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Originally published In Press as doi:10.1074/jbc.M409884200 on November 10, 2004

J. Biol. Chem., Vol. 280, Issue 5, 3269-3274, February 4, 2005
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Spectroscopic and Redox Properties of a CooA Homologue from Carboxydothermus hydrogenoformans*

Sayaka Inagaki{ddagger}§, Chiaki Masuda¶, Tetsuhiro Akaishi¶, Hiroshi Nakajima¶||, Shiro Yoshioka§, Takehiro Ohta§, Biswajit Pal§, Teizo Kitagawa§, and Shigetoshi Aono§**

From the {ddagger}Department of Structural Molecule Science, The Graduate University for Advanced Studies, 38 Nishigo-naka, Myodaiji, Okazaki 444-8585, the §Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, and the School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan

Received for publication, August 27, 2004 , and in revised form, November 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CooA is a CO-sensing transcriptional activator that contains a b-type heme as the active site for sensing its physiological effector, CO. In this study, the spectroscopic and redox properties of a new CooA homologue from Carboxydothermus hydrogenoformans (Ch-CooA) were studied. Spectroscopic and mutagenesis studies revealed that His-82 and the N-terminal {alpha}-amino group were the axial ligands of the Fe(III) and Fe(II) hemes in Ch-CooA and that the N-terminal {alpha}-amino group was replaced by CO upon CO binding. Two neutral ligands, His-82 and the N-terminal {alpha}-amino group, are coordinated to the Fe(III) heme in Ch-CooA, whereas two negatively charged ligands, a thiolate from Cys-75 and the nitrogen atom of the N-terminal Pro, are the axial ligands of the Fe(III) heme in Rr-CooA. The difference in the coordination structure of the Fe(III) heme resulted in a large positive shift of redox potentials of Ch-CooA compared with Rr-CooA. Comparing the properties of Ch-CooA and Rr-CooA demonstrates that the essential elements for CooA function will be: (i) the heme is six-coordinate in the Fe(III), Fe(II), and Fe(II)–CO forms; (ii) the N-terminal is coordinated to the heme as an axial ligand, and (iii) CO replaces the N-terminal bound to the heme upon CO binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The heme-based sensor proteins play an important role in biological signal transduction systems such as two-component signal transduction, chemotaxis control, production of a second messenger, and the regulation of transcription (1–6). These proteins have a protoheme, which is used to sense effector gas molecules such as oxygen (O2), nitric oxide (NO), or carbon monoxide (CO). An effector gas molecule is bound to the heme in these sensor proteins, which induces a conformational change of these proteins. Their physiological functions are regulated by a conformational change induced by binding of their effector molecules (1–6).

CooA from Rhodospirillum rubrum (Rr-CooA)1 is a transcriptional activator that regulates the expression of coo operons encoding the CO-oxidizing system in response to CO (1–4). Only CO-bound Rr-CooA can recognize and bind to its target DNA sequence and promote the transcription of the coo operons (1–4). The amino acid sequence and the crystal structure of Rr-CooA are homologous to those of CRP, cAMP receptor protein from Escherichia coli, revealing that CooA belongs to the CRP/FNR family of transcriptional regulators (7, 8).

Rr-CooA has a b-type heme that has some unique properties: (i) the N-terminal Pro-2 residue is coordinated to the Fe(III) and Fe(II) hemes via its nitrogen atom (8–10); (ii) redox-controlled ligand exchange takes place between Cys-75 and His-77, that is, Cys-75, one of the axial ligands of the Fe(III) heme, is replaced by His-77 upon reduction of the heme iron, and vice versa (11); and (iii) the Fe(II) heme in Rr-CooA, however, is six-coordinate with no vacant site for an exogenous ligand, CO-bound Rr-CooA is formed easily upon the reaction of Fe(II) Rr-CooA with CO under physiological conditions (9–11). Although these properties have been characterized in detail, it is not clear if they are essential for CooA function.

Recently, some genes have been discovered as possible candidates for CooA homologues by a homology search on DNA databases (12, 13). H. Youn et al. (13) have reported that cooA homologue genes from Carboxydothermus hydrogenoformans 2340, Azotobacter vinelandii, and Desulfovibrio vulgaris express a hemeprotein in E. coli and that the CooA homologue in C. hydrogenoformans (Ch-CooA) is a CO-dependent transcriptional regulator as is Rr-CooA. However, detailed properties of these CooA homologues have not yet been clarified.

