From the Department of Biochemistry, Uppsala
University, Box 576, 75123 Uppsala, Sweden, the ¶ Department of
Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU,
United Kingdom
Received for publication, August 11, 2000, and in revised form, October 16, 2000
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ABSTRACT |
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The heme ligation in the isolated c
domain of Paracoccus pantotrophus cytochrome
cd1 nitrite reductase has been characterized in
both oxidation states in solution by NMR spectroscopy. In the reduced
form, the heme ligands are His69-Met106, and
the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome
cd1 nitrite reductase. In the oxidized state,
however, the structure of the isolated c domain is
different from the structure seen in oxidized crystals of intact
cytochrome cd1, where the c heme
ligands are His69-His17. An equilibrium mixture
of heme ligands is present in isolated oxidized c domain.
Two-dimensional exchange NMR spectroscopy shows that the dominant
species has His69-Met106 ligation, similar to
reduced c domains. This form is in equilibrium with a
high-spin form in which Met106 has left the heme iron.
Melting studies show that the midpoint of unfolding of the isolated
c domain is 320.9 ± 1.2 K in the oxidized and
357.7 ± 0.6 K in the reduced form. The thermally denatured forms
are high-spin in both oxidation states. The results reveal how redox
changes modulate conformational plasticity around the c
heme and show the first key steps in the mechanism that lead to ligand
switching in the holoenzyme. This process is not solely a function of
the properties of the c domain. The role of the
d1 heme in guiding His17 to the
c heme in the oxidized holoenzyme is discussed.
The x-ray structures of Paracoccus pantotrophus
(formerly Thiosphaera pantotropha (1)) cytochrome
cd1 nitrite reductase in the oxidized and
reduced forms have revealed a remarkable heme-ligand switching event at
both hemes of this enzyme (2). Upon reduction of the
cd1 enzyme in the crystal, Tyr25,
which ligates the d1 heme in the active site of
the oxidized protein is released to allow substrate binding.
Concomitantly, the c domain refolds, resulting in a change
in c heme coordination from
His17-His69 to
His69-Met106 (see Fig. 1, see also Ref. 2).
During the conversion of nitrite to nitric oxide, both the c
heme, which is the site of electron entry and the
d1 heme, the site where catalysis takes place,
of cd1 nitrite reductase are sequentially
oxidized and the enzyme returns to its
His17-His69 ligated oxidized state in the
crystal when all reducing equivalents are exhausted.
Similar heme-ligand switching has been observed since in other systems.
The transcriptional activator CooA from Rhodospirillum rubrum contains a b-type heme that acts as a CO
sensor in vivo. Under physiological conditions, CO can
easily replace one of the ligands to the ferrous heme, thereby causing
conformational changes around the heme and triggering activation of the
transcriptional regulator (3). The heme in CooA is in the
six-coordinate form in both oxidation states, with Cys75
being a heme ligand in the oxidized form (E0 = Another example of a redox-driven heme-ligand switching event has been
reported for the yeast F82H/C102S iso-1-cytochrome c variant
(7, 8). In the reduced state of the protein, the heme iron is
coordinated by His18 and Met80, similar to the
wild-type protein, whereas the heme iron is ligated by
His18 and His82 in the oxidized protein. The
redox potential of the His-Met form is E0 = 247 mV and that of the His-His form is E0 = 47 mV.
Electrochemical examination has revealed that the oxidized
His18-His82 form is highly disordered, and it
is proposed that this high level of disorder facilitates rapid
rearrangement to His18-Met80 upon reduction
(8).
Heme-ligand exchange reactions between methionine and histidine
residues were first observed during the in vitro folding and unfolding reactions of oxidized (9-11) and reduced (12, 13) horse
heart cytochrome c. His18 and Met80
are the axial heme ligands in native cytochrome c in both
oxidation states. In unfolded cytochrome c, the proximal
histidine (His18) remains an axial ligand by virtue of its
proximity to Cys15 and Cys17, which form the
thioether linkages to the porphyrin ring (14-16). The sixth
coordination site can either be vacant or occupied to various degrees
by His33, which is the predominant non-native heme iron
ligand (17), His26 or the N-terminal amino group (18).
During in vitro folding and refolding, cytochrome
c molecules can be trapped transiently in intermediate
structures with various His-His ligation (9, 11, 19).
Structural changes induced by changes in the redox state of a heme
group in a protein play a central role in channeling redox energy into
conformational energy in biology. For cytochrome
cd1 nitrite reductase, the factors that drive
the switch between His-His and His-Met coordination of the
c-type heme center in the enzyme (Fig.
1) have not been elucidated. An open
question is whether ligand switching at the c heme is a
property of the c heme domain or of the whole
cd1 molecule. To understand the molecular
details of this process, we present here the characterization of the
heme ligation in the isolated c domain of the P. pantotrophus cytochrome cd1 nitrite
reductase enzyme in both oxidation states in solution.
Protein Expression and Purification
The isolated c domain consisting of the first 133 amino acid residues of the mature P. pantotrophus cytochrome
cd1 nitrite reductase was expressed in
Escherichia coli, as described
elsewhere.1 Six or
eight-liter cultures of E. coli in LB medium were grown overnight at 37 °C and centrifuged at 7000 × g for
15 min at 4 °C. The protein was removed from the periplasm using an
osmotic shock procedure. The cell pellet was resuspended in a
0.4-culture volume of 30 mM Tris-HCl, pH 8, 20% sucrose, 1 mM EDTA, incubated 5-10 min at room temperature, and
centrifuged at 10,000 × g for 10 min at 4 °C. The
supernatant was removed, and the pellet was resuspended in a
0.4-culture volume of ice-cold 5 mM MgSO4,
stirred on ice for 10 min, and centrifuged at 10,000 × g at 4 °C. The red-colored supernatant was loaded onto a
fast-flow Q-Sepharose column (Amersham Pharmacia Biotech, Uppsala,
Sweden) equilibrated using 50 mM Tris-HCl, pH 8, and 50 mM NaCl. The c domain was isolated in the
reduced form.
