The heme environment in barley hemoglobin.

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm-1, with relative intensities that are pH sensitive. The 534 cm-1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogen-bonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm-1 is assigned as being associated with the 534 cm-1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm-1, respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm-1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.

Barley (Hordeum sp.) hemoglobin (Hb), 1 the first nonsymbiotic plant Hb to be isolated and characterized from a monocotyledonous plant (1), is expressed in seed and root tissues under anaerobic conditions. Because the level of Hb in barley aleurone tissue is of the order of only 20 M, an expression system in Escherichia coli was developed to produce a barley Hb fusion protein that is indistinguishable from the native protein in most properties but differs in having five extra amino acids at the N terminus (1).
Nonsymbiotic plant Hbs constitute a new class of protoheme proteins that are expressed at low concentrations (on the order of 1-20 mol/kg wet tissue weight) in roots, stems, or germinating seