
and
299 (a rotation of up to 22° was applied). Apart from a few
contacts between the N-terminal residues of the
-subunit and domain
I' of the
-subunit (see below), the hinge movement was considered to
be a rigid body rotation of the
-subunit alone with minor effects on
the
-subunit (Fig. 4c), the latter playing a major role
in tetramerization. However, since some of the helices in domain III of
the
-subunit help to stabilize the tetramer interface (5), an
influence on the arrangement of the two
dimer pairs cannot be
excluded. Furthermore, due to the likely nature of a flexible hinge,
the possibility of multiple conformers cannot be ruled out. The result
from the scattering pattern simulation for the model of Kp11/2 (together with the result for Kp1) is given in Fig. 2. The simulated profile based on the Kp1 crystal structure (10) agrees very well with
the scattering curve of fully loaded Kp1 in solution. The profile from
the theoretical model of Kp11/2 effectively reproduces the
characteristic features of the experimental curve (this is also
reflected in the goodness of fit (
2 value); see Table
I). Deviations for 0.015 Å
1
s
0.022 Å
1 may be
rationalized, given that this only represents one (i.e. a
model that has been obtained by rigid body movement of domain III of
one of the
-subunits only) of several possible conformations. Besides, parts of the surrounding environment and even the tetrameric conformation are probably affected. Ribbon drawings representing the
structure of Kp1 and the modeled structure of Kp11/2 have been
superimposed on the shapes displayed in Fig. 4b.
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Solution Structure of 1:1 Complex and Changes in the Fe
Protein--
The x-ray solution scattering curve and pair distribution
function for the isolated 1:1 complex, purified as described under "Experimental Procedures," is compared with the 1:2 complex data (21) in Fig. 5a. The
scattering results for both complexes are considerably different (see
also Table I). Analysis of the I(0) intensities of 1:2 and
1:1 complex revealed a ratio of 1.3, confirming the proposed
stoichiometry for both complexes, since an intensity ratio of 1.21 would be expected based on the difference in molecular masses of the
two complexes. The distance distribution function (Fig. 5a,
inset) of 1:1 complex computed for infinite dilution shows a
decrease of approximately 30 Å in long distances compared with the 1:2
complex being consistent with only one Kp2 bound to Kp1. For shape
calculations, again no symmetry was assumed, and an envelope with
harmonics up to L = 4 could be restored. The fits to
the experimental data are superimposed in Fig. 5a and
yielded R factors of 2.2% (1:1 complex) and 1.8% (1:2
complex). Two views of the molecular shape thus obtained for the 1:1
complex are shown in Fig. 5b. This is superimposed with the
model built for Kp11/2 (see above) and one Fe protein. Although
this comparison clearly demonstrates that only one Fe protein can be
included in the restored shape for the 1:1 complex, a scattering
pattern simulation (distinguishing between the two known structural
states of the Fe protein) confirms that the Fe protein undergoes a very
similar conformational change (see simulated curves in Fig.
5a and 2 values given in Table I) as that
documented for the 1:2 complex (20, 21). Interestingly, in all
simulations the goodness of fit improves when the Fe protein from the
Av1-Av2 complex (20) is considered (Table I). This is also emphasized
visually by a better agreement with the restored molecular envelope
(see Fig. 5b) overlaying the compact conformation for the Fe
protein when in complex with the MoFe protein (20) rather than the less
tight conformation of the free Fe protein as suggested in the original docking model (5). Deviations between experimental and simulated scattering results for the 1:1 complex (in particular concerning the
scattering range 0.018 Å
1
s
0.023 Å
1 (see Fig.
5a) as well as the Rg value (Table I))
indicate, however, that the missing cofactor in Kp11/2, together with the formation of the 1:1 complex, causes further structural rearrangements compared with the model presented here (based
solely on an open
-subunit in one of the
subunit pairs of
Kp11/2). Additional structural modifications regarding the
subunit pairs offer an attractive option also in view of the
recent evidence for long range conformational changes in the MoFe
protein upon Fe protein binding (26).
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The absence of the second Fe protein from the opposite side of the
molecule that lacks the cofactor provides support to the idea that the
Fe protein is able to recognize the altered conformation due to the
missing cofactor from the MoFe protein (Fig. 4b). At this
point, it has to be mentioned that the "open" conformation of
Kp11/2 (here modeled simply as rigid body rotation of domain
III in one of the -subunits) does not directly affect the surface
area implicated in Fe protein docking. However, as a result of certain
contacts between the N-terminal residues (
1-
56) in the
-subunit (forming part of domain III) and residues
111-
140 in
domain I' of the
-subunit, this particular region of the
-interface is likely to be modified. Most importantly, the latter
polypeptide segment of the
-subunit contains one of the two helices
for Fe protein docking (see Ref. 20; see also Fig. 4c). It
is therefore most plausible to assume that destabilization or
reorientation of this docking helix in the
-subunit (as a
consequence of a hinge movement in the
-subunit due to the missing
FeMo cofactor) leads to rearrangements of a considerable section in the
interaction surface with the Fe protein. It is assumed that the other
docking helix (located in domain I) in the
-subunit is unaffected
due to stabilizing effects of the bound P cluster. This is an appealing
structural scenario, considering that the gel permeation data presented
above show the binding of a second Fe protein to Kp11/2, albeit
with lower affinity. Interestingly, besides binding the Fe protein,
additional functional roles of the
docking helix have been inferred
from the structure of Kp1 (10). Our examination may even suggest
that this helix is able to sense the absence of the FeMo cofactor.
Conformational Change of the Isolated Fe Protein upon Nucleotide Binding-- The conformational changes observed for both Av2 and Kp2 in the transition state complexes are of significant functional importance, since they enable the Fe4S4 cluster of Fe protein and MoFe protein to approach significantly closer together to typical electron transfer distances. This is likely to result in an efficient electron transfer between the Av2/Kp2 Fe4S4 cluster to the Av1/Kp1 P cluster, which in the complex is same distance away from the FeMo cofactor, the site of nitrogen reduction.
There is a body of experimental data indicating that the binding of nucleotides to the Fe protein results in changes in the spectroscopic properties and reactivity of the Fe4S4 center (see Ref. 4) and in the sensing of the redox level of the cluster by bound nucleotide (42). Both MgATP and MgADP, competitive inhibitors of electron transfer, are expected to bind at the same site, which is located some ~20 Å away from the Fe4S4 cluster, thus their effect on the properties of the cluster has been rationalized to result from a conformational change in the Fe protein. It is of interest to see if this conformational change in the isolated protein is similar to that observed for Kp2 or Av2 in the transition state complex analogues. A preliminary x-ray scattering study has been reported on the effects of nucleotide binding on Av2, where the radius of gyration (Rg) deduced from the Guinier region alone has been determined (22). Recent advances in the x-ray scattering technique, particularly the ability to use a wider data range, have proved very powerful in studying conformational changes in proteins (14, 35, 43).
Fig. 6a shows the calculated
scattering profiles for Av2 obtained using the crystallographic
structures of Av2 on its own (6, 9) and that observed in the
Av1-(ADP·AlFRg = 1.9 Å) and Dmax (
Dmax = 6 Å). The changes in the scattering profile over the extended scattering
range are similar in nature to those observed in the half-molecule of
transferrin upon binding of iron (35) and thus should be easily
accessible by x-ray scattering. Fig. 6b illustrates the
experimental x-ray scattering data for Kp2 on its own, with MgADP and
with MgATP. The profiles are practically indistinguishable. The lack of
change compared with the one seen in Fig. 6a is apparent.
This experiment has been repeated for the Fe protein of the vanadium
nitrogenase system (see Ref. 44), and identical results were obtained
(data not shown). In addition, data collection for the set of Kp2 (Kp2
plus MgATP and Kp2 plus MgADP) has been made on three different
occasions, and very similar results have been obtained (data not
shown). It is clear from these data that free Kp2 does not undergo a
significant structural change upon nucleotide binding. Thus, the
extensive changes observed in Kp2 and Av2 in the transition state
complexes must primarily arise from the interaction with the MoFe
protein; the role of the nucleotide may thus be to prime the
interaction region of the Fe protein to respond to the MoFe protein.
Consistent with this, Duyvis et al. (24) recently proposed
from pre-steady state kinetic data that MgATP interacts with the
A. vinelandii nitrogenase complex to trigger the
conformational changes that are essential for effective electron
transfer. We note that the surface incompatibility of Av1 and Av2
necessitates the large scale conformational changes in Av2 to
accommodate specific interactions between the two proteins (20).
|
The excellent agreement between structures in solution and crystalline
state is illustrated when the scattering data for Kp2 are compared
(Fig. 6c, solid line) with the
scattering profile calculated from the crystallographic structure of
free Av2 (9) surrounded by a water layer (40). The
Rg from the crystal structure increases from 23.9 to
25.5 Å when hydration effects are taken into account and agrees neatly
with the experimental Rg value. The inclusion of a
hydration shell improves the fit to the experimental x-ray scattering
pattern considerably (45, 46). In fact, the value for the hydrated
structure of free Av2 is equivalent to what was observed in an earlier
study by Chen et al. (22) for Av2 with MgATP but is
substantially different from their values for native Av2 and Av2 plus
MgADP. Their Rg values (>27 Å) for the latter two
states would suggest an Fe protein structure even less compact compared
with the structures reported for the free Fe protein (6, 9).
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CONCLUSION |
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Our earlier kinetic work (15) showed that preparations of Kp1
containing nonintegral amounts of molybdenum did not contain apoprotein
but were a mixture of species with different reactivity containing one
or two molybdenum atoms. The gel filtration data presented here show
that, depending on the molybdenum content, these species react with Fe
protein to form both 1:1 and 2:1 complexes with different stabilities.
These differences are consistent with our demonstration that the
missing FeMoco in an subunit pair of Kp1 affects its molecular
shape; the
subunit pair with FeMoco bound has a more compact
structure compared with that of the empty
subunit pair
(Kp11/2). The open structure of Kp11/2 provides a
potential route for insertion of FeMoco from its precursor carrying
proteins in MoFe protein maturation (4). The difference in conformation influences the interaction with the Fe protein significantly. Kp11/2 forms an inactive 1:1
ADP·AlF
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FOOTNOTES |
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* This work was supported by the Biotechnology and Biological Research Council as part of the Competitive Strategic Grant to the John Innes Center. Facilities were provided by the Daresbury Synchrotron Radiation Source.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.: 44-1925-603441; Fax: 44-1925-603748; E-mail, J.G.Grossmann@dl.ac.uk.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M005350200
1 Throughout, we use the standard nitrogenase nomenclature, i.e. a two-letter abbreviation indicating the genus and species, followed by either 1 or 2 denoting the component type (e.g. Kp2 stands for the Fe protein of K. pneumoniae).
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ABBREVIATIONS |
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The abbreviations used are: FeMoco, iron-molybdenum cofactor; Kp11/2, Kp1 with only half the FeMo cofactors present.
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REFERENCES |
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