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J. Biol. Chem., Vol. 277, Issue 16, 13528-13538, April 19, 2002

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Resonance Raman and Ligand Binding Studies of the Oxygen-sensing Signal Transducer Protein HemAT from Bacillus subtilis^*

Shigetoshi Aono^§, Toshiyuki Kato, Mayumi Matsuki, Hiroshi Nakajima, Takehiro Ohta^¶, Takeshi Uchida^¶, and Teizo Kitagawa^¶

From the  School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292 and the ^¶ Center for Integrative Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki, Aichi 444-8585, Japan

Received for publication, December 21, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be k_on = 32 µM¹ s¹ and k_off = 23 s¹, respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 °C was 0.06 h¹, which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the _Fe-O2 band at a noticeably low frequency (560 cm¹), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the _Fe-His band at a higher frequency (225 cm¹) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the _Fe-CO and _C-O bands at 494 and 1964 cm¹, respectively, which fall on the same _C-O versus _Fe-CO correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Motile bacteria are known to swim toward or away from specific environmental stimuli such as nutrients, oxygen, or light (1, 2). This behavior, termed chemotaxis, is mediated by a signal transduction system consisting of methyl-accepting chemotaxis proteins (MCPs),¹  a histidine kinase CheA, a response regulator CheY, a coupling protein CheW, and the two enzymes that mediate sensory adaptation by covalently modifying the MCPs, CheR and CheB (3-6). Typical MCP is an integral membrane protein with an N-terminal periplasmic substrate binding domain and a C-terminal cytoplasmic signaling domain (7, 8). The binding of a substrate, a repellent, or attractant to the periplasmic substrate binding domain is believed to induce a change in the MCP conformation that allows it to activate CheA (7, 8). The activated CheA phosphorylates CheY, and, consequently, the phosphorylated CheY binds to the switch complex at the base of the flagella to control the direction of flagellar rotation (3-6). In this signal transduction system, MCP acts as a sensor for the external signal and as a signal transducer.

Hou et al. (9) have recently reported that Bacillus subtilis and Halobacterium salinarum have a signal transducer protein, HemAT-Bs and HemAT-Hs, respectively, for aerotaxis, the migratory response toward or away from oxygen. HemAT-Bs and HemAT-Hs are soluble proteins, and their C-terminal regions, residues 222-489 of HemAT-Hs and 198-432 of HemAT-Bs, are 30% identical to the cytoplasmic signaling domain of Tsr, an MCP from Escherichia coli (9). Their N-terminal regions, residues 1-184 in HemAT-Hs and 1-175 in HemAT-Bs, show limited homology to myoglobin (9). Recombinant HemAT-Bs and HemAT-Hs are hemoproteins containing a b-type heme as a prosthetic group and show similar electronic absorption spectra to those of myoglobin (9). HemAT-Bs and HemAT-Hs bind oxygen reversibly as myoglobin does. The residues 1-195 for HemAT-Hs and 1-176 in HemAT-Bs retain the heme- and oxygen-binding properties of the respective native proteins, which represent a globin-coupled sensor motif (10). These results suggest that the heme in HemAT acts as an oxygen sensor and that the binding of oxygen to the heme in HemAT triggers the signal transduction for aerotaxis in B. subtilis and H. salinarum.

Sensing gas molecules such as O₂, NO, and CO is a novel function of hemoproteins (11-14), whereas hemoproteins exhibit a wide variety of functions such as oxygen storage/transport, electron transfer, and redox reactions of various substrates. Recently, reports on hemoprotein sensors in which a heme prosthetic group acts as a sensor for a gas molecule such as O₂, NO, or CO are on the increase. FixL (15), direct oxygen sensor (16), and phosphodiesterase A1 (17) for O₂ sensors, soluble guanylate cyclase (sGC) (18) for a NO sensor, and CooA (19-21) for a CO sensor are typical examples for hemoprotein sensors. In these sensor proteins, the binding of O₂, NO, or CO to the heme regulates the function of these proteins. The hemes in these sensor proteins play a central role not only for sensing their effector molecules but also for regulating the functional properties with a conformational change induced by the ligand binding.

The heme in HemAT is thought to be the active site for sensing O₂, and the binding of O₂ to the heme will be responsible for triggering the aerotaxis signal transduction. Therefore, the elucidation for the coordination structure of the heme, especially in the O₂-bound form and for the interaction between the bound O₂ and the heme pocket, are required to understand the mechanism of the O₂ sensing and signal transduction for HemAT. Although the electronic absorption spectra of HemAT-Hs and HemAT-Bs are reported (9), very little information is available for the structure and ligand binding properties of the heme in HemAT. In this work, we present results of the kinetic analysis for oxygen binding with HemAT-Bs, autoxidation kinetics of oxygen-bound HemAT-Bs, and resonance Raman measurements with the deoxy, oxy, and CO-bound forms of HemAT-Bs. Using resonance Raman spectroscopy, we show a unique heme environment for HemAT-Bs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of HemAT-Bs in E. coli-- An expression vector for HemAT-Bs was constructed as follows. The hemAT-Bs gene was prepared by polymerase chain reaction (PCR) with the chromosomal DNA of B. subtilis and the two synthetic deoxyoligonucleotides (5'-gaaggagatccatatgttatttaaaaaagacagaaaacaagaaacagc-3' and 5'-ggatcccatatgttattattcttctgtcaggatgacaagcgaatcaacgg-3' for the sense and antisense primers, respectively) as the template and primers, respectively. Although the original translational initiation codon for HemAT-Bs is TTG in B. subtilis (9), the translational initiation codon was changed to ATG in the sense primer, which is underlined in the above sequence. The PCR product was cloned into a pCR4-TOPO vector with the TOPO TA cloning kit (Invitrogen). The plasmid containing the hemAT-Bs gene with the correct direction relative to the lac promoter was selected as an expression vector for HemAT-Bs and named pCR-HemAT.

