| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 37, 35384-35393, September 12, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|| **
From the
Department of Chemistry, Princeton
University, Princeton, New Jersey 08544 and
¶Department of Bacteriology, University of
Wisconsin, Madison, Wisconsin 53706
Received for publication, January 29, 2003 , and in revised form, May 16, 2003.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
CooA is isolated from the bacterium Rhodospirillum rubrum, which grows anaerobically on CO as sole energy source (3). CooA regulates the expression of genes, termed coo for CO oxidation, whose products are associated with CO metabolism. The protein is a homodimer (222 amino acids per monomer) containing two b-type hemes that reversibly bind CO. The CO binding enables the protein to bind a specific DNA sequence and induce transcription of the coo genes. CooA contains DNA-binding and signal domains analogous to those found in the catabolite gene activator protein CAP1 (or CRP) (1, 2) (Fig. 1). The heme is bound within the signal domain, where it is ligated by a histidine (His77) side chain (although this ligand is replaced by the Cys75 side chain when the Fe[II] is oxidized to Fe[III] (4)). The sixth Fe[II] ligand is unprecedented; it is the N-terminal proline residue (Pro2) from the opposite chain, which is displaced by CO binding (5).
|
The structure of CO-bound CooA is unknown, but on the basis of the CAP structure (6) it is reasonable to assume that the DNA-binding domains are brought into proper position for DNA binding by movement of the C-helix, which runs the length of the protein and passes close to the heme (1, 2, 7). It has been shown that a repositioning of the C helices at the dimer interface is a major communication pathway between the hemes and the DNA-binding domains (8), but the basis for this repositioning upon CO binding remains unclear. To elucidate the nature of the CO-bound form of CooA and the mechanism of its CO activation, we have prepared a series of CooA variants by site-directed mutagenesis and examined in vivo activity, specific DNA binding, and the Fe–CO and C–O stretching resonance Raman (RR) bands. The residues chosen for mutagenesis are in the region of the heme groups, as defined by the Fe[II]CooA structure. Fig. 2 gives a close up view of this region.
|
The data permit us to identify the approximate position of the heme-CO in CooA and propose a specific model for how that positioning affects CooA activation and leads to the C-helix repositioning. An important aspect of the active form involves the breakage of a critical interaction that stabilizes the inactive structure: an H-bond from the His77 proximal ligand to the side chain of Asn42.
These structural inferences also provide a basis for understanding the cooperative binding and complex kinetics of CooA.2 Finally, we propose a structural model for the heme movement that is consistent with the data and will serve as a working hypothesis for future work.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
-galactosidase reporter system in the
chromosome was described previously
(8), and in vivo
activities were quantitated using the standard protocol
(10). All the site-directed
and region-randomized CooA mutations were constructed in a
pEXT20-based expression plasmid, which provides tight control of CooA
expression (11).
CooA Purification—The purification of WT CooA and most CooA
variants was performed with our standard method as described previously
(12). Some CooA variants such
as C75S, L116K, and M124R CooA were purified with modified methods, which are
also described elsewhere
(12–14).
The purity of WT CooA and CooA variants was estimated to be >95% based on
SDS-PAGE (in the case of L116K CooA, 
90%). The heme content of CooA
preparations was estimated using the extinction coefficient of Fe[III] WT CooA
(3) or by a modified reduced
pyridine-hemochromogen method
(3), and protein concentration
was measured using the BCA assay (Pierce).
In Vitro DNA Binding Assays—In vitro DNA binding assays of WT CooA and CooA variants were performed using the method described elsewhere (11). As a fluorescence probe, a 26-bp target DNA containing PcooF was labeled with Texas Red on one end of the duplex and used at the concentration of 6.4 nM. Salmon sperm DNA was used as the nonspecific DNA competitor. Dissociation constants (Kd) were calculated by fitting of the binding data to a nonlinear equation with correction of the fluorescence quenching as described elsewhere (15).
Sample Preparation—Purified CooA was diluted in buffer (25
mM Mops/0.1 M NaCl, pH 7.4) to a heme concentration of
20 µM. Other pH values were investigated using 0.1
M Tris, pH 7–9, 0.1 M glycine, pH 9.5–10,
and 0.1 M NaOH, pH 11–12). CooA samples were purged with
N2 for 20 min and then with CO for 3 min, followed by
reduction of the Fe[III] to the Fe[II] form with sodium dithionite (final
concentration 63 mM). Reduction and CO binding were monitored
by changes in the absorption spectra.
RR Spectroscopy—RR spectra were obtained with excitation
wavelengths of 406.7 and 568.1 nm from a Kr+ laser (2080-RS;
Spectra Physics) and 441.7 nm from a He-Cd (Liconix) laser in a 270°
backscattering sample geometry. Photodissociation of the bound CO was
minimized by using low laser power (5 milliwatts at the sample) and by
focusing it with a cylindrical lens onto a spinning sample. The scattered
light was collected and focused onto a single spectrograph (Chromex) equipped
with a notch filter (Kaiser Optical) and a CCD detector (Roper Scientific)
operating at 77 K. Spectra were calibrated with dimethyl formamide and
dimethyl sulfoxide-d6.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
The results of this study are organized in the following manner. 1) There
are actually two different forms of CooA–CO, with different C–O
stretching frequencies, as revealed by reanalysis of RR spectra. This
observation has an effect on the interpretation of RR data in this manuscript
and resolves a discrepancy in RR data reported previously. 2) Backbonding
analysis of the RR data reveals that the Fe–His proximal bond is
weakened in the major form of CooA–CO. Mutational data point to breaking
of the His77-Asn42 H-bond as the cause of this weakening
and is consistent with a significant role for this process in CooA activation.
3) The bound CO is certainly in close proximity to Ile113,
Leu116, and Gly117, which has implications for the
movement of the heme and the protein upon CO binding. This proximity was
revealed by the RR analysis of mutational variants substituted at these
positions that show striking perturbations and is generally consistent with
functional characterization of these variants. 4) RR analysis of pH effects of
WT CooA, and of variants containing deletions at the N terminus that includes
Pro2, suggest that CO displacement of Pro2 leads to its
expulsion from the hydrophobic CO binding pocket, because of its positive
charge when protonated. The previously reported depression of the Fe[II]CooA
11 heme RR band is found, via the effects of variants, to stem
from the strong ligand field of the Pro2 ligand.
Structurally Distinct Populations of CO-bound CooA—We have
previously reported RR spectra of CooA–CO showing a Fe–CO
stretching band (FeC) at 487 cm–1 and an
unusually high CO stretching band (CO) at 1982
cm–1
(16). In contrast, Uchida
et al. (17) reported
CO = 1969 cm–1 for their CooA–CO
preparation although they reported the same FeC value as ours, 487
cm–1. To resolve this discrepancy we reexamined
the CO and FeC regions of the RR spectrum at high amplification
(Fig. 3), using 13CO
shifts to identify the bands definitively. FeC was measured at 487
cm–1, as before, and we also reproduced
the 1982-cm–1 CO band. However the newly
measured CO band has a pronounced shoulder at 1962
cm–1. Upon 13CO substitution, this
1962-cm–1 band shifted appropriately, indicating a
second CO band.
|
The 487-cm–1 FeC band is a single band.
Deconvolution from the 465-cm–1 band of excess
dithionite (Fig. 3) yields a
bandwidth, 14.5 cm–1, which is less than that of
the CO adduct of myoglobin (data not shown), 18
cm–1, ruling out multiple FeC contributions in
CooA–CO. Therefore, we conclude that the molecules having CO = 1982
and 1962 cm–1 both have the same FeC = 487
cm–1.
The CO reported by Uchida et al.
(17), 1969
cm–1, is closer to the lower of the two values we
observe, but their CooA–CO preparation also had a fraction with CO =
1979 cm–1; this is the frequency reported in
picosecond FTIR experiments
(18) for CooA–CO
molecules undergoing photolysis. Thus, CooA–CO has two populations of
molecules, having distinct CO values (1962 and 1982
cm–1 in our hands), but the same FeC value,
487 cm–1. The relative size of the populations
appears to be preparation-dependent. In our preparation the
1962-cm–1 fraction is small; the species is
labeled CooA–CO'. We suggest that the species represented by 1982
cm–1 is the active form and that
CooA–CO' is a form that has not rearranged to the active, DNA
binding structure. There may be an equilibrium between active and inactive
forms, or else a fraction of the molecules are stuck in the inactive
configuration because of an energy barrier of some type.
Conformational Change of CooA upon CO Binding Weakens the
Fe–His77 Bond by Breaking the
His77-Asn42 H-bond—The anomalous CO of
the major form of CooA–CO, 1982 cm–1, is
produced by a weakened Fe–His77 bond. This can be seen in a
plot of FeC against CO (Fig.
