From the University of Crete, Department of
Chemistry, 71409 Heraklion, Crete, Greece, and § Paul
Scherrer Institut, Life Sciences, OSRA/008,
CH-5232 Villigen PSI, Switzerland
Received for publication, October 8, 2002, and in revised form, February 13, 2003
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ABSTRACT |
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We report the first study of
O2 migration in the putative O2 channel
of cytochrome ba3 and its effect to the
properties of the binuclear heme
a3-CuB center of cytochrome
ba3 from Thermus thermophilus. The
Fourier transform infrared spectra of the
ba3-CO complex demonstrate that in the presence
of 60-80 µM O2, the Cytochrome ba3 from
Thermus thermophilus is a member of the large family of
structurally related heme-copper oxidases (1, 2). It catalyzes both the
four-electron reduction of O2 to H2O,
converting the energy of this reaction to a transmembrane proton motive
force, and the two-electron reduction of NO to N2O (1-4).
Based on the crystal structure, the enzyme contains a homodinuclear
copper (CuA), a low-spin heme b, and a heme
a3-CuB binuclear center (1). The
ba3-oxidase retains the electron transport chain
functional under low oxygen concentration in the medium. In both
structurally characterized aa3-type heme-copper oxidases, three possible O2 channels have been suggested
(5, 6). One of them leads from a trapped lipid pocket in subunit III to
the active site and contains Glu-278 (Paracoccus
denitrificans numbering), a residue near CuB (5, 6).
In the presence of three O2 channels in
aa3-type oxidases, it is not known yet whether a
facilitated oxygen channel is necessary, because the O2
concentrations normally far exceed the O2 affinity of the
enzyme; thus, excess O2 would not be rate-limiting. The
proposed oxygen input channel in ba3 contains
Ile-235 instead of Glu-278 (conserved to aa3
oxidases), which optimizes the formation of a hydrophobic pore (1, 2). The evolutionary development of an optimized oxygen channel is appropriate for organisms such as T. thermophilus
ba3 oxidase, the archaeal Sulfolobus
acidocaldarius aa3-quinol oxidase, and Natronomonas pharaonics, which grow under low oxygen
tension and at high temperatures (2). However, no data exist in the
literature to demonstrate the nature of the conformational changes that
occur in the binuclear center in the presence of O2 in the
channel. Because of the unusual ligand-binding and kinetic properties
of the binuclear center, cytochrome ba3 oxidase
is unique among the heme-copper oxidases in that it is susceptible to a
detailed kinetic analysis of its ligand dynamics (4, 7). The binding of
CO to the binuclear center of ba3 follows that
found in all heme-copper oxidases and proceeds according to the Scheme
1 (7-12).
(C-O) of
CuB1+-C-O at 2053 cm
1 (complex A)
shifts to 2045 cm
1 and remains unchanged in
H2O/D2O exchanges and in the pH 6.5-9.0 range.
The frequencies but not the intensities of the C-O stretching modes of
heme a3-CO (complex B), however, remain
unchanged. The change in the
(C-O) of complex A results in an
increase of k
2, and thus in a higher affinity
of CuB for exogenous ligands. The time-resolved step-scan
Fourier transform infrared difference spectra indicate that the rate of
decay of the transient CuB1+-CO complex at pH
6.5 is 30.4 s
1 and 28.3 s
1 in the presence
of O2. Similarly, the rebinding to heme
a3 is slightly affected and occurs with
k2 = 26.3 s
1 and 24.6 s
1 in the presence of O2. These results
provide solid evidence that in cytochrome ba3,
the ligand delivery channel is located at the CuB site,
which is the ligand entry to the heme a3
pocket. We suggest that the properties of the O2 channel
are not limited to facilitating ligand diffusion to the active site but
are extended in controlling the dynamics and reactivity of the
reactions of ba3 with O2 and NO.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Scheme 1.
In our previous work (7), we identified the C-O stretching
mode of the equilibrium CuB1+-CO species
(complex A) at 2053 cm1 and concluded that the
environment in the binuclear center does not alter the protonation
state of the CuB histidine ligands. Understanding the
conformational transitions that are associated with
protonation/deprotonation of labile residues is essential because
ionizable groups whose pKa values are near
physiological pH are involved in proton uptake or release. A
hydrogen-bonded connectivity between the propionates of heme
a3, Asp-372, and H2O was also
reported. Accordingly, plausible mechanisms of proton pathway(s)
directly associated with the propionates of the heme a3 redox center and the proton-labile side chain
of Asp-372 were suggested.
