Heme Ligation and Conformational Plasticity in the Isolated c Domain of Cytochrome cd1 Nitrite Reductase*

Elles SteensmaDagger §, Euan Gordon, Linda M. ÖsterDagger ||, Stuart J. Ferguson, and Janos HajduDagger

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 = -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).

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.



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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -20 °C before use.

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 Delta 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.

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°-tau -180°-tau -90°) (29) with tau  = 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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (beta  band), and 548 nm and 554 nm (split alpha  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).

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-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

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 -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



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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 Hdelta 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).


                              
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Table I
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 
T1 values are reported for non-overlapping hyperfine shifted resonances only.

The exchangeable Hdelta 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 Hepsilon 1 and Hdelta 2 protons were assigned (Table I). Weak NOEs between the His69 Hepsilon 1 proton and the heme meso proton 15 and between the His69 Hdelta 2 proton and the heme meso proton 5 were identified (Fig. 4).

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 -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 beta  and gamma  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).

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 Hepsilon 1 and Hdelta 1 protons and the methyl group of the axial methionine, and between the tryptophan Hepsilon 1 and Hzeta 2 protons and the heme meso proton 5 (see Fig. 4).

Around 30 intense cross peaks in the NH (8.6-7.7 ppm)-Halpha region (4.7-4.0 ppm) were observed with clearly narrower linewidths than the surrounding NH-Halpha 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.

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 -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).

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 Hdelta 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 Hdelta 2 proton of His69.

The remaining hyperfine-shifted resonances appearing at high field between -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 beta  protons and one gamma  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).

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)-Halpha region (4.7-4.0 ppm) with clearly narrower linewidths than the surrounding NH-Halpha cross peaks at other chemical shift positions in the spectra (data not shown).

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 Hdelta 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).

Fig. 5 shows that Curie plots (chemical shift versus T-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.

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 alpha /beta absorption bands resulting from pi -pi * 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 Delta 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.


                              
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Table II
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
The reduced protein samples contained 10 mM dithionite as well.

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 beta  band, and the alpha  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 Delta 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 alpha  and beta  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 alpha  and beta  bands are not resolved (Fig. 2B) indicate that the unfolded reduced c domain is a high-spin species (14, 39).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-Halpha 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).

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-Halpha 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).

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- 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.

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.


    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.


    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.


    ABBREVIATIONS

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; delta , chemical shift (ppm); Delta Cp, heat capacity change upon denaturation; Delta delta , difference in chemical shift; Delta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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