Activation of Heme-regulated Eukaryotic Initiation Factor 2 (cid:1) Kinase by Nitric Oxide Is Induced by the Formation of a Five-coordinate NO-Heme Complex

Heme-regulated eukaryotic initiation factor 2 (cid:1) kinase (HRI) regulates the synthesis of hemoglobin in reticulocytes in response to heme availability. HRI contains a tightly bound heme at the N-terminal domain. Earlier reports show that nitric oxide (NO) regulates HRI catalysis. However, the mechanism of this process remains un-clear. In the present study, we utilize in vitro kinase as-says, optical absorption, electron spin resonance (ESR), and resonance Raman spectra of purified full-length HRI for the first time to elucidate the regulation mechanism of NO. HRI was activated via heme upon NO binding, and the Fe(II)-HRI(NO) complex displayed 5-fold greater eukaryotic initiation factor 2 (cid:1) kinase activity than the Fe(III)-HRI complex. The Fe(III)-HRI complex exhibited a Soret peak at 418 nm and a rhombic ESR signal with g values of 2.49, 2.28, and 1.87, suggesting coordination with Cys as an axial ligand. complex, neutral Cys thiol and His coordinate with the heme iron. The conforma- tional changes induced lead to a 2.4-fold increase in kinase activity, compared with the Fe(III)-HRI complex. Following binding of NO to the Fe(II)-HRI complex, a five-coordinate Fe(II)-HRI(NO) complex is formed. Conformational changes in- duced by dissociation of both endogenous axial ligands (His/neutral Cys) and for- mation of the five-coordinate NO-heme complex transduce a signal to the kinase domain and increase HRI activity up to 5-fold, compared with the Fe(III)-HRI complex.

Heme-regulated inhibitor kinase (HRI), 1 a member of the eukaryotic initiation factor 2␣ (eIF2␣) kinase family, binds heme as a prosthetic group (1)(2)(3). The family of eIF2␣ kinases (including HRI, double-stranded RNA-activated protein kinase (PKR), PKR-like endoplasmic reticulum-related kinase (PERK), and general control of amino acid biosynthesis kinase (GCN2) (4)) catalyzes phosphorylation of eIF2␣. Phosphorylated eIF2 binds eIF2B and inhibits the guanine nucleotide exchanging activity required for protein synthesis. HRI regulates globin synthesis in reticulocytes in response to heme availability. Earlier studies show that HRI contains two hemebinding sites. One heme is tightly bound to the N-terminal domain, whereas the other interacts weakly with the catalytic domain (5). Under conditions of heme deficiency, HRI becomes active and phosphorylates the ␣-subunit of eIF2 at Ser 51 . It is proposed that interaction of the second heme with the catalytic domain suppresses catalysis, whereas heme deficiency leads to dissociation of heme from the catalytic domain and initiation of catalysis. In iron-deficient HRI Ϫ/Ϫ mice, globins are devoid of the heme aggregate within red blood cells and precursors, resulting in hyperchromic, normocytic anemia with decreased red cell blood count, compensatory erythroid hyperplasia, and accelerated apoptosis in bone marrow and spleen (6).
HRI is additionally activated by other environmental and chemical stimuli, including nitric oxide (NO), heat shock, oxidative stress, denatured proteins, and sulfhydryl-reactive reagents (3). Specifically, NO affects reticulocyte HRI in situ and in vitro complexed with heat shock protein (HSP90) and its partner proteins (7,8). To determine the mechanism of NOinduced regulation of HRI, the heme environment of the isolated N-terminal heme-binding domain (HRI-NTD) was investigated using physicochemical methods and site-directed mutagenesis (9 -11). Ishikawa et al. (10) demonstrated that NO-bound HRI-NTD forms a six-coordinate NO-heme complex and concluded that the molecular mechanism of NO-induced regulation of HRI is distinct from that of soluble guanylate cyclase (sGC), whereby formation of the five-coordinate NOheme complex is essential for activity. However, to date, there have been no reports on the biochemical and physicochemical characterization of the HRI holoenzyme. Elucidation of the roles of heme and NO in catalysis as well as the structure of full-length HRI in the absence of the second heme should be particularly useful to determine the molecular mechanism of globin synthesis in reticulocytes.
In the present report, we examine, for the first time, the effects of the redox state of heme and NO on catalysis by full-length HRI enzyme in the absence of the second heme, using an in vitro kinase assay. Various spectroscopic methods (including optical absorption, ESR, and resonance Raman) are applied to the system. Our data show that one of the axial ligands of the Fe(III) complex is thiolate and that NO-induced activation is accompanied by the formation of a five-coordinate NO-heme complex in HRI. These findings are distinct from the results obtained for HRI-NTD. We further discuss the heme environment of HRI and the possibility that HRI is a novel heme-based sensor protein (12)(13)(14).
Protein Expression and Purification-His 6 HRI was expressed from E. coli BL21 (DE3) Codon Plus harboring pET-28a/HRI (15) and purified as described previously (16,17) with some modifications. Cell lysates containing apo-His 6 HRI were reconstituted with 50 M hemin dissolved in 0.1 M NaOH for 30 min at 4°C (except for preparation of the apo form), fractionated with ammonium sulfate, and subjected to DEAE-cellulose (DE52, Whatman, Maidstone, UK) and Ni 2ϩ -nitrilotriacetic acid-agarose (Qiagen K.K., Tokyo, Japan) chromatography. Concentrations were determined using the Coomassie Brilliant Blue dye binding method for protein (Nakalai Tesque, Kyoto, Japan) and the pyridine hemochromogen method for heme. His 6 eIF2␣ was expressed and purified according to a previous report (18).
In Vitro Protein Kinase Assay-The in vitro protein kinase assay was conducted as described previously, with some modifications (19). The kinase reaction mixture (20 l) containing 20 mM Tris-HCl, pH 7.7, 2 mM Mg(OAc) 2 , 60 mM KCl, 2 g of His 6 eIF2␣, 500 ng of His 6 HRI, and 50 M ATP was incubated at 15°C for 10 min. This process was performed under anaerobic conditions by monitoring absorption spectra. NO solutions were prepared using the NO donor (NOC9; Sigma) in kinase reaction buffer. The reaction was stopped by adding Laemmli's sample buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% 2-mercaptoethanol, 2% SDS, 0.002% bromphenol blue) for 10 min at 95°C and subjected to 10% SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad). Phosphorylated proteins were detected by immunoblotting with an anti-phosphorylated eIF2␣ antibody.
Optical Absorption Spectra-Spectral experiments under aerobic conditions were performed on a Shimadzu Multispec 1500 spectrophotometer maintained at 25°C. Anaerobic spectral experiments were conducted on a Shimadzu UV-1650 spectrophotometer at 15°C in an anaerobic glove box (Hirasawa, Tokyo, Japan).
ESR Measurements-Fe(III)-HRI complexes were prepared in 50 mM Tris-HCl, pH 8.0, containing 12% glycerol at 25°C for ESR measurements. ESR spectra were recorded on a JEOL FE-3X spectrometer (Tokyo, Japan) at 20 K. The magnetic field was calibrated using an NMR gauss meter (Echo Electronics; model EFM-2000), and temperature was controlled with an Oxford ITC4 cryosystem.
The Fe(II)-NO complex was formed by adding NO gas to the sodium dithionite-reduced anaerobic Fe(II)-HRI sample (360 M) in 50 mM Tris-HCl, pH 8.0. NO gas was formed under anaerobic conditions by reducing NaNO 2 with an aqueous solution of sodium ascorbate in a Thunberg-type tube, with a side arm on a vacuum line. ESR spectra were recorded on a JEOL JES-TE200 spectrometer at 77 K, using a liquid nitrogen Dewar.
Resonance Raman Measurements-The Fe(III)-HRI complex (25 M, in 50 mM Tris-HCl, pH 8.0) was placed in an air-tight spinning cell with a rubber septum and reduced by the addition of sodium dithionite at a final concentration of 10 mM. 15 NO and 13 C 18 O (Shoko, Tokyo, Japan) gas was collected from a gas cylinder.
Raman scattering was performed by excitation at 413.1 or 406.7 nm with a krypton ion laser (Spectra Physics; model 2016). Excitation light was focused into the cell at laser power of 5 mW for the Fe(II)-HRI and Fe(III)-HRI complexes, and 0.1-0.2 mW for the Fe(II)-HRI(CO) and Fe(II)-HRI(NO) complexes to avoid photolysis. Raman spectra were detected with a CCD camera attached to a single polychrometer (Ritsu Oyokagaku; model DG-1000). Raman shifts were calibrated with indene.