The nature of heme-copper oxidases is to bring in O2
through ligand-entry channels to the binuclear center and then remove protons and H2O from the active site. Because it has been
proposed that 1) CuB is a way-stop for ligand entry to heme
a3 (8) and 2) the O2 channel is
located at the CuB site, we sought to determine the
properties of the binuclear center by applying our FTIR approach (7, 9,
11) to study the CO bound cytochrome ba3 at room temperature in the presence of low-oxygen concentrations in the medium
(~70 µM), preventing spontaneous replacement of CO by
O2. In cytochrome ba3, the
exceptionally high affinity for CO binding to CuB
(K1 > 104) has allowed us to
perform such experiments. We have also used time-resolved step-scan
Fourier transform infrared spectroscopy (FTIR)1 to investigate the
ligand dynamics subsequent to CO photolysis at room temperature in the
presence of ~70 µM O2 and compare the results with those obtained in the absence of O2, which is
essential for elucidating the unique chemical mechanisms of the redox
processes catalyzed by the enzyme and the dynamics of the binuclear center.
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EXPERIMENTAL PROCEDURES |
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Cytochrome ba3 was isolated from T. thermophilus HB8 cells according to previously published
procedures (3). The samples used for the FTIR measurements had an
enzyme concentration of ~1 mM and were placed in a
desired buffer (pD 5.5-6.5, MES; pD 7.5, HEPES; pD 8.5-9.5 CHES). The
pD solutions prepared in D2O buffers were measured by using
a pH meter and assuming pD = pH (observed) + 0.4. Dithionite
reduced samples were exposed to 1 atmosphere CO (1 mM) in
an anaerobic cell to prepare the CO adduct and transferred to a tightly
sealed FTIR cell under anaerobic conditions (pathlength l = 15 and 25 µm for the experiments in H2O and D2O,
respectively). CO gas was obtained from Messer (Frankfurt, Germany) and
isotopic CO (13CO) was purchased from Isotec (Miamisburg,
OH). The ba3 carbonmonoxy/O2 adduct
was prepared by addition of aliquots of 5 µl of an oxygen-saturated buffer solution (1.2 mM O2) to make the final
O2 concentration of 60-80 µM. The 532 nm
pulse from a Continuum neodymium-yttrium aluminum garnet (Nd-YAG) laser
(7-ns width, 3 Hz) was used as a pump light (10 mJ/pulse) to photolyze
the ba3-CO complex. The time-resolved step-scan
FTIR spectra were obtained with spectral resolution of 8 cm1 and 100-µs time resolution for the 0-75-ms
measurements. A total of 10 co-additions per retardation data point was
collected. Changes in intensity were recorded with a mercury cadmium
telluride detector, amplified (dc-coupled), and digitized with a
200-kHz, 16-bit, analog-to-digital converter. Blackman-Harris
three-term apodization function with 32 cm
1 phase
resolution and the Mertz phase correction algorithm were used.
Difference spectra were calculated as
A =
log(intensity of sample/intensity of reference). The detailed
experimental set-up for the time-resolved step-scan FTIR has been
described previously (7, 9, 11). The rate constants for each phase of
the decay of the CuB1+-CO complex and for CO
rebinding to heme a3 were calculated, assuming first-order kinetics, with three-parameter exponential fits to the
experimental data. Optical absorbance spectra were recorded before and
after FTIR measurements to assess sample stability with a PerkinElmer
Lamda 20 UV-visible spectrometer.
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RESULTS AND DISCUSSION |
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The FTIR spectrum of the CO-bound cytochrome
ba3 complex at neutral pH exhibits peaks at
1967, 1973, 1982, and 2053 cm1 (Fig.
1, trace A). In the
13C16O derivative, these peaks shift to 1923, 1928, 1937, and 2007 cm
1, respectively (Fig.
1, trace B). The peaks at
1967, 1973, and 1982 cm
1 have been assigned to the C-O
stretching modes of heme a3-CO (complex
B), and the peak at 2053 cm
1 to the C-O
stretching mode of CuB1+-CO (complex
A). As shown in Fig. 1, trace C, addition of 70 µM buffered O2 to the
ba3-CO complex leads to a shift of the CO mode
of CuB1+ to 2045 cm
1. Addition of
oxygen-saturated buffer (>300 µM) causes the spontaneous replacement of CO by O2, as evidenced by the full
disappearance of both the heme a3-CO modes at
1967, 1973, and 1982 cm
1 and the CuB-CO mode
at 2053 cm
1. Confirmation that the newly developed 2045 cm
1 mode is CO-sensitive is shown in the 13CO
spectrum (Fig. 1, trace D), where it shifts to 1999 cm
1. These data also demonstrate a significant growth of
the 2045 cm
1 conformation on the expense of intensity
loss of the CO modes of heme a3 (complex
B), indicating an increase of k
2. The optical absorption spectrum of the ba3-CO
complex, shown in the inset (trace a), displays
Soret maxima at 427 and visible maxima at 529, 559, and 592 nm.
Addition of 70 µM O2 over the ba3-CO enzyme results in trace b with
a reduced intensity at 427 and a shoulder at 444 nm, indicating an
increase of k
2. No evidence for any oxidized
contribution from hemes b/a3 is
observed. The conversion of the 2053 cm
1 conformer to
that of 2045 cm
1 is stable over a wide pH range. Fig. 2,
trace A, shows the spectrum of the
ba3-CO complex at pH 6.5 in the absence of
O2. Addition of 70 µM
O2-equilibrated buffer yields trace B, where
both 2053 and 2045 cm
1 conformers are present. The
O2-induced conformational change undergoes a very slow
(t1/2 ~ 65 min) conformational change (trace
C). The absence of any additional changes in trace C
indicates that equilibrium has been reached. Trace D is that
of ba3-CO at pH 7.0 in the absence of
O2. and traces E and F are in the
presence of 70 µM of O2 until no additional changes were observed (trace G). Trace H is
obtained in the presence of 70 µM of O2 at pD
7.0. Traces I (ba3-CO)-K
(ba3-CO in the presence of O2) are
the analogous spectra at pH 9. The absence of pH/pD-induced spectral
changes in the 2045 cm
1 conformer indicates that is not
coupled to proton-dependent conformational changes.
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The insensitivity of the 2045 cm1 mode to pH/pD and to
the pH 6.5-9.0 range indicates that the degree of back-donation of
electron density from the d orbitals of CuB to
the antibonding
* orbitals of CO is not altered under these
conditions. The same conclusions have been reached for the 2053 cm
1 mode. As we have demonstrated previously for complex
A (2053 cm
1 mode), a change in the protonation
state of one of the CuB-His ligands would have significant
changes in the back donation and thus on the frequency of
(C-O) (7).
Because we see no change in the frequency of the 2045 cm
1
mode, we conclude that the CuB-His environment is very
rigid and not subject to conformational transitions that are associated with protonation/deprotonation events of the CuB-His
ligands. We suggest that low concentrations of O2 (60-80
µM) in the putative O2 channel play a role in
modulating the structure of the CuB-CO complex that
facilitates the transition from 2053 to 2045 cm
1. We
attribute the transition to an increased electron density in the CO
antibonding orbitals that results in weakening the C-O bond strength
(lower
C-O). It remains to be determined whether the O2 is hydrogen-bonded to one of the CuB-His
ligands, causing the weakening of the C-O bond, or the newly developed
conformer is the result of a dynamic effect in the delivery channel
generating a conformational change in the CuB environment.
It becomes intriguing to speculate that the properties of the
O2 channel are not limited to facilitate ligand diffusion
to the active site but are extended in controlling the dynamics and
reactivity of the reactions of ba3 with
O2 and nitric oxide and other ligands, including
cyanide. The increased value of k
2
indicates that CuB undergoes structural changes to behave
as an efficient trap for CO. We suggest that this behavior is extended
to the physiological function of the enzyme. This way, conformational
changes associated with CuB occur, facilitating the entry
and the later coordination of O2 to the binuclear center.
Fig. 3A shows the
time-resolved step-scan FTIR difference spectra
(td = 0-75 ms, 8 cm1 spectral
resolution) of fully reduced ba3-CO subsequent
to CO photolysis by a nanosecond laser pulse (532 nm). Upon photolysis, CO is transferred from heme a3 to
CuB. It should be noted that the CuB-CO complex
(complex A) is not photolabile and thus remains a spectator
in the photodynamic events occurring to complex B. Under our
8 cm
1 spectral resolution, the heme
a3 Fe-CO peaks at 1967, 1973, and 1982 cm
1 are not resolved; thus, a single negative peak at
1976 cm
1 indicates the photolyzed heme
a32+-C-O complex. The positive peak
that appears at 2053 cm
1 is attributed to the C-O stretch
(
C-O) of the transient CuB1+-CO
complex, and its frequency is the same as that obtained at pD 8.5 (7)
and that of the equilibrium CuB1+-CO complex.
