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J. Biol. Chem., Vol. 277, Issue 37, 33616-33623, September 13, 2002
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,,§,
From the Department of Bacteriology and the
¶ Department of Chemistry, University of Wisconsin,
Madison, Wisconsin 53706
Received for publication, April 16, 2002, and in revised form, July 9, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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CooA is the CO-sensing transcriptional
activator from Rhodospirillum rubrum, in which CO binding
to its heme prosthetic group triggers a conformational change of CooA
that allows the protein to bind its cognate target DNA sequence. By a
powerful in vivo screening method following the
simultaneous randomization of the codons for two C-helix residues, 113 and 116, near the distal heme pocket of CooA, we have isolated a series
of novel CooA variants. In vivo, these show very high
CO-independent activities (comparable with that of wild-type CooA in
the presence of CO) and diminished CO-dependent activities.
Sequence analysis showed that this group of variants commonly contains
lysine at position 116 with a variety of residues at position 113. DNA-binding analysis of a representative purified variant, L116K CooA,
revealed that this protein is competent to bind target DNA with
Kd values of 56 nM for Fe(III), 36 nM for Fe(II), and 121 nM for Fe(II)-CO CooA
forms. Electron paramagnetic resonance and electronic absorption
spectroscopies, combined with additional mutagenic studies, showed that
L116K CooA has a new ligand replacing Pro2 in both Fe(III)
and Fe(II) states. The most plausible replacement ligand is the
substituted lysine at position 116, so that the ligands of
Fe(III) L116K CooA are Cys75 and Lys116 and
those in the Fe(II) form are His77 and Lys116.
A possible explanation for CO-independent activity in L116K CooA is that ligation of Lys116 results in a repositioning
of the C-helices at the CooA dimer interface. This result is consistent
with that repositioning being an important aspect of the
activation of wild-type CooA by CO.
Rhodospirillum rubrum, a photosynthetic
bacterium, can grow with CO as a sole energy source. The presence of
CO is sensed by CooA, and CO-bound CooA activates the
transcription of a series of genes encoding the CO oxidation system in
R. rubrum (1). CooA contains a heme prosthetic
group, as do other sensors for gaseous molecules such as soluble
guanylyl cyclase (sGC),1
FixL, DOS, and HemAT (2-5). The heme moieties are directly involved in
binding their respective effector molecules and thereby regulate the
activity of the sensor proteins. In CooA, the signal of CO binding to
the heme is transmitted to the DNA-binding domain, the consequence of
which is a conformational change allowing CooA to bind its target DNA sequence.
The crystal structure of Fe(II) CooA revealed a novel subunit-swapped
N-terminal Pro2 as a heme ligand trans to His77
in the homodimer (6). Because the Fe(II) form of CooA is 6-coordinate and low spin, the CO-sensing mechanism of CooA necessarily
involves the displacement of one of these two ligands. Nuclear magnetic resonance and time-resolved resonance Raman studies identified Pro2 and His77 as the displaced and retained
ligands, respectively, in the Fe(II)-CO form of CooA (7, 8). CooA is a
redox sensor as well as a CO sensor because only the Fe(II) form of
CooA is competent to bind the physiological effector, CO.
Interestingly, CooA undergoes a redox-dependent ligand switch,
in which Cys75 replaces His77 in the Fe(III)
form (9, 10). Although the physiological role and exact mechanism of
the ligand switch is still elusive, this switch indicates that the
position of the heme prosthetic group of CooA is highly flexible
relative to surrounding protein matrix.
CooA belongs to the family of transcriptional activators containing the
cAMP receptor protein (CRP) (11). In addition to similar target DNA
sequences, CooA and CRP exhibit similar overall topologies, in which
each protein is a dimer and each monomer contains two functionally
distinct domains (Fig. 1). The
effector-binding domain of each protein binds its respective small
molecule (CO for CooA, cAMP for CRP). The end result of effector
binding to this domain is a precise repositioning of the DNA-binding
domain, which allows each protein to bind its cognate target DNA
sequence. The two domains are connected through a long
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helix
(designated as C-helix) that also serves as a subunit dimerization
interface (6). Importantly, the comparison of the structure of effector (CO)-free CooA with that of effector (cAMP)-bound CRP (6) reveals a
repositioning of these two C-helices with respect to each other, suggesting a role of this repositioning in the activation of these proteins in response to their respective effectors. Consistent with
this concept, alterations of particular amino acids within the
C-helices exert a variety of effects on the activity in CRP (12, 13)
and in the fumarate and nitrate reductase activator protein (14),
another member of this transcriptional activator family. It is
therefore our working hypothesis that CO binding to the heme of CooA
alters the positioning of the C-helices and that this repositioning
effectively transmits the effector-binding signal through the protein
and leads to the reorganization of the DNA-binding domains.
View larger version (37K):
[in a new window]
Fig. 1.
Structural comparison of effector-free CooA
(PDB no. 1ft9) with effector-bound CRP (PDB no. 1g6n). Two
functionally distinct domains (effector-binding domain and DNA-binding
domain) are noted. The C-helices that form the dimerization interface
in each protein and the F-helices that bind target DNA sequences are
marked by arrows.
It is unknown how the displacement of Pro2 by CO might
affect C-helix positioning as proposed above. Although the release of Pro2 from the heme iron by incoming CO triggers the
sequential events that result in the activation of CooA, a variety of
Pro2 variants including a 5-residue-deleted variant
(
P3-I7 CooA) are CO-responsive (15, 16), indicating that the
released Pro2 is not critical for activation of CooA in
response to CO. Rather, the role of Pro2 appears to be to
keep CooA inactive in the absence of
CO,2 which has led to the
hypothesis that the release of the heme from Pro2 might
allow a steric interaction between the heme and the C-helices, resulting in their repositioning. Consistent with this view,
Gly117 on the C-helix is important for CO activation (16).
To better understand the importance of heme/C-helix interactions in
CooA activation, we have investigated other C-helix residues,
Ile113 and Leu116, which together with
Gly117 lie near the distal heme pocket of CooA. In this
report, we address a novel CooA variant that, in the absence of
effector (CO), effectively mimics the active conformation of wild-type
(WT) CooA in the presence of CO. A possible underlying mechanism behind
such a novel phenotype is discussed from a structural viewpoint.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Strains, Plasmids, and in Vivo Assays--
The construction of
strains overexpressing WT CooA and CooA variants in an
Escherichia coli background having a 
-galactosidase reporter system in the chromosome was described previously, and in vivo activities were quantitated using the standard
protocol (18). All the site-directed and region-randomized
cooA mutations were constructed in a pEXT20-based expression
plasmid, which provides tight control of CooA expression (15).
Creation of cooA Mutations--
Site-directed mutagenesis
involved polymerase chain reaction amplification of cooA
with primers designed to incorporate the desired nucleotide change, as
described elsewhere (19). The method used for dual randomization was
essentially identical to the method used for site-directed mutagenesis,
except that the primers contained randomized codons for both 113- and
116-positions. Screening of CooA variants involved the analysis of
their ability to cause -galactosidase accumulation on agar plates
incubated under different growth conditions as described previously
(15). Based on colony color, CooA variants could be classified as
active, weakly active, and inactive. Selected variants were examined
quantitatively in an in vivo -galactosidase assay, after
which the cooA genes were sequenced to determine the
causative residue changes.
Purification of WT CooA and CooA Variants--
The purification
of WT CooA and the P3R4 CooA variant (>95% homogeneity) were
performed as described previously (9). The purification of L116K and
L116K P3R4 CooA involves the published method through the ammonium
sulfate precipitation following the polyethyleneimine precipitation
(9). However, preliminary work indicated that CooA variants with the
L116K substitution had lower stability than did WT CooA at normal salt
levels but were substantially stabilized with 0.5 M NaCl.
Therefore, the resulting ammonium sulfate (final 50%) pellet was
dissolved in 25 mM MOPS, pH 7.4, 0.5 M NaCl,
5% glycerol and applied to a hydroxylapatite Bio-Gel HTP
column (Bio-Rad). This column was extensively washed with 10 mM potassium phosphate, 25 mM MOPS, pH 7.4, 0.5 M NaCl, 5% glycerol and eluted with a linear gradient of
10-100 mM potassium phosphate in 25 mM MOPS,
pH 7.4, 0.5 M NaCl, 5% glycerol. The resulting
CooA-containing fractions were precipitated with 50% ammonium sulfate,
and the samples were applied to Sephacryl S-100 (Amersham Biosciences)
size exclusion column in 25 mM MOPS, pH 7.4, 1 M NaCl, 5% glycerol. Again, the resulting CooA fractions were precipitated with ammonium sulfate and resuspended in 25 mM MOPS, pH 7.4, 0.5 M NaCl. The residual
ammonium sulfate was removed on a Sephadex G-25 fine (Amersham
Biosciences) column equilibrated in 25 mM MOPS, pH 7.4, 0.5 M NaCl, and the final sample was stored at 
80 °C until
use. The heme content of CooA preparations was estimated using the
extinction coefficient of WT CooA (20) or by a modified reduced
pyridine-hemochromogen method (20), and protein concentration was
measured using the BCA assay (Pierce).