RESULTS
Protein Purification-Purified His 6 HRI was more than 95% homogenous, as determined by SDS-PAGE followed by staining with Coomassie Brilliant Blue R250 (Fig. 1A). Purified HRI reconstituted with hemin displayed an apparent molecular mass of 91,700 Da and contained 0.84 mol of heme/mol of protein, indicating a stoichiometry of one heme at the N-terminal domain per subunit. Gel filtration analysis using Superose 6 (Amersham Biosciences) revealed that heme-reconstituted His 6 HRI has a molecular mass of nearly 560 kDa (data not shown), similar to previously described His 6 HRI (640 kDa) and native HRI (420 kDa) (17). It is well established that native HRI from reticulocyte forms a homodimer with elongated shape, although the molecular mass estimated by gel filtration appears variable, depending on experimental conditions. The difference in molecular mass (80 kDa) between the present and previous results may be due to different conditions of gel filtration column chromatographies. The yield of HRI was low at 0.2 mg/liter of culture.
Optical Absorption Spectra-We examined the absolute absorption spectra of highly purified HRI under various conditions. Absorption spectra of the reconstituted Fe(III), Fe(II), and Fe(II)-CO complexes of HRI are depicted in Fig. 1B. Spectral parameters are compared with those of other heme proteins in Table I. Soret absorption maxima of the Fe(III), Fe(II), and Fe(II)-CO forms of HRI were observed at 418, 426, and 421 nm, respectively. Spectral data on the Fe(III) complex revealed characteristic six-coordinate low spin heme complexes similar to those of CooA (a CO-sensing transcription factor from Rhodospirillum rubrum) (20,21), CBS (22)(23)(24), and P450cam (25). The Soret absorption of the Fe(III) complex appeared comparatively broad with a shoulder at around 360 nm, distinct from that of the isolated heme binding domain, HRI-NTD (Fig. 1, B and C). The optical absorption spectrum of the Fe(II)-HRI complex displayed peaks at 426, 531, and 560 nm, signifying six-coordinate low spin heme. The spectrum of the Fe(II)-HRI complex is similar to those of CooA (20,21) and the acidic form (pH 6.0) of CBS (23) that contains neutral Cys thiol and His ( Table I). The peak of the Fe(II)-HRI(CO) complex in the Soret region was located at around 420 nm, compared with 450 nm for cytochrome P450, suggesting that the proximal ligand is unlikely to be thiolate as in HRI-NTD (11).
Upon the addition of NO to the Fe(II)-HRI complex, the Soret band shifted from 426 to 398 nm and decreased in intensity (Fig. 1B). The addition of NO to the Fe(III)-HRI complex resulted in a shift in the Soret peak to 398 nm, indicating reductive nitrosylation of the complex, leading to the formation of a Fe(II)-HRI(NO) complex (data not shown), as reported for Hb (26). The Soret band of the Fe(II)-HRI(NO) complex at around 400 nm is characteristic of a five-coordinate NO-heme complex, analogous to sGC (27), CBS (28), CooA (29), and cytochrome cЈ from denitrifying bacteria (30,31), and is distinct from spectra of the six-coordinate NO-heme, which contain Soret bands at around 415-425 nm ( Table I). The spectral pattern of HRI in response to NO is different from that of HRI-NTD, which displays a Soret band at 422 nm characteristic of a six-coordinate NO-heme (Fig. 1C). Despite the loss of both endogenous axial ligands following NO binding to the Fe(II)-HRI complex, the five-coordinate NO-heme did not dissociate from HRI on a gel filtration column.
A spectrum of the Fe(II)-O 2 complex was not obtained due to its high autoxidation rate.
In Vitro eIF2␣ Kinase Assay-A previous study on HRI in rabbit reticulocyte lysates showed that NO binds to the heme iron and stimulates kinase activity, whereas binding of CO to the heme iron suppresses kinase activity (7). It is possible that in this system, regulation of HRI by NO or CO is mediated through interaction with HSP90 and its partner protein or modification of cysteine by NO. The effects of NO and CO on eIF2␣ kinase activities of highly purified HRI require examination to confirm these hypotheses. In this report, we investigate whether CO and NO directly affect eIF2␣ kinase activity of HRI via interactions with heme.
To determine the effects of NO and CO on HRI activity, we employed highly purified protein (expressed in E. coli at 15°C) in an in vitro kinase assay, using His 6 eIF2␣ as a substrate. As shown above, purified HRI contains only one heme in the N-terminal domain and releases the heme from the second binding site in the catalytic domain. Heme redox and coordination states were simultaneously monitored with a spectrophotometer. The Fe(II)-HRI complex displayed 2.4-fold higher kinase activity than the Fe(III)-HRI complex oxidized with potassium ferricyanide ( Fig. 2A). Upon binding of NO to the Fe(II)-HRI complex, kinase activity was increased by 2-fold and eventually up to 5-fold, compared with the Fe(III)-HRI complex, at a concentration of 10 M NOC9. NO-induced activation was not observed in the heme-deficient apo form of HRI (Fig.  2C). Rather, NO inhibited eIF2␣ activity in a dose-dependent manner. Therefore, it is evident that NO-induced activation is closely associated with the heme prosthetic group. The Fe(II)-HRI(CO) complex displayed 1.2-fold higher activity than the CO-free Fe(III)-HRI complex (Fig. 2B).
Notably, purified HRI did not incorporate 32 P i in the in vitro kinase assay using [␥-32 P]ATP as a substrate, suggesting that the protein is already hyperphosphorylated in E. coli (15). Therefore, in comparison with previous reports, basal activity on eIF2␣ was high and not significantly down-regulated upon the addition of heme (7). This finding indicates that purified HRI is less sensitive to inhibition induced by binding of the second heme. An earlier investigation shows that HRI expressed in E. coli at 13°C is hyperphosphorylated (particularly at Thr 485 in the activation loop) (15).
We additionally examined HRI activity on eIF2␣ following digestion of the His tag with thrombin. No significant differences in HRI activity were observed between substrate proteins with and without the tag, thus eliminating the possibility of His tag interaction.
ESR Spectra-The ESR spectrum of the Fe(III)-HRI-NTD complex displayed major signals at g ϭ 3.05, 2.20 and 1.46, implying that low spin heme has His/His as axial ligands, analogous to bovine liver cytochrome b 5 (g ϭ 3.03, 2.23, and 1.43) (10). As shown in Fig. 3A, the ESR spectrum of the Fe(III)-HRI complex contained signals at g ϭ 2.49, 2.28, and 1.87, characteristic of low spin heme. However, these g values are similar to those of CBS (23,32) and CooA (33)(34)(35), which contain cysteine thiolate and nitrogenous ligands as axial ligands to the heme (Table II). Crystal field analyses of g values for low spin Fe(III) hemoproteins and model complexes are useful to identify heme axial ligands. Crystal field data ob- tained using Bohan's method (convention I) (36) applied to HRI and other related hemoproteins are listed in Table II. A crystal  field diagram based on data from Table II is depicted in Fig. 3B. It is clear that HRI belongs to a family with His/Cys ligands (area i), whereas HRI-NTD is part of a family with His/His ligands (area ii). Cytochrome P450 proteins with Cys and water/hydroxyl anion as axial ligands are identified in area iii.
Resonance Raman Spectra-To further clarify the nature of the heme environment of HRI, resonance Raman spectra of the Fe(III), Fe(II), Fe(II)-CO, and Fe(II)-NO complexes were analyzed. Spectra of the Fe(II)-HRI and Fe(III)-HRI complexes in the high frequency region are depicted in Fig. 5. Bands at 1360 and 1374 cm Ϫ1 for the Fe(II)-HRI and Fe(III)-HRI complexes, respectively, were assigned as redox-sensitive 4 bands. Spinand coordination state marker bands ( 3 ) were observed at 1470 and 1493 cm Ϫ1 for the Fe(II)-HRI complex, which represent the five-coordinate high spin and six-coordinate low spin states, respectively (Fig. 5a). On the other hand, in the Fe(III)-HRI complex, 3 was located at 1503 cm Ϫ1 , signifying the six-coordinate low spin state (Fig. 5b). A shoulder at 1494 cm Ϫ1 ascribed to a five-coordinate high spin complex was observed as a minor component in Fe(III)-HRI. Upon binding of CO and NO to the Fe(II)-HRI complex, 3 bands shifted to 1496 and 1507 cm Ϫ1 , respectively (data not shown).
To characterize the proximal ligand in the Fe(II)-HRI complex, a resonance Raman spectrum of photolyzed Fe(II)-HRI(CO) was obtained with 435.7-nm 10-ns pulses. The spectrum of the photolyzed Fe(II)-HRI(CO) complex displayed features characteristic of a transient five-coordinate high spin Fe(II)-HRI complex. The iron-His stretching band ( Fe-His ) was observed at 219 cm Ϫ1 in Fe(II)-HRI (Fig. 5, inset) and 226 cm Ϫ1 in Fe(II)-HRI-NTD (data not shown). The Fe-His band (corresponding to iron-His bond tension) in this case is compared with those of other heme proteins in Table III. The data imply that iron-His bond tension of HRI is lower than those of sGC (204 cm Ϫ1 (40)), CooA (211 cm Ϫ1 (41)), and SmFixL* (soluble truncated domain of Sinorhizobium meliloti (formerly Rhizobium meliloti) FixL protein (46 kDa)) (209 cm Ϫ1 (42)) but comparable with that of sperm whale myoglobin (Mb) (220 cm Ϫ1 ).
Since the Fe-CO and C-O stretching frequencies are sensitive to the electrostatic and steric interactions of surrounding groups, resonance Raman spectra of the CO adducts of heme proteins provide valuable information of the heme environment. Low and high frequency regions of the resonance Raman spectra of the Fe(II)-HRI(CO) complex are shown in Fig. 6  ingly, we assigned the 492-and 1967-cm Ϫ1 bands to Fe-CO and C-O modes. In Fig. 6, the Fe-C-O bending mode, ␦ Fe-C-O , was identified at 571 cm Ϫ1 , indicating a slightly bent structure. Comparison of the CO environments between HRI and HRI-NTD revealed that the Fe-CO (492/494 cm Ϫ1 ) and C-O (1967/ 1963 cm Ϫ1 ) stretching modes are not significantly different. Thus, CO binding environments of HRI and HRI-NTD are similar, whereas the original ligands expelled by CO are different (Cys and His for HRI and HRI-NTD, respectively).

HRI as a Heme-based NO Sensor Protein
A new class of hemoproteins has been identified that comprise a heme-containing sensor domain and an effector domain catalyzing enzymatic reactions or binding to DNA as transcription factors. Among these, heme-based sensor proteins sense diatomic gas molecules. For example, HemAT (a heme-containing aerotaxis transducer from Bacillus subtilis) and FixL sense O 2 , CooA and a gas-sensitive transcription factor identified in the forebrain (NPAS2) sense CO, and sGC senses NO (13,14,43). A heme-based sensor is defined as a protein in which a heme-binding domain controls the activity of another domain within the protein. In the present report, we demonstrate that HRI regulates activity in response to heme redox and coordination states with NO. However, this regulation is not observed for apo-HRI in the absence of heme. Since HRI is activated upon NO binding to heme, protein conformational changes at the heme-binding domain induced by the formation of five-coordinate NO-heme and/or dissociation of both endogenous axial ligands may be transduced to the kinase domain, leading to increased kinase activity. Base on these findings, we propose that HRI is a novel heme-based NO sensor protein.

Heme Coordination Structure and Heme
Environment of HRI Fe(III)-HRI Complex-Optical absorption, ESR, and resonance Raman spectra reveal that the Fe(III)-HRI complex is a six-coordinate low spin heme with Cys as one of axial ligands. In the crystal field diagram (Fig. 3B), HRI is located within the solid line area (i), HRI-NTD is present within the dashed line area (ii), and cytochrome P450 proteins are observed in the dotted line area (iii). The diagram clearly shows that HRI and CBS have the same coordination structure, which is distinct from that of CooA. CooA contains Cys and an N-terminal Pro residue as axial ligands, whereas P450 has Cys and water at the resting state. Therefore, we speculate that the Fe(III)-HRI complex has His and Cys residues as axial ligands, similar to CBS. The coordination structure differences between HRI (His/ Cys) and HRI-NTD (His/His) appear to be a result of deletion of the C-terminal catalytic domain in HRI-NTD. Truncation of the C terminus may lead to different heme environments, due to loss of interactions between NTD and other domains.
The Soret band of HRI is broad, with a peak at 418 nm. The peak position is close to that of the heme complex with water or hydroxyl anion as an axial ligand trans to Cys, as deduced from  3-7), and the Fe(III)-HRI complex (lane 8). B, effect of CO on catalysis by heme-bound HRI. eIF2␣ kinase activities were obtained using Fe(II)-HRI complexes in the presence of CO-saturated (ϳ1 mM) buffer (lane 9) and the Fe(III)-HRI complex (lane 10). C, effect of NO on catalysis by heme-free HRI. Kinase activities were assayed using the conditions employed in A.  in C correspond to lanes 1-8 in A. Note that heme-free HRI has basal eIF2␣ kinase activity, since hyperphosphorylated HRI was used in the present study (cf. in vitro eIF2␣ kinase assay under "Results"). spectra of low spin P450 complexes (Cys and water: axial ligands) with a 417-nm peak (Table I) (25). Binding of 2phenylimidazole, benzimidazole, or indole to P450 leads to optical absorption spectra characteristic of low spin complexes coordinated with "abnormal nitrogen" (25). Soret bands of low spin complexes bound to abnormal nitrogen are observed between 416 and 420 nm. However, the crystal structure of 2-phenylimidazole-bound P450 reveals that water is bound to the heme iron (91). An additional shoulder is observed at around 395 nm in the Soret region of the Fe(III)-HRI complex, which is ascribed to five-coordinate Cys-Fe(III) (44) (Fig. 1B). Moreover, resonance Raman spectra indicate that a small portion of a five-coordinate heme complex at 1494 cm Ϫ1 is mixed with the major six-coordinate Fe(III)-HRI complex (Fig. 5b). Thus, the possibility that the peak at 418 nm is observed as a result of mixing an absorption peak of the Cys-Fe(III)-His complex at around 425 nm with that of the five-coordinate Cys-Fe(III) complex at around 395 nm cannot be discounted.
To account for the inconsistencies between ESR and optical absorption spectra of the Fe(III)-HRI complex, we speculate that temperature-or concentration-dependent ligand switching occurs in HRI. ESR spectra were obtained at 20 K for concentrated enzymes (100 M) in the presence of glycerol, whereas optical absorption spectra were obtained at 25°C for diluted enzymes (less than 1 M). Similar facile ligand switching has been observed for other heme sensor enzymes. For example, optical absorption bands of the Fe(III) complex of CooA were reported at 418 nm (20) or 424 nm (21), due to differences in purification or sample preparation for spectroscopy. Additionally, coordination structural changes sensitive to glycerol concentration were suggested for CBS in terms of ESR spectroscopy (23).
Earlier studies report that partially purified HRI from reticulocytes has an absorption peak at around 418 nm (45). However, baculovirus-expressed HRI has been purified as a hemoprotein with absorption peaks at 424 nm in the Soret region and around 550 nm in the visible region (2). E. coliexpressed HRI exhibits absorption peaks at 422 and 550 nm in "difference spectra" (17). Spectral data obtained in the present study are similar to those of native purified proteins (45). These results confirm the flexibility of HRI with regard to facile ligand switching, with equilibrium between Cys-Fe(III)-His, Cys-Fe(III)-H 2 O/OH Ϫ , and five-coordinate Cys-Fe(III) species.
Fe(II)-HRI Complex-Data from optical absorption spectra suggest that Fe(II)-HRI is a six-coordinate low spin heme complex, similar to CooA (20,21) and the acid form of CBS (23). The axial ligands of the acid form of Fe(II)-CBS are suggested to be His and neutral Cys thiol. Recently, Perera et al. (46) demonstrated that neutral thiol can bind as a ligand to several Fe(II) heme proteins. Accordingly, in view of the observed Soret peak of Fe(II)-HRI at 426 nm, we suggest that neutral Cys thiol binds as the axial ligand trans to His (Fig. 8).
Resonance Raman spectra indicate that the Fe-His frequency of Fe(II)-HRI (219 cm Ϫ1 ) is lower than that of Fe(II)-HRI-NTD (226 cm Ϫ1 ) (Table III). In general, the Fe-His frequency is sensitive to a number of factors, including the hydrogen-bonding status of N ␦ nitrogen, strain imposed on the axial His by the protein moiety, and the geometry of bound imidazole (47)(48)(49)(50). For example, the high Fe-His frequency at 245 cm Ϫ1 for cytochrome c peroxidase (a result of the strong hydrogen bond between the axial His and Asp 235 ) shifts to 205 cm Ϫ1 following disruption of the hydrogen bond (51,52). Mb has a weak hydrogen bond on the axial His, which displays a Fe-His frequency at around 220 cm Ϫ1 . Breakage of the hydrogen bond of Mb leads to a shift in the Fe-His frequency to 223 cm Ϫ1 (53). On the other hand, for the cavity mutant of Mb, in which the axial His 93 is replaced with Gly in the presence of exogenous imidazole (H93G), the exogenous imidazole acts as the proximal ligand, and thus the strain on coordinated imidazole should be absent. The Fe-imidazole frequency of the cavity mutant shifted to 225 cm Ϫ1 , and a geometrical change of imidazole was observed concomitantly in that dihedral angles () between the projection of imidazole plane and the nearest N(pyrrole)-Fe-N(pyrrole) axis altered from 0°(wild type) to 45°(H93G) (54 -56). Therefore, the geometry of His bound to the heme iron is also an important factor in the origin of the Fe-His frequency associated with hydrogen-bonded imidazole coordination (49). We propose that the higher frequency of the Fe-His mode in Fe(II)-HRI-NTD (226 cm Ϫ1 ) compared with Fe(II)-HRI (219 cm Ϫ1 ) is collectively imposed by several factors that affect the Fe-His bond upon isolation of the N-terminal heme-binding domain.