C. hydrogenoformans is a CO-utilizing thermophilic anaerobic bacterium that utilizes CO as the sole energy and carbon source (14). Carbon monoxide dehydrogenase, which contains a nickel-iron-sulfur cluster as a prosthetic group, plays a central role for CO metabolism in C. hydrogenoformans, as is the case with R. rubrum (15). Therefore, Ch-CooA, a homologue of Rr-CooA in C. hydrogenoformans, regulates the expression of carbon monoxide dehydrogenase in response to CO (13).

The amino acid sequence of Ch-CooA, which is deduced from the DNA sequence, shows significant identity (31%) and similarity (55%) to that of Rr-CooA (13). Cys and His, which are the fifth ligand of the Fe(III) and Fe(II) hemes (Cys-75 and His-77 in Rr-CooA), are conserved in Ch-CooA at the corresponding positions (Cys-80 and His-82 in Ch-CooA). However, the sixth ligand of the Fe(III) and Fe(II) hemes, Pro-2 in Rr-CooA, is not conserved in Ch-CooA. This suggests that the coordination structure of the heme will be different between Rr-CooA and Ch-CooA. Therefore, elucidating the detailed properties of Ch-CooA and comparing between Ch-CooA and Rr-CooA will provide useful information of what is essential for CooA function among the unique properties of Rr-CooA described above. In this study, we carried out spectroscopic and mutagenesis studies of Ch-CooA and found that Ch-CooA has a different coordination structure of the heme, which results in a different redox property of Ch-CooA compared with Rr-CooA. We propose essential factors for CooA function based on the comparison of Ch-CooA and Rr-CooA.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
C. hydrogenoformans (DMS 6008) was obtained from the German Collection of Microorganisms and Cell Cultures. The sequence data of the ch-cooA gene were obtained from The Institute for Genomic Research website at www.tigr.org. The ch-cooA gene was prepared by PCR with the chromosomal DNA of C. hydrogenoformans and two synthetic deoxyoligonucleotides (5'-AGGAGAGGACTATGGCCACCCAAATGAGATTAACCGAC-3' and 5'-TTACTAAACGCCTGAGGAAAACTC-3') as the template, a sense primer, and an antisense primer, respectively. The PCR product was cloned into a pCRTM2.1 vector by using a TOPO TA Cloning kit (Invitrogen) to obtain pCR-Ch-CooA. For the following manipulation, an EcoRI site (GAATTC, 36–41) in the ch-cooA gene was changed to GAACTC by using a QuikChange site-directed mutagenesis kit (Stratagene) to obtain pCR-Ch-CooA ({Delta}EcoRI). The encoding amino acid is not changed by this mutation. The DNA fragment, containing the ch-cooA gene excised from pCR-Ch-CooA ({Delta}EcoRI) by digestion with EcoRI, was ligated with EcoRI-treated pKK223–3 to construct pKK-Ch-CooA. The DNA sequence was analyzed using a ABI PRISM® 310 Genetic Analyzer (Applied Biosystems).

E. coli JM109 was used as a host for the expression of Ch-CooA. pKK-Ch-CooA/E. coli JM109 was pre-cultured for 5 h at 37 °C in LB medium containing 50 µg/ml ampicillin. The pre-cultured cells (0.4 ml) were inoculated into 400 ml of TB medium containing 50 µg/ml ampicillin in a 2-liter cultivation flask, which was then cultured for 14 h at 37 °C with a rotary shaker. Isopropyl-{beta}-D-thiogalactopyranoside (0.5 mM in final concentration) was then added, and the culture continued for 4 h at 37 °C. After the harvested cells were washed once with 20 mM MES-NaOH buffer (pH 6.0) (buffer A), they were frozen in liquid nitrogen and stored at –80 °C until use.

The frozen cells were suspended in buffer A containing 1 mM of phenylmethylsulfonyl fluoride, which was sonicated on ice. The resulting suspension was centrifuged at 75,600 x g for 30 min at 4 °C. The supernatant was applied to a Q-Sepharose column equilibrated with buffer A. After loading the supernatant, the column was washed with buffer A. The adsorbed proteins were eluted with a linear gradient of NaCl from 0 to 1 M in buffer A. The eluted fractions containing Ch-CooA were combined, and ammonium sulfate fractionation was carried out. The precipitate obtained at 40–65% saturation of ammonium sulfate was collected by centrifugation and dissolved in buffer A. To remove the ammonium sulfate, the resulting solution was applied to a Sephadex G-25 column pre-equilibrated with buffer A containing 0.15 M NaCl. The fractions containing Ch-CooA were combined and then applied to a HiTrap heparin column pre-equilibrated with buffer A. The column was washed with 5 bed-volumes of buffer A, and then adsorbed proteins were eluted with a linear gradient of NaCl from 0 to 1 M in buffer A. The fractions containing Ch-CooA were combined and concentrated by ultrafiltration. The purified protein was stored at 4 °C.

The type of heme in Ch-CooA was determined by the pyridine ferrohemochrome method (16). The heme concentration was determined by means of the molar extinction coefficient for the {alpha} band of pyridine ferrohemochrome derived from a b-type heme (34 mM–1 cm–1). A molecular mass of Ch-CooA in native conditions was estimated by gel filtration with a Sephacryl S-100 column. The buffer used for gel filtration was 20 mM MOPS-NaOH (pH 7.0) containing 0.1 M NaCl.

The N-terminal amino acid sequence was determined using a protein sequencer (ABI Procise 494HT). The electronic absorption spectra were recorded on a Type 8453 UV-visible spectrophotometer (Agilent Technologies) at 20 °C. Ch-CooA was dissolved in 100 mM MES-NaOH buffer (pH 6.0) containing 0.1 M NaCl for the measurement of absorption spectra. Electrochemical redox titrations were carried out as previously reported (9). Resonance Raman spectra were measured as previously reported (17, 18). Briefly, the spectra were obtained with laser excitation at 413.1 nm using a Kr+ laser (Spectra Physics, model 2016) and at 428.7 nm using a diode laser. The scattered light was collected at right angles using a collecting lens and dispersed with a single monochromator (Ritsu Oyokogaku, DG100). A charge-coupled device detector (Astromed) cooled with liquid nitrogen was used for signal detection. Raman shift was calibrated using indene as a frequency standard, providing an accuracy of ±1 cm–1 for intense isolated lines.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of Ch-CooA—Ch-CooA was expressed as a hemeprotein in cytosolic fraction in E. coli as is Rr-CooA. Purified Ch-CooA migrated as a single band of 26.0-kDa molecular mass on a SDS-PAGE gel, which is consistent with the calculated value (25.1 kDa) based on the amino acid sequence of Ch-CooA. Gel filtration column chromatography revealed that the molecular mass of Ch-CooA was about 46.0 kDa (data not shown). These results indicate that Ch-CooA is a homo-dimer of an identical subunit as is Rr-CooA.

The pyridine hemochromogen prepared from Ch-CooA showed the Soret, {alpha}, and {beta} bands at 417.5, 556.0, and 524.0 nm, respectively, indicating that Ch-CooA contains a b-type heme as a prosthetic group. Quantitative analysis of the heme content by pyridine hemochromogen analysis revealed that the Ch-CooA monomer contained one protoheme.

The N-terminal amino acid sequence of Ch-CooA was determined to be ATQMRLTDTN, which is identical to the N-terminal amino acid sequence deduced from the DNA sequence except for the first Met. The N-terminal Met was removed by post-translational modification.

The Electronic Absorption Spectra of Ch-CooA—The electronic absorption spectra of the Fe(III), Fe(II), and Fe(II)–CO forms of Ch-CooA are shown in Fig. 1. Fe(III) Ch-CooA showed the Soret, {alpha}, and {beta} bands at 415, 559, and 529 nm, respectively. Fe(II) Ch-CooA, formed by reducing the Fe(III) form with sodium dithionite, showed the Soret, {alpha}, and {beta} bands at 424, 559, and 529 nm, respectively. Upon the reaction of Fe(II) Ch-CooA with CO, Fe(II)–CO Ch-CooA was formed. Fe(II)–CO Ch-CooA showed the Soret, {alpha}, and {beta} bands at 421, 569, and 538 nm, respectively. These spectra of Ch-CooA are typical of six-coordinate, low spin heme proteins.



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  		FIG. 1.
Electronic absorption spectra of Fe(III) (solid line), Fe(II) (dashed line), and Fe(II)–CO (dashed and dotted line) of Ch-CooA. Ch-CooA was dissolved in 0.1 mM MES-NaOH buffer (pH 6.0) containing 0.1 M NaCl. The molar extinction coefficients of the Soret band are 121, 163, 209 mM–1 cm–1 for Fe(III), Fe(II), and Fe(II)–CO forms Ch-CooA, respectively.

 
The absorption peaks are summarized in Table I compared with those of Rr-CooA. Although the spectra of Fe(II) and Fe(II)–CO Ch-CooA were nearly identical to those of Rr-CooA, the spectrum of Fe(III) Ch-CooA was different from that of Rr-CooA. The Soret peak of Fe(III) Ch-CooA was blue-shifted by 9 nm compared with that of Fe(III) Rr-CooA. Fe(III) Rr-CooA shows clear {delta} and CT bands at 363 and 760 nm (11), respectively, typical of a thiolate-bound hemeprotein (19). In Fe(III) Ch-CooA, however, neither the clear {delta} band nor the CT band due to the Fe(III)-S charge transfer was observed. These results suggest that the coordination structure of the heme iron in Fe(II) and Fe(II)–CO Ch-CooA is similar to that of Rr-CooA, whereas Fe(III) Ch-CooA has a different coordination structure from that of Rr-CooA.


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  		TABLE I
Electronic absorption peaks of Ch-CooA and Rr-CooA

 
The Resonance Raman Spectra of Ch-CooA—The resonance Raman spectra in the high frequency region are shown in Fig. 2, and the frequencies of selected marker bands are summarized in Table II. In this frequency region, {nu}2, {nu}3, and {nu}4 can be used as sensitive markers of the oxidation state ({nu}4) and spin and coordination states ({nu}2 and {nu}3) of the heme iron (20). The {nu}2, {nu}3, and {nu}4 of Fe(III) Ch-CooA were observed at 1581, 1505, and 1371 cm–1, respectively, indicating that Fe(III) Ch-CooA has a six-coordinate, low spin heme. The {nu}2 and {nu}4 frequencies of Fe(III) Ch-CooA were 4 cm–1 lower than those of Fe(III) Rr-CooA. These results of electronic absorption and resonance Raman spectroscopy suggest that the heme environmental structure of Fe(III) Ch-CooA is obviously different from that of Fe(III) Rr-CooA.



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  		FIG. 2.
High frequency resonance Raman spectra of Fe(III) (a), Fe(II) (b), and Fe(II)–CO Ch-CooA (c).

 


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  		TABLE II
Raman frequencies of WT and C80A Ch-CooA compared with those of Rr-CooA

 
The {nu}2, {nu}3, and {nu}4 bands of Fe(II) Ch-CooA were observed at 1585, 1492, and 1361 cm–1, respectively. Fe(II)–CO Ch-CooA showed the {nu}2, {nu}3, and {nu}4 bands at 1581, 1497, and 1371 cm–1, respectively. These frequencies of {nu}2, {nu}3, and {nu}4 bands for Fe(II) and Fe(II)–CO Ch-CooA were also typical of six-coordinate, low spin state hemeproteins. The resonance Raman spectra are consistent with the conclusions drawn from electronic absorption spectroscopy.

Fig. 3 shows the resonance Raman spectra in the Fe–CO stretching ({nu}Fe–CO) and the C=O stretching ({nu}C=O) frequency regions of 12CO (Fig. 3a) and 13CO-labeled CO (Fig. 3b) adducts of Fe(II) Ch-CooA. A line at 483 cm–1 (12CO) was shifted to 480 cm–1 upon the 13CO substitution. The isotope shift of 3 cm–1 is consistent with the assignment of the band at 483 cm–1 to the Fe–CO-stretching mode. The other CO isotope-sensitive band appeared at 1977 cm–1 for Fe(II)-12CO Ch-CooA and at 1934 cm–1 for Fe(II)-13CO Ch-CooA. The isotope difference spectra are delineated in Fig. 3c. Based on the frequency shift, the 1977 cm–1 band is assigned to the C=O stretching mode ({nu}C=O).



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  		FIG. 3.
Resonance Raman spectra of Fe(II)–CO Ch-CooA. 12CO-bound Ch-CooA (a), 13CO-bound Ch-CooA (b), and the difference spectrum (a–b) (c).

 
It is well known that {nu}Fe–CO and {nu}C=O display an inverse linear correlation (21–23), and this relationship is a useful tool for analyzing the heme ligand. The {nu}Fe–CO versus {nu}C=O correlation depends on the nature of the proximal ligand, i.e. the CO-bound hemes with an imidazole/histidine as a trans ligand exhibit a correlation line different from those with a thiolate ligand. The {nu}Fe–CO and {nu}C=O frequencies of Fe(II)–CO Ch-CooA are plotted in Fig. 4 together with those of His-coordinated hemeproteins. The data point for Ch-CooA fell on the imidazole/histidine correlation line. This indicates that the axial heme ligand trans to the bound CO is a histidine residue in Ch-CooA. The {nu}Fe–CO and {nu}C-O frequencies suggest that the environment around the oxygen atom of the heme-bound CO is less hydrophobic than that of Rr-CooA.



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  		FIG. 4.
The correlation between {nu}(Fe–CO) and {nu}(C-O). The circle and squares represent the data of Ch-CooA and the variety of heme proteins with a proximal histidine, respectively. Labels: 1, sGC-1 (Ref. 24); 2, EcDOS PAS (25); 3, Rr-CooA (17); 4, HemAT-Bs (26); 5, RmFixLT (27); 6, HO-1 (28); 7, RmFixLH (27); and 8, SwMb (29), respectively.

 
Properties of C80A and H82A Mutants—The axial ligands of the heme in Fe(III), Fe(II), and Fe(II)–CO Rr-CooA are Cys-75 and Pro-2, His-77and Pro-2, and His-77 and CO, respectively. The Cys and His are conserved in Ch-CooA at positions 80 and 82 corresponding to the positions of Cys-75 and His-77, respectively, in Rr-CooA. To elucidate if Cys-80 and His-82 are axial ligands of the heme in Ch-CooA, C80A and H82A mutants were prepared.

H82A Ch-CooA, in which His-82 is replaced by Ala, was expressed as an apo-form that did not contain a heme, suggesting that His-82 is an axial ligand of the heme in Ch-CooA. On the other hand, C80A Ch-CooA, in which Cys-80 is replaced by Ala, was expressed as a holo-form containing a heme. The electronic absorption and resonance Raman spectra of C80A Ch-CooA were identical to those of wild-type (WT) Ch-CooA, as shown in Fig. 5 and Table II, respectively. In the resonance Raman spectra of Fe(III), Fe(II), and Fe(II)–CO forms of C80A Ch-CooA, the {nu}2, {nu}3, and {nu}4 bands appeared at the same frequencies as in the case of WT Ch-CooA. These results indicate that the Cys-80 mutation did not change the coordination structure of the heme in Ch-CooA, indicating that Cys-80 is not coordinated to the heme iron in WT Ch-CooA.



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  		FIG. 5.
Electronic absorption spectra of Fe(III) (solid line), Fe(II) (dashed line), and Fe(II)–CO (dashed and dotted line) of Ch-CooA C80A. Ch-CooA C80A was dissolved in 0.1 mM MES-NaOH buffer (pH 6.0) containing 0.1 M NaCl. The molar extinction coefficients of the Soret band are 110, 148, 190 mM–1cm–1 for Fe(III), Fe(II), and Fe(II)–CO forms C80A Ch-CooA, respectively.

 
Redox Titrations of Ch-CooA—Electrochemical redox titration revealed that the redox property of Ch-CooA was completely different from that of Rr-CooA. Fig. 6 shows the Nernst plots of the electrochemical redox titrations of Ch-CooA. The apparent oxidation and reduction midpoint potentials of Ch-CooA were determined to be +230 and +150 mV (versus NHE), respectively, which were positively shifted by ~500 mV compared with those of Rr-CooA.



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  		FIG. 6.
Electrochemical redox titrations of Ch-CooA. The squares and circles represent the data points in reductive and oxidative titrations, respectively. The solid lines are theoretical Nernst curves for one-electron oxidation and reduction with a midpoint potential of +230 and +150 mV, respectively.

 
Although the redox potentials were completely different between Ch-CooA and Rr-CooA, similar hysteresis was observed in the redox titrations of Ch-CooA as observed in the case of Rr-CooA. The apparent oxidation potential was shifted toward the positive side by +80 mV compared with the apparent reduction potential in the case of Ch-CooA. A similar positive shift of the oxidation potential is observed in Rr-CooA, where the apparent oxidation and reduction potentials were –260 and –320 mV (versus NHE), respectively (9).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have characterized Ch-CooA, a CooA homologue from C. hydrogenoformans, by spectroscopic and mutagenesis studies. Ch-CooA contains a protoheme as a prosthetic group, as does Rr-CooA, and the heme in Ch-CooA is in a six-coordinate, low spin state in the Fe(III), Fe(II), and Fe(II)–CO forms. Although these properties are the same as those of Rr-CooA, the electronic absorption spectrum of the Fe(III) form and the redox potential of Ch-CooA were different from those of Rr-CooA. These differences are caused by a different coordination structure of the heme in Ch-CooA from that of Rr-CooA (8–10).

The fifth ligand of the Fe(III) heme in Rr-CooA is Cys-75 (8–10). Cys is conserved in Ch-CooA at position 80 that corresponds to the position of Cys-75 in Rr-CooA, and is predicted to be a fifth ligand of Fe(III) Ch-CooA. However, electronic absorption and resonance Raman studies of wild-type and C80A Ch-CooA reveal that Cys-80 is not coordinated to the Fe(III) heme in Ch-CooA. Instead, the mutation on His-82 in Ch-CooA results in the formation of an apo-form of Ch-CooA. The position of His-82 corresponds to that of His-77 in Rr-CooA, which is the axial ligand of the Fe(II) and Fe(II)–CO forms. Resonance Raman spectroscopy reveals that histidine is the proximal ligand of CO-bound Ch-CooA. These results indicate that His-82 is the fifth ligand of Fe(III), Fe(II), and Fe(II)–CO hemes in Ch-CooA.

We examined the sixth ligand of Ch-CooA by comparing 1H NMR of Ch-CooA with that of Rr-CooA. Ch-CooA showed similar 1H NMR signals to Fe(II) Rr-CooA {Delta}N5 mutant, in which the first four amino acid residues were deleted from the N terminus in the upfield region (data not shown). In the case of Rr-CooA {Delta}N5, the N-terminal {alpha}-amino group is thought to be the sixth ligand of the Fe(III) and Fe(II) hemes (10). These results suggest that the N-terminal {alpha}-amino group will be the sixth ligand of the heme in Ch-CooA.

Based on the results discussed above, we propose here a coordination structure of the heme in Ch-CooA, as shown in Fig. 7. The coordination structure of the heme is not fully conserved between Ch-CooA and Rr-CooA. His-82 is the fifth ligand of the Fe(III) heme in Ch-CooA, whereas Cys-75 as the fifth ligand is coordinated to the Fe(III) heme in Rr-CooA. The sixth ligand of the Fe(III) and Fe(II) hemes in Rr-CooA and Ch-CooA is the nitrogen atom of the N-terminal Pro and the N-terminal {alpha}-amino group, respectively (Fig. 7). Although the N-terminal amino acid residue is not conserved between Rr-CooA and Ch-CooA, it is conserved between them: the nitrogen atom of the main chain at the N terminus is coordinated to the Fe(III) and Fe(II) hemes. The coordination of the N terminus is therefore essential for CooA function. In the case of Rr-CooA, the replacement of Pro-2 by CO is a trigger for Rr-CooA to gain specific DNA-binding activity via conformational change. Rr-CooA mutants with a deletion of several amino acid residues from the N terminus retain the coordination of the N terminus to the heme and CO-dependent activity (9). These results indicate that the release of the N terminus from the heme upon CO binding is a trigger of the activation of CooA by CO.



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  		FIG. 7.
The proposed coordination structure of the heme in Ch-CooA (a) and Rr-CooA (b).

 
Ch-CooA does not show redox-controlled ligand exchange as does Rr-CooA between Cys-75 and His-77, because the coordination of His-82 is retained among the Fe(III), Fe(II), and Fe(II)–CO forms. The physiological significance of this difference between Ch-CooA and Rr-CooA is discussed below.

Ch-CooA shows elevated reduction and oxidation midpoint potentials of +150 and +230 mV (versus NHE), respectively, in contrast with those of Rr-CooA (oxidation potential, –260 mV (versus NHE); reduction potential, –320 mV (versus NHE) (9)). Although Rr-CooA shows very low oxidation and reduction potentials, the oxidation and reduction potentials of Ch-CooA were higher by ~500 mV than those of Rr-CooA. The difference of the redox potential between Ch-CooA and Rr-CooA can be explained by the different coordination structure of the Fe(III) heme. In the case of Rr-CooA, both of the axial ligands of Fe(III) heme are negatively charged, i.e. Cys-75 and Pro-2 are deprotonated to coordinate to the heme (9–11). These negative charges stabilize the Fe(III) form to decrease the redox potential by an electron-push effect caused by the electron-donating capability of axial ligands. In contrast, the axial ligands of Fe(III) Ch-CooA are His-82 and the N-terminal {alpha}-amino group, both of which are electrically neutral. A neutral histidine and {alpha}-amino group have lower electron-donating capability than deprotonated cysteine and proline. Therefore, Ch-CooA shows more positive reduction and oxidation potentials than those of Rr-CooA.