To ensure full reduction or oxidation, a small excess of ascorbic acid
or ferricyanide in the solid form was added to the purified protein
immediately before it was put on a Novarose PrePac S.E.-100/17
gel-filtration column (Inovata, Bromma, Sweden), which was equilibrated
with 50 mM sodium phosphate buffer and 100 mM NaCl, pH 7. The protein was either directly frozen in liquid nitrogen or concentrated to 1-2 mM using 3K Centriprep (Millipore,
Bedford, MA) and 3K Microsep concentrators (Filtron Technology Corp.,
Northborough, MA), subsequently lyophilized, and stored at Characterization of the Purified Protein
The purity of c domain samples was checked by native
and SDS-PAGE2 on a
PhastSystem (Amersham Pharmacia Biotech, Uppsala, Sweden) and
silver-stained. Care was taken to minimize proteolytic cleavage of the
c domain samples by purifying and concentrating the protein at 4 °C as quickly as possible and by freezing the samples in liquid
nitrogen between handling steps.
The mass of the isolated c domain was determined by
MALDI-TOF spectrometry. N-terminal analysis was performed to check
whether the periplasmic leader sequence was cleaved off at the correct amino acid position.
Equilibrium sedimentation studies were performed on 10 mM
oxidized c domain samples in 100 mM sodium
phosphate buffer, pH 7, 300 K, using a Centriscan 75 ultracentrifuge.
The absorbance at 280 nm was recorded.
Kinetic Studies and UV-visible Spectroscopy
UV-visible absorbance spectra between 250 and 750 nm were
recorded on a Hewlett-Packard 8453 diode array spectrophotometer. Kinetic experiments to follow the ascorbate reduction of the isolated c domain were performed using an RX2000 stopped-flow cell
(Applied Photophysics, Leatherhead, UK) coupled to the HP 8453 spectrophotometer. The reaction and storage chambers of the
stopped-flow cell were kept at 4.5 °C. In the experiments, 2.5 µM oxidized c domain in 50 mM
phosphate buffer, pH 7, was used. Pseudo first order rate constants
were determined from exponential curves of the absorbance at 417 nm
versus time at 5, 10, 20, 30, 40, and 50 mM
ascorbate in 50 mM phosphate buffer, pH 7. From these, the
second order rate constant for ascorbate reduction of the isolated
c domain was determined.
Unfolding of the oxidized (16-22 µM) and reduced (8-10
µM) isolated c domain was monitored by
absorbance spectroscopy as a function of the sample temperature in 50 mM sodium phosphate and 100 mM NaCl, pH 7. Thermal denaturation curves were measured between 277 and 340 K for
oxidized and between 316 and 368 K for reduced protein samples,
respectively. The isolated c domain was reduced with solid
dithionite (end concentration 10 mM) inside an anaerobic glove box and kept overnight in the glove box before it was pipetted in
an airtight cuvette and sealed. The sample temperature was measured
with a thin NiCr-NiAl thermocouple (Testo 925, Göteborgs Termometerfabrik, Göteborg, Sweden), which was in direct contact with the protein solution inside the sealed cuvette. The precision of
the temperature reading was within ±0.1 °C. The heating rate was
manually controlled and varied between 0.25 and 1 °C per minute to
ensure that thermal unfolding of the protein was at equilibrium, i.e. a change in heating rate within this regime did not
result in a change of the Tm. Thermal unfolding
curves were measured at different wavelengths and corrected with the
absorbance measured at 900 nm, a wavelength at which no protein
absorbance is expected. The corrected unfolding curves were normalized
and subsequently analyzed according to a two-state mechanism of
unfolding (21, 22) and assuming a linear dependence of the pre- and
post-unfolding baselines with the temperature. The data were fitted as
described in Ref. 23 to obtain values for Tm,
except that no linear dependence of the post-unfolding baseline was
assumed and that NMR Spectroscopy
NMR Samples--
NMR samples were prepared by dissolving
lyophilized protein in either 99.9% 2H2O or
90% H2O/10% 2H2O to yield 1-2
mM protein solutions in 50 mM sodium phosphate buffer and 100 mM sodium chloride, pH* 6.7. Solutions were
prepared in an anaerobic glove box, and the NMR tubes were sealed with gas-tight caps. All samples contained 0.1-0.2 mM DSS as an
internal standard. The c domain has a tendency to
auto-oxidize and to auto-reduce, and 100% oxidized or 100% reduced
samples, as detected by NMR spectroscopy, could not be made without the
addition of oxidizing and reducing agents, respectively. The phenomenon
of auto-reduction has been observed for other cytochromes c
at high pH (24).3 Samples
were oxidized or reduced in the NMR tubes by addition of small amounts
of ferricyanide or sodium ascorbate, respectively, in 50 mM
sodium phosphate, 100 mM sodium chloride, pH 7, using a
Hamilton syringe until subsequent additions did not result in changes
in the NMR spectrum. Before and after each two-dimensional 1H NMR experiment, one-dimensional 1H NMR
spectra of the NMR sample were recorded and compared to ensure that the
sample had not deteriorated during the NMR experiment. Furthermore, an
aliquot of each c domain sample was taken before and after
each series of NMR experiments (lasting for several days) and checked
with SDS-PAGE to see whether degradation had occurred during
acquisition of the NMR experiments.
NMR Spectra--
All 1H NMR spectra were recorded on
a Bruker DRX 500 spectrometer either at 500.13 or at 500.03 MHz. One-
and two-dimensional experiments on isolated oxidized and reduced
c domain were performed over the range of 278-308 K. One-dimensional 1H NMR experiments were recorded with a
spectral width of 95 ppm. In two-dimensional 1H NMR
experiments, the spectral width in both dimensions was 17 or 18 ppm
when information on the diamagnetic region of spectra of the isolated
c domain was to be obtained. To obtain information on
hyperfine shifted resonances of the isolated oxidized c
domain, a spectral width of 95 ppm was used in two-dimensional
1H NMR experiments. Routinely, 2048 complex points were
acquired in the direct dimension, whereas 256 complex points were
recorded in the indirect dimension of two-dimensional 1H
NMR experiments. Quadrature detection in the indirect dimension was
accomplished using the time proportional phase incrementation method.
Presaturation of the water signal was always employed during the
relaxation period.
Ordinary 30- and 150-ms NOESY, 31-ms clean-TOCSY (25), using an mlev17
(26) sequence, and DQF-COSY experiments were recorded on the
diamagnetic regions of spectra of the isolated c domain in
both oxidation states. The relaxation delay was between 1 and 2 s.
T1 inversion recovery experiments were performed on the oxidized
protein in 100% 2H2O at 278, 295, and 298 K
and in 90% H2O/10% 2H2O at 278 K
using a relaxation delay of 5 s between individual free induction
decays. Peak volumes of resolved peaks were plotted with respect to
recovery delay time, and T1 values were extracted from exponential fits
to the data using the XWINNMR package unless stated otherwise.
In DEFT-NOESY and DEFT-TOCSY experiments (27, 28), the first 90°
pulse in the ordinary NOESY and TOCSY pulse sequences was replaced with
the modified DEFT sequence (90°-
Two-dimensional exchange spectroscopy (EXSY) 1H NMR
experiments (30, 31) were performed on samples containing mixtures of
the oxidized and reduced c domain in
2H2O (278 and 298 K) and in 90%
H2O/10% 2H2O (278 K). Under the
experimental conditions used, all 1H resonances are
broadened to some extent due to intermediate exchange between oxidized
and reduced molecules. NOE mixing times of 5 and 15 ms were used in the
DEFT-NOESY pulse sequences to ensure that even cross peaks arising from
resonances with the shortest T1 values could be observed.
Data Processing and Analysis--
Two-dimensional 1H
NMR data were processed using the XWINNMR package. One-dimensional and
two-dimensional 1H NMR data sets were apodized using
an exponential decay in the direct dimension and using a 30-40°
shifted sine-bell-squared window function in the indirect dimension.
After phase correction, the spectra were baseline corrected in both
dimensions. Two-dimensional 1H NMR spectra were analyzed
using the program XEASY (ETH Zurich, Switzerland (32)). One-dimensional
1H NMR spectra were processed and analyzed using the
program Gifa (33). 1H chemical shifts were referenced using
internal DSS as a standard.
Biochemical Characterization of the Isolated c Domain in
Solution--
The isolated c domain is expressed in the
reduced form in the periplasm of E. coli1 and
can be purified in the reduced state. At pH 8, the isolated c domain remains reduced for weeks under aerobic conditions
at 4 °C, as determined by absorbance spectroscopy. The absorbance spectrum of the reduced c domain (gray curve in
Fig. 2A) shows characteristic
maxima at 418 nm (Soret band), 522 nm (
The c domain appeared as a monomer on native gels, and
mass/charge MALDI-TOF spectra did not show any indication of dimers. Sedimentation equilibrium studies with 10 µM protein
solution confirmed that the isolated c domain is monomeric
in solution.
Although absorbance spectra of reduced c domain samples did
not change over a period of weeks when stored at 4 °C, SDS-PAGE shows that the protein underwent a degradation over such periods of
time (data not shown). N-terminal amino acid sequencing of an aged
c domain sample, which was stored in the reduced state for a
month at 4 °C, gave the sequence APEGVSALSD, showing that the first
41 residues of the c domain had been cleaved off. Mass spectrometric analysis gave a mass of 10,422 Da, confirming that most
of the aged protein consisted of residues 42-133 of the c domain sequence and that no full-length c domain was present anymore.
The isolated c domain can reversibly be oxidized
by ferricyanide and re-reduced by dithionite or ascorbate. The oxidized
c domain is readily reduced by ascorbate, with a second
order rate constant of 20.9 ± 1.4 M Characterization of the Heme Environment in the Isolated Reduced c
Domain by NMR Spectroscopy--
The one-dimensional 1H NMR
spectrum of the reduced c domain in
2H2O (green curve, Fig.
3A) and H2O
(black curve, Fig. 3A) shows several outstanding
features typical for c-type cytochromes. The resonances of
the reduced protein exhibit a broad chemical shift dispersion brought
about by the presence of the aromatic heme group (Fig. 3A).
The four downfield resonances between 9 and 10.5 ppm in the
2H2O spectrum arise from the four meso protons
(numbered 5, 10, 15, and 20 in Fig. 4), which reside within the heme
plane. Many resonances between ~1 and
The exchangeable H
In the isolated reduced c domain, a methionine is expected
to be the sixth ligand to the heme. The methyl chemical shift of the
axial methionine in reduced cytochromes c is always shifted upfield and ranges from
The typical cross peak pattern of a tryptophan spin system (35),
present in two-dimensional NOESY, TOCSY, and COSY 1H NMR
spectra of the reduced c domain recorded in H2O
and 2H2O, allowed the assignment of the
side-chain resonances of the single tryptophan, Trp109
(Table I). NOE contacts were identified between the tryptophan H
Around 30 intense cross peaks in the NH (8.6-7.7
ppm)-H Chemical Exchange Studies between the Reduced and Oxidized Form of
the Isolated c Domain--
NMR spectroscopy was used to obtain
structural information at atomic details on the nature of the ligands
to the paramagnetic heme iron in the oxidized c domain. The
upfield and downfield regions of one-dimensional 1H NMR
spectra of the paramagnetic oxidized c domain in
H2O at different temperatures are shown in Fig.
3B. The two resonances in the region 30-45 ppm are readily
recognized as heme methyl resonances, whereas resonances in the region
below
The assignment of the hyperfine-shifted signals of the oxidized
c domain was carried out using two-dimensional EXSY
1H NMR experiments (30) performed on samples containing
approximately equal amounts of the oxidized and reduced forms of the
c domain. If chemical exchange between the two oxidation
states is fast compared with the cross relaxation rate and slow
compared with the chemical shift difference of the corresponding NMR
resonances, the electron transfer effect dominates a two-dimensional
NOESY 1H NMR spectrum at short NOE mixing times.
Fig. 3C shows 5-ms DEFT-NOESY 1H NMR spectra of
the oxidized c domain, of the reduced c domain,
and of a mixture of oxidized and reduced c domains in
2H2O at 278 K. Spectra obtained from the pure
reduced or oxidized c domains only show cross peaks due to
cross relaxation of resonances in the c domain in either
oxidation state, respectively. In the 5-ms DEFT-NOESY 1H
NMR spectrum recorded on a mixture of reduced and oxidized c domains, however, solely EXSY cross peaks are seen and no cross peaks
due to cross relaxation are observed. The chemical shifts of the EXSY
cross peaks enable the identification of hyperfine-shifted resonances
in the oxidized protein using the known resonances of the reduced
c domain (Table I).