The Fe-His frequency at 219 cm Ϫ1 in the Fe(II)-HRI complex is comparable with that of Mb (220 cm Ϫ1 ). Interestingly, Mb forms a six-coordinate NO-heme, whereas HRI forms a fivecoordinate NO-heme complex upon NO binding. Among the heme proteins that form the five-coordinate NO-heme complex, the Fe-His frequencies of sGC (203 and 204 cm Ϫ1 ), CooA (211 cm Ϫ1 ), and SmFixL* (209 and 212 cm Ϫ1 ) are relatively low, whereas those of Alcaligenes xylosoxidans cytochrome cЈ and ovine prostaglandin endoperoxide H synthase (oPGHS) are observed at 231 and 222 cm Ϫ1 , respectively. X-ray crystallographic analyses of A. xylosoxidans cytochrome cЈ reveal that the imidazole ring of His 120 is oriented such that is ϳ33°in the Fe(II) state (57). In the crystal structure of ovine prostaglandin endoperoxide H synthase, the imidazole ring of His 388 is oriented such that is ϳ26°in the Fe(III) state (50). Therefore, it is speculated that iron-His bond cleavage of HRI upon NO binding is associated, at least in part, with the geometry of the proximal His residue. However, a contribution of the hydrogen bond with the proximal His N ␦ proton and protein strain to iron-His cleavage cannot be ruled out. Structural differences between the heme distal sides trans to His of HRI, Mb, A. xylosoxidans cytochrome cЈ, and ovine prostaglandin endoperoxide H synthase should also be considered. Specifically, a distal mutant, G117I, of CooA (with Fe-His frequency at 220 cm Ϫ1 ) forms a five-coordinate Fe(II) complex, whereas the wildtype protein ( Fe-His frequency at 211 cm Ϫ1 ) forms a six-coordinate Fe(II) complex (41,58,59).
Fe(II)-HRI(CO) Complex-The correlation between Fe-CO and C-O frequencies is shown in Fig. 6B. The Fe(II)-HRI(CO) complex contains a neutral His as the proximal ligand trans to CO (60). The CO molecules of Fe(II)-CO complexes, which contain resonance Raman bands in the central part of the Fe-CO versus C-O plot, do not interact strongly with nearby amino acid residues. Therefore, bound CO of HRI is unlikely to interact directly or strongly with distal amino acids, despite the observed dissociation of Cys from the heme upon CO binding. The Cys residue should dissociate from the heme plane follow-ing CO binding. The observed C-O stretching frequency (1967 cm Ϫ1 ) implies that the CO molecule is located in a somewhat hydrophobic environment.    Table III that contain histidine as the proximal ligand. Open triangles are specific for cytochrome P450s and nitric-oxide synthases that contain cysteine as a proximal ligand (60).  Table III; closed squares, phenyl-modified derivatives of the Fe(II)-TPP(NO) complexes (62).  (62)) are present. Kinase Activity and Coordination Structure-Our in vitro eIF2␣ kinase assay data indicate that the Fe(II)-HRI complex has higher activity than the Fe(III)-HRI complex, and formation of the five-coordinate NO-heme complex further enhances catalysis. The heme coordination structures proposed in this study are shown in Fig. 8. In the Fe(III)-HRI complex, the heme iron is possibly coordinated with endogenous Cys and His or water/hydroxyl anion. When the Fe(III)-HRI complex is reduced to Fe(II)-HRI, His and neutral Cys thiol appear to coordinate to the heme iron and to form a six-coordinate low spin complex. In Fe(II)-HRI(CO), Cys is dissociated from the heme iron. Upon binding to the Fe(II)-HRI complex, NO interacts with the heme iron either distal or proximal to the heme plane, resulting in the formation of a five-coordinate NO-heme complex.
To identify the axial ligand for heme, we prepared Ala mutants of His 75 , His 78 , and His 80 (His 78 , His 81 , and His 83 for rabbit, respectively) and performed preliminary in vitro kinase assay experiments. The H75A protein was expressed similarly to the wild-type protein but did not display kinase activity, whereas the H80A mutant exhibited activity comparable with that of the wild-type enzyme. The H78A mutant was degraded at the expression stage. In view of the data, we speculate that His 75 is an axial ligand of HRI. To identify the axial ligands of the Fe(III)-HRI and Fe(II)-HRI complexes, further site-directed mutagenesis and spectroscopic studies are currently under way.

Mechanism of NO-induced Activation of HRI
Conformational changes induced by ligand binding to the heme are crucial for effector sensing and signal transduction in heme-based sensor proteins. Previous spectral studies on HRI-NTD show that the five-coordinate NO-heme complex formed via cleavage of the Fe-His bond is not necessary for HRI activation (10). In this study, we use full-length HRI for in vitro kinase assay and spectral measurements and demonstrate that HRI is activated by NO, forming the five-coordinate NO-heme complex. These results imply a similar activation mechanism to that of sGC.