The large difference of the redox potential between Ch-CooA and Rr-CooA is related to the difference in phenotype of their original host strains (C. hydrogenoformans and R. rubrum). R. rubrum can grow aerobically and anaerobically. CooA activates the expression of coo operons only in the presence of CO under anaerobic conditions to gain the energy for growth on CO (1–4). In the presence of O2, the CO metabolism pathway is shut off and the oxygen respiratory chain starts to work. Rr-CooA is deactivated under aerobic conditions to stop the expression of coo operons. Therefore, the redox potential of Rr-CooA is thought to be very low so that it can detect oxygen in the cell via autoxidation of the heme. Redox-controlled ligand exchange between Cys-75 and His-77 in Rr-CooA is responsible for tuning the redox potential of the heme suitable for oxygen detection. On the other hand, because C. hydrogenoformans is a strict anaerobic bacterium (14), Ch-CooA is not required to detect the presence of oxygen in the cell and should sense only CO under anaerobic conditions. Therefore, the redox potential of Ch-CooA is +150 and +230 mV, and Ch-CooA is maintained to take the Fe(II) form ready to bind CO in the cell.

In summary, we have characterized a CooA homologue from C. hydrogenoformans, Ch-CooA. The comparison of Ch-CooA and Rr-CooA properties elucidates what is essential for sensing CO by CooA protein. The essential factors for CooA function are (i) the heme is six-coordinate in the Fe(III), Fe(II), and Fe(II)–CO forms; (ii) the N terminus is coordinated to the heme as an axial ligand; and (iii) CO replaces the N terminus bound to the heme upon CO binding. Redox potentials of Ch-CooA are higher by ~500 mV than those of Rr-CooA, whose difference is caused by the different axial ligands of the heme. The difference of redox potentials is concerned with a different phenotype of R. rubrum and C. hydrogenoformans, which are the original host strains of Rr-CooA and Ch-CooA, respectively.


   FOOTNOTES
 
* This work was supported by a Grant-in-aid for Scientific Research of Priority Areas on Metal Sensors (12147203) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan and by a Grant-in-Aid for Scientific Research B (16370065) from the Japan Society for the 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

|| Present address: Dept. of Chemistry, Nagoya University, Furo, Chikusa, Nagoya 454-8602, Japan. Back