Using EXSY cross peaks, heme proton resonances in the oxidized form of
the c domain were assigned (Table I). NOE contacts observed
in the two-dimensional DEFT-NOESY 1H NMR spectra of the
oxidized c domain support these assignments. The
exchangeable H
The remaining hyperfine-shifted resonances appearing at high field
between
Two-dimensional NOESY and TOCSY 1H NMR spectra of the
diamagnetic region of the oxidized c domain in
H2O show around 30 intense cross peaks in the
NH (8.8-8.1 ppm)-H Effect of Temperature on NMR Spectra of the Isolated c
Domain--
Most of the hyperfine-shifted resonances of the oxidized
c domain obey Curie temperature dependence in the sense that
their chemical shifts decrease with increasing temperature,
i.e. they shift in the direction of the diamagnetic region
(Figs. 3B and 5). However, the heme methyl groups 2 and 12 and the exchangeable H
Fig. 5 shows that Curie plots (chemical
shift versus T
NMR spectra of the diamagnetic reduced c domain at
temperatures between 278 K and 303 K show the expected line sharpening at higher temperatures (data not shown).
These results suggest a temperature-dependent unfolding of
the c domain, which is influenced by the oxidation state of
the heme group in the protein.
Thermal Unfolding of the Isolated c Domain Followed by Absorbance
Spectroscopy--
The temperature-induced unfolding of the isolated
oxidized c domain was studied by absorbance spectroscopy to
investigate whether the observed anomalous
temperature-dependent behavior of the hyperfine-shifted
resonances of the oxidized protein was related to thermal unfolding of
the protein. Fig. 2B shows spectra of thermally unfolded
oxidized protein with absorbance maxima at 403, 523, and around 620 nm.
The maximum around 620 nm indicates the presence of a high spin species
(38, 39).
Fig. 6A shows thermal
unfolding curves for the oxidized c domain at wavelengths of
280, 398, 416, 527, and 695 nm. The results show very similar midpoints
for the temperature-dependent unfolding of the oxidized
c domain at all wavelengths tested (Table
II). The absorbance at 280 nm was used as
a probe for the absorbance of the aromatic residues and the heme group.
Changes in the Soret band and in the
Fig. 6B shows that the reduced c domain is much
more resistant to thermal denaturation than the oxidized c
domain. Thermal unfolding curves of the reduced protein were measured
at 417, 432, 522, and 554 nm (Fig. 6B). The absorbances at
417, 522, and 554 nm monitor changes in the Soret band, the The Isolated c Domain Is Monomeric in Solution--
Experimental
data on the isolated c domain indicate that the protein is
monomeric in solution. This is in contrast to the arrangement of
c domains in intact cytochrome cd1
nitrite reductase where they adopt a dimeric structure (2, 40-42).
Inspection of the crystal structures (2, 41, 42), however, shows that none of the 11 hydrogen bonds involved in subunit-subunit interactions comes from the c domain (residues 1-133), and less than 10 and 20% of the total surface area buried between c and
d1 domains derives from the dimer interface
between the c domains in the oxidized and reduced
cd1 enzyme, respectively, as calculated with the
program GRASP (43). This suggests that the dimeric cytochrome cd1 holoenzyme derives its stability mainly from
interactions between the d1 domains.
Establishment of the Tertiary Structure Around the Heme in the
Isolated Reduced c Domain--
Mass spectrometry, N-terminal amino
acid sequence analysis, and absorbance spectroscopy on an aged isolated
reduced c domain have shown that 41 N-terminal residues of
the protein can be removed without loss or change of the characteristic
features in its absorbance spectrum and that the remaining 10-kDa
protein is relatively stable against proteolytic cleavage. This
together with earlier x-ray data on ligand switching shows that the
N-terminal segment of the molecule is mobile. The ~30
NH-H
We assigned the axial histidine resonances in NMR spectra of the
reduced c domain to protons of the proximal
His69 from the Cys-Ala-Gly-Cys-His motif and not to
His17 in the flexible N terminus, because the proximal
histidine ligand in c-type cytochromes is known to be
strongly bound to the heme by virtue of its position adjacent to the
Cys-X-X-Cys sequence in which both cysteines form
thioether linkages with the porphyrin ring (14-16). The NOEs observed
between His69 side-chain resonances and heme protons (Fig.
4) indicate that the orientation of this axial ligand relative to the
porphyrin ring is similar to that observed in the crystal structure (2) of the c domain in the reduced cd1
nitrite reductase (Fig. 4), which is the orientation commonly observed
in c-type cytochromes (44).
The NOEs between side-chain protons of Trp109, the axial
ligands, and the heme as observed in the NMR spectra of the isolated reduced c domain in solution agree well with the distances
between side-chain protons of these residues and the heme as determined in the crystal structure of the reduced cd1
nitrite reductase enzyme (see Fig. 4). Based on these NOE contacts, we
identify Met106 as the axial ligand to the heme in the
isolated reduced c domain and not Met123, the
only other methionine residue present in the isolated c domain. Thus, the NMR results indicate that the tertiary structure around the heme in the isolated reduced c domain is highly
similar to that of the c domain in the crystal structure of
the reduced cytochrome cd1 nitrite reductase.
Determination of the Axial Ligands to the Heme Iron in the Isolated
Oxidized c Domain--
The NMR data obtained on the isolated oxidized
c domain show around 30 NH-H
The NMR (Fig. 3, B and C) and absorbance
spectroscopy (Fig. 2A) data on the isolated oxidized
c domain demonstrate that His69 and
Met106 are the axial ligands in the majority of the
isolated oxidized c domain molecules. The EXSY cross peaks
(Fig. 3C) clearly show that Met106, which is an
axial ligand in the isolated reduced c domain, also is the
axial ligand in the oxidized protein. Note, however, that a potential
population of His69-His17-ligated c
domain molecules comprising less than an estimated 5% of the molecules
present in our oxidized protein samples would escape detection by NMR.