Preparation of Hydroxylapatite Batch-treated CooA Samples-- Preparation of hydroxylapatite batch-treated CooA samples was carried out using the procedure described previously (16). By this method, heme-containing CooA accounted for ~10% of total protein, in the case of WT CooA. These samples were used for the comparison of electronic absorption spectra and heme accumulation of CooA variants.
Electronic Absorption Spectroscopy-- Electronic absorption spectroscopy of CooA samples was performed using a Shimadzu UV-2401PC spectrophotometer, at room temperature in quartz cuvettes with a sample interval of 0.5 nm, a slit width of 1 nm, and monochromator grating of 1600 lines/mm. Electronic absorption spectra of CooA samples were obtained using 25 mM MOPS, pH 7.4, 0.5 M NaCl, if not stated. For the spectra taken at pH 4, the protein samples were dissolved in 0.1 M sodium acetate buffer, pH 4, 0.1 M NaCl.
EPR Spectroscopy--
Fe(III) CooA samples, purged with argon,
were frozen and stored at 77 K in 25 mM MOPS, pH 7.4, and
0.5 M NaCl, except where noted. For EPR analyses, the final
heme concentrations of WT, L116K, and L116K P3R4 CooA were 200, 49, and 120 µM, respectively. Spectra were recorded on a
Bruker ESP 300E spectrometer equipped with an Oxford ESR 900 continuous
flow cryostat and an Oxford ITC4 temperature system to monitor and
regulate the temperature. The microwave frequency was monitored using a
Varian EIP model 625A CW frequency counter. The spectra were recorded
at X-band and 10 K; each reported spectrum represents the average of 4 (WT CooA) or 16 (L116K and L116K P3R4 CooA) scans, each comprising 1024 points. The only EPR signals that were observed between 0 and 4000 gauss are those reported. Specific conditions for the recording
of each spectrum are given in the legend to Fig. 7.
In Vitro DNA-binding Assays--
In vitro DNA-binding
assays of WT CooA and CooA variants were performed using the
fluorescence polarization technique with a Beacon 2000 fluorescence
polarization detector (PanVera Corp., Madison, WI) as described
previously (15). As a fluorescence probe, a 26-base pair target DNA
containing PcooF was labeled with Texas Red on
one end of the duplex and used at the concentration of 6.4 nM. Binding assays were performed in 40 mM Tris-HCl, pH 8.0, 6 mM CaCl2, 50 mM
KCl, 5% (v/v) glycerol, and 1 mM dithiothreitol. 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 (21).
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Unique Functional Properties of L116K CooA--
As described in
the Introduction, it is a reasonable hypothesis that CO binding to the
heme of CooA causes a perturbation of the C-helices. Under such a
scenario, one would expect that residues on the surface of the
C-helices that are in the vicinity of the heme would be important for
that process. We therefore examined the effects of the perturbation of
C-helix residues near the heme. Ile113 and
Leu116 together with Gly117 are the C-helix
residues within 7 Å of the heme iron of CooA. These residues lie near
the heme on Pro2 side, the ligand that is displaced by CO
(Fig. 2). To investigate the functional
importance of these hydrophobic residues in the CooA activation
mechanism, we completely randomized the codons for both
Ile113 and Leu116 and screened the resulting
library of ~6,000 clones for functionally interesting variants. The
screening involved the detection of -galactosidase activity in
colonies of an E. coli strain in which CooA regulates
lacZ expression (15). One class included CO-responsive variants, a behavior rather like that of WT CooA. This class of CooA
variant will be described in another report. Of relevance to this
report, ~1.6% of the clones displayed significant activity under
anaerobic conditions without CO, a behavior distinctly different from
that of WT CooA (Table I). These
CO-independent CooA variants were then reanalyzed in a quantitative
liquid assay of in vivo -galactosidase activity, and a
number of them were sequenced to reveal the causative
substitutions.