E. coli JM109 was used as a host for the expression of HemAT-Bs. pCR-HemAT/E. coli JM109 was grown in LB medium containing 50 µg/ml ampicillin at 37 °C. The expression of HemAT-Bs was induced by 1 mM isopropyl--D-thiogalactopyranoside. The cultivation was continued at 25 °C for 12 h after adding isopropyl--D-thiogalactopyranoside. The cells were harvested by centrifugation and stored at 80 °C until use.

Purification of the Recombinant HemAT-Bs-- Purification of the recombinant HemAT-Bs was carried out as follows. The cells were resuspended in 50 mM Tris-HCl buffer, pH 8.5, containing 1 mM phenylmethylsulfonyl fluoride and disrupted by sonication. After unbroken cells and cell debris were removed by centrifugation, the supernatant was applied to a column (2.6 × 30 cm) of Q-Sepharose (Amersham Biosciences, Inc.) equilibrated with 50 mM Tris-HCl buffer, pH 8.5. The adsorbed proteins were eluted by a linear gradient of NaCl from 0 to 0.6 M at a flow rate of 1 ml/min after washing the column with a 4-bed volume of 50 mM Tris-HCl buffer, pH 8.5. The fractions containing HemAT-Bs were combined and applied to a column (1.6 × 30 cm) of hydroxylapatite (Seikagaku Co.) equilibrated with 50 mM Tris-HCl containing 1 M KCl, pH 8.5. The adsorbed proteins were eluted at a flow rate of 1 ml/min by increasing linearly the concentration of K₂HPO₄ in the elution buffer from 0 to 0.3 M. The fractions containing HemAT-Bs were combined and concentrated by a Centricon 50 (Amicon). The concentrated sample was applied to a column (1.6 × 80 cm) of Superdex 200 pg (Amersham Biosciences, Inc.) equilibrated with 50 mM Tris-HCl buffer containing 0.1 M NaCl, pH 8.5. The column was run at 0.2 ml/min using 50 mM Tris-HCl buffer containing 0.1 M NaCl, pH 8.5, as an elution buffer. 1-2 mg of purified HemAT-Bs was obtained from approximately 10 g of wet cells of pCR-HemAT/E. coli JM109.

Characterization of HemAT-Bs-- The reduction of HemAT-Bs was carried out by adding a few grains of sodium dithionite into the isolated HemAT-Bs. CO gas was introduced into HemAT-Bs solution by a gas-tight syringe.

The type of the heme in HemAT-Bs was determined by the pyridine ferrohemochrome method. The value of 34 mM¹ cm¹ at the absorption maximum of the  band for the pyridine ferrohemochrome derived from the protoheme was used to calculate the concentration of the heme in HemAT-Bs. Protein concentration was determined by the Coomassie Protein Assay Reagent (Pierce) or the Advanced Protein Assay Reagent (Cytoskeleton Inc.) using bovine serum albumin as a standard.

The molecular mass of HemAT-Bs was determined by gel filtration, using a Superdex 200-pg gel filtration column (1.6 × 80 cm). The system was calibrated with -amylase (molecular weight, 200,000), alcohol dehydrogenase (150,000), bovine serum albumin (66,000), carbonic anhydrase (29,000), and cytochrome c (12, 400), using 50 mM Tris-HCl buffer containing 0.1 M NaCl, pH 8.5, as the eluent. These proteins used in the calibration were obtained from Sigma Chemical Co.

Spectral Measurement-- The electronic absorption spectra were measured on a Hitachi U-3300 UV-visible spectrophotometer. Resonance Raman spectra were obtained with laser excitation at 413.1 nm by a Kr⁺ laser (Spectra Physics, model 2016). The excitation light was focused into the cell, and the laser power was 1 milliwatt (mW) at the cell for the oxy and deoxy forms of HemAT-Bs but 0.1 mW for CO-bound HemAT-Bs so as to avoid photolysis of coordinated CO. The sample solutions for the Raman measurements were sealed in quartz cells, which were rotated at 500 rpm at room temperature. Typically, 30 µl of the sample aliquots containing 30 µM protein in 50 mM Tris-HCl buffer, pH 8.5, were put into the cell. The scattered light was dispersed with a single polychromator (Ritsu, DG-1000) equipped with a liquid nitrogen-cooled charge-coupled device camera. The spectral slit width was 6 cm¹. Raman shifts were calibrated using indene and an aqueous solution of potassium ferrocyanide as frequency standards, providing accuracy of ±1 cm¹ for intense isolated lines.

Ligand Binding Kinetics-- The bimolecular on-rate (k_on) of oxygen was determined at room temperature after photolysis of oxygen-bound HemAT-Bs, HemAT-Bs(O₂), with a 20-ns Nd-YAG laser pulse at 532 nm (Quanta Ray GCR-3). A Xe-lamp (ILC Technology, LX-300) was used as a monitor light source. The transient kinetic curves were observed at 433 nm, which were measured on a digital storage oscilloscope (Tektronix 11401).

The off-rate (k_off) of oxygen was determined by stopped-flow spectrophotometry (Unisoku USP-526) at room temperature. About 3 µM HemAT-Bs(O₂) was mixed with 5 mM sodium dithionite solution in 50 mM Tris-HCl buffer, pH 8.0, as reported previously (22). The reaction was followed at 413 nm.

Autoxidation of HemAT-Bs-- The rate of autoxidation of HemAT-Bs(O₂) was measured in 50 mM Tris-HCl buffer (pH 8.5) at 37 °C under air. HemAT-Bs(O₂) as isolated was maintained in an optical quartz cell with 1-cm optical path length at 37 °C, and spectra were recorded at regular intervals of time with an Hitachi U-3300 UV-visible spectrophotometer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HemAT-Bs was expressed as a soluble hemoprotein in the expression system constructed in this study. A nearly homogeneous HemAT-Bs was obtained by column chromatography described under "Experimental Procedures," as evident from SDS-polyacrylamide gel electrophoresis (Fig. 1a). The pyridine ferrohemochrome derived from HemAT-Bs gave the  band at 556 nm (data not shown), which means that HemAT-Bs contains a protoheme (b-type heme) as a prosthetic group. The molecular mass of the purified HemAT-Bs was estimated to be about 188 kDa by gel filtration (Fig. 1b), suggesting that the purified HemAT-Bs is a homo-tetramer of an identical subunit (48.7 kDa).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE of purified HemAT-Bs (a) and estimation of molecular weight by gel filtration column chromatography (b). The circle and squares represent HemAT-Bs and standard proteins for molecular weight determination (1, -amylase; 2, alcohol dehydrogenase; 3, bovine serum albumin; 4, carbonic anhydrase; 5, cytochrome c), respectively.

Kinetics Constants for the Reaction of Ferrous HemAT-Bs with Oxygen-- The binding of oxygen to the heme is the first and crucial step of the oxygen sensing and signal transduction by HemAT-Bs. Therefore, elucidation of the oxygen binding properties is essential to understand the functional properties of HemAT-Bs. In this work, the reaction rate constants for oxygen binding and dissociation, k_on and k_off, were determined by laser-flash photolysis and stopped-flow spectroscopies, respectively. The k_on and k_off values for HemAT-Bs thus determined were 32 µM¹ s¹ and 23 s¹, respectively, which were similar to those of sperm whale Mb (Sw Mb) (Table I). HemAT-Bs exhibits a moderate oxygen affinity, similar to that of Sw Mb but different from another oxygen sensor protein FixL. FixL exhibits an extremely low oxygen affinity due to a very low k_on value (k_on = 0.14 µM¹ s¹), although the k_off of FixL is comparable to that of HemAT-Bs (Table I).

The dissociation equilibrium constant (K_d) given by the ratio k_off/k_on for HemAT-Bs was 719 nM (Table I), which is comparable to the K_m values of terminal oxidases for oxygen respiration. That the K_d value of HemAT-Bs for O₂ is comparable to K_m values of terminal oxidases seems reasonable from the functional point of view for HemAT-Bs. Because HemAT-Bs acts as an oxygen-sensing signal transducer in aerophilic response of B. subtilis (9), it should detect the most suitable oxygen concentration for the bacterium, i.e. that for oxygen respiration. If HemAT-Bs has an extremely high oxygen affinity as do Mt Hb, Asacaris Hb, and Synechocystis Hb (Table I), it would be disadvantageous, because a signal transduction would occur to induce chemotaxis toward a too low concentration of oxygen unsuitable for oxygen respiration. The K_d value of 719 nM for HemAT-Bs, a moderate oxygen affinity, reveals that HemAT-Bs can detect an oxygen concentration suitable for oxygen respiration.

Autoxidation of Oxy HemAT-Bs-- HemAT-Bs(O₂) as isolated showed a rate constant for autoxidation of 0.06 h¹ at 37 °C, which is similar to that of Sw Mb (Table II). FixL, another oxygen sensor protein, shows a 20- to 40-fold lager autoxidation rate constant compared with that of HemAT-Bs (29, 30). The kinetics parameters for autoxidation and oxygen binding are different between HemAT-Bs and FixL, which suggests some qualitative difference in the heme environmental structure between the two oxygen sensor proteins.

