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Originally published In Press as doi:10.1074/jbc.M003972200 on November 28, 2000

J. Biol. Chem., Vol. 276, Issue 10, 7055-7061, March 9, 2001
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Redox Properties and Coordination Structure of the Heme in the CO-sensing Transcriptional Activator CooA*

Hiroshi Nakajima, Yumiko Honma, Toshifumi Tawara, Toshiyuki Kato, Sam-Yong ParkDagger , Hideyuki MiyatakeDagger , Yoshitsugu ShiroDagger , and Shigetoshi Aono§

From the School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa 923-1292 and Dagger  RIKEN Harima Institute/Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan

Received for publication, May 10, 2000, and in revised form, November 15, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys75 and His77 are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro2 is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (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). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys75 and His77 is thought to cause the difference between the reduction and oxidation midpoint potentials.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Hemeproteins are the most popular metalloproteins exhibiting a wide variety of functions such as oxygen storage/transport, electron transfer, and redox reactions of various substrates (1, 2). In addition to these traditional heme proteins, a new class of heme proteins termed the heme-based sensor proteins have been reported recently in which the heme acts as a sensor of an effector molecule (3). There are five such proteins known, soluble guanylate cyclase (sGC)1 (4-13), FixL (14-23), DOS (24), HemAT (25), and CooA (26-37). These proteins contain a heme that acts as a sensing site of NO (sGC), O2 (FixL, DOS, and HemAT), and CO (CooA).

CooA from Rhodospirillum rubrum is a heme-based CO-sensing transcriptional activator. Although the function of the heme in CooA is as a sensor of a gaseous effector molecule, similar to other heme-based sensor proteins, the coordination structure of the heme in CooA is quite different from that in FixL and sGC. In the case of FixL and sGC, the heme is five-coordinate with a histidine as a proximal ligand in the resting state and binds the effector, O2 or NO, at the distal side trans to the proximal histidine (4-23). On the other hand, the heme in CooA is six-coordinate in the ferric, ferrous, and CO-bound forms (26, 28, 29, 34-37). Therefore, CO must replace one of the axial ligands in the ferrous heme when the heme in CooA binds its effector, CO (28, 29, 34-36). The exchange of the axial ligand upon CO binding is functionally relevant for CooA, as described below.

CO is a physiological effector regulating the activity of CooA; i.e. only the CO-bound form of CooA can be bound to the target DNA and is active as a transcriptional activator (27, 29, 31-35). Replacement of one of the axial ligands of the ferrous heme with CO triggers the activation of CooA (28, 29, 34-36). The release of the axial ligand from the heme upon binding CO causes conformational changes around the heme and subsequently in the whole molecule (28, 29, 35). These conformational changes induced by the interchange of the axial ligand are the principal part of the activation of CooA by CO.

Because the heme in CooA plays a central role in sensing CO and in regulating the transcriptional activator activity of CooA, elucidation of the coordination structure of the heme in CooA is required to understand the mechanisms of CO sensing and of the activation of CooA by CO. Mutagenesis studies, EPR, resonance Raman, and uv/vis spectroscopies have revealed that the heme is in the six-coordinate and low-spin state in ferric, ferrous, and CO-bound CooA, and that Cys75 and His77 are the fifth ligand of the ferric and ferrous hemes in CooA, respectively (28, 29, 35-38). These results suggest the possibility that exchange of the axial ligand takes place between Cys75 and His77 during the change in the oxidation state of the heme in CooA (29, 35, 36).

Recently, Lanzilotta et al. (39) have reported the crystal structure of ferrous CooA, which shows that the N-terminal Pro2 and His77 are the axial ligands of the ferrous heme in CooA and that the Pro2 of one subunit provides one ligand to the heme of the other subunit in the CooA homodimer (39). Although the crystal structure of ferrous CooA has been reported, the sixth ligand of the ferric heme in CooA remains to be elucidated. The proximal ligand of CO-bound CooA also remains controversial. We have proposed that His77 is the proximal ligand of the CO-bound heme in CooA (28, 29), whereas Vogel et al. (34) and Dhawan et al. (38) have pointed out the possibility that CO displaces His77 upon CO binding to the ferrous heme in CooA.