The results show that heme ligation in the isolated oxidized
c domain is similar to that in the oxidized semi-apo form of the P. pantotrophus cd1 enzyme (34, 45, 46) and
in the oxidized Pseudomonas aeruginosa cytochrome
cd1 nitrite reductase structure (47, 48), but
notably different from the His69-His17 ligation
in the oxidized P. pantotrophus cytochrome
cd1 nitrite reductase structure (34, 41, 42, 45,
46). However, the results also indicate that in a readily measurable
fraction of the oxidized molecules, Met106 is released from
the ferric iron, and the iron is five-coordinate. There is a markedly
different temperature dependence in the release of Met106
from the heme iron in the oxidized and reduced form of the c domain (Fig. 6).
Effect of Temperature on the Ligation State of the Heme in the
Isolated c Domain--
Thermal unfolding studies on both oxidized and
reduced c domain show that the protein unfolds, giving rise
to a penta-coordinated heme species (Figs. 2B and 6).
Measured midpoint temperatures of unfolding (Tm)
reveal huge differences between the oxidized (Tm = 320.9 ± 1.2 K) and the reduced forms (Tm = 357.7 ± 0.6 K). The stability of the oxidized c
domain is also much lower than that of horse heart ferricytochrome
c, which has a Tm of 341 K as
determined by absorbance spectroscopy at 280 and 420 nm (49). The
native fraction of oxidized molecules already starts to decrease slightly above room temperature, whereas no such change is visible in
the reduced protein. Such an early change in the spin state of the
oxidized c domain with temperature is not common among His-Met-ligated cytochromes c, although it has been observed
in Wolinella succinogenes cytochrome c (14,
50).
Unfolding of the oxidized protein is accompanied by an anomalous line
broadening in the NMR spectrum (Fig. 3B). Similar anomalous behavior has also been observed for the hyperfine-shifted resonances of
horse heart ferricytochrome c, but only at temperatures
above 330 K at pH* 5.3 (51). For horse heart cytochrome c,
the behavior at pH* 5.3 has been attributed to the presence of a
high-spin species with large paramagnetic shifts and large linewidths,
which is in fast chemical exchange with the native low-spin form (51). From the line-broadening effects (Fig. 3B) and the effects
on the chemical shifts of the hyperfine-shifted resonances in NMR spectra of the isolated oxidized c domain (Fig. 5) in
combination with the thermal unfolding data measured with absorbance
spectroscopy (Fig. 6A), we infer the presence of a high-spin
species of the isolated oxidized c domain, which is in fast
chemical exchange with the native low-spin species around and above
room temperature. Such a species with high plasticity could adopt
various conformational states depending on interactions with its local environment.
The affinity of the reduced heme iron for methionine ligation is much
higher than that of the oxidized heme iron (14, 38, 39, 52), which
explains why reduced His-Met-ligated cytochromes c are
generally more stable than their oxidized counterparts (see e.g. Refs. 14, 38, 39, 52-54). The reduced heme can retain a methionine ligand even at high temperatures. For the folded c domain described in this paper, the reduced protein is in
the folded state at 340 K, whereas the oxidized protein has lost its key spectral features at this temperature (Fig. 6) and is completely unfolded. The reduced c domain has a significantly smaller
transition region, and seems to unfold much more abruptly and in a more
cooperative manner than the oxidized protein.
We have shown that the unfolded forms of both the reduced and oxidized
c domain are high-spin at neutral pH (Fig. 2B).
This indicates that the c heme probably adopts a
penta-coordinated form in both oxidation states during unfolding (or
there may be a hexa-coordinated form present with a weak field ligand
such as H2O or OH Heme-Ligand Switching and Conformational
Plasticity--
Heme-ligand switching in the transcriptional activator
R. rubrum CooA takes place between the residues
Cys75 and His77, whereas residues
Met80 and His82 exchange upon oxidation and
reduction in the yeast F82H/C102S iso-1-cytochrome c
variant. In both proteins, the heme-ligand switching involves the
exchange of residues that are only two amino acids apart in the amino
acid sequence and that are thus relatively close in space even in a
partially unfolded state.
In contrast, the heme-ligand switching event in P. pantotrophus cytochrome cd1 nitrite
reductase involves a large rearrangement of the c domain
between the His69-His17-oxidized form and the
His69-Met106-reduced form of the c
heme (Fig. 1). For religation, each of the exchangeable
ligands must be able to search the available conformational space via
diffusion-like processes, and these processes may be modulated through
interactions with the solvent and the protein envelop. Religation of
the c heme in cytochrome cd1 nitrite reductase resembles the diffusion-like processes occurring during (un)folding of horse heart cytochrome c, which give rise to
the His-His-ligated folding intermediates.
In the intact cytochrome cd1 holoenzyme, the
equilibrium between His69-His17- and
His69-Met106-ligated c hemes is
shifted with changing oxidation states of cytochrome
cd1 nitrite reductase. The present study shows
that in the isolated c domain this equilibrium is shifted to
His69-Met106 ligation in both the oxidized and
the reduced form of the protein around and below room temperature. This
is similar to the situation in oxidized and reduced horse heart
cytochrome c. However, the oxidized c domain is
much more unstable than the oxidized horse heart cytochrome
c and has a high propensity to release the methionine ligand
from the ferric iron. We have demonstrated that, upon increasing the
temperature, the heme ligation in the isolated oxidized c domain changes from a low-spin hexa-coordinated His-Met form to a
non-native high-spin species where the Met has left the iron. However,
His17 fails to rebind to the oxidized heme in the isolated
c domain, probably due to the lack of guiding interactions,
which would be present in the cd1 holoenzyme.
These guiding interactions include the binding of Tyr25 to
the d1 heme, which positions His17
on the N-terminal arm ready for rebinding if the c heme is
in the ferric state. The observation that the heme ligation in the oxidized semi-apo cd1 enzyme is His-Met (34, 45,
46), just like in the isolated oxidized c domain, suggests
that the d1 heme plays a crucial role in
orchestrating ligand switching on the c heme in the
holoenzyme. The importance of the d1 heme also
is supported by studies of mixed-valence crystal structures of P. aeruginosa cytochrome cd1 nitrite
reductase, which suggest that the reduction of the
d1 heme is responsible for the structural movements seen upon reduction of this protein (55).