At early times (0-3000 µs), the intensity of the 1976 and 2053 cm
1 modes in the transient difference spectra remains
unchanged, suggesting that dissociation of CO from CuB does
not occur on this time scale. At later times (3-75 ms), there is a
decrease in intensity of the 2053 cm
1 mode that is
accompanied by an increased intensity of the 1976 cm
1
mode. At 75 ms after CO photolysis, the intensities of both the 1976 and 2053 cm
1 modes are almost diminished. The intensity
ratio (~2) of the Fe-CO/CuB-CO remains constant for all
data points, indicating that no significant fraction of CO escapes the
binuclear center. This is also consistent with both the low-temperature
experiments (21 K) and those obtained at pD 8.5 at room temperature (7, 8). The high signal-to-noise ratio in the time-resolved FTIR difference
spectra has allowed us to monitor the decay and reappearance of the
2053 and 1976 cm
1 modes, respectively. Fig.
3A, inset, compares the decay of the CuB1+-CO complex, as measured by
A of the 2053 cm
1 mode shown in Fig.
3A, with the formation of the heme
a32+-CO complex by measuring
A of the 1976 cm
1 modes. The rate of decay
of the transient CuB1+-CO complex is 30.4 s
1, and the observed rate of rebinding to heme
a3 is 26.3 s
1. Both rates are 10%
lower than those observed at pD 8.5 (7). The pH/pD dependence of the
rates of decay of the CuB complex and the rebinding to heme
a3 will be presented
elsewhere.2
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Fig. 3B shows the time-resolved step-scan FTIR difference
spectra (td = 0-75 ms, 8 cm1
spectral resolution) of fully reduced ba3-CO in
the presence of 70 µM O2 after CO photolysis
by a nanosecond laser pulse (532 nm). The results are very similar to
those obtained in the absence of O2, including the
intensity ratio (~2) of the Fe-CO/CuB-CO modes. This
observation indicates that O2 is not coordinated to CuB in complex B, before photolysis.
Importantly, the observed rates for the decay of the transient
CuB1+-CO complex (28.3 s
1) and
the heme a32+ recombination
(k2 = 24.6 s
1) that we have
determined, shown in Fig. 3B, inset, indicate
that the presence of O2 in the delivery channel has no
direct control in the CO ligation/dissociation dynamics. The
observation of the C-O stretch (
C-O) of the transient
CuB1+-CO complex at 2053 cm
1 and
not at 2045 cm
1, as observed in the equilibrium
CuB1+-CO complex in the presence of
O2, indicates the absence of an equilibrium between the
transient CuB1+-CO complex and O2
at the time-scale of our observations (0-75 ms).
This work presents a very peculiar and unexpected observation. However,
the ba3 oxidase is expressed under limited
amounts of oxygen; thus, an
O2-concentration-dependent behavior, such that
presented in this work, is feasible. Several control experiments with
Mb-CO (data not shown) indicate that under the same experimental conditions (O2 concentration, pH-range, temperature),
O2 spontaneously replaces CO. The experiments reported here
have been repeated with different enzyme preparations to avoid some
sort of artifact and clearly demonstrate O2 migration in
the delivery channel that is located at the CuB site. The
presence, but not coordination, of O2 at the
CuB site results in a structural reorientation of the
CuB environment and concomitantly to an increase of
k2. This effect can be either steric or
electrostatic involving either one of the CuB-His ligands
(polar) or the CuB atom directly (electrostatic), producing
an increased electron density in the CO antibonding orbitals that
results in weakening the C-O bond strength (lower
C-O).
Our data also support the developing consensus that CuB is
a way stop for O2 en route to its heme
a3 binding site (8). Whether the presence of
Glu-278 in other heme-copper oxidases (5, 6) interrupts the putative
oxygen channel provided by Ile-235 in ba3, and
thus the efficient diffusion of low O2 concentrations to
the binuclear center, remains to be determined. Moreover, it is
important to establish whether the conformational changes induced to
CuB environment by O2, which is present in low
concentrations at physiological conditions because of the reduced gas
solubility at higher temperatures, in the putative channel of
ba3, are extended to the superfamily of
cytochrome oxidases. Experiments toward this goal are in progress in
our laboratory.
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ACKNOWLEDGEMENT |
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We thank E. Pinakoulaki for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by the Greek Ministry of Education.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Fax: 30-2810-393601; E-mail: varotsis@edu.uoc.gr.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M210293200
2 K. Koutsoupakis, T. Soulimane, and C. Varotsis, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: FTIR, Fourier transform infrared; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid.
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