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As seen in Table I, several patterns are apparent. As expected, all the
selected variants showed high in vivo activity (comparable with that of WT CooA in the presence of CO) under anaerobic conditions (when CooA is in the Fe(II) state), the same conditions used in the
initial screen. The variants also displayed substantial in vivo activity under "aerobic" and "anaerobic + CO"
conditions, but these activities were dependent upon the residue at
position 113. Although the activities under "anaerobic + CO" are
generally lower than the anaerobic activities, they showed nearly
identical activities when there was a large hydrophobic residue at
position 113. Because both oxidation/reduction and CO binding occur at the heme moiety in CooA, this activity modulation suggests that local
conformational change of the heme moiety may be the molecular mechanism
behind this unusual phenotype. The loss in activity upon the
introduction of CO is particularly striking in this group of variants
and is clearly different from the behavior of WT CooA. Such a phenotype
has not been found previously for CooA variants despite extensive
studies of a variety of variants altered in the heme vicinity on either
side including those altered at the heme ligands (Pro2,
Cys75, and His77 variants) (9) or in the distal
heme pocket (Gly117 variants) (16) (Fig.
3). This novel behavior suggests that the
mechanism of activation in these variants might be rather different
from that of WT CooA.
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A second obvious pattern among the CO-independent variants is the presence of Lys at position 116, although a variety of residues were acceptable at position 113. Moreover, the activity of this group of variants under anaerobic conditions is almost invariant with respect to the residue at position 113, although the activities under "aerobic" and "anaerobic + CO" conditions are somewhat modulated by residues at that position. The basis of this modulation is unknown, but might reflect differential accumulation of heme-containing CooA in different position 113 variants. There is clearly an effect under aerobic conditions in rich medium (Table I), but other conditions have not been examined. The failure to detect variants of this phenotype with Arg at position 116 suggested that the behavior was not merely the result of the introduction of positive charge in the distal heme pocket of CooA. This view was confirmed by the construction of L116R CooA and I113K CooA. Consistent with the results of randomization, neither L116R nor I113K resulted in CO-independent CooA activation; L116R CooA was inactive (0.5, 0.7, and 0.8% for "aerobic," "anaerobic," and "anaerobic + CO" conditions) and I113K CooA was CO-responsive, though to a lower level than WT CooA (1, 4, and 58% for aerobic, anaerobic, and anaerobic + CO conditions). These results strongly imply that Lys116 is central to the CO-independent activity in these CooA variants.
Purification of L116K CooA--
To understand the biochemical
basis for the activity in the absence of CO, we isolated a
representative variant, L116K CooA. With the procedures described under
"Experimental Procedures," the isolated material was ~90% pure
based on SDS-PAGE. For spectral comparisons, we also isolated L116K
P3R4 CooA to a purity of ~70%. The column purification step of
these CooA variants always contained high levels of salt (0.5-1 M NaCl) because such conditions stabilized the protein
(data not shown); for the same reason, all the analyses were carried
out in the presence of 0.5 M NaCl if not otherwise
specified. However, the use of high salt did not completely eliminate
the heme loss observed during purification. The final preparation
contained only 50% of the heme expected for that amount of CooA.
Because the DNA binding activity of L116K CooA is modulated by the
oxidation or CO binding to the heme moiety (see below), heme-containing
L116K CooA is responsible for the activity. Therefore, all the CooA
concentrations used here were based on the concentration of heme rather
than protein.
