Oxygen-linked Equilibrium CuB-CO Species in Cytochrome ba3 Oxidase from Thermus thermophilus

IMPLICATIONS FOR AN OXYGEN CHANNEL AT THE CuB SITE*

Konstantinos KoutsoupakisDagger , Stavros StavrakisDagger , Tewfik Soulimane§, and Constantinos VarotsisDagger

From the Dagger  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

    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 nu (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 nu (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.

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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).


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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 cm-1 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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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 cm-1 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 Delta 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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The FTIR spectrum of the CO-bound cytochrome ba3 complex at neutral pH exhibits peaks at 1967, 1973, 1982, and 2053 cm-1 (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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Fig. 1.   FTIR spectra of the cytochrome ba3-CO complex in the absence (A), (13CO, B), and in the presence of O2 (C), (13CO, D) at pH 7.5 and 293 K. Inset, UV-visible spectra of the cytochrome ba3-CO complex in the absence (a) and presence (b) of O2. Enzyme concentration was approx 1 mM and the pathlength was 15 µm. The spectral resolution was 2 cm-1.


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Fig. 2.   FTIR spectra of the cytochrome ba3-CO complex at pH 6.5 (A-C), pH 7.0 (D-G), pD 7.0 (H), and pH 9.0 (I-K) in the absence (A, D, I) and in the presence of O2 (B-C, E-H, J-K) at 293 K. The experimental conditions were the same as those in Fig. 1.

The insensitivity of the 2045 cm-1 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 pi * 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 nu (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 nu 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 cm-1 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 (nu 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 Delta A of the 2053 cm-1 mode shown in Fig. 3A, with the formation of the heme a32+-CO complex by measuring Delta 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. 3.   Time-resolved step-scan FTIR difference spectra of the CO-bound form of fully reduced cytochrome ba3 oxidase (pH 6.5) at 1, 2, 4, 5, 6, 8, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, and 75 ms after CO photolysis, in the absence (A) and in the presence (B) of O2. The spectral resolution was 8 cm-1, the time resolution was 100 µs, and 10 co-additions were collected per data point. The excitation wavelength was 532 nm (10 mJ/pulse), and each data set is the average of three measurements. Insets, kinetic analysis of the 2053 cm-1 (CuB-CO) (squares) and 1976 cm-1 (Fe-CO) (circles) modes versus time after CO photolysis in the absence (A) and in the presence (B) of O2. Delta A was measured from the intensity of the corresponding modes at times between 0 and 75 ms after the photolysis of CO from heme a3. The curves are three-parameter exponential fits to the experimental data, according to first-order kinetics.

Fig. 3B shows the time-resolved step-scan FTIR difference spectra (td = 0-75 ms, 8 cm-1 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 (nu 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 k-2. 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 nu 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.

    ACKNOWLEDGEMENT

We thank E. Pinakoulaki for helpful discussions.

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: FTIR, Fourier transform infrared; CHES, 2-(cyclohexylamino)ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Soulimane, T., Buse, G., Bourenkov, G. P., Bartunik, H. D., Huber, R., and Than, M. E. (2000) EMBO (Eur. Mol. Biol. Organ.) J. 19, 1766-1776[Abstract/Free Full Text]
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