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Originally published In Press as doi:10.1074/jbc.M407766200 on August 23, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47320-47325, November 5, 2004
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The C-helix in CooA Rolls upon CO Binding to Ferrous Heme*

Taku Yamashita{ddagger}, Yohei Hoashi{ddagger}, Yoshikazu Tomisugi, Yoshinobu Ishikawa, and Tadayuki Uno§

From the Graduate School of Pharmaceutical Sciences, Kumamoto University, Oehonmachi, Kumamoto 862-0973, Japan

Received for publication, July 9, 2004 , and in revised form, August 3, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CooA is a homodimeric transcriptional activator from Rhodospirillum rubrum containing one heme in each subunit. CO binding to the heme in its sensor domain activates CooA, facilitating the binding to DNA by its DNA-binding domain. The C-helix links the two domains and shapes an interface between the subunits. To probe the nature of CO activation, residues at positions 112–121 on the C-helix were replaced by Asn or Gln and their effects were evaluated by resonance Raman spectroscopy and by the measurements of CO binding affinity. The {nu}(Fe-CO) stretching Raman line in CO-bound wild-type CooA was up-shifted by 6 cm-1 in the L116Q, G117N, and L120Q mutants, indicating unequivocally that these residues are close to the bound CO. Residues Leu116 and Leu120 from each subunit form contacts with the corresponding residues in the opposite subunit, enabling hydrophobic interactions in the inactive ferrous form. Thus, in the CO-bound activated form, both C-helices appear to roll to direct these residues toward the heme, forming a hydrophobic pocket for the bound CO. The CO affinity is approximately one order of magnitude higher in the L112Q, I115Q, L116Q, G117N, L120Q, and T121N mutants but reduced in A114N mutant. The variation indicates that these residues are close to the heme in the ferrous and/or CO-bound forms and are responsible for CooA activation. A roll-and-slide mechanism is proposed for CO activation of CooA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CooA from the photosynthetic bacterium Rhodospirillum rubrum is a heme-based transcription factor that is activated upon binding CO (1, 2). CooA regulates the expression of genes whose products are associated with CO metabolism (3). CooA is a homodimer (221 amino acids/monomer) containing a DNA-binding and a sensor domain analogous to those found in the cAMP receptor protein (CRP)1 (4, 5). The crystal structure of ferrous CooA revealed (4) that a b-type heme is contained within the sensor domain where it is ligated to the His77 side chain (Fig. 1). This ligand is replaced by the Cys75 side chain in the ferric state (6, 7). The ferrous heme ligand trans to His77 is the N-terminal proline residue (Pro2) from the partner subunit in the dimer (4). We have recently established (8) that Pro2 is the ligand that is displaced by the incoming CO as had been proposed previously (9, 10) and that Pro2 coordination finetunes the sensing of CO in the media. Because CooA is the first example of a CO sensor protein (1, 2), its CO sensing and activation mechanisms have been attracting the attention of many researchers (11–14).



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  		FIG. 1.
Structure of CooA and CRP. A, effector-free inactive CooA (Protein Data Bank code 1FT9 [PDB] ) (4). B, effector-bound active CRP (Protein Data Bank code 1G6N [PDB] ) (18). Sensor domain, DNA-binding domain, and the C-helices that form the dimerization interface are shown in blue, green, and brown, respectively. The hemes in CooA and cAMPs in CRP are shown in red. C, view of the C-helices (brown) and heme (red) in CooA. The Pro2-His77 axial ligands as well as the side chains of Ala114 (magenta), Leu116 (cyan), and Leu120 (green) are shown. The drawing was generated using PyMOL (41).

 
The signal of CO binding to the heme is transmitted to the DNA-binding domain, directing the protein to its target DNA sequence, which is palindromic (15–17). Because the two domains are linked by a long {alpha}-helix (C-helix), the residues on the C-helix must play a key role in signal transmission from the heme-containing domain to the DNA-binding domain and hence CO activation of CooA. The C-helices form a coiled-coil structure, which shapes the dimer interface in the inactive ferrous form (Fig. 1). As summarized in Table I, the C{alpha} atoms of Ile113, Leu116, Leu120, Cys123, and Ile127 on the respective subunits are in close proximity to each other (<7.0 Å) in CooA as in the optimal leucine zipper alignment. To reveal the activation mechanism, it is potentially informative to inspect the structural difference between CooA and CRP, which is homologous to CooA and whose cAMP-bound activated structure is available (18). The C{alpha} atoms in the C-helix of CRP (residues at position 112–136) are organized in a similar way to the corresponding region of CooA (Table I), and the difference in the distances between the inactive ferrous CooA and active cAMP-bound CRP is not apparent. Thus, it is unclear what kind of structure is required for the activation of these transcription factors.