** To whom correspondence should be addressed. Tel.: 81-564-59-5575; Fax: 81-564-59-5576; E-mail: aono{at}ims.ac.jp.

1 The abbreviations used are: Rr-CooA, CooA from R. rubrum; Ch-CooA, CooA from C. hydrogenoformans; CRP, cAMP receptor protein; FNR, regulator of fumarate and nitrate reduction; WT, wild type; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; NHE, normal hydrogen electrode. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Y. Yamamoto (Tsukuba University) for the measurement of NMR spectra of Ch-CooA. Preliminary sequence data was obtained from The Institute for Genomic Research website at www.tigr.org. The sequencing of C. hydrogenoformans was accomplished with support from the United States Department of Energy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aono, S. (2003) Acc. Chem. Res. 36, 825–831[CrossRef][Medline] [Order article via Infotrieve]
  2. Aono, S., and Nakajima, H. (2000) Prog. React. Kinet. Mech. 25, 65–107
  3. Aono, S., and Nakajima, H. (1999) Coord. Chem. Rev. 190–192, 267–282[CrossRef]
  4. Roberts, G. P., Thorsteinsson, M. V., Kerby, R. L., Lanzilotta, W. N., and Poulos, T. L. (2001) Prog. Nucleic Acids Res. Mol. Biol. 67, 35–63[Medline] [Order article via Infotrieve]
  5. Rodgers, K. R. (1999) Curr. Opin. Chem. Biol. 3, 158–167[CrossRef][Medline] [Order article via Infotrieve]
  6. Chan, M. K. (2001) Curr. Opin. Chem. Biol. 5, 216–222[CrossRef][Medline] [Order article via Infotrieve]
  7. Shelver, D., Kerby, R. L., He, Y., and Roberts, G. P. (1995) J. Bacteriol. 177, 2157–2163[Abstract/Free Full Text]
  8. Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876–880[CrossRef][Medline] [Order article via Infotrieve]
  9. Nakajima, H., Honma, Y., Tawara, T., Kato, T., Park, S.-Y., Miyatake, H., Shiro, Y., and Aono, S. (2001) J. Biol. Chem. 276, 7055–7061[Abstract/Free Full Text]
  10. Yamamoto, K., Ishikawa, H., Takahashi, S., Ishimori, K., Morishima, I., Nakajima, H., and Aono, S. (2001) J. Biol. Chem. 276, 11473–11476[Abstract/Free Full Text]
  11. Aono, S., Ohkubo, K., Matsuo, T., and Nakajima, H. (1998) J. Biol. Chem. 273, 25757–25764[Abstract/Free Full Text]
  12. Voordouw, G. (2002) J. Bacteriol. 184, 5903–5911[Abstract/Free Full Text]
  13. Youn, H., Kerby, R. L., Conrad, M., and Roberts, G. P. (2004) J. Bacteriol. 186, 1320–1329[Abstract/Free Full Text]
  14. Svetlichny, V. A., Sokolova, T. G., Gerhardt, M., Ringpfeil, N. A., Kostrikina, N. A., and Navarzin, G. A. (1991) System. Appl. Microbiol. 14, 254–260
  15. Dobbek, H., Svetlitchnyi, V., Germer, L., Huber, R., and Meyer, O. (2001) Science 293, 1281–1285[Abstract/Free Full Text]
  16. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370–2378[Free Full Text]
  17. Uchida, T., Ishikawa, H., Takahashi, S., Ishimori, K., Morishima, I., Okubo, K., Nakajima, H., and Aono, S. (1998) J. Biol. Chem. 273, 19988–19992[Abstract/Free Full Text]
  18. Uchida, T., Ishikawa, H., Ishimori, K., Morishima, I., Nakajima, H., Aono, S., Mizutani, Y., and Kitagawa, T. (2000) Biochemistry 39, 12747–12752[CrossRef][Medline] [Order article via Infotrieve]
  19. Dawson, J. H., and Sono, M. (1987) Chem. Rev. 87, 1255–1276[CrossRef]
  20. Spiro, T. G., and Li, X.-Y. (1988) in Biological Applications of Raman Spectroscopy (Spiro, T. G., ed) Vol. III, pp. 1–37, John Wiley & Sons, New York
  21. Hu, S., Smith, K. M., Spiro, T. G. (1996) J. Am. Chem. Soc. 118, 12638–12646[CrossRef]
  22. Yu, N.-T., and Kerr, E. A. (1988) in Biological Applications of Raman Spectroscopy (Spiro, T. G., ed) Vol. III, pp. 39–95, John Wiley & Sons, New York
  23. Oldfield, E., Guo, K., Augspurger, J. D., and Dykstra, C. E. (1991) J. Am. Chem. Soc. 113, 7537–7541[CrossRef]
  24. Pal, B., Li, Z., Ohta, T., Takenaka, S., Tsuyama, S., and Kitagawa, T. (2004) J. Inorg. Biochem. 98, 824–832[CrossRef][Medline] [Order article via Infotrieve]
  25. Sato, A., Sasakura, Y., Sugiyama, S., Sagami, I., Shimizu, T., Mizutani, Y., and Kitagawa, T. (2002) J. Biol. Chem. 277, 32650–32658[Abstract/Free Full Text]
  26. Aono, S., Kato, T., Matsuki, M., Nakajima, H., Ohta, T., Uchida, T., and Kitagawa, T., (2002) J. Biol. Chem. 277, 13528–13538[Abstract/Free Full Text]
  27. Miyatake, H., Mukai, M., Adachi, S., Nakamura, H., Tamura, K., Iizuka, T., Shiro, Y., Strange, R. W., and Hasnain, S. S. (1999) J. Biol. Chem. 274, 23176–23184[Abstract/Free Full Text]
  28. Takahashi, S., Wang, J., Rousseau, D. L., Ishikawa, K., Yoshida, T., Takeuchi, N., and Ikeda-Saito, M. (1994) Biochemistry 33, 5531–5538[CrossRef][Medline] [Order article via Infotrieve]
  29. Ramsden, J., and Spiro, T. G. (1989) Biochemistry 28, 3125–3128[CrossRef][Medline] [Order article via Infotrieve]



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