In Vitro DNA-binding Analysis of L116K CooA--
Substantial
in vivo activity of a CooA variant does not always mean that
it has high DNA binding activity. A specific group of variants, with
changes in the "activating regions" that interact with RNA
polymerase, display increased in vivo activity but without an increase in the DNA-binding affinity (22). These CooA variants have
a higher affinity for RNA polymerase, which has the effect of trapping
the small population of CooA in the active form without effector at the
appropriate promoter. To determine whether the high in vivo
activity of Fe(III) and Fe(II) L116K CooA is correlated with increased
DNA binding activity, we tested the DNA binding of L116K CooA directly
with fluorescence anisotropy using WT CooA as control. To eliminate
possible nonspecific DNA binding of L116K CooA, 100 times excess of
salmon sperm DNA was used for this assay. When the Texas-Red-labeled
target DNA was titrated with increasing amounts of purified L116K CooA,
all three states of the protein (Fe(III), Fe(II), and Fe(II)-CO L116K)
showed an increase in anisotropy values reflecting
Kd values of 56, 36, and 121 nM,
respectively (Fig. 4). In contrast, WT
CooA showed DNA binding activity corresponding to a
Kd of 12 nM only in the presence of CO
(Fig. 4); Fe(III) or Fe(II) WT CooA did not show any DNA binding up to
500 nM. These results indicate that, unlike WT CooA, L116K
CooA in all three forms has an easily detectable population in a
reasonable conformation for DNA binding. The ability of L116K CooA to
bind PcooF DNA with approximately the same
affinity as WT CooA, as well as its ability to induce in
vivo activity, demonstrates that DNA binding by L116K CooA is
substantially normal. This implies that L116K CooA recognizes DNA
through the same mechanism as does WT CooA, which requires significant
repositioning of F-helices containing DNA-binding domains. We were
concerned that the Lys substitutions at position 116 might affect CooA
dimerization, and therefore DNA binding, because Leu117
lies on the C-helices that form the dimer interface. However, gel
filtration analysis of Fe(III) L116K CooA showed that it migrated as a
single peak at the position of dimeric WT CooA (data not shown). For
technical reasons, it has not been possible to show that the DNA-bound
form of CooA is a dimer, although the DNA affinities and dimerization
of the DNA-free forms make that the most likely possibility.
Nevertheless, it is a formal possibility that the poorer affinity DNA
affinity of Fe(II)-CO L116K CooA might result from a modest shift to
the monomeric form.
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Although all three forms of L116K CooA show substantial DNA binding in vitro, there are some modest differences between these results and those obtained in vivo. Most strikingly, the Fe(III) form has a greater affinity for DNA than does the Fe(II)-CO form (Fig. 4), yet displays lower activity in vivo (Table I). We assume that some of these differences reflect differences in the precise positioning of the activating regions, as noted above. Specifically, we assume that the DNA-binding domains are properly positioned to bind DNA in Fe(III) form of L116K CooA, but that the mis-positioning of the heme in Fe(III) L116K CooA (relative to that typically seen in Fe(II)-CO WT CooA, for example) improperly positions the activating regions and therefore results in lower in vivo activity. Moreover, we also note that there are differences in the levels of accumulation of heme-containing CooA under these three conditions (data not shown), which might also affect the activities detected in vivo.
A Different Endogenous Ligand Replaces Pro2 in Fe(III)
L116K CooA--
Given the close proximity of the position 116 residue
to the heme and the unusually high aerobic DNA binding activity of
L116K CooA, we expected that Fe(III) L116K would be structurally
perturbed around the heme center and that this perturbation might be
revealed by electronic absorption spectroscopy. Because of increased
stability of L116K CooA in the presence of 0.5 M NaCl, all
the spectra were taken at this salt concentration. As shown in Fig.
5, the electronic absorption spectrum of
Fe(III) L116K CooA showed a slightly blue-shifted Soret maximum of 422 nm compared with Fe(III) WT CooA (Table
II), but was consistent with a typical
6-coordinate low spin heme. The electronic absorption spectrum of
Fe(III) WT CooA in 0.5 M NaCl gave the same 6-coordinate
low spin heme spectrum as in 0.1 M NaCl. The observation of
6-coordinate low spin heme in Fe(III) L116K CooA was surprising because
all the aerobically active CooA variants studied so far have shown
significant perturbation of Pro2 ligation (16, 23), which
was revealed as a significant amount of 5-coordinate high spin heme
with displacement of Pro2 (the spectrum of 5-coordinate
high spin heme is like that in Fig. 5D). This correlation
between DNA binding and the presence of high spin heme led to the
working hypothesis that such deligation might be necessary to mimic the
deligation of Pro2 by CO in the activation of WT CooA. We
therefore tested whether Pro2 is still a ligand in Fe(III)
L116K by investigating the spectral properties of Fe(III) L116K P3R4
CooA. The P3R4 alteration of CooA has been shown to severely perturb
the ability of Pro2 to serve as a ligand in Fe(III) CooA
(16), as is evident by the significant population of 5-coordinate high
spin heme present in Fe(III) P3R4 CooA (Fig. 5). In contrast,
Fe(III) L116K P3R4 CooA showed 6-coordinate low spin spectral
features similar to that of Fe(III) L116K CooA (Fig. 5). Considering
the severe spectral perturbation in Fe(III) P3R4 CooA, it is highly
unlikely that Pro2 is the ligand in Fe(III) L116K P3R4
CooA. Moreover, the close spectral similarity between Fe(III) L116K
P3R4 CooA and Fe(III) L116K CooA (Fig. 5 and Table II) is consistent
with the hypothesis that both CooA variants have the same ligand
replacing Pro2. Because of the proximity of
Lys116 to the heme and other arguments listed below, we
suggest that the new ligand is actually the introduced
Lys116 in both Fe(III) L116K P3R4 CooA and Fe(III) L116K
CooA.