The c domain sequence is designed such that the protein has
a long and flexible N-terminal tail and that the His-Met-ligated oxidized c domain is highly unstable while the
His-Met-ligated reduced form is much more stable, as shown in the
present study. The release of Met106 from the oxidized
c heme and religation by His17 in the fully
oxidized resting form of the cd1 holoenzyme
seems to be caused by two key factors: (i) the different binding
propensities of sulfur, nitrogen, and oxygen ligands to oxidized and
reduced heme irons (sulfur ligands stabilize the Fe(II) state, whereas nitrogen and oxygen ligands stabilize the Fe(III) state (14, 38)) and
(ii) a "directed diffusion" of the N-terminal arm, which can bring
His17 to the vicinity of the c heme in the
oxidized holoenzyme. As suggested in Ref. 56, a cooperative binding of
Tyr25 to the d1 heme with the
binding of His17 to the oxidized c heme is a
chelate effect and minimizes the entropy change. Such a religation is
not necessarily part of the catalytic cycle of the
cd1 holoenzyme. Recent quantum mechanical calculations on catalysis by cytochrome cd1
nitrite reductase (57) indicate that a rebinding of Tyr25
to the d1 heme may be bypassed during fast
steady-state catalysis, although both His17 and
Tyr25 eventually rebind to the c and
d1 heme centers, respectively, when the supply
of electrons is exhausted. A potential function of the rebinding of
His17 and Tyr25 to the c and
d1 heme centers in the
cd1 enzyme is to shut off access to the iron
centers when the supply of electrons is not sufficient to sustain
catalysis but the oxygen concentration is high. Under such
circumstances dioxygen, superoxides, or peroxides may react with
unprotected heme irons, creating radicals through Fenton chemistry.
Shutting off access to the c and d1
hemes in the resting enzyme prevents uncontrolled oxygen reactions. If, however, there is a good supply of external reducing equivalents, the
active site stays open, and dioxygen is reduced to water through the
cytochrome c oxidase activity (58) of the enzyme.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
320 mV), whereas His77 appears to be an axial ligand in
the reduced protein (E0 =
260 mV) and is
essential for activation of the protein by CO (3-6).
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Fig. 1.
Structure of the c-type
cytochrome domain in the oxidized (A) and reduced
(B) cytochrome cd1
nitrite reductase holoenzyme (see also Ref. 2). The first 8 residues are disordered in A, and the first 25 residues are disordered in B. The protein is rainbow
colored starting at the N terminus with blue. The
present study examines heme ligation at different redox states in the
isolated c domain, i.e. residues 1-133
overexpressed in E. coli.1
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C
before use.
Cp was kept constant at an estimated value of 2513 cal.mol
1.K
1 (22) in fits to the data of the
isolated reduced c domain.
-180°-
-90°) (29) with
= 100 ms. In two-dimensional DEFT 1H NMR
experiments, the relaxation delay was set to 200 ms. A 5-ms DEFT-TOCSY
1H NMR experiment was recorded on the isolated oxidized
c domain. NOE mixing times of 5 and 15 ms were used in
DEFT-NOESY 1H NMR experiments.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
band), and 548 nm and 554 nm
(split
bands), which are indicative of a low-spin six-coordinated
heme iron. The oxidized c domain has absorption maxima at
410 and 527 nm (black curve in Fig. 2A). Furthermore, at high protein concentrations (millimolar) a small band
is observed at 696 nm (see inset in Fig. 2A)
indicative of methionine ligation to the heme (14). For comparison, the
absorbance spectra of reduced and oxidized cytochrome
cd1 nitrite reductase are shown in Fig.
2C.
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Fig. 2.
A, absorbance spectrum of a 12 µM sample of the isolated c domain of
cytochrome cd1 nitrite reductase from P. pantotrophus in the reduced (gray) and oxidized
(black) state in 50 mM sodium phosphate and 100 mM sodium chloride, pH 7.0, 296 K. The inset
shows the 700-nm region of oxidized and reduced spectra of a 170 µM protein sample under identical experimental
conditions. The absorption band around 695 nm in the oxidized spectrum
is indicative of methionine ligation to the heme (14). B,
absorbance spectra of thermally unfolded samples (8 µM)
of the isolated c domain in the oxidized state at 340 K
(black) and in the reduced state at 368 K (gray)
in 50 mM sodium phosphate and 100 mM sodium
chloride, pH 7.0. The reduced sample also contained 10 mM
sodium dithionite. The inset shows the 620-nm region of
oxidized and reduced spectra of the same samples. The absorption bands
around 420 nm in the spectrum of the reduced protein and around 620 nm
in the spectrum of the oxidized protein (see inset) are
indicative of non-native high-spin species in the two oxidation states
(14, 39). C, absorbance spectra of the dimeric cytochrome cd1
nitrite reductase (the holoenzyme) in the reduced (gray) and
oxidized state (black) in 50 mM sodium
phosphate; 100 mM sodium chloride, pH 7.0; 296 K. Enzyme
concentration: 3.5 µM sample (7 µM per
monomer).
1.s
1 in 50 mM
phosphate buffer, pH 7.0, 4.5 °C. This is in contrast to the
oxidized cd1 nitrite reductase enzyme, which can
only be reduced slowly by ascorbate
(34).4
3 ppm arise from protons of
the axial ligands and from other protons located close to the center of and perpendicular to the heme plane. Using two-dimensional NOESY, TOCSY, and COSY 1H NMR
spectra, most of the heme proton were assigned (Table
I).5
View larger version (18K):
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Fig. 3.
A, one-dimensional 1H NMR
spectrum of the isolated reduced c domain of cytochrome
cd1 nitrite reductase from P. pantotrophus in 90% H2O/10%
2H2O (black curve) and
2H2O (green curve) in 50 mM sodium phosphate buffer and 100 mM sodium
chloride, pH* 6.7, 300 K. To ease comparison of resonances in the
upfield and downfield areas, the region between 0.5 and 4.5 ppm of the
2H2O spectrum is not shown. B,
downfield (left) and upfield (right) regions of
one-dimensional 1H NMR spectra of the isolated oxidized
c domain in 90% H2O/10%
2H2O in 50 mM sodium phosphate
buffer and 100 mM sodium chloride, pH* 6.7 recorded at 277, 282, 288, 295, 299, 306, and 312 K, respectively. C,
two-dimensional 5-ms DEFT-NOESY 1H NMR spectrum recorded on
the isolated oxidized c domain (blue), on the
reduced c domain (yellow), and on a mixture of
oxidized and reduced c domain (red) in 50 mM sodium phosphate buffer and 100 mM sodium
chloride in 2H2O, pH* 6.7, 278 K. Only EXSY
cross peaks (red) are seen in the spectrum recorded ona
mixture of oxidized and reduced c domain. The
inset shows the region between 9 and 19 ppm of the 5-ms
DEFT-NOESY spectra recorded on the oxidized (blue) and on a
mixture of the oxidized and reduced c domain
(red) in 90% H2O/10%
2H2O (278 K). The EXSY cross peak of the
His69 H 1 proton, not present in
the 2H2O spectra, is present in the
inset.