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  		TABLE I
Distances between specific atoms of residues in the C-helix region All of the values are calculated with atomic coordinate sets of ferrous inactive CooA (Protein Data Bank code 1FT9 [PDB] ) (4) and cAMP-bound active CRP (Protein Data Bank code 1G6N [PDB] ) (18).

 
A previous study (19) revealed that the side chains of Ile113 and Leu116 are close to the CO in the ferrous CO-bound CooA. When Leu116 is replaced by the heme-coordinating lysine residue, CooA is active both in the presence and absence of CO (20). The importance of the nearby Gly117 to CooA activation has also been stressed (21). Thus, it is believed that residues in this region are crucial for CooA activation. Although these pieces of evidence are informative, the mutation studies have not been systematic so far and the role of the C-helix in CO activation remains to be fully elucidated.

In our strategy, we at first inspected the distances between the heme iron and each C{alpha} atom on the C-helix with the atomic coordinate set of the inactive ferrous form (4) (Table I). Because CooA is homodimeric, the two C{alpha}-Fe distances within the respective subunits (d(C{alpha}-Fe)) are averaged and the deviation is given. Similarly, two distances from iron to C{alpha} atoms on the opposite subunit (d(C{alpha}-Fe')) are evaluated. Although the deviations in the C-terminal half of the C-helix are relatively large, the distances in the N-terminal half are well conserved between the subunits (the deviations are <0.1 Å). The C{alpha} atoms of Leu112 and Leu116 are relatively close to the heme iron within the same subunit (<10 Å), whereas those of Ile113, Ala114, Gly117, Arg118, and Thr121 are close to the iron in the opposite subunit. Thus, in this study, 10 residues at positions from 112 to 121 have been systematically mutated and the mutated proteins have been examined by resonance Raman spectroscopy and by the measurement of CO binding affinity. We conclude that the C-helix rolls to direct residues Leu116, Gly117, and Leu120 toward the heme and that there is a concomitant sliding of the heme with respect to the C-helix. Therefore, we propose a roll-and-slide model for CO activation of CooA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of CooA Mutants—We have reported a high expression system for CooA, which employs a synthetic gene optimized for expression in Escherichia coli (8). The QuikChange system (Stratagene) was used to introduce mutations into the CooA coding sequence, and the DNA sequence of the plasmid products were confirmed with a Li-Cor Model 4200S2 DNA sequencer. Cell growth and protein purification were performed as described previously (6) with slight modifications. Soluble fractions of the cell extract were applied to a Q-Sepharose FF column (Amersham Biosciences), and the adsorbed proteins were eluted with linear gradient of NaCl in 50 mM Tris-HCl (pH 8.0). The CooA fractions were subsequently purified by Sephacryl S-100 column chromatography (Amersham Biosciences) and concentrated (Amicon Ultra-15, Millipore). Proteins were purified to homogeneity as judged by SDS-PAGE criteria. CooA concentrations were evaluated from the heme content, which was measured by a pyridine hemochrome assay, and hence were based on a monomer unit.

Resonance Raman Spectroscopy—The spectra were recorded using a double monochromator (Jasco R-800) with a slit width of 6 cm-1 following excitation by a krypton ion laser (406.7-nm line, Coherent I-302). A photomultiplier detector was used (Hamamatsu Photonics, R595), and the frequencies were calibrated with indene. The frequencies reported are accurate within 1 cm-1 for sharp and discrete Raman lines. A spinning Raman cell was used throughout the measurements to minimize local heating and sample damage. The samples contained 100 µM protein in 0.1 M Tris-HCl (pH 8.0). Ferrous proteins were prepared by the addition of sodium dithionite after purging extensively with nitrogen gas. The carbonmonoxy forms were prepared by the addition of sodium dithionite under 1 atm CO. The spectra of the CO forms were obtained with a defocused laser beam.