4). Because of backbonding, FeC and CO are negatively
correlated in heme proteins
(19,
20), as illustrated for a set
of Mb variants in Fig. 4 (solid
line). Changes in the polarity of the binding pocket augment or diminish
backbonding and shift the points along the line
(19,
20). In addition, the
correlation is displaced upward when the Fe-proximal ligand bond is weakened,
because of diminished 
bonding competition between the proximal ligand
and the CO (19,
21). The upper dashed
line in Fig. 4 represents
data for Fe[II] porphyrin CO adducts without any trans ligand
(21).
|
When the CooA–CO values are plotted on the graph, the minority form,
CooA–CO', is seen to fall near Mb variants with hydrophobic
pockets, in which the distal histidine is replaced by hydrophobic residues
(cluster of points marked –H64 in
Fig. 4). However, the
distinctly higher CO of the dominant form, CooA–CO, places it above
the Mb line by 7 cm–1, implying a weakened bond
from the Fe to the proximal ligand, His77 in CooA (see
Fig. 2).
This displacement from the Mb backbonding line was missed in our earlier
correlation (16), because a
more varied group of heme proteins was used, having greater scatter of the
points. The high CO was instead attributed to negative polarity in the CO
binding pocket (16). However,
examination of the pocket region, defined by the mutagenesis results to be
described below, fails to reveal any candidate residue with a negative charge
or even a lone pair of electrons that could provide a negative dipole. In
principle, peptide dipoles
(22) could interact with the
bound CO, but this is unlikely, because the residues that form the binding
pocket are part of a helix.
The inference of a weakened Fe–His77 bond in CooA–CO
is strongly supported by the picosecond RR study by Uchida et al.
(23), which identified the
Fe–His stretching band at 211 cm–1 in the
immediate photoproduct of CooA–CO. This band, a direct measure of the
Fe–His bond strength, is enhanced in five coordinate Fe[II] hemes, and
is found at 220 cm–1 in deoxyMb
(24). The distinctly lower
value found by Uchida et al.
(23) shows that the
Fe–His77 bond is weaker than in Mb for the immediate
photoproduct, consistent with a weaker bond in the CO adduct itself. The
minority form, CooA–CO', which lies on the Mb backbonding line
(Fig. 4), does not have a
weakened Fe–His77 bond and is expected to have an Mb-like
photoproduct; however, its Fe–His would escape detection because of
the low population.
How strong is the Fe–His77 bond in Fe[II]CooA itself?
Because Fe[II]CooA is six-coordinate, the Fe–His vibration is not
enhanced (25), and its
frequency cannot be measured. However, we took advantage of a significant
five-coordinate fraction in the G117I variant of Fe[II]CooA
(7), in which the bulky Ile
side chain causes partial dissociation of the Pro2 ligand (see
below). The five-coordinate heme Soret absorption band is red-shifted from the
six-coordinate band, allowing us to use laser wavelength tuning (442 nm) to
enhance the five-coordinate RR signals
(Fig. 5). Selection of the
five-coordinate species is confirmed by the position of the strong
4 band at 1355 cm–1, the standard
five-coordinate frequency
(25). The Fe–His
band is seen at 220 cm–1, the same frequency as in
deoxyMb. Thus the Fe–His bond is as strong in G117I variant of
Fe[II]CooA as in Mb. We infer that the Fe–His bond is similarly strong
in WT Fe[II]CooA (there is no reason that steric displacement of
Pro2 should make the proximal Fe–His connection stronger) and
that it weakens significantly in the major form of CooA–CO, as well as
its immediate photoproduct. At later times, the photo-product Fe–His
position should increase to 220 cm–1 as the
protein relaxes to the Fe[II]CooA structure, but this process cannot be
followed in photolysis experiments because of rapid geminate rebinding of CO
(18,
23).
|
These results establish that CO binding weakens the Fe–His77 bond. We therefore asked what could be the mechanism responsible for this property of CooA. It has been known that H-bond donation increases the anionic character of the imidazole ring and strengthens the Fe–His bond (26). For example, the Fe–His frequency is much higher, 240 cm–1, in five-coordinate Fe[II] forms of the peroxidases that have a negative Asp side chain in position to accept the proximal His H-bond (24). In Mb the proximal His is H-bonded to a neutral OH group of a Ser residue, as well as to a backbone carbonyl (Fig. 4, inset).
In the Fe[II]CooA structure (Fig.