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The comparison of the electronic absorption spectra of the CooA
variants at pH 4 gives a much clearer picture about the ligation structure of Fe(III) L116K CooA (at this pH, 0.5 M NaCl
leads to the precipitation of Fe(III) L116K CooA, so 0.1 M
NaCl was used for all samples). Under low pH conditions, both Fe(III)
L116K and L116K P3R4 CooA displayed the spectral features of
6-coordinate low spin hemes (Fig. 6),
whereas Fe(III) WT has the features of a 5-coordinate high spin heme
(the pKa of the transition from 6- to 5-coordinate
in Fe(III) WT CooA was previously reported to be ~5.5; Ref. 16). This
result strengthens the above suggestion that Fe(III) L116K CooA and
Fe(III) L116K P3R4 CooA share a common ligation structure, which is
clearly different from that of Fe(III) WT CooA or of Fe(III) P3R4
CooA. The inset in Fig. 6D shows the pH-dependent spectral change of Fe(III) L116K CooA, giving
a pKa of 3 for the 6- to 5-coordinate transition of
the protein. Although it is not known if this pKa
value is of the suggested Lys116 or the trans-ligand, it
has been reported that the pKa of Lys could be
lowered by as much as 7 pH units when it is a heme ligand (24).
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Electron paramagnetic resonance (EPR) spectroscopy of Fe(III) L116K
CooA confirms that the heme is low spin and reveals the presence of a
cysteine thiolate ligand. The EPR spectrum of L116K CooA (Fig.
7) exhibits two overlapping sets of
rhombic signals, with g values of 2.47, 2.24, and 1.89, and
2.42, 2.24, and 1.91, both characteristic of low spin Fe(III) heme. The
g anisotropies are clearly indicative of the presence of a
thiolate ligand; the rhombicity and tetragonality parameters place both
signals in the P region of a Blumberg-Peisach plot (25). To verify that the thiolate ligand in the L116K variant is Cys75, a C75A
substitution was introduced into L116K CooA. The electronic absorption
spectrum of Fe(III) L116K C75A CooA was distinct from that of Fe(III)
WT CooA or that of Fe(III) L116K CooA, with a blue-shifted Soret
maximum (Table II). The altered spectrum in the double variant suggests
that Cys75 is indeed a ligand to the heme in Fe(III) L116K
CooA. Furthermore, the specific ligand environment present in L116K
CooA, including Cys75, is necessary for substantial
in vivo activity in the Fe(III) form. In contrast to L116K
CooA, the double variant, Fe(III) L116K C75A CooA, shows little
in vivo activity (Table II).
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The heme coordination environment of Fe(III) L116K CooA is distinct
from that of WT CooA, as evidenced by the unique low spin g
values and EPR signal intensities observed. Comparison of the EPR
spectra of Fe(III) L116K CooA, and the WT CooA (Fig. 7) reveals small
but significant shifts in the g values, and substantial changes in the relative intensities of the two observed signals. The
two signals observed for Fe(III) L116K CooA are similar in g
anisotropy to the major signal in WT CooA, but the precise g values of Fe(III) L116K CooA are unique, suggesting that the ligand environment in the variant is similar but not identical to that of the
WT CooA. The intensities of the two signals in Fe(III) L116K CooA are
approximately equal to one another. These observations suggest that the
coordination environment in Fe(III) L116K CooA is perturbed in some way
from that of the native protein. Consistent with the small changes in
g values and in g anisotropy is the hypothesis
that the primary amine ligand of Lys116 had replaced the
secondary amine of Pro2 in the L116K CooA variant. To
further test this hypothesis, the EPR spectrum of Fe(III) L116K P3R4
CooA was recorded (Fig. 7). The simple N-terminal deletion variant
Fe(III) P3R4 CooA is high spin, with observed high spin g
values of 8.07, 7.08, and 4.91 (16). In contrast, Fe(III) L116K P3R4
CooA is low spin. Only a minimal high spin signal at approximately
g = 4.3 was observed, which could be the result of
non-heme iron. Although the spectrum of Fe(III) L116K P3R4 CooA is
less well resolved than that of Fe(III) L116K CooA, the g
values and g anisotropy appear to be the same in both
spectra. These observations suggest that the ligation environment in
the two variants is the same and support the conclusion that
Lys116 has replaced Pro2 as the ligand trans to
Cys75 in the L116K CooA variants.
The combination of EPR spectroscopy, electronic absorption spectroscopy, and mutant analysis provides compelling evidence that Fe(III) L116K CooA has a new ligand trans to Cys75, and is consistent with that ligand being Lys116.
An Endogenous Ligand Replaces Pro2 in Fe(II) L116K CooA-- Because L116K CooA has functionally novel properties in the Fe(II) form, it was also important to determine the heme ligands in that form. It has been demonstrated that WT CooA undergoes a ligand switch upon reduction, with His77 replacing Cys75 (9, 10). The electronic absorption spectrum of Fe(II) L116K CooA showed a 6-coordinate low spin heme, but lacked the Soret maximum near 450 nm, a diagnostic feature of thiolate-ligated Fe(II) heme proteins (Fig. 5). This result suggests that Fe(II) L116K CooA lacks a cysteinate ligand and that the ligand switch also occurs in this variant. This notion is further supported by the observation that the introduction of the C75A substitution fails to perturb the spectrum of Fe(II) L116K CooA (Table II). Finally, we constructed a strain with L116K H77A CooA and measured the effect of the H77A substitution on the electronic absorption spectrum and in vivo activity. Relative to that of Fe(II) L116K CooA, the Soret maximum of Fe(II) L116K H77A CooA was blue-shifted with concomitant lowering of the in vivo activity (Table II), consistent with the notion that His77 is one of the two ligands in Fe(II) L116K CooA, just as in Fe(II) WT CooA.
We then asked if Lys116 could be a ligand in Fe(II) state
of L116K CooA as was suggested for the Fe(III) state. Fig. 6 shows the
electronic absorption spectrum of Fe(II) L116K CooA at pH 4 compared
with those of Fe(II) WT CooA and L116K P3R4 CooA. Fe(II) WT CooA
showed a featureless spectrum at pH 4. Both Fe(II) L116K CooA and
Fe(II) L116K P3R4 CooA showed essentially identical spectra, with
6-coordinate low spin heme evident, although continual denaturation of
Fe(II) L116K CooA or L116K P3R4 CooA was observed. The above results
suggest that Fe(II) L116K CooA and Fe(II) L116K P3R4 CooA may share
a common ligand that is stronger than Pro2, the ligand
trans to His77 in Fe(II) WT CooA. The similarity in
functional properties between Fe(II) L116K CooA and L116K P3R4 CooA
is also consistent with the spectral results. The simplest hypothesis
for these results is that Lys116 serves as the ligand in
Fe(II) L116K CooA.
The Fe(II)-CO State Is Also Perturbed in L116K CooA--
The
CO-bound form of L116K CooA was stable for up to 30 min at room
temperature (but see below) and showed a characteristic blue-shifted
Soret maximum compared with that of Fe(II)-CO WT CooA. The 418-nm Soret
maximum for Fe(II)-CO L116K CooA was observed in all the variants
containing the L116K substitution, including L116K C75A CooA, L116K
H77A CooA, and L116K P3R4 CooA (Table II). This invariant Soret
maximum might reflect CO binding to the same side of the heme in all
the variants containing the L116K substitution, although it is unclear
whether CO binding displaces His77 or Lys116 in
these proteins. As noted previously, the activity of the Fe(II)-CO form
of L116K CooA is lower than that of the Fe(II) form. The less effective
DNA binding activity of Fe(II)-CO L116K CooA could be rationalized by
either binding to the "wrong side" of the heme or by perturbation
of the heme position when CO was bound on the "Pro2
side," given the fact that Fe(II) L116K CooA effectively mimics the
active conformation of Fe(II)-CO WT CooA.
Although Fe(II)-CO L116K CooA was stable, a small portion of the CooA (~5%) was irreversibly precipitated upon the initial addition of CO to the Fe(II) form, as reflected in a reduced ratio of AFe(II)-CO/AFe(II) compared with WT CooA (Table II). This small amount of precipitation cannot be responsible for the 4-fold increase of Kd value for DNA binding, so we do not believe that it significantly perturbs the reported results, although the cause of the phenomenon is unknown.
Working Hypothesis--
The data presented here show that
Pro2 has been replaced by another ligand in the Fe(III) and
Fe(II) states of L116K CooA and Lys116 is an excellent
candidate for the replacement ligand. Fig.
8 shows a representation of the position
of Lys116 relative to the known Fe(II) WT CooA structure as
viewed with the SwissPro program. Among 16 possible conformers, we
chose the Lys116 conformer that gives the shortest distance
from the N-
of Lys116 to the nitrogen ligand of
His77. The distance from heme iron to the nitrogen ligand
of His77 is 2.12 Å in the known Fe(II) WT CooA and the
distance between the nitrogen ligand of His77 and the N-
of Lys116 was calculated to be 5.39 Å for the
Lys116 conformer (Fig. 8), if the protein backbones
remained unchanged. The difference of 3.27 Å is apparently too far for
a Lys116-Fe ligation, considering that the only
structurally known ligation of this type is the 2.1-Å Lys-Fe bond
length in cytochrome c nitrite reductase (17).
Lys116 ligation therefore would require a significant
change in the relative positions of the heme and the C-helix region
compared with their positions in the Fe(II) WT CooA structure. Because Lys116 ligation suggests that the heme is attached to
His77 and Lys116, it would also alter the
relative position of the C-helix of one subunit with respect to its own
effector-binding domain of the same subunit (intrasubunit
reorientation). However, accepting the uncertainty in the precise
ligand arrangement in L116K, it is still appropriate to ask how this
ligation might result in the novel DNA binding activities of the
protein. Structural comparison between effector-free CooA and
effector-bound CRP (6) showed that there is a difference in the
relative conformations of the long C-helices in these two proteins and
led to the hypothesis that this conformational change might serve as
the signal transduction link between the CO binding to the heme and the
movement of the DNA-binding regions. Consistent with this, we have
found that altering the relative position of the C-helices in the
121-126 region by improving its quality as a leucine zipper leads to
CooA variants that display activity in the absence of
effector.2 Therefore, we assume Lys116 ligation
in Fe(II) L116K CooA causes a similar repositioning of the C-helices,
but quite possibly by a different mechanism from that of CO with WT
CooA. For example, the intrasubunit reorientation mentioned above can
contribute to C-helix repositioning by altering the interactions
between the effector-binding domain (Ile63 through
Asp72) and portions of the C-helix (Arg118
through Thr126) occurring in the known Fe(II) CooA
structure. In short, we hypothesize that Lys116 ligation in
Fe(II) L116K CooA effectively mimics a local heme environment change of
WT CooA upon CO binding by a mechanism involving a spatial
reorientation of the heme moiety relative to the C-helix.
|
Conclusion--
The major conclusions of this paper are as
follows. (i) The fact that L116K CooA actually loses activity upon CO
binding is interesting and thus far novel among CooA variants. (ii) The
variant has perturbed the ligation of Pro2, and the ligand
that replaces it is apparently Lys116, a ligand that is
relatively unusual among heme proteins. This conclusion is supported by
a variety of spectral and mutational results. (iii) A reasonable
hypothesis for the CO-independent activity in L116K involves a
repositioning of the C-helices, which is consistent with the current
view of CO activation of WT CooA, though the precise mechanism for the
repositioning in the two proteins must be different.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Mary Conrad for helpful discussion and Jose Serate, Melissa Killen, John Beack, and Cristin Heyroth for technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported by the College of Agricultural and Life Sciences (University of Wisconsin, Madison, WI), by National Institutes of Health Grants GM53228 (to G. P. R.) and HL65217 (to J. N. B.), and by a National Research Service Award fellowship (to M. V. T.).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.
§ Current address: Dept. of Vaccine Bioprocess Engineering, Merck & Co., Inc., West Point, PA 19486.
To whom correspondence should be addressed. Tel.:
608-262-3567; Fax: 608-262-9865; E-mail:
groberts@bact.wisc.edu.
Published, JBC Papers in Press, July 16, 2002, DOI 10.1074/jbc.M203684200
2 R. L. Kerby, H. Youn, M. V. Thorsteinsson, and G. P. Roberts, submitted for publication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: sGC, soluble guanylyl cyclase; CRP, cAMP receptor protein; EPR, electron paramagnetic resonance; WT, wild-type; MOPS, 3-(N-morpholino)propanesulfonic acid; PDB, Protein Data Bank.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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