CO Titration—CO titration of ferrous CooA was performed in 0.1 M Tris-HCl (pH 8.0) at 25 °C as described previously (8). A cuvette was filled with a CooA solution, which was purged extensively with nitrogen gas stoppered with a rubber septum, and the protein was reduced by an excess of sodium dithionite solution, which was injected by a syringe. Full reduction of CooA was confirmed by absorption spectroscopy (Beckman DU640). Aliquots of CO saturated in 0.1 M Tris-HCl (pH 8.0) and dithionite were added to solutions containing ~2 µM protein, and the absorption spectra were recorded. The CO concentrations were calibrated using pig myoglobin. The absorbance changes at the Q-band maxima were traced and analyzed after normalization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ferric and Ferrous CooAs—In our mutation strategy, those residues having long and hydrophobic side chains (Leu and Ile) were replaced by Gln, which has long but polar side chain. On the other hand, small residues (Ala, Gly, and Thr) were replaced by the short and polar Asn, which is analogous to Gln. Arg118, the sole charged residue in our target set, was replaced by the hydrophobic Leu. The effect of these mutations was evaluated by resonance Raman spectroscopy, which is a powerful tool for revealing the coordination states of heme iron (22, 23). In the ferric state, the spectral profiles of these mutants are essentially the same as that of WT CooA (Fig. 2). {nu}2, {nu}38, and {nu}3 lines, which are sensitive to the iron coordination state, are observed at 1583, 1549, and 1501 cm-1, respectively, indicating that the Cys75 and Pro2 ligands in the WT CooA are unperturbed by the mutations. However, in the A114N, I115Q, and A119N mutants, the {nu}38 line appears stronger than the {nu}2 line and it is also slightly up-shifted. In these mutants, a relatively strong line is observed at 268 cm-1, which is assignable to the {delta}(C{beta}C1) bending vibration of peripheral carbon atom of the heme (24).



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  		FIG. 2.
Resonance Raman spectra of the ferric CooAs. Listed from top are WT, L112Q, I113Q, A114N, I115Q, L116Q, G117N, R118L, A119N, L120Q, and T121N. The samples contained 100 µM proteins in 0.1 M Tris-HCl buffer (pH 8.0). Asterisks indicate a plasma line of a laser. Spectral condition: slit width, 6 cm-1; laser, 406.7 nm at 30 milliwatts.

 
In the ferrous CooA mutants, the resonance Raman spectra are again essentially the same as those of the WT protein (Fig. 3). The {nu}2, {nu}38, and {nu}3 lines are observed at 1581, 1555, and 1493 cm-1, respectively, corresponding to six-coordination with axial ligands contributed by the His77 and Pro2 side chains (Fig. 1). However, in the L116Q, G117N, and L120Q mutants, the {nu}2 and {nu}38 lines are slightly up-shifted and a weak line appears around 1470 cm-1 ({nu}3). A similar profile was detected in the P2H and H77A mutants of CooA in which one of the Pro2-His77 axial ligands is replaced (8). Thus, some of the C-helix residues affect the axial coordination of the ferric and ferrous hemes in CooA but the effects are slight.



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  		FIG. 3.
Resonance Raman spectra of the ferrous CooAs. Listed from top are WT, L112Q, I113Q, A114N, I115Q, L116Q, G117N, R118L, A119N, L120Q, and T121N. The samples contained 100 µM proteins in 0.1 M Tris-HCl buffer (pH 8.0). Spectral condition: slit width, 6 cm-1; laser, 406.7 nm at 30 milliwatts.