The activation mechanism of binding of NO to sGC has been discussed for A. xylosoxidans cytochrome cЈ, in view of crystallographic and spectroscopic data (57,63,64). One proposal is that NO binds to the vacant distal side of the heme trans to proximal His ligand, forming a six-coordinate NO-heme complex. Due to the repulsive trans effect of bound NO, proximal His dissociates from the heme, forming a five-coordinate NOheme with 300-fold increased activity (63,65). Therefore, it is FIG. 8. Hypothetical model for the activation of HRI and proposed heme coordination structures. In the Fe(III)-HRI complex, heme iron is coordinated to Cys on one side and to His, water/hydroxyl anion or is vacant on the other side. Upon reduction of the Fe(III)-HRI complex, neutral Cys thiol and His coordinate with the heme iron. The conformational changes induced lead to a 2.4-fold increase in kinase activity, compared with the Fe(III)-HRI complex. Following binding of NO to the Fe(II)-HRI complex, a five-coordinate Fe(II)-HRI(NO) complex is formed. Conformational changes induced by dissociation of both endogenous axial ligands (His/neutral Cys) and formation of the five-coordinate NO-heme complex transduce a signal to the kinase domain and increase HRI activity up to 5-fold, compared with the Fe(III)-HRI complex.
likely that HRI is also activated by NO via cleavage of the proximal iron-His bond.
Absorption spectra clearly show that both Fe(III)-HRI and Fe(II)-HRI complexes have the six-coordinate low spin heme. This observation is inconsistent with the finding that both the Fe(III) and Fe(II) complexes of sGC and cytochrome cЈ are five-coordinate high spin hemes. However, in all of these cases, dissociation of endogenous ligand(s) triggers signal transduction. It is proposed that in the case of HRI, upon binding of NO, both heme-ligated His and neutral Cys thiol residues dissociate from the heme at the same time. This dissociation may induce conformational change(s) in the heme environment, which in turn transduce a signal to the catalytic domain to trigger activation. In contrast, upon binding of CO, ligated Cys is dissociated from the heme iron, whereas the proximal bond between Fe-His remains unchanged. Thus, the structural changes induced upon NO binding are not generated by CO, and no enhancement in catalysis is observed.

Physiological Functions
Although NO-mediated inhibition of protein synthesis is established, the present study shows for the first time that NO directly affects HRI activity via heme following eIF2␣ phosphorylation. Previous analyses reveal that NO-induced activation of HRI in rabbit reticulocyte lysates requires high concentrations of NOC9 (0.25-0.5 mM) (7). In the present investigation, HRI was activated at a range of physiological NO concentrations (ϳ20 M). This inconsistency is due to the presence of Hb in experiments with reticulocyte lysates. HRI is mainly expressed in reticulocytes, where precursors of red blood cells contain a large quantity of Hb molecules. Hb is the most abundant intraerythrocytic protein and reacts rapidly with NO in both the deoxy and oxy forms. Under such conditions, it is unlikely that HRI senses the NO molecule in the reticulocyte at the expense of sensing heme concentrations. However, if NO binding affinity to HRI is higher than that to Hb, HRI should sense the NO molecule in reticulocytes.
Notably, HRI is expressed not only in reticulocytes but also in numerous tissues and cultured cells (7,16). Although only a limited quantity of HRI is present in the brain, reperfusion after brain ischemia results in the suppression of protein synthesis. Recent reports show that brain ischemia and reperfusion mainly activate the other eIF2␣ kinase, PERK (66), but it is possible that HRI simultaneously phosphorylates eIF2␣ in response to NO. In hippocampal sclerosis, immunoreactive neuronal nitric-oxide synthase and phosphorylated eIF2␣ are located in the immediate vicinity of each other (about 10 m) (67). NO-induced activation occurs in the hippocampus, with HRI possibly playing a role in eIF2␣ phosphorylation. Further studies are required to elucidate the physiological functions of NO-induced active HRI.
In summary, we show that 1) one of the axial ligands of Fe(III)-HRI is Cys, 2) HRI needs the heme iron for activation by NO, 3) the Fe(II)-HRI(NO) complex displays 5-fold greater activity than the Fe(III)-HRI complex, and 4) the Fe(II)-HRI(NO) complex has a five-coordinate NO-heme. In particular, heme coordination structures of HRI are distinct from those of the isolated N-terminal heme-binding domain, HRI-NTD.