Electronic Absorption Spectra of HemAT-Bs-- The electronic absorption spectra of HemAT-Bs are shown in Fig. 2. HemAT-Bs as isolated gave the Soret, , and  peaks at 414, 578, and 543 nm, respectively, as shown in Fig. 2a. This spectrum is typical of six-coordinate, low spin hemoproteins and resembles that of the oxy form of Mb, as described previously (9). When CO was reacted with the isolated HemAT-Bs without any reductant, CO-bound HemAT-Bs was formed (data not shown). These results strongly suggest that HemAT-Bs is purified as the oxy form in which O₂ is bound to the ferrous heme. Resonance Raman spectroscopy revealed that this was the case, as described below. Upon deoxygenation with sodium dithionite, HemAT-Bs showed a spectrum with the Soret peak at 431 nm and a single peak at 563 nm in the Q-band region, as shown in Fig. 2b. This spectrum is typical of five-coordinate, high spin ferrous hemoproteins, which show the formation of deoxy HemAT-Bs. CO-bound HemAT-Bs was formed upon the reaction of dithionite reduced HemAT-Bs with CO, which showed the Soret, , and  peaks at 422, 567, and 543 nm, respectively, as shown in Fig. 2c. These electronic absorption spectra of HemAT-Bs were similar to those of Mb as reported previously (9), which is consistent with the fact that the N-terminal region of HemAT-Bs shows an amino acid sequence homology to Mb (9). The values of the absorption maxima observed in this study are slightly different from those reported in Ref. 9. Hou et al. have noted from their absorption spectra the presence of a significant population of deoxy species for both the CO- and O₂-bound forms of HemAT-Bs (9). In our preparation of HemAT-Bs, however, such an incomplete ligand binding was not noticed, which was confirmed by resonance Raman spectroscopy as described below. These differences may cause the difference of the absorption maxima, although the reasons of the difference are not clear at present.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Electronic absorption spectra of oxy (a), deoxy (b), and CO-bound HemAT-Bs (c). HemAT-Bs was dissolved in 50 mM Tris-HCl buffer, pH 8.5. HemAT-Bs as isolated was in the oxy form. Deoxy HemAT-Bs was prepared by adding sodium dithionite into oxy HemAT-Bs solution. CO was introduced into deoxy HemAT-Bs solution to prepare CO-bound HemAT-Bs. The molar extinction coefficients for the heme in HemAT-Bs are shown in this figure.

The heme in HemAT-Bs is thought to play an important role for sensing O₂. The ligand conformation, the interaction between the bound O₂ and the amino acid residue(s) in the heme pocket, and/or the heme environmental structure will be responsible for triggering the signal transduction upon the O₂ binding to the heme in HemAT-Bs. To characterize these properties, we measured the resonance Raman spectra of HemAT-Bs in the oxy, deoxy, and CO-bound forms.

Resonance Raman Spectra of Oxy HemAT-Bs-- The resonance Raman spectrum in the high frequency region (1300-1700 cm¹) of HemAT-Bs(O₂) is shown in Fig. 3a. In Table III, the observed frequencies of the major bands are compared with those of Mb (32, 33), heme-bound heme oxygenase (32, 33), and gas-sensing hemoproteins such as the O₂, CO, and NO sensors of FixL (34-37), CooA (38-40), and sGC (41-43), respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   High-frequency resonance Raman spectra of oxy (a), deoxy (b), and CO-bound HemAT-Bs (c).

It is established that resonance Raman spectra in the high frequency region contain a few marker bands sensitive to the oxidation state (₄) and the spin and coordination states (₂ and ₃) of the heme iron (44). HemAT-Bs(O₂) gave two bands at 1471 and 1501 cm¹ in the ₃ band region, indicative of a mixture of five- and six-coordinate heme species. The relative intensities of these bands depended on the laser power, i.e. the intensity of the band at 1471 cm¹ due to a five-coordinate heme increased as the laser power increased. These results show that the bound O₂ in HemAT-Bs(O₂) was photodissociated to form the five-coordinate form at the higher laser power. Therefore, the band at 1501 cm¹ was assigned to the ₃ band of HemAT-Bs(O₂). The ₂ band of HemAT-Bs(O₂) was observed at 1578 cm¹.

The ₂ and ₃ modes of HemAT-Bs(O₂) observed at 1578 and 1501 cm¹, respectively, are slightly lower than the corresponding ones of Mb(O₂) (₂ = 1584 cm¹ and ₃ = 1507 cm¹) (33) but coincide with the corresponding ones of FixL(O₂) (₂ = 1577 cm¹ and ₃ = 1502 cm¹) (34). Because the ₂ and ₃ frequencies are linearly correlated with the Ct-N distance (distance between the center and a nitrogen atom of a porphyrin ring) (45, 46), the lower frequencies of the ₂ and ₃ modes in HemAT-Bs(O₂) may be indicative of an expanded porphyrin core compared with Mb(O₂), which is also suggested for FixL(O₂) (34).

The resonance Raman spectra of HemAT-Bs(O₂) in the low frequency region are shown in Fig. 4. The spectra of the ¹⁶O₂- and ¹⁸O₂-bound forms of HemAT-Bs are shown in the top and middle, respectively, and the ¹⁶O₂-¹⁸O₂ difference spectrum is shown in the bottom. A large isotope shift was observed for the band at 560 cm¹ where the derivative pattern of the difference spectrum was observed. Therefore, this band is assigned to the Fe-O₂ stretching mode (_Fe-O2). However, the peak-to-valley frequency difference in Fig. 4c is as large as 26 cm¹, which is slightly larger than the frequency shift expected for an Fe-O₂ diatomic oscillator (23 cm¹). The 561 cm¹ band in Fig. 4a is broader than those of other bands, implying inhomogeneous broadening. Accordingly, a too large isotopic frequency shift might be caused by the existence of more than two bands and the difference of their intensity distributions between ¹⁶O₂ and ¹⁸O₂ derivatives. Although this should be clarified in later studies, it is no doubt that the 560 cm¹ band primarily involves the _Fe-O2 character.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   The isotope shift in the low frequency resonance Raman spectra of HemAT-Bs(O2). 16O2 (a), 18O2 (b), and 16O2-18O2 (c) represent the Raman spectra of HemAT-Bs(16O2), HemAT-Bs(18O2), and HemAT-Bs(16O2)-HemAT-Bs(18O2), respectively.