 
CO Probe—In Fig. 4, resonance Raman spectra of ferrous CO-bound CooA are shown. The {nu}(Fe-CO) stretching frequency is sensitive to the polarity of the residues surrounding the bound CO, and it therefore represents an excellent probe of the distal environment (25–29). The {nu}(Fe-CO) stretching line is observed at 490 cm-1 in WT CooA as reported previously (8, 19, 30), indicating that the bound CO is in a hydrophobic distal environment. This line is clearly affected by mutations of some of the C-helix residues, and especially noted are the large up-shifts of 6 cm-1 in the L116Q, G117N, and L120Q mutants. A small but measurable up-shift of 3 cm-1 in the L112Q and A119N mutants is also noted. In these mutants, hydrophobic residues are replaced by polar Gln or Asn residues. The effect of hydrophobic to polar substitutions on the {nu}(Fe-CO) stretching frequency has been reported in myoglobin (31). In ferrous CO-bound myoglobin, the bound CO is close to the distal His64 and the {nu}(Fe-CO) stretching line is observed at ~510 cm-1 (32, 33). The line is observed at 490 cm-1 in H64L and at 492 cm-1 in H64I mutants that have apolar replacements, but it is restored to 508 cm-1 in H64Q (31). Thus, it is clear that Leu116, Gly117, and Leu120 in WT CooA are close to the bound CO, contributing to a hydrophobic distal heme pocket. Leu112 and Ala119 may also locate close to the CO but in a rather remote fashion. The frequency of the {nu}(Fe-CO) stretch and the shift values relative to that in WT protein are summarized in Table II.



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  		FIG. 4.
Resonance Raman spectra of ferrous-CO bound CooAs. Listed from top are WT, L112Q, I113Q, A114N, I115Q, L116Q, G117N, R118L, A119N, L120Q, and T121N. The samples contained 100 µM proteins in 0.1 M Tris-HCl buffer (pH 8.0). Spectral condition: slit width, 6 cm-1; laser, 406.7 nm at 0.3 (A114N) or 1 milliwatt (other mutants) with defocused laser beam.

 


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  		TABLE II
{nu}(Fe-CO) frequencies and CO dissociation constants of ferrous CO-Bound CooA

 
Along with the frequency shift, the band shape of the {nu}(Fe-CO) stretch is slightly affected by the mutations of some of the C-helix residues (Fig. 4). The {nu}(Fe-CO) lines are relatively broad in the I113Q, A114N, L116Q, and L120Q mutants, suggesting the fluctuation in the Fe-CO geometry. A minor band seems to exist at ~520 cm-1 in the I115Q mutant, and the presence of multiple Fe-CO conformers is suggested. Similar multiple {nu}(Fe-CO) stretching modes were detected in some of the His77 axial ligand mutants of CooA (8), and hence, the Fe-His bond may partly be affected in the I115Q mutant. The main {nu}(Fe-CO) stretching frequency at 489 cm-1, however, is almost the same as that in WT CooA; hence, Ile115 is suggested to be remote from the heme in the CO-bound state.

Because some of the C-helix residues affect the bound CO, the effect of these residues on the CO binding affinity was evaluated. The CO dissociation constants (Kd) for the ferrous-CO bound CooA mutants are given in Table II. The CO affinity of WT CooA (Kd = 11 µM) is similar to that for FixL (9.0 µM) (34) and Dos (10 µM) (35), which are heme-based gas sensor proteins. Although the CO binding kinetics is reported to be multi-step (36, 37), the equilibrium CO binding is a one-step process (8). Thus, binding was analyzed in terms of a simple equilibrium between the ferrous and CO-bound forms of the protein. The Kd values generally decreased in the C-helix mutants, indicating that polar side chains in the mutants facilitate CO binding to the ferrous heme. The logarithm of the ratio of the Kd values for the mutant and WT CooA (i.e. {Delta}log Kd) is calculated and also summarized in Table II. The CO affinity is less affected in I113Q and R118L, indicating that Ile113 and Arg118 are remote from the heme. It should be noted here that the Kd value greatly increased in the A114N mutant. Thus, Ala114 is suggested to play a key role in CO activation of CooA. Because of this low affinity, resonance Raman measurement of CO-bound A114N was very difficult (Fig. 4) and the laser power had to be reduced to 0.3 milliwatts. Even under this condition, the ferrous heme is not fully coordinating CO because saturated CO concentration is 1 mM. This is the main reason for the relatively weak {nu}(Fe-CO) stretching line in this mutant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Inactive Ferrous Form—In Fig. 5, the positions of C-helix residues are schematically drawn in rectangle and in helical wheel projection models. Because the C-helix shapes a coiled-coil structure in the inactive ferrous form (4), a 3.5 residue/turn scheme was adopted. As summarized in Table I, the C{alpha} atoms of Ile113, Leu116, Leu120, Cys123, and Ile127 residues on the respective subunits are located close to each other (<7.0 Å) and these residues are marked with small pink circles (Fig. 5, top). It is clear that these residues align on one face of the C-helix and shape a dimer interface. The C{alpha} atoms of Leu112 and Leu116 (marked with cyan circles) are close to the ferrous heme within the same subunit, whereas those of Ile113, Ala114, Gly117, Arg118, and Thr121 (marked with green circles) are close to the heme within the opposite subunit. Pro2, one of the heme axial ligands, is provided by the opposite subunit and is shown in green. In the ferric state, the A114N, I115Q, and A119N mutants affected the relative intensity of the {nu}38 and {delta}(C{beta}C1) bending modes (Fig. 2) and, hence, these residues are probably close to the heme periphery in the WT CooA. In the crystal structure of ferrous CooA (4), the side chain of Ile115 is close to the heme 1-methyl and 2-vinyl groups and Ala119 is close to the 1-methyl group, although Ala114 is remote from the heme periphery (Fig. 1). The proximal ligand in the ferric state is the side chain of Cys75, which is replaced by that of His77 in the ferrous state (6, 7). The redox-linked ligand exchange in CooA may reposition the heme, providing a close contact of heme periphery with Ala114 side chain.