We report herein the coordination structure of the heme in CooA revealed by alanine-scanning mutagenesis and EXAFS analyses. In this study, we also examined the redox properties of the heme in CooA by means of electrochemical redox titrations.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Site-directed mutagenesis was carried out by using the Quick-change site-directed mutagenesis kit (Stratagene). The expression and purification of the recombinant CooA were carried out as reported previously (26, 29). A Sephacryl S-100 (Amersham Pharmacia Biotech) gel filtration column equilibrated with 50 mM Tris-HCl buffer, pH 8.0 containing 100 mM NaCl was used for the final step of the purification. The purified CooA was concentrated to about 30 µM and about 2 mM by ultrafiltration with a YM-10 membrane (Amicon, Inc.) for electrochemical redox titration and EXAFS measurements, respectively.

The gene encoding CooADelta N5 was synthesized by polymerase chain reaction using pKK3CO5 (26) as a template and the following primers: primer 1, 5'-AGGAGACTCGTATGAACATCGCCAATGTCCTGTTG-3' and primer 2, 5'-TCATTAATCGTCGTCGTCGTCGCGGTC-3'.

The PCR product containing the cooADelta N5 gene was cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen) to construct pCR-CODelta N5. An expression vector of CooADelta N5, pKK-CODelta N5, was constructed by inserting the EcoRI fragment containing the cooADelta N5 gene, which was cut from pCR-CODelta N5, into the EcoRI site of pKK223-3 (Amersham Pharmacia Biotech). The activity of CooADelta N5 was measured as reported previously (27, 29).

The electrochemical experiments were made using an electrochemical cell equipped with a quartz optical cell (40 (height) × 10 (width) × 1 mm (thickness)). The optical path length of the optical cell was 1 mm. A working electrode of gold mesh (40 × 9 × 0.7 mm) was immersed in the optical cell. A platinum wire and Ag2+/AgCl (3 M KCl) electrodes (RE-1B, BSA) were used as auxiliary and reference electrodes, respectively. The potential was controlled by a potentiostat (HA-301, Hokuto Denko Co.). The electronic absorption spectra were measured on a U-3300 Hitachi uv/vis spectrophotometer.

For electrochemical redox titrations, the following redox mediator dyes (2 µM each) were added to the sample solution (the values of E1/2 (versus NHE) of the mediator dyes are shown in parentheses): phenazine methosulfate (+80 mV, Ref. 40), gallocyanine (+20 mV, Ref. 40), indigo trisulfonate (-80 mV, Ref. 40), 2-hydroxy-1, 4-naphthoquinone (-120 mV, Ref. 41), anthraquinone 2-sulfonate (-230 mV, Ref. 40), benzyl viologen (-350 mV, Ref. 40), methyl viologen (-440 mV, Ref. 42), and N, N'-dimethyl-2,2'-bipyridinium hexafluorophosphate (-720 mV, Ref. 42). CooA solution containing the mediator dyes was repeatedly degassed and flushed with argon prior to the measurement and then ~2.5 ml of the sample solution was transferred into the electrochemical cell, which was sealed by a rubber septum, by using a gas-tight syringe under argon atmosphere. The electrochemical cell was kept in a thermoelectric cell holder of a U-3300 Hitachi uv/vis spectrophotometer at 15 °C during the electrochemical titration. The redox reaction of CooA was followed by recording the absorbance change in the regions of the Soret and Q bands.

EXAFS spectra at the iron K-edge were measured at liquid nitrogen temperature using monochromatized synchrotron radiation at the BL 12C of the Photon Factory in the National Laboratory for High Energy Physics (Tsukuba, Japan). The EXAFS spectra were measured as fluorescence excitation spectra using a bent cylinder type focusing mirror and a silicon (111) double-crystal monochromator. The sample was placed at an angle of 45 degrees against the incident x-ray beam, and the fluorescent x-ray intensity perpendicular to the beam was measured using a solid state 19-element detector (Canberra Industries, Inc.) The analysis of the EXAFS data was performed using the program XFIT (43) as previously reported (44-46). The k-windows used for the EXAFS analyses are shown in figures. The multiple scattering from the outershell atoms of the porphyrin ring and axial ligand molecules were taken into account. In constrained refinement, the number of parameters was reduced by treating a set of scattering atoms as a unit (47).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Mutagenesis of CooA

Alanine-scanning Mutagenesis-- Mutagenesis and spectroscopic studies have revealed that the heme in CooA is six-coordinate in the ferric, ferrous, and CO-bound forms (28-30, 34-36). To date, the sixth ligand of the ferric and ferrous hemes in CooA is not known, whereas Cys75 and His77 are assigned to be the fifth ligand of the ferric and ferrous hemes, respectively (29, 35). We have reported that His, Met, Cys, and Lys are not candidates for the axial ligands of the heme in CooA, except for Cys75 and His77 (29). To identify the unknown ligand of the heme in CooA, we carried out alanine-scanning mutagenesis on all Arg, Asp, Glu, Asn, Gln, Ser, Thr, and Tyr residues in the heme-binding domain of CooA in the present work. These side chains could potentially coordinate to the heme iron, located in the N-terminal region from Met1 to Met131 (27). The mutant CooA proteins constructed in this work are summarized in Table I. The individual mutant proteins were partially purified by means of a Q-Sepharose column and their uv/vis spectra were measured in the ferric, ferrous, and CO-bound forms. All the mutants thus prepared exhibited almost the same electronic absorption spectra as did wild-type CooA (data not shown), indicating that no candidate for the sixth ligand of the heme in CooA was found by alanine-scanning mutagenesis.2 These results suggest that side chains of any amino acid residues, except for Cys75 and His77, do not coordinate to the heme iron as an axial ligand in CooA.


                              
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Table I
Mutant CooA proteins constructed in this work
The numbers in the Table stand for the positions of the residues at which the mutation was introduced. The individual residues were replaced by Ala in these mutants except for Tyr. For the Y55F and Y67F mutants, Tyr was replaced by Phe.

Truncated Mutant Lacking Four Residues in the Amino-terminal of CooA-- To elucidate whether or not the amino group of the amino-terminal residue is coordinated to the heme in CooA, a truncated mutant (CooADelta N5), in which four residues were deleted from the amino-terminal of CooA, was constructed and its electronic absorption spectra and transcriptional activator activity were measured. The amino-terminal amino acid sequences of wild-type and CooADelta N5, which were deduced from the DNA sequences, are 1MPPRFNIANV and 1MNIANV, respectively. The four underlined residues in this sequence were deleted in CooADelta N5. CooADelta N5 showed the Soret peak at 422, 424, and 422 nm in the ferric, ferrous, and CO-bound forms, respectively, as shown in Fig. 1. The spectral features of CooADelta N5 were almost the same as the corresponding ones in the wild-type though the ferric CooADelta N5 showed a slightly blue-shifted and broad Soret peak with a shoulder at 399 nm compared with wild-type. The shoulder at 399 nm and a CT-band at 638 nm in the spectrum of ferric CooADelta N5 suggest that a high-spin form of the ferric heme exists to some extent in ferric CooADelta N5.



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  		Fig. 1.   Electronic absorption spectra of CooADelta N5. CooADelta N5 was dissolved in 50 mM Tris-HCl buffer, pH 8.0.

The transcriptional activator activity of CooADelta N5 was 13.2 and 0.29 units/mg of protein in the presence or absence of CO, respectively. These values were comparable with those for wild-type CooA; the activity of wild-type CooA is 15.7 and 0.23 units/mg of protein in the presence or absence of CO, respectively (27, 29). CooADelta N5 was also a CO-dependent transcriptional activator as is wild-type CooA. The deletion of four amino acid residues from the amino-terminal of CooA did not perturb the electronic absorption spectra and CO-dependent transcriptional activator activity of CooADelta N5. A similar result has been reported recently for the different N-terminal truncated mutants of CooA (48).

The above results suggested that the first five residues from the amino terminus are not responsible for the coordination of the heme in CooA. However, this conclusion is now known to be incorrect. During the course of the reviewing process of this paper, Lanzilotta et al. (39) have reported the crystal structure of the reduced form of CooA in which His77 and the nitrogen atom from Pro2 are the axial ligands of the heme (39). Though Pro2 is deleted in CooADelta N5, CooADelta N5 showed almost the same uv/vis spectra and activity as those of wild-type CooA. These results indicate that some compensation for the mutation takes place to maintain CooADelta N5 in order to have a six-coordinate heme and to be active as the CO-dependent transcriptional activator. However, it is not known at present what is coordinated to the heme in place of Pro2 in the case of CooADelta N5.

EXAFS Analyses of CooA

To analyze the coordination atoms of the heme iron more directly, we measured the iron K-edge EXAFS of CooA in the ferric, ferrous, and CO-bound states. The k3-weighted EXAFS raw data and the corresponding Fourier transforms are shown in Figs. 2 and 3, respectively. The following models for the axial ligands of the heme were used to fit the experimental data: i.e. Cys and Pro, His and Pro, and His and CO were assumed to be the axial ligands of the ferric, ferrous, and CO-bound hemes in CooA, respectively. The atomic coordinates of ferrous CooA (39) were used as a starting model for refinement of the fitting in the case of ferric and ferrous CooA. The structural parameters obtained by the fitting are shown in Table II.



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  		Fig. 2.   Iron K-edge k3-weighted EXAFS curves of the ferric (A), ferrous (B), and CO-bound CooA (C). Experimental data are shown by solid lines and the fitting results by broken lines. The windows used in Fourier filter are also shown in the figures.



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  		Fig. 3.   Fourier transformed EXAFS data of the ferric (A), ferrous (B), and CO-bound CooA (C). Experimental data are shown by solid lines and the fitting results by broken lines. The windows used in Fourier filter are also shown in the figures.


                              
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Table II
Final refined parameters obtained from restrained refinement of EXAFS
The abbreviations used are: Fe-Npyr, bond distance between iron and porphyrin pyrrol nitrogen atom; Fe-SCys, bond distance between iron and Cys sulfur atom; Fe-NHis, bond distance between iron and His imidazole nitrogen atom; Fe-NPro, bond distance between iron and Pro nitrogen atom; Fe-CCO, bond distance between iron and CO carbon atom. Deby-Waller factors of the ligand atom coordinating iron are shown in this table, where Npyr, SCys, NHis, NPro, and CCO stand for porphyrin pyrrol nitrogen atom, Cys sulfur atom, His nitrogen atom, Pro nitrogen atom, and CO carbon atom, respectively. The definitions of S<UP><SUB>0</SUB><SUP>2</SUP></UP> and Ni/p are the same as those described in Refs. 42, 64, and 65.

The model of the coordination structure of the heme in CooA elucidated by EXAFS analyses is shown in Fig. 4. Cys75 and the nitrogen atom from Pro2 are thought to be coordinated to the ferric heme in CooA. The distances of the Fe-SCys and Fe-NPro bonds in the ferric heme were estimated to be 2.25 and 2.19 Å, respectively. In the ferrous heme, His77 and the nitrogen atom from Pro2 are thought to be the axial ligands. The distances of the Fe-NHis and Fe-NPro bonds in the ferrous heme were estimated to be 2.02 and 2.16 Å, respectively. Good fitting results were obtained for CO-bound CooA when His77 was thought to be retained to be the proximal ligand of the CO-bound heme, as shown in Figs. 2 and 3. The distances of the Fe-NHis and Fe-CCO bonds in the CO-bound CooA were estimated to be 1.98 and 1.79 Å, respectively. Details of the coordination structure of CooA in each state are discussed in the following sections.



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  		Fig. 4.   Coordination structure of the heme in CooA elucidated by EXAFS analyses.

Coordination Structure of Ferrous CooA-- Lanzilotta et al. (39) have reported the crystal structure of ferrous CooA, which reveals that His77 and Pro2 are the axial ligands of the ferrous heme in CooA (39). As shown in Figs. 2B and 3B, the EXAFS curves can be fitted by using the atomic coordinates of ferrous CooA as a starting model for refinement of the fitting. Compared with the crystallographic result, the EXAFS analysis in this study reveals a slightly short Fe-NHis and a slightly long Fe-NPro bond distances by 0.17 and 0.04 Å, respectively. Although the bond distance of Fe-NPro is slightly longer than that of Fe-NHis, a similar bond distance for Fe-NHis and Fe-NPro does not show that one bond is more labile to be replaced with CO than the other (39). However, EXAFS and time-resolved resonance Raman spectroscopies reveal that His77 remains coordinated to the heme when CO reacts with ferrous CooA, as described below.

Coordination Structure of the Ferrous CO Complex of CooA-- EXAFS analysis of the CO-bound CooA indicated that His77 would be the proximal ligand of the CO-bound CooA, which is consistent with our model reported previously (28, 29), i.e. the mixed coordination of CO and the His77 imidazole, as shown in Fig. 4.

Picosecond time-resolved resonance Raman spectroscopy reveals that a new intense line because of nu (Fe-His) at 211 cm-1 is observed immediately after photolysis of CO-bound CooA (49). The transient 211 cm-1 band is completely absent in the case of H77Y CooA in which His77 is replaced by Tyr (49). These results are consistent with the notion that His77 is the proximal ligand of the CO-bound CooA.

Resonance Raman spectroscopy has revealed the absence of any significant interactions between the bound CO and the distal heme pocket (28). These results indicate that the sixth ligand (Pro2, Ref. 39) replaced by CO moves far away beyond the coordination sphere of the heme.

CO reacts with the six-coordinate ferrous heme under physiological conditions to form CO-bound CooA, by which CooA is activated as the transcriptional activator. In other words, CO replaces one of the axial ligands of the ferrous heme to induce a conformational change required for the activation of CooA (29). The replacement of the axial ligand is functionally relevant for the activation of CooA by CO. It seems reasonable for us to anticipate that a large conformational change, including a concomitant movement of the main chain, is induced by the replacement of Pro2 with CO. Replacement of Pro2 will cause rotation of the C-helices about the dimer interface, as proposed by Lanzilotta et al. (39).

Coordination Structure of Ferric CooA-- The mutagenesis analyses suggested that side chains of any amino acid residues, except for Cys75 and His77, do not coordinate to the heme iron as an axial ligand in CooA. It has been reported that His77 is not coordinated to the ferric heme in CooA (29, 35, 38). Therefore, a possible candidate for the sixth ligand would be either an exogenous molecule such as H2O or OH-, or the nitrogen atom from Pro2 that is the sixth ligand of the ferrous heme (39).

Given that an exogenous molecule is coordinated to the heme as the sixth ligand, it might be H2O or OH-. This is mainly because the EPR spectral feature of the ferric CooA bears good resemblance to that of cytochrome P450 in the low spin state with the Cys-Fe3+-H2O/OH- coordination (29, 37). However, the coordination of H2O or OH- to the ferric heme is unlikely because all the two axial ligands must be changed upon reduction of the heme, if this is the case. Furthermore, experiments on ligand binding with imidazole have shown that the heme center in CooA is remarkably resistant to exogenous ligand binding (35). The electronic absorption spectra of CooA have been reported to be unchanged between pH 6.5 and 11 (35). These results apparently indicate that the sixth ligand of the heme in CooA will be an endogenous ligand, not an exogenous one.

Dawson et al. (50) have reported spectroscopic investigations of ferric cytochrome P450cam ligand complexes. The spectroscopic properties of ferric CooA are similar to those of the nitrogen donor complexes of ferric cytochrome P450cam rather than those of the oxygen donor complexes. Dhawan et al. (38) have reported that magnetic circular dichroism (MCD) spectroscopy reveals the coordination of a neutral nitrogen atom as the sixth ligand to the ferric heme in CooA. As shown in Figs. 2A and 3A, good fitting results were obtained for the EXAFS spectra of ferric CooA when Cys75 and Pro2 were assumed to be the axial ligands of the ferric heme. These results suggest that the nitrogen atom from Pro2 is the most plausible candidate for the sixth ligand of the ferric heme in CooA.

Electrochemical Properties of CooA

Electrochemical Redox Titrations-- The present mutagenesis and EXAFS experiments showed ligand switching of the heme iron in CooA coupled with the change in its oxidation state. Then, we examined the effect of the ligand switching on the redox properties of CooA by electrochemical techniques. In the presence of redox mediator dyes, electron transfer proceeded between a gold mesh electrode and CooA. In electrochemical redox titrations, the electronic absorption spectra of CooA were measured at each potential after an electric current reached <= 0.5 µA. Fig. 5A shows a typical spectral change of CooA in the visible region during reductive titration of ferric CooA, when the potential was varied from -100 to -600 mV with a step width of 25 or 50 mV. The electronic absorption spectrum at a potential of -100 mV was identical to that of ferric CooA as isolated, giving the Soret peak at 423.5 nm. As the applied potential was shifted negatively, the Soret peak at 423.5 nm decreased in intensity, whereas a new Soret peak at 424.5 nm increased in intensity, with a concomitant appearance of the alpha  (558.5 nm) and beta  (528.0 nm) bands. No spectral change was observed below -600 mV. The final spectrum was identical to the spectrum of ferrous CooA obtained by the chemical reduction of the ferric protein with sodium dithionite.



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  		Fig. 5.   Typical spectral change of CooA during reductive (A) and oxidative (B) titrations. CooA (15 µM in dimer) was dissolved in 50 mM Tris-HCl buffer, pH 8.0 containing 0.1 M NaCl. A, the difference spectra, the electronic absorption spectra at the appropriate potential minus the spectrum of oxidized CooA, are shown. The spectra were measured at potentials of -200 (a), -250 (b), -275 (c), -300 (d), -325 (e), -350 (f), -375 (g), -400 (h), -450 (i), -500 (j), and -550 mV (versus NHE) (k). No spectral change was observed at -100 mV. The spectra measured at potentials of -600 and -650 mV were identical to spectrum k. B, the difference spectra, the electronic absorption spectra at the appropriate potential minus the spectrum at a potential of -650 mV (the spectrum of reduced CooA), are shown. The spectra were measured at potentials of -400 (a), -375 (b), -350 (c), -300 (d), -275 (e), -250 (f), -225 (g), -200 (h), -150 (i), and -100 mV (versus NHE) (j). No spectral change was observed at potentials of -600, -550, -500, and -450 mV.

After the reductive titration, the potential was shifted positively to reoxidize CooA in the electrochemical oxidative titration. As shown in Fig. 5B, the spectral changes of CooA during the oxidative titration were the reverse of those observed in the reductive titration. At the end point of the oxidative titration, the electronic absorption spectrum of ferric CooA was restored. In the spectral change during the redox titrations, isosbestic points were observed at 411.0, 434.5, 510.0, and 569.5 nm, as shown in Fig. 5. These results show that the redox reaction of CooA proceeded quantitatively and without any side reaction in the electrochemical redox titration system described herein.

Nernst plots to determine the reduction and oxidation midpoint potentials of wild-type CooA are shown in Fig. 6A. The reduction and oxidation midpoint potentials of wild-type CooA were calculated from the plots to be -320 mV and -260 mV, respectively. In the case of wild-type CooA, the reduction and oxidation midpoint potentials were not identical; i.e. the oxidation midpoint potential was shifted positively by 60 mV compared with the reduction midpoint potential.



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  		Fig. 6.   Reductive (black-triangle) and oxidative () titrations of wild-type (A) and H77G CooA (B). The fraction of the ferric form was plotted as a function of potential. The individual data points are an average of at least three independent measurements. The solid lines are theoretical Nernst curves for one-electron reduction (black-triangle) and oxidation () with midpoint potentials of -320 and -260 mV for wild-type CooA and with those of -420 mV for H77G CooA, respectively.

If a simple redox reaction of the heme in CooA takes place during the electrochemical redox titrations, the midpoint potentials should be identical to each other. However, this is not the case for CooA. On the basis of the coordination structure described in this work, the reduction and oxidation reactions of the heme iron are accompanied by the ligand exchange and are thought to proceed according to the scheme as shown in Scheme 1. A similar scheme has been reported for analyzing the redox titrations of a mutant (F82H/C102S) of yeast iso-1-cytochrome c (51) and of cytochrome cd1 from Paracoccus pantotrophus (52). A similar hysteresis in the redox titrations has been observed for cytochrome cd1 from Paracoccus pantotrophus (52). The oxidized Paracoccus pantotrophus cytochrome cd1 has His/His axial ligation at the c heme iron and Tyr/His axial ligation at the d1 heme iron (53, 54). Upon reduction, the ligation of the c heme iron switches to His/Met concomitant with dissociation of the Tyr from the d1 heme iron (53, 54). Koppenhöfer et al. (52) have reported that the hysteresis in the redox titrations is observed in the case of Paracoccus pantotrophus cytochrome cd1, because the true equilibrium is not reached for the ligand switching reactions during the redox titrations.



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  		Scheme 1.  

In the case of CooA, the change in the oxidation state of the heme iron accompanies the axial ligand switching between Cys75 and His77, which is similar to ligand switching for Paracoccus pantotrophus cytochrome cd1. The reaction intermediates 2 and 4 shown in Scheme 1 seem to be formed during the reduction and oxidation of CooA, respectively, whereas the attempts to observe these intermediates by uv/vis spectroscopy with stopped flow method were unsuccessful so far. The reduction and oxidation of CooA should proceed with the same midpoint potential, if all the reaction steps shown in Scheme 1 are in equilibrium. However, this is not the case for wild-type CooA as shown in Fig. 6A, i.e. the apparent reduction and oxidation midpoint potentials are different from each other. Because the equilibrium is not reached for the ligand switching reactions during the redox titrations, the hysteresis in the redox titrations is thought to be observed also in the case of CooA, as is the case for Paracoccus pantotrophus cytochrome cd1.

To support the above suggestion, we also examined the redox titrations of the H77G mutant of CooA in which His77, which is one of the axial ligands of the ferrous heme, is replaced by Gly. The results of the electrochemical redox titrations for H77G CooA are shown in Fig. 6B. The redox properties of H77G CooA were significantly different from those of wild-type CooA in which the reduction and oxidation midpoint potentials (-420 mV) were identical to each other. Because H77G CooA lacks His77, only redox reactions of the heme iron seem to take place during the redox titrations. The ligand switching during the redox titrations, the vertical transitions in Scheme 1, does not take place in the case of H77G CooA, which is the reason why the hysteresis in the redox titrations was not observed for H77G CooA.

Redox Properties of CooA Relevant to its Functional and Structural Properties-- The reduction midpoint potential of ferric CooA (-320 mV) is comparable with that of the low-spin form of Pseudomonas putida d-camphor hydroxylase cytochrome P450cam (E1/2 = -300 mV, Ref. 55), whose axial ligands are a thiolate derived from Cys and H2O/OH-. This observation is consistent with the coordination of the Cys75 thiolate to the ferric heme iron in CooA, because the low reduction potential is thought to be caused by the strong electron-donating nature of the Cys thiolate (56). The thiolate coordination is apparently supported by EPR results, in which the spectral features of ferric CooA are similar to those of the low-spin form of P450cam (29, 37).

It is also characteristic of CooA that the heme having the histidyl imidazole as the axial ligand gives -260mV for the oxidation midpoint potential. Compared with the redox potentials of other heme proteins having a histidyl imidazole, the value is similar to that of horseradish peroxidase (-250 mV, Ref. 57) rather than those of myoglobin (+50 mV, Refs. 58, 59)) and cytochrome b562 (+113 mV, Ref. 60). The difference in the redox potentials has been explained in terms of the difference in the character of the ligand imidazole group; i.e. the imidazole ring of peroxidase has an anionic character, resulting from the strong hydrogen bond of the NH with carboxylate (61), whereas those in myoglobin and cytochrome b562 have a neutral character (58, 59, 62). In the case of CooA, deprotonation of Pro2 that is coordinated to the ferrous heme may be responsible for the low oxidation potential of CooA.

The low oxidation potential of CooA could be concerned with the regulation of CooA activation. CooA is required for CO-dependent expression of the coo operons encoding a CO-oxidizing system that allows R. rubrum to grow on CO as the sole energy source (31). CooA is activated only in the presence of CO under anaerobic conditions to induce the expression of the coo operons (30, 32, 33). Even in the presence of CO, however, CooA must not be activated under aerobic conditions because CO dehydrogenase, a key enzyme for the CO-oxidizing system encoded in the coo operons, is labile to oxygen (63). The low oxidation potential of CooA would facilitate the oxidation of the heme to prevent CooA from being activated in vivo, once oxygen is present in the cells.


    FOOTNOTES

* This work was supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture in Japan 11116212 and 11132219 (to S. A.) and by a grant from the Asahi Glass Foundation (to S. A.). EXAFS data collection at Photon Factory was performed under approval of the Program Advisory Committee (Proposal No. 97G059).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 81-761-51-1681; Fax: 81-761-51-1149; E-mail: aono@jaist.ac.jp.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M003972200

2 Among the mutants constructed in this work, N6A and D72A CooA showed a slightly blue-shifted Soret peak at 420 nm in the ferric state, whereas the ferrous and CO-bound spectra of these mutants were identical to those of wild-type CooA. However, when Asn6 or Asp72 was replaced by Met, the resulting mutants showed almost the same electronic absorption spectra in the ferric, ferrous, and CO-bound forms as those of wild-type CooA.


    ABBREVIATIONS

The abbreviations used are: sGC, soluble guanylate cyclase; EXAFS, extended x-ray absorption fine structure; EPR, electron paramagnetic resonance; NHE, normal hydrogen electrode.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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