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Fig. 4.
NOEs observed in two-dimensional
1H NMR spectra of the isolated reduced c
domain are indicated as red lines in the crystal
structure of the reduced P. pantotrophus cytochrome
cd1 nitrite reductase (Protein Data Bank
file 1AOF; hydrogens were generated using the program InsightII
(Biosym/MSI, San Diego, CA). The c heme is also shown
to the right with the four mesoprotons numbered according to
the IUPAC recommendations 1999 (59). The c heme is
covalently attached to the protein via the two sulfur atoms of
Cys65 and Cys68, which are colored
yellow. The figures were drawn using a modified version of
MOLSCRIPT (61) and were rendered using Raster3D (20).
Chemical shifts of the 1H resonances of the heme protons and of
the side-chain protons of the two axial ligands, His69 and
Met106, and of Trp109 of the P. pantotrophus-isolated c domain in the reduced and
oxidized state in 50 mM sodium phosphate and 100 mM sodium chloride in 100% 2H2O, pH*
6.7
1 side-chain resonance of
the proximal histidine, His69, is readily identified at
10.38 ppm in the H2O spectrum (black curve, Fig.
3A) of the reduced c domain at 300 K, since it is the most downfield-shifted resonance. Using two-dimensional NOESY and
TOCSY 1H NMR spectra, the resonances arising from the
histidine H
1 and
H
2 protons were assigned (Table I). Weak
NOEs between the His69 H
1 proton
and the heme meso proton 15 and between the His69
H
2 proton and the heme meso proton 5 were
identified (Fig. 4).
2.7 to
3.7 ppm (14). The most
upfield-shifted resonance in the one-dimensional 1H NMR
spectrum of the reduced c domain at 300 K occurs at
2.54 ppm (Fig. 3A). This resonance is assigned to the methyl
group of the axial methionine ligand, because it does not show any COSY contacts. Furthermore, NOE contacts of the methyl resonance to other
upfield one-proton resonances and the identification of COSY cross
peaks between those established the assignments of the
and
resonances of the axial methionine in the reduced state (Table I). The
methyl resonance shows a medium NOE contact to one of the heme meso
protons, proton 20 (Fig. 4).
1 and H
1 protons
and the methyl group of the axial methionine, and between the
tryptophan H
1 and
H
2 protons and the heme meso proton 5 (see
Fig. 4).
region (4.7-4.0 ppm) were observed with clearly
narrower linewidths than the surrounding
NH-H
cross peaks at other chemical shift
positions in two-dimensional TOCSY and NOESY 1H NMR spectra
of the reduced c domain in H2O (data not shown). This indicates that around 30 residues in the protein are flexible.
5 ppm arise from heme-ligand protons (14). T1 values were
determined for nonoverlapping hyperfine-shifted resonances (see Table
I). Short T1 values were observed for protons less than 7-8 Å from
the paramagnetic iron and are a measure for the distance of the
respective protons to the heme, because the spin nuclear relaxation
depends on the sixth power of the electron-proton distance (36).
1 proton of His69
can readily be identified (at 12.21, 10.47 ppm) in the two-dimensional EXSY 1H NMR spectrum recorded on a H2O sample
at 278 K (see inset in Fig. 3C). As expected, the
resonance at 10.47 ppm is absent in the one-dimensional 1H
NMR spectra recorded on the oxidized c domain in
2H2O. A weak EXSY peak observed at (
12.23,
0.50) ppm at 278 K and at (
11.30, 0.48) at 295 K is attributed to
the H
2 proton of His69.
9 and
34 ppm arise from protons of an axial ligand, which
is identified to be a methionine residue in the isolated oxidized
c domain. For example, the methyl resonance of the axial methionine in the reduced c domain at
2.51 ppm shows a
strong EXSY cross peak at (
12.23,
2.51) ppm in the two-dimensional EXSY 1H NMR spectrum (Fig. 3C). The EXSY cross
peak thus reveals that the same methionine residue is an axial ligand
to the heme in both oxidation states of the isolated c
domain. Furthermore, the hyperfine-shifted resonances arising from two
protons and one
proton of the axial methionine are assigned
using EXSY cross peaks as well as using cross peaks in two-dimensional
5-ms DEFT-NOESY and two-dimensional 5-ms DEFT-TOCSY 1H NMR
spectra recorded on the oxidized c domain (Table I).
region (4.7-4.0 ppm)
with clearly narrower linewidths than the surrounding
NH-H
cross peaks at other chemical shift
positions in the spectra (data not shown).
1 proton of the axial
histidine exhibit anti-Curie behavior, a peculiar effect observed
before in many ferricytochromes c and explained by a
redistribution of the two highest (partially) filled molecular orbitals
at higher temperatures (37).
1) for most of the
hyperfine-shifted resonances deviate from linearity (Fig. 5). Moreover,
all hyperfine-shifted resonances undergo line broadening at higher
temperature (Fig. 3B) and the linewidths of oxidized
resonances in the diamagnetic region are also broader at 312 K than at
278 K. The changes in the spectra of the oxidized c domain
are totally reversible over the temperature range measured.
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Fig. 5.
Temperature dependence of 1H NMR
chemical shifts of hyperfine-shifted resonances of the isolated
oxidized c domain of P. pantotrophus
cytochrome cd1 nitrite
reductase. For experimental conditions see the legend of Fig.
3B.
/
absorption bands resulting
from
-
* transitions in the porphyrin molecule were monitored at
416 and 527 nm, respectively. The absorbance at 695 nm was used as a
diagnostic of methionine ligation to the heme iron (14). The appearance
of a non-native high spin species was probed at 398 nm and also at 620 nm (39). The latter data are not shown, because a strong increase in
absorbance at 620 nm of the unfolded protein at higher temperatures
made calculations less reliable. However, the estimated
Tm at 620 nm was 322 K, i.e. similar
to values from the other measurements. Tm values
at all wavelengths were calculated assuming a two-state mechanism of
unfolding as described in Ref. 23. The average Tm for the oxidized c domain from
these data is 320.9 ± 1.2 K and the average
Hm is 47 ± 6 kcal.mol
1.
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Fig. 6.
A, thermal unfolding curves of the
isolated oxidized c domain as monitored using absorbance
spectroscopy at 280, 398, 416, 527, and 695 nm (experimental conditions
as in Fig. 2B). B, thermal unfolding curves of
the isolated reduced c domain as monitored using absorbance
spectroscopy at 417, 432, 522, and 555 nm (experimental conditions as
in Fig. 2B). Results of the fits for the changes in
absorbance calculated assuming a two-state mechanism of unfolding are
shown as straight lines in A and
B.
The midpoint of unfolding (Tm) of isolated oxidized and
reduced c domain of P. pantotrophus in 50 mM sodium
phosphate buffer and 100 mM sodium chloride, pH 7, as
determined from thermal unfolding curves measured at different
absorbance wavelengths
band,
and the
absorption band, respectively, whereas the absorbance
around 430 nm monitors the presence of a non-native high spin species. The data show (Fig. 6B, Table II) very similar
Tm values for all wavelengths with an average
Tm of 357.7 ± 0.6 K and an average
Hm of 111 ± 7 kcal.mol
1. Tm values of unfolding
were calculated assuming a two-state mechanism (23). Fig. 2B
shows the absorbance spectrum of the thermally unfolded reduced protein
at 368 K with maxima at 421 and 553 nm. The figure also shows that the
and
bands have become broader and are no longer resolved. The
broadening of the Soret band at higher temperatures, the shift of the
Soret band to 421 nm, and the fact that the
and
bands are not
resolved (Fig. 2B) indicate that the unfolded reduced
c domain is a high-spin species (14, 39).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cross peaks with narrow linewidths
observed in two-dimensional NOESY and TOCSY 1H NMR spectra
of fresh samples of isolated reduced c domain are likely to
arise from backbone protons of residues constituting the flexible
N-terminal arm. Crystallographic data of the reduced cd1 enzyme also indicate that the N terminus of
the enzyme is flexible, because no electron density is detected for the
first 35 residues of monomer A (2).
cross peaks with narrow linewidths in two-dimensional NOESY and TOCSY
1H NMR spectra. This suggests that around 30 residues in
the isolated oxidized c domain are flexible, very much like
in the reduced c domain. These NMR results are difficult to
reconcile with the crystal structure of the oxidized
cd1 enzyme in which electron density is found
from residue 9 of the c domain and in which
His17 and Tyr25 are axial ligands to the
c and d1 heme iron, respectively (41, 42). Furthermore, the notable difference in the kinetics of ascorbate
reduction between the isolated oxidized c domain and the
oxidized P. pantotrophus cytochrome
cd1 nitrite reductase (34)4 points
to a difference in redox potential of these two proteins and thus to a
probable difference in c heme ligands analogous to the
oxidized semi-apo cd1 enzyme (from which the
noncovalently bound d1 heme has been removed),
which reacts fast with ascorbate and has a His-Met-ligated c
heme (34).
on the iron). Apparently,
the methionine axial ligand leaves the heme crevice, and no significant
mis-ligation occurs on the heme in the thermally denatured proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. J. Chattopadhyaya for giving us time on the NMR spectrometers at the Biomedical Center in Uppsala; Dr. Tania Maltseva for expert technical assistance with NMR data collection; Dr. Petra Franzén at the Expression Facility funded by SBNet, Sweden and Dr. Jan Saras for helpful discussions regarding protein expression and purification; Dr. Åke Engström for performing MALDI-TOF experiments and N-terminal analysis; Dr. Gunnar Johansson and students from the Molecular Biotechnology program at Uppsala University for performing sedimentation equilibrium studies; Remco Wouts for computer assistance; Amir Fathejalali, who was involved in part of this research as an undergraduate student; Tove Sjögren for providing cytochrome cd1 nitrite reductase; and Drs. Carlo P. M. van Mierlo and David van der Spoel for critically reading the manuscript.
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FOOTNOTES |
---|
* This research was supported in part by grants from the Swedish Natural Science Research Council (NFR), EU-BIOTECH, and the British Biotechnology and Biology Research Council (B05860).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.
An undergraduate student at the Uppsala Graduate School in
Biomedical Research. Present address: Dept. of Molecular Biology, SLU,
Uppsala, Sweden.
§ To whom correspondence should be addressed: Tel.: 46-18-471-4642; Fax: 46-18-511-755; E-mail: elles@xray.bmc.uu.se.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007345200
1 E. Gordon, E. Steensma, and S. J. Ferguson, manuscript in preparation.
3 M. Ubbink, personal communication.
4 T. Sjögren, personal communication.
5 The NMR data on the isolated c domain in the reduced and oxidized state are deposited in the BioMagResBank web site and have accession numbers 4800 and 4801, respectively.
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ABBREVIATIONS |
---|
The abbreviations used are:
PAGE, polyacrylamide
gel electrophoresis;
, chemical shift (ppm);
Cp, heat capacity change upon
denaturation;
, difference in chemical shift;
Hm, enthalpy change upon unfolding
at the midpoint of unfolding;
DEFT, driven equilibrium Fourier
transform;
DSS, sodium 2,2-dimethyl-2-silapentane-5-sulfonate;
DQF-COSY, double-quantum filtered coherence spectroscopy;
EXSY, exchange spectroscopy;
MALDI, matrix-assisted laser
desorption/ionization;
TOF, time-of-flight;
NOE, nuclear Overhauser
enhancement;
NOESY, nuclear Overhauser enhancement spectroscopy;
pH*, glass-electrode reading of the pH meter at room temperature,
uncorrected for deuterium isotope effects;
T, absolute
temperature;
T1, spin-lattice relaxation time;
Tm, temperature at the midpoint of unfolding;
TOCSY, total correlation spectroscopy.
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