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  		FIG. 5.
Roll-and-Slide model for the CO activation of ferrous CooA. The C-helix is drawn schematically as a rectangle or helical wheel projection models. Top, inactive ferrous form. The residues having d(C{alpha}-C{alpha}') <7 Å (boldface in Table I) are marked with small pink circles. The residues having d(C{alpha}-Fe) and d(C{alpha}-Fe') of <10 Å (boldface in Table I) are marked with cyan and green circles, respectively. Pro2 is shown in green to indicate that the ligand is provided by the opposite subunit. Bottom, active ferrous-CO form. The residues that showed {Delta}{nu}(Fe-CO) stretching frequencies greater than 3 cm-1 (boldface in Table II) by the mutation are marked with red circles. The residues that showed Kd values of one order of magnitude greater or smaller than that for WT CooA (boldface in Table II) by the mutation are marked with small purple circles.

 
In the ferrous state, the mutation of residues Leu116, Gly117, and Leu120 slightly affected the axial coordination of the heme (Fig. 3). Because His77 ligand is opposite to the heme plane when viewed from the C-helix, relatively weak coordination by Pro2 ligand (8) should be affected by the mutation. It has been reported that the replacement of Gly117 with Ile sterically perturbs Pro2 coordination (21), and this is similar to that observed in our G117N mutant. In these Leu116, Gly117, and Leu120 mutants, the Kd values for CO dissociation increased by ~10-fold (Table II). Although many factors may affect CO affinity, this increase may partly be attributed to the weaker Pro2 coordination in these mutants. Pro2 coordination greatly reduces the CO affinity, and this coordination is the way in which CooA regulates its CO-sensing level (8). Leu116 and Leu120 locate in the dimer interface in the inactive ferrous state, and the side chain of Leu120 is too far from the Pro2 ligand for direct interaction (Fig. 5, top). Thus, the C-helices must roll relative to each other at least in the L120Q mutant to weaken the Fe-Pro2 bond in the ferrous state. The replacement of these aliphatic residues by the polar Gln should weaken the hydrophobic interaction between the helices. This may promote the rolling of the helices, mimicking the active CO-bound form as described below and facilitating CO binding with high affinity.

Active Ferrous-CO Form—As we revealed previously (8), the Pro2 ligand is replaced by the incoming CO molecule and this replacement triggers CooA activation (11–14). The residues affecting the {nu}(Fe-CO) stretch (Fig. 4) by >3 cm-1 are marked with red circles (Fig. 5). Among these residues, the effects of Leu116, Gly117, and Leu120 mutation were dominant again; hence, these three residues appear to be located close to the CO molecule. Because Leu116 and Leu120 locate at the dimer interface in the ferrous state, the C-helices must roll to direct these residues to the CO molecules. To maximize the interaction between these residues and CO, the hemes must slide along the C-helices. Because Pro2 is provided from the opposite subunit, CO replacement should heave the Pro2 anchor in CooA. This should increase the freedom for relative movement of C-helices and hemes. Regardless, the movement of both helices as well as both hemes must be symmetrical because CooA is homodimeric and the recognition sequence of CooA is palindromic (15–17).

The residues that affected the CO affinity more than one order of magnitude are marked by small purple circles in Fig. 5 (bottom). The CO affinity increased predominantly in the L112Q, I115Q, L116Q, Gl117N, L120Q, and T121N mutants. The Leu116, Gly117, and Leu120 side chains are close to one another on one face of the C-helix, thus lining the distal heme pocket and greatly affecting the CO affinity. The remaining Leu112, Ile115, and Thr121 residues are directed outward from the dimer interface in the ferrous form (Fig. 5, top) and may not interact directly with the bound CO. These residues surround the Leu116, Gly117, and Leu120 triad, possibly forming an entrance allowing CO to access the heme.

Among the CooA mutants examined, A114N is the sole mutant, which greatly reduced the CO affinity, although Gly117 and Thr121, which lie on the same C-helix face, enhanced the CO affinity when replaced by Asn. It is interesting to note that Ala114 is sandwiched by Gly111 and Gly117, which have small side chains. Such small residues may facilitate the helical roll in the CO activation. When the small Ala114 is substituted by the polar and bulkier Asn, it may inhibit C-helix rolling. This inhibition may stabilize the ferrous inactive form so that CO affinity is reduced.

Roll-and-Slide Model—As discussed above, the C-helices must roll, and here, we propose a roll-and-slide model for the CO activation of CooA (Fig. 5, bottom). In this section, we validate the model based on the experimental evidence. Since A114N showed low CO affinity, we propose that Ala114 could be the fulcrum of the helix rolling to shape the CO-bound form. This means that the heme must slide to the C-terminal side on the helix, and this view is consistent with the close proximity of the Leu116, Gly117, and Leu120 triad to the CO-bound heme. Leu116 is reported to be crucial for CooA activity, and the L116K mutant is active in both ferrous and CO-bound states (20). Leu116 is close to the heme in our model (Fig. 5, bottom), enabling the coordination of Lys side chain in L116K.

It is proposed (19) that the C-helix relatively moves upon the CO binding and that the heme also moves to the N-terminal side. In our systematic mutation study, however, we favor the movement of the heme toward the C-terminal portion of the C-helices for the reasons outlined below. In the I113Q mutant, the Kd value and {nu}(Fe-CO) stretching frequency are less affected than in the other mutants examined (Table II), although Ile113 is one helical turn up from Leu116 and Gly117 and is close to the heme (marked with green circle in Fig. 5, top). In contrast, Met124 is reported to affect iron coordination in the activated form (38). This residue is one helical turn down from Leu120, supporting the notion of translation of the heme toward the C terminus of the helix.

Three major factors contribute to cofactor retention in heme proteins (39): (i) covalent bonding between the heme iron and axial ligand residues; (ii) apolar interactions between the heme and surrounding hydrophobic residues; and (iii) polar interactions between the heme propionates and surrounding hydrophilic residues (40). In ferrous CooA, the bonds to the Pro2-His77 ligands may be enough to anchor the heme, although the heme propionate is not surrounded by amino acid side chains. However, in the CO-bound state, Pro2 is replaced by CO, requiring a compensating mechanism for the heme retention. In our model, the helical roll may allow the positively charged Arg118 to interact favorably with the negatively charged heme propionate in the opposite subunit. Although Arg118 interacts with Asp72 in the ferrous form (4), this residue may partly contribute electrostatically to the stabilization of the slipped heme.

In summary, the C-helix in CooA rolls upon CO binding to the heme as revealed by resonance Raman spectra and CO dissociation constants. We propose a roll-and-slide model for the CO activation mechanism of CooA. This model satisfactorily accounts for our observations as well as those reported earlier. Although further studies on the interactions between the C-helices and the DNA-binding domains are required, our roll-and-slide model may be a good starting point for fully understanding the CO activation mechanism.


   FOOTNOTES
 
* This work was supported by Grant 13470480 for Science Research (to T. U.) from the Japan Society for Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed. Tel./Fax: 81-96-371-4350; E-mail: unot{at}gpo.kumamoto-u.ac.jp.

1 The abbreviations used are: CRP, cAMP receptor protein; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Anthony J. Wilkinson (University of York) for valuable discussion.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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