The ligand modes of various hemoproteins are listed in Table IV. The ligand modes get resonance Raman intensity from the electronic coupling of the ligand orbital to the metal and porphyrin orbitals. In particular, the assignment of a ligand-Fe stretching mode is useful, because it directly reflects the strength of the Fe-ligand bond and thus the nature of interactions of the ligand with the surrounding amino acid residues.

Resonance Raman Spectra of Deoxy HemAT-Bs-- The high frequency resonance Raman spectrum of deoxy HemAT-Bs is shown in Fig. 3b. Deoxy HemAT-Bs showed the ₂, ₃, and ₄ modes at 1558, 1469, and 1352 cm¹, respectively, at typical frequencies of the deoxy form of five-coordinate heme species in the high spin state.

The resonance Raman spectrum of deoxy HemAT-Bs in the low frequency region is shown in Fig. 5a. The intense line at 225 cm¹, which was undetectable with the O₂- and CO-bound forms, was observed for deoxy HemAT-Bs. In general, the Fe-His stretching Raman band of hemoproteins is observable in the region of 200-250 cm¹ for the five-coordinate ferrous species (52). We thus assign the 225 cm¹ band to the Fe-His stretching mode of deoxy HemAT-Bs. This frequency is higher than that of deoxy Mb, and it will be discussed later.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   The 200-400 cm1 region resonance Raman spectra of deoxy HemAT-Bs (a) and HemAT-Bs(CO) (b) and (c). The power of the 413.1-nm laser for the measurement of deoxy HemAT-Bs was 1 mW (a), whereas 5 mW (b) and 0.1 mW (c) laser power were applied for HemAT-Bs(CO).

Resonance Raman Spectra of CO-bound HemAT-Bs-- The resonance Raman spectrum of CO-bound HemAT-Bs, HemAT-Bs(CO), in the high frequency region is shown in Fig. 3c. HemAT-Bs(CO) was photolabile and showed two ₃ bands at 1468 and 1495 cm¹, corresponding to a five-coordinate, high spin and a six-coordinate, low spin heme species, respectively. The former species is thought to be formed by photodissociation of the bound CO upon the laser excitation. Thus, the CO-bound form gives the ₂, ₃, and ₄ bands at 1578, 1495, and 1368 cm¹, respectively. The ₄ frequency of the CO-bound form (1368 cm¹) is lower than that of the O₂-bound form (1372 cm¹), indicating that the heme itself distinguishes O₂ from CO as a ligand. Because the ₄ frequency becomes higher for a heme with larger delocalization of  electrons from the e_g orbital of porphyrin to the * orbital of the bound ligand through the d orbital of Fe, the electron delocalization is larger for O₂ than for CO.

The resonance Raman spectra of HemAT-Bs(CO) in the low frequency region are shown in Fig. 5 (b and c). The 225 cm¹ band was also observed in HemAT-Bs(CO) under increased laser power conditions, as shown in Fig. 5b. This suggests that photodissociation of CO from HemAT-Bs(CO) generates a five-coordinate ferrous HemAT-Bs, whereby the _Fe-His band appears at 225 cm¹.

The resonance Raman spectra of the HemAT-Bs(CO) with ¹²C¹⁶O and ¹³C¹⁶O in the low frequency (300-700 cm¹) and high frequency (1800-2100 cm¹) regions are shown in Fig. 6. The isotope shifts of 3, 19, and 46 cm¹ were observed for the bands at 494, 570, and 1964 cm¹, respectively, upon substitution of ¹³C¹⁶O for ¹²C¹⁶O. Based on the frequencies of these bands and sizes of the isotope shifts, we assign the 494, 570, and 1964 cm¹ bands to the Fe-CO stretching (_Fe-CO), the Fe-C-O bending, and the C-O stretching (_C-O) modes, respectively.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   The isotope shift in the low and high frequency resonance Raman spectra of HemAT-Bs(CO). 12C16O (a), 13C16O (b), and 12C16O-13C16O (c) represent the Raman spectra of HemAT-Bs(12C16O), HemAT-Bs(13C16O), and HemAT-Bs (12C16O)-HemAT-Bs(13C16O), respectively.

It is established that the _Fe-CO and _C-O frequencies display an inverse linear correlation (53, 54) because of the -electron back donation mentioned above. This -back donation results in strengthening of the Fe-CO bond and concomitant weakening of the C-O bond (or vice versa), leading to the empirical _Fe-CO versus _C-O linear inverse line (53, 54). The _Fe-CO versus _C-O correlation depends on the nature of the proximal ligand; the CO-bound hemes with an imidazole/histidine as the trans ligand exhibit a correlation line different from those with a thiolate or imidazolate. The _Fe-CO and _C-O bands of HemAT-Bs(CO), detected at 494 cm¹ and 1964 cm¹, respectively, fell on the imidazole/histidine correlation curve as illustrated in Fig. 7. This fact means that proximal His is neutral like Mb, and the high frequency of the Fe-His stretching frequency cannot be ascribed to deprotonation of proximal His. The _Fe-CO and _C-O frequencies suggest that the environment around the oxygen atom of bound CO is less hydrophilic than that of native Mb like that in the open form of Mb. The negligibly weak enhancement of the Fe-C-O bending band observed for HemAT-Bs(CO), as shown in Fig. 6, suggests an undistorted Fe-C-O geometry (54).

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Plot of Fe-CO frequencies against C-O frequencies observed for CO-bound hemoproteins. Data obtained in the present study for HemAT-Bs are shown by an open circle. Solid squares represent data points of other CO adducts taken from Table II. Data for human Hb A and Mb (pH 2.6 and 8.4) were from Ref. 49.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structural Characteristics of Heme Pocket of HemAT-Bs-- The _Fe-O2 frequency of HemAT-Bs(O₂) in the ¹⁶O₂ spectrum (Fig. 4a, 560 cm¹) is one of the lowest Fe-O₂ stretching frequencies among O₂-bound hemoproteins having a histidine as an axial ligand (33, 34, 47, 55-61). Recently, Yeh et al. (47) have reported a low Fe-O₂ stretching frequency (560 cm¹) for hemoglobin from Mycobacterium tuberculosis (Mt Hb). In conjunction with the mutagenesis studies, they have proposed a unique hydrogen bonding pattern between the bound oxygen and the distal tyrosine at the B10 position (the tenth residue in the B helix) through the sp² orbital of the proximal oxygen atom and have pointed out that this unique hydrogen bonding plays a crucial role in the low frequency of the _Fe-O2 mode for Mt Hb (47). Considering the low frequency of the _Fe-O2 mode of HemAT-Bs(O₂) comparable to that of Mt Hb, such a unique hydrogen bonding as seen for Mt Hb would also be involved between one or more distal amino acid residues and the proximal oxygen atom of the bound O₂ in HemAT-Bs(O₂). Specification of the hydrogen bonding counterpart residue is under progress in this laboratory.

The important determinants of the _Fe-His frequency is the hydrogen bonding status of the His N hydrogen (52), the strain imposed on the axial His by the protein moiety (62), and geometry of bound imidazole (63). In practice, a high frequency shift as large as 25 cm¹ was reported to be caused by deprotonation of bound imidazole in imidazole-coordinated high spin Fe(II) porphyrin (64). In cytochrome c peroxidase, the strong hydrogen bond between the axial His and Asp²³⁵ enhances the anionic character of the imidazole ring of the axial His, resulting in the higher _Fe-His frequency (245 cm¹) (65). When this hydrogen bond is disrupted by the Asp²³⁵ to Asn mutation, the _Fe-His band is shifted to a lower frequency (205 cm¹) (66, 67). Deoxy Mb, which has a weak hydrogen bond on the axial His, gives the _Fe-His mode around 220 cm¹ (48). Thus, deoxy HemAT-Bs may possibly have a stronger hydrogen bond compared with deoxy Mb.

The effect of strain from the protein on the _Fe-His frequency is most clearly seen for deoxy Hb, which gives different _Fe-His frequencies between the T (_Fe-His = 215 cm¹) and R (_Fe-His = 221 cm¹) structures (62). The low _Fe-His frequency is a consequence of the strain in the T quaternary structure, and the magnitude of strain is directly related to oxygen affinity (68). In the absence of strain, the _Fe-His frequency is close to that of deoxy Mb. The high _Fe-His frequency is also observed for a cavity mutant of Mb (226 cm¹), in which the axial His is replaced with Gly in the presence of exogenous imidazole (69). In this case, an exogenous imidazole acts as the proximal ligand, and the strain on the coordinated imidazole should be absent. Such a cavity mutant was also be prepared for sGC (70), heme-bound heme-oxygenase (71), and CooA (39), which show similar phenomena. Accordingly, the higher vibrational frequency of the _Fe-His mode in deoxy HemAT-Bs is indicative of less strain being imposed on the Fe-His bond compared with other hemoproteins listed in Table IV except for Mt Hb.

Desbois and coworkers (63) investigated the geometry dependence of _Fe-His frequencies and found that _Fe-His frequencies fall on a straight line with the inclination of 0.5 cm¹/deg when they are plotted against dihedral angles () formed by the imidazole plane and the nearest N(pyrrole)-Fe-N(pyrrole) axis. This correlation is different between the imidazolate and imidazole groups, although both give higher frequency for smaller  values. On the basis of this correlation, Desbois and co-workers (63) interpreted the high _Fe-His frequency (228-231 cm¹) of cytochrome c' in terms of a large  angle for strongly hydrogen-bonded imidazole coordination.

Recently, Andrew et al. (72) observed a relatively high _Fe-His frequency (231 cm¹) for Alcaligenes xylosoxidas cytochrome c'. According to the x-ray structure of this protein in the oxidized form, the  angle is 33°, and accordingly, the high _Fe-His frequency seemed to be due to deprotonation of proximal His. However, the x-ray structure for the oxidized form did not exhibit a hydrogen bonding counterpart of proximal His. Furthermore, the _Fe-CO versus _C-O correlation of this protein indicated the coordination of neutral imidazole at the trans position of CO. Therefore, Andrew et al. assumed that the structure in the proximity of heme changes with the oxidation state of iron as well as upon ligand binding. Although the x-ray crystallographic structure is not available for HemAT, its _Fe-His frequency is too high to interpret it in terms of a neutral imidazole coordination even for  = 0°, but its _Fe-CO versus _C-O correlation suggests the coordination of a neutral imidazole. Thus, the same kind of dilemma as seen for A. xylosoxidas cytochrome c' is present. Also in the case of HemAT-Bs, a structural change would take place in the proximal side of the heme upon the ligand binding, as is suggested for A. xylosoxidas cytochrome c'. Such a conformational change in the proximal heme pocket might be concerned with signal transduction in HemAT-Bs.