2), the H-bond acceptor is the amide side chain carbonyl of
Asn42 (Fig. 2). The
O ... N distance is short, 2.7 Å
(1,
2), making it reasonable that
the H-bond is equivalent to the proximal H-bonds in Mb. Because the
Fe–His bond strength depends on the His H-bond status, the observed
weakening of the Fe–His77 bond in CooA–CO implies
weakening of the His77-Asn42 H-bond when CO binds. This
inference is strongly supported by the nearly unaltered FeC or CO
positions when Asn42 is replaced by Ala, Lys, or Asp, which have
different capability of forming H-bond
(Fig. 6). This result indicates
that neither Asn42 nor its replacements are within H-bonding range
of His77 in the major CooA–CO form. If Asn42
remained H-bonded to His77 in CooA–CO, then its replacement
by Ala, which would eliminate the H-bond, would have raised FeC and
CO, whereas replacement by Asp, a stronger H-bond acceptor, would have
lowered them. The absence of either effect implies that His77 has
moved away from residue 42 in CooA–CO.
|
With regard to the minor CooA–CO' form, we note that the low
frequency shoulder on the N42A CO band is attenuated relative to the
wild-type 1962-cm–1 shoulder. Curve resolution
gives a higher frequency, 1966 cm–1 for the N42A
shoulder, suggesting a weakened H-bond in the N42A CooA–CO'
structure.
Although Asn42 interacts with His77 in the Fe[II]
form and not the CO-bound form, the nature of the residue is of some
importance to activation. N42A, N42D, and N42K CooA all displayed poorer DNA
affinity in the presence of CO (and poorer -galactosidase activity) than
did WT CooA (Table I),
suggesting some additional role for this residue in the active form. The basis
for that effect is unknown. Note that the activity assays that measure DNA
affinity are somewhat easier to interpret than the in vivo assays
reporting -galactosidase activity, because the former only measures DNA
binding, whereas the latter measures interaction with RNA polymerase and has
other complications (8).
These results indicate that an element of the activation mechanism is the perturbation of the His–Fe bond. The data and arguments below strongly suggest that the His–Fe bond is perturbed by displacement of the heme upon CO binding, which is critical for CooA activation.
Hydrophobic C-helix Residues, but Not Pro2, Form the CO Pocket of CooA That Is Necessary for Activation—The distal heme pocket has an important role in ligand discrimination in many heme proteins (27). CooA activation is specific to CO, and an understanding of the CO binding pocket should be valuable in understanding both specificity and the activation in response to CO. We have already shown that hydrophobic residues at positions 113 and 116 are critical for normal CO activation (28) and that Gly117 is also necessary for this response (7). However, the positioning of these residues with respect to the bound CO was not investigated. We therefore measured the RR spectra of a variety of CooA variants that were either novel in terms of activity and might therefore be altered in their sensing of the bound CO (Table I) or assumed to be near bound CO based on the structure of Fe[II]CooA.
Pro2 is the ligand of Fe[II]CooA displaced by CO, so we tested
the hypothesis that the displaced Pro2 helped form the CO pocket.
As shown in Table I, neither
replacement of the Pro2 terminus by tyrosine (P2Y CooA) nor
deletion of the two penultimate residues (
P3R4 CooA) showed noticeable
effect on the FeC or CO band. This implies that Pro2 in WT
CooA does not form the CO pocket and is consistent with the previous
observation that Pro2 is not essential for activation of CooA by CO
(11). We note, however, that
the DNA binding affinities of these two Pro2 variants are
5–10-fold poorer than WT CooA, suggesting a minor role for the displaced
Pro2 in the active form of CooA. Properties of the Pro2
variants are further considered below.
Because Pro2 is not near the bound CO, then the structure of
Fe[II]CooA suggests that the most probable candidates for CO pocket residues
are on the C-helix, with Ile113, Leu116, and
Gly117 being particularly attractive
(1,
28). The RR analysis of CooA
variants altered at these positions establishes significant influences on
FeC and CO consistent with a positioning of these residues close to
the bound CO (see Fig. 7 and
Table I). When these residues
are replaced by H-bond donors, histidine and lysine FeC shifts up, and
CO shifts down from the position in WT CooA, as expected for a positive
polar
interaction.3
|
The effect is particularly marked for the Gly117 position. G117H
CooA displayed an 11-cm–1 upshift of FeC and
an 18-cm–1 downshift of CO. These shifts are
opposite in sign and comparable in magnitude to those observed when the distal
histidine of Mb is replaced by hydrophobic residues
(19,
20). G117S CooA also showed a
sizeable, though more modest effect on the RR spectrum, shifting FeC up by
7 cm–1 and CO down 12
cm–1. These results indicate that the newly
introduced His117 or Ser117 (albeit to a smaller extent)
lie sufficiently close to the CO to exert a positive potential, via H-bond
donation. However, the introduced His117 in G117H CooA is not well
ordered and exerts a heterogeneous effect on the CO as evidenced by broad
CO and FeC bands (Fig.
7). Consistent with the polarity interpretation, introduction of
steric hindrance at 117 position (G117I CooA) had very little effect on
FeC and CO (Table I),
because the CO environment remains non-polar. The importance of
Gly117 to CooA activation has already been described
(7) and is reflected in the
dramatically reduced activity of Gly117 variants seen in
Table I. Because
Gly117 is critical for activity, and its substitution perturbs the
RR spectra, it would be tempting to conclude that its role is to contact the
bound CO. Although this hypothesis cannot be discounted, it is also possible
that Gly117 is an important determinant of the C-helix structure
per se and simply happens to be near the bound CO.
At position 113, the effect of mutation on RR spectra is smaller, as
FeC shifts up only 3 cm–1 and CO down 11
cm–1 in I113H CooA (see
Fig. 7 and
Table I). Interestingly,
essentially the same effect was seen in I113A and I113D CooA. A possible
interpretation is that in these cases water molecules may be brought into
contact with the CO, filling the cavity that results from substituting the
smaller Ala residue, or attracted to the negatively charged Asp side chain.
These results suggest that Ile113 is close to the bound CO but not
as close as Gly117. In the case of I113Y CooA, the RR spectrum
gives a slight indication of negative polarity, shifting FeC down 2
cm–1 and CO up 3
cm–1. This effect could result from proximity to
the 
electron cloud of the Tyr ring to the CO. Substitutions at position
113 typically have modest effects on activity, as reported previously
(28) and shown in
Table I. The exception is
I113D, and we have already shown that hydrophilic residues at positions 113
and 116 severely perturb activation
(28). It is our working
hypothesis that the effect is because the hydrophobic pocket is important for
eliciting the proper conformational change in the C-helices upon displacement
of the Pro2 ligand by CO.
At position 116, Lys and Phe substitutions were tested. Other possible
H-bond donors were not examined, because they have been shown to accumulate
heme-containing CooA very poorly, presumably because of disruption of the
hydrophobic pocket (28). L116K
CooA variant was chosen, because it showed very high CO-independent DNA
binding activity (comparable with that of wild-type CooA in the presence of
CO) and diminished CO-dependent DNA binding activities (see Ref.
14 and
Table I). As shown in
Fig. 7, the introduction of Lys
at position 116 exerted an almost identical effect on the RR spectrum as did
G117H, shifting FeC by 13 cm–1 up and CO
by 18 cm–1 down
(Table I). This result is
consistent with the hypothesis that the L116K substitution, like G117H,
introduces positive polarity into what is otherwise a non-polar environment
around the CO. This CooA variant also showed broad CO and FeC bands.
CooA-CO activity is impaired by the L116K substitution but is still
considerable, indicating a modest perturbation of the active structure. The
high activity of L116K CooA in the absence of CO has already been analyzed,
and the result strongly implies that the substituted Lys residue serves as a
ligand in both the Fe[III] and Fe[II] forms. We have suggested that this
ligation leads to a similar helix repositioning as that seen in WT CooA when
bound to CO (14). The absence
of significant FeC or CO shifts in L116F is again consistent with
maintenance of a hydrophobic environment for the bound CO.
Importantly, in the G117H and L116K CooA variants, the FeC/CO point
shifts up and to the left in the backbonding plot
(Fig. 4, only L116K is shown,
for clarity) but remains displaced from the Mb line by the same amount as WT
CooA-CO. This behavior indicates that the Fe–His77 bond is
unaffected by the distal residue replacement, although backbonding is enhanced
by the distal polarity. CO binding weakens the Fe–His77 bond
in these variants, just as in WT CooA–CO.
It should be noted that these observations apply only to the major
population of CooA-CO molecules as described at the beginning of the
"Results." It is impossible to tell how the substitutions affect
the minor CooA–CO' fraction because of its weak signal and the
breadth of the perturbed bands. However, a non-polar binding pocket is also
indicated for the minor WT CooA–CO by its position in the FeC/CO
correlation (Fig. 4), close to
those of Mb variants with non-polar pockets.
In summary, the RR data establish that Gly117, Leu116, and Ile113 are all in or near the CO binding pocket. However, the data also indicate that substitutions of the Ile113 and Leu116 side chains are much more permissive with respect to CO activation of CooA than are those at Gly117. A possible interpretation is that the CO directly contacts the Gly117 and that any steric hindrance destabilizes the DNA binding conformation of the protein. However, it is also possible that Gly117 induces a critical bend in the C-helix, which is disrupted by substitutions.
We also examined other candidate residues for their effect on RR spectra. These included C-helix variants M124R and W110L and B-helix variants E99L and I95W (the B-helix is the short helix on the "bottom" of CooA as shown in Fig. 1). M124R (13) was chosen because of its significant impact on CO-sensing function of CooA. The others were chosen because of their potential proximity to the heme iron. As shown in Table I, all these variants except M124R had little effect on RR spectra, suggesting that these residues are not near the bound CO and consistent with the pocket being formed primarily by Gly117, Leu116, and Ile113.
The M124R replacement has little effect on the CooA-CO activity
(Table I) but produces a double
peaked CO band; one component is at the WT CooA position, 1982
cm–1, whereas the other is 17
cm–1 lower. The FeC band is lowered slightly
and is broadened (see Table I
and Fig. 7). Although
Met124 is further from the heme iron than are Gly117,
Leu116, and Ile113, it is possible that the long Arg
side chain reaches the vicinity of the bound CO in a fraction of the
CooA–CO molecules, producing the downshifted CO component. Modeling
of the introduced arginine in Fe[II]CooA
(13) suggested two favored
conformations of the side chain, the guanidine cation interacting either with
the seven-propionate of the heme or with the backbone carbonyl of
Ser78. It is possible that in the M124R-CO structure one of the Arg
conformers interacts instead with the CO.
The Strongly Basic Pro2 Ligand Is Expelled from the
Hydrophobic CO Pocket in the CO-bound Form of CooA at Physiological
pH—Extensive mutational studies have shown that Pro2 is
not critical for the response of CooA to CO
(11). Nevertheless, the
Pro2 ligand is a central aspect of cooperativity in CO binding by
CooA.2 Because the global conformational change of CooA upon CO
binding is initiated by the displacement of the Pro2 ligand by CO,
it would be informative to trace the fate of the displaced Pro2. We
have shown above that Pro2 does not appear to be near the bound CO
at normal pH, but when the solution pH was increased above 7, the FeC and
CO of WT CooA–CO were affected significantly
(Fig. 8). The FeC band at
487 cm–1 was replaced by one at 497
cm–1 whereas the CO band at 1982
cm–1 was replaced by one at 1964
cm–1. Although the latter frequency is close to
that of CooA–CO', the FeC frequency is distinctly higher than
in CooA–CO'. Thus the change at high pH is not simply an increase
in the CooA–CO' fraction, but instead a new species is formed in
basic solution.
|
The high pH FeC and CO values are similar to those displayed by the
L116K and G117H variants (see Fig.
7 and Table I). The
FeC/CO point remains well above the MbCO backbonding correlation
(Fig. 4), indicating little
change in the Fe–His77 bond. We infer that the effect of
raising the pH is to bring a H-bond donor into proximity with the bound CO. As
in L116K and G117H, the high pH CO and FeC bands are broadened,
suggesting heterogeneity in the H-bond donor position.
What might this H-bond donor be? Key evidence comes from the P3R4
CooA variant, whose FeC and CO bands are unperturbed when the pH is
raised, up to 11 (Fig. 8). We
conclude that the H-bond donor is the N-terminal Pro2, interacting
with the bound CO via its secondary amine NH group. When the two penultimate
residues, Pro3 and Arg4, are removed, the N terminus is
constrained from interacting with the CO.
The Pro2 interaction in WT CooA requires pH elevation, presumably because the secondary amine becomes protonated when it is displaced from the Fe[II] at pH 7. Because the CO binding pocket is hydrophobic, the protonated N terminus would be expelled from the binding site, because it is positively charged, just as the distal histidine is expelled from the MbCO binding pocket upon protonation at low pH (29, 30). At high pH, the displaced Pro2, being neutral, can remain in contact with the CO and enhance backbonding, just as the distal histidine does in Mb.
What is the pKa of the displaced Pro2? In
aqueous solution the pKa of the Pro amine is 10.5, but it
should be lowered in CooA–CO because of the energy cost of expelling
Pro2 from the hydrophobic pocket. In Mb, the
pKa of the distal His is 4.3
(29,
30), nearly three units lower
than the aqueous His pKa. We examined the pH dependence of
WT CooA–CO by decomposing the FeC band into 487- and
497-cm–1 components and plotting their intensities
against the solution pH (Fig.
9). The resulting titration curves yield a pKa
of 8.6, two units lower than the aqueous pKa of proline.
If the assignment of the pKa to Pro2 is
correct, then the energy cost for expelling the distal H-bond donor is about
two-thirds as high in CooA as it is in Mb.
|
We note that the absorption spectrum is not sensitive to the pKa 8.6 process. The Soret band position, which is 422 nm at pH 7, only shifts significantly when the pH is raised to values greater than 10; at pH 12 the band maximum is 419 nm. We found that the RR spectral changes could not be fully reversed for solutions whose pH was raised to 10 or higher, indicating some irreversible change in the protein structure. However the Soret band shift could be reversed, even from pH 12, showing that there is a different physical mechanism for perturbation of the absorption spectrum.
These results indicate that Pro2 does not lie near the bound CO
under physiological conditions, and the role of Pro2 in the active
form of CooA is uncertain. The DNA affinity of the CO-bound forms of P2Y and
P3R4 CooA is 5- to 10-fold poorer than that of WT CooA, implying some
small perturbation of the active structure when the N terminus is altered.
However the primary roles of Pro2 appear to be to maintain the
inactive form of CooA in the absence of CO
(8) and to provide a mechanism
for the cooperative binding of CO when it is present.2
The ligating nitrogen atom of Pro2 is part of a strongly basic
secondary amine and is expected to be a strong donor to the Fe[II]. Evidence
for strong donation is seen in the RR spectrum of Fe[II]CooA, which reveals a
depressed value, 1532 cm–1, for the
11 porphyrin ring stretching vibration
(25)
(Fig. 10, 568-nm excitation
was used to enhance 11 via resonance with the heme Q bands
(25)). This mode is known to
be sensitive to the strength of the axial ligand field in low spin Fe[II]
porphyrin complexes. For example, the band shifts from 1547 to 1533
cm–1 when methionine is replaced by lysine in
Fe[II] cytochrome c
(31) and from 1539 to 1527
cm–1 upon deprotonation of the imidazole complexes
of microperoxidase or of bisimidazole Fe[II] protoporphyrin
(32,
33). The sensitivity to
imidazole deprotonation was the basis of our earlier suggestion
(16) that His77
might be deprotonated or strongly H-bonded in Fe[II]CooA. However,
His77 is now revealed by the crystal structure to have a normal
H-bond with Asn42, and the position of 11 is
unaffected when Asn42 is replaced by the non-H-bonding Ala
(Fig. 10).
|
We conclude that the 11 depression is unrelated to
His77 but instead reflects the strong ligand field of
Pro2. This interpretation is supported by the observation
(Fig. 10) that
11 shifts up, by 4 cm–1, in the P2Y
and P3R4 variants. When Pro2 is replaced by Tyr, the
secondary amine is replaced by a primary amine at the terminus, producing a
somewhat weaker ligand field. In the P3R4 variant, the penultimate pair
of residues is deleted, shortening the N terminus. The Fe[II] protein remains
mainly six-coordinate, but a small fraction of the heme is five-coordinate, as
judged by a shoulder on the 4 porphyrin band at the expected
five-coordinate position, 1355 cm–1
(25). Pro2 might
remain the ligand in P3R4, but the shortening of the polypeptide chain
would strain the Pro2–Fe bond, again weakening the field.
This interpretation is consistent with the very rapid binding of CO to the
P3R4 variant.2
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Two principal findings are central to our model. 1) The C-helix residues Ile113, Gly117, and Leu116 become part of the CO binding pocket in CooA–CO. This is revealed by the spectral perturbations observed when their side chains are replaced with H-bond donors. This result implies repositioning of the hemes relative to the C-helices, because all three residues are at substantial distances from the heme iron in the Fe[II]CooA crystal structure (1).
Measured Fe–C
distances are 9.4 (10.2) Å for
Leu116, 13.0 (7.0) Å for Gly117, and 11.2 (9.3)
Å for Ile113. The first number refers to the residue on the
same subunit as the heme, whereas the second number refers to the residue on
the opposite subunit. Although not required crystallographically this part of
the structure is nearly 2-fold symmetric; the Fe–C
distances from the second heme are within 0.1 Å of those from the first.
It is notable that only in the case of Leu116 does the closer
residue reside on the same subunit as the heme. For both Ile113 and
Gly117, the residue from the opposite subunit is much closer than
is the residue from the same subunit. Thus a simultaneous rearrangement of
both hemes and both C-helices is suggested to bring all three residues close
enough to allow H-bond donor replacements to interact with the bound CO. 2)
The His77-Asn42 H-bond, which is evident in the
Fe[II]CooA crystal structure, appears to be broken upon CO binding. The
strongest evidence is that the CO and FeC RR bands are unaffected when
Asn42 is replaced by the non-H-bonding residue Ala (or by any other
residue), despite the known sensitivity of these bands to the H-bond status of
the proximal histidine ligand in heme-CO adducts
(19,
20). Moreover, the position of
the CO and FeC on the backbonding correlation reveal that the
Fe–His bond is weaker in CooA–CO than in MbCO, consistent with
His77 having no H-bond acceptor. Yet the Fe–His bond is
equally strong in Mb and in Fe[II]CooA as revealed by equal Fe–His
stretching frequencies (revealed for Fe[II]CooA in the five-coordinate heme of
the G117I variant), indicating the same extent of proximal His H-bonding. Thus
CO binding evidently leads to loss of the His77-Asn42
H-bond in CooA.
These are the findings that flow from the present study. Together with the Fe[II]CooA crystal structure, they lead to a model for the initial motions leading to activation of CooA. The proposed motions are shown as dashed arrows in Fig. 2.
Upon binding CO, the heme is suggested to slide upward, bringing the FeCO
unit closer to the side chain of Leu116 on the same subunit. As
discussed above, the L116K variant is active in the absence of CO, suggesting
that a Lys at this position induces the same heme displacement by displacing
Pro2 as the distal ligand. We modeled a Lys side chain in the
orientation of the Leu116 side chain and measured a 4.0-Å
separation from the iron to the amine nitrogen. Thus a 2.0-Å
displacement of the heme group would be required to form a bond. This
displacement provides an explanation for loss of the
His77-Asn42 H-bond, because His77 remains
bound to the heme and is displaced with it. It is likely that this heme
displacement is also involved in the His77/Cys75 ligand
switch that is known to occur upon oxidation to Fe[III]CooA
(4), because the shifted heme
position would accommodate Cys75 ligation. Similar heme movement
and ligand switching has been described for cytochrome cd1
(35). Although WT Fe[III]CooA
is not active, there are a number of mutations that induce activity in the
Fe[III] form of the protein
(13).
It is further suggested that when the heme is displaced upward, it is approached more closely by the C-helix from the second subunit, whose Gly117 and Ile113 residues help form the hydrophobic binding pocket for the CO. A modest motion of the second C-helix would permit H-bond donors at these positions to perturb the CO, whereas a much larger rearrangement of the first C-helix would be required to produce this effect. Thus the overall motion that initiates the conformation change to the active CooA-CO structure is viewed as a clockwise rotation of both hemes and both C-helices, in the plane of Fig. 2.
Some CooA molecules do not undergo this motion. The minority
CooA–CO' fraction represented by the
1962-cm–1 CO shoulder appears to have an
intact His77-Asn42 H-bond (the FeC/CO point
falls on the Mb backbonding line), indicating that the heme is undisplaced
from its position in the Fe[II]CooA structure. Presumably these molecules are
not active in binding DNA. We do not know whether they are in a dynamic
equilibrium with the active molecules or else are prevented from undergoing
activation by some kind of energy barrier.
What is the driving force for the proposed heme and C-helix displacements?
We propose that hydrophobic interactions are responsible
(28), perhaps abetted by the
electronic reorganization of the heme that results from a acceptor ligand
(CO) replacing a donor ligand (the N terminus). Examination of the
Fe[II]CooA crystal structure shows the heme to be partially exposed to
solvent, whereas it would become more buried by sliding into the cavity
(Fig. 11). Hydrophobic
interactions would be enhanced by the suggested displacement of the opposite
C-helix toward the heme. The hydrophobic character of the CO binding pocket is
evidenced by the two-unit pKa reduction of the
displaced Pro2, relative to the expected aqueous
pKa.
|
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
FOOTNOTES |
|---|
Present address: Dept. of Chemistry, University of Texas at San Antonio,
San Antonio, TX 78249.
|| Present address: Dept. of Physics and Astronomy, University of Aarhus, Ny
Munkegade, DK-8000 Aarhus C, Denmark.
** To whom correspondence should be addressed. Tel.: 609-258-3907; Fax: 609-258-0348; E-mail: spiro{at}princeton.edu.
1 The abbreviations used are: CAP, catabolite gene activator protein; RR,
resonance Raman; Mb, myoglobin; WT, wild-type; Mops,
4-morpholinepropanesulfonic acid.
2 M. Puranik, S. B. Nielsen, H. Youn, J. L. Bourassa, M. A. Case, A. N.
Hvitved, C. Tengroth, G. Balakrishnan, M. V. Thorsteinsson, J. S. Olson, G. P.
Roberts, J. T. Groves, G. L. McLendon, and T. G. Spiro, manuscript in
preparation.
3 An alternative interpretation is that these donors ligate the iron in place
of Pro2 and that CO displaces His77 instead. However,
there is no reason to suppose that distal replacements would labilize the
Fe-His77 bond. When His77 itself is replaced by Tyr, the
FeC/CO values (Table I)
are significantly different, and there is a five-coordinate CO-bound fraction,
implying displacement of both ligands
(16).
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
) with data taken from the larger data set
reported in Refs.