Porphyrin Skeletal Vibrational Modes in the Low Frequency Region-- In Table V, the low frequency porphyrin skeletal modes of HemAT-Bs(O₂), HemAT-Bs(CO), and Mb are listed. The peak assignment was mainly based on the study of Hu and co-workers (73).

The ₈ mode, which has been reported to involve the bending motions of peripheral substituents of porphyrin (74), is believed to be sensitive to changes that occur in the distal side of the heme pocket upon binding of a ligand (75). For the replacement of O₂ by CO, the largest shift of 11 cm¹ was observed for the ₈ mode of HemAT-Bs. The corresponding frequency shift in Mb is 2 cm¹. This suggests that binding of O₂ to the heme of HemAT-Bs appreciably changes a structure of the distal side of heme pocket through strong hydrogen bonding to the bound oxygen but binding of CO does little.

Relation between Structure and Kinetic Characteristics-- Resonance Raman spectroscopy of HemAT-Bs(O₂) has revealed that a distal amino acid residue is hydrogen bonding to the proximal atom of bound oxygen molecule as described above. Although Mt Hb (46) and Ascaris Hb (77) have a hydrogen bonding network similar to that of HemAT-Bs, they have an oxygen affinity very different from that of HemAT-Bs. The difference in oxygen affinity is mainly due to very small k_off values for Mt Hb and Ascaris Hb compared with that for HemAT-Bs (Table I), whereas all have similar k_on values except for Ascaris Hb. Indeed, the k_off value for HemAT-Bs(O₂) is greater than those for Mt Hb and Ascaris Hb by 100- and 5000-fold, respectively. We note here that k_off depends on the free energy of the whole protein in the oxygenated form relative to that of the transient state. When the strong hydrogen bond between the bound oxygen and distal residues causes some strain in other parts of the protein, the stabilization of the oxygenated form by the strong hydrogen bonds would be partially canceled. Such a feature is really seen for deoxy Hb with T structure, in which the inter-subunit hydrogen bonds are stronger and free energy is lower in the T than R state, but the Fe-His bond is weaker in the T than R state due to the strain imposed on the proximal His by the protein. In other words, the globin moiety is stabilized whereas the heme group is destabilized by the hydrogen bonds between subunits. The binding of a ligand to one heme of Hb is conveyed to another heme via changes in the inter-subunit hydrogen bonds. Accordingly, it is highly likely that HemAT-Bs utilizes a similar character of protein for signal transduction and thus to work as an O₂ sensor.

It is interesting to note that Paramecium caudatum (Pc) Hb exhibits similar kinetics and equilibrium parameters for oxygen binding to those of HemAT-Bs (23). Although Pc Hb possesses a distal E7 glutamine and a B10 tyrosine that form a hydrogen bond to the proximal and terminal oxygen atoms, respectively (23), it gives a k_off value (25.2 s¹) larger than those for Mt Hb and Ascaris Hb but close to that of HemAT-Bs (k_off = 23 s¹). The _Fe-O2 mode of Pc Hb appears at 563 cm¹ (23), which is similar to those of HemAT-Bs and Mt Hb (Table IV). Das et al. (23) have proposed that the oxy complex of Pc Hb is an equilibrium mixture of a hydrogen-bonded closed structure and non-hydrogen bonded open structure and that oxygen will dissociate preferentially from the open structure. Because the Fe-O₂ stretching Raman band of HemAT-Bs(O₂) is broad and likely to be composed of multiple bands as mentioned above, we cannot rule out the possibility that there are two oxygenated forms in HemAT-Bs(O₂), that is, hydrogen-bonded and non-hydrogen-bonded forms, and that oxygen is dissociated from the latter.

HemAT-Bs(O₂) shows an autoxidation rate significantly slower than those of LegHb and FixL as shown in Table II. The autoxidation rate of HemAT-Bs(O₂) is close to that of Mb. It is well known that the bound O₂ of Mb is stabilized by a hydrogen bond from the distal His, and accordingly, the bound O₂ of HemAT-Bs would be stabilized to a similar extent. An extremely small autoxidation rate constant for Mt Hb is probably caused by hydrogen bonding to the proximal oxygen atom within a closed distal heme pocket (26). The increased autoxidation rate of HemAT-Bs compared with Mt Hb, despite the strong hydrogen bonding to the proximal oxygen atom similar to that in Mt Hb, might be due to the presence of the second conformer with a relatively open distal heme pocket.

Primary Structure of HemAT-Bs-- It has been reported that the N-terminal region of HemAT-Bs and HemAT-Hs exhibit an amino acid sequence homology to Mb (9). Alignment of the heme binding domain of HemAT-Bs (10) with some globin sequences is shown in Fig. 8. Among these proteins, the proximal histidine at the F8 position is conserved, but amino acid residues at the B10 and E7 positions are not. It has been recently confirmed that His¹²³ corresponding to a histidine at the F8 position does indeed act as the proximal ligand of the heme in HemAT-Bs (10). A tyrosine at the B10 position in Mt Hb and Pc Hb hydrogen bonds to the bound oxygen (23, 47). In HemAT-Bs, however, the residue at the B10 position would be a leucine, an amino acid not capable of hydrogen bonding, as is the case for Sw Mb. The distal residue at the E7 position in Sw Mb and Pc Hb, His⁶⁴ and Gln⁴², respectively, also hydrogen bonds to the bound oxygen (23, 78). In HemAT-Bs, the residue at the E7 position would be a serine, Ser⁸⁷. Although a serine is a possible candidate for an amino acid residue hydrogen bonding to the bound oxygen, there is not any direct evidence at present that the Ser⁸⁷ is involved in the hydrogen bonding network in HemAT-Bs(O₂).

View larger version (37K): [in this window] [in a new window]   Fig. 8.   Alignment of the heme-binding domain of HemAT-Bs with some globin sequences. The distal positions at B10 and E7, and the proximal histidine at F8 position are indicated. Abbreviations used are: Sw Mb, sperm whale myoglobin; Mt Hb, M. tuberculosis hemoglobin; Pc Hb, P. caudatum hemoglobin.

In a preliminary resonance Raman study of S87A HemAT-Bs, in which Ser⁸⁷ is replaced by Ala, the _Fe-O2 mode was observed at 563 cm¹. In the case of Chlamydomonas eugametos Hb, the mutation of the B10 Tyr or E7 Gln, both of which hydrogen bond to the bound oxygen, results in the up-shift of the _Fe-O2 mode by 7 or 15 cm¹, respectively (79). Compared with the case for C. eugametos Hb, the _Fe-O2 mode is not perturbed as much by the mutation of Ser⁸⁷ to Ala in the case of HemAT-Bs. Thus we need additional experiments to find out if Ser⁸⁷ hydrogen bonds to the bound oxygen and to determine the distal amino acid residue that hydrogen bonds to the bound oxygen.

Oxygen Sensing and Signal Transduction Mechanisms for HemAT-Bs-- A conformational change induced by the ligand binding to the heme is a crucial step for the effector sensing and signal transduction in the heme-based sensor proteins. For the CO sensor protein CooA, CO replaces one of the endogenous axial ligands, Pro², upon the binding to the six-coordinated ferrous heme, which results in a conformational change in the N-terminal region and/or in a relocation of the heme with a conformational change of the proximal heme pocket (39, 80, 81). The ligand exchange between Pro² and CO is the first step of the CO sensing in the case of CooA. This sensing mechanism would be specific for CooA among CooA, FixL, and HemAT-Bs, because only the heme in CooA is six-coordinate in the resting state (20, 80). FixL and HemAT-Bs contain a five-coordinate heme in their resting state, and oxygen binds to a vacant distal site of the heme.

In the case of FixL, the sensing mechanism is completely different from that for CooA. Gong et al. (82) have reported that oxygen binding to the heme in FixL induces rearrangements of the hydrogen bonding network between the heme 6- and 7-propionates and amino acid residues (Arg²⁰⁶ and His²¹⁴) in the F/G loop region that results in the flattening of the heme plane upon the ligand binding. They propose that the signal transduction might be mainly driven by the motion of the heme plane induced by oxygen binding (82). On the other hand, Mukai et al. (83) have proposed that the interaction between Ile²⁰⁹ (and/or Ile²¹⁰), which are located on the F/G loop region, and the iron-bound oxygen is essential for oxygen sensing by FixL.

Although both of FixL and HemAT-Bs are oxygen sensor proteins, they show no structural homology. HemAT-Bs is a member of globin-coupled sensor proteins (10), whereas FixL is a member of the PAS domain superfamily (84). Furthermore, oxygen binding and autoxidation kinetics are completely different between FixL and HemAT-Bs, as described in this work. These results suggest that the oxygen-sensing mechanism for HemAT-Bs will be different from that for FixL.

Oxygen binding to the heme iron in Hb results in a movement of the iron ion into the heme plane along with the movement of the proximal histidine and F helix, which is a trigger of the allosteric control for Hb. As described in this work, HemAT-Bs shows amino acid sequence homology and similar spectroscopic and ligand binding properties to those of Mbs and Hbs. These results suggest that a similar conformational change, i.e. a movement of the heme iron with the proximal ligand and F helix, upon the oxygen binding could be a trigger of the signal transduction for HemAT-Bs. Given that this is the case, however, CO should have the same effect as O₂. Because CO is not a physiological effector of HemAT-Bs, HemAT-Bs should discriminate between O₂ and CO. Hydrogen bonding to the bound oxygen is considered to play a crucial role for the ligand discrimination as discussed above, but further studies are required to determine if this is the case.

	ACKNOWLEDGEMENTS

We thank Professors I. Okura and T. Kamachi of Tokyo Institute of Technology for help in laser flash photolysis experiments.

	FOOTNOTES

^* This work was supported by a Grant-in-Aid for Scientific Research of Priority Areas on Metal Sensors 12147203 and by 12680631 (to S. A.), 12740361 (to H. N.), and 12045264 (to T. K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.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 may be addressed: Tel.: 81-761-51-1681; Fax: 81-761-51-1149; E-mail: aono@jaist.ac.jp.

To whom correspondence may be addressed: Tel.: 81-564-55-7340; Fax: 81-564-55-4639; E-mail: teizo@ims.ac.jp.

Published, JBC Papers in Press, January 30, 2002, DOI 10.1074/jbc.M112256200

	ABBREVIATIONS

The abbreviations used are: MCP, methyl-accepting chemotaxis protein; HemAT-Bs, HemAT from B. subtilis; HemAT-Hs, HemAT from H. salinarum; HemAT-Bs(O₂), the oxy form of HemAT-Bs; HemAT-Bs(CO), the CO-bound form of HemAT-Bs; sGC, soluble guanylate cyclase; Mb, myoglobin; Hb, hemoglobin; Mb(CO), the CO-bound form of myoglobin; FixL(O₂), the oxygen-bound form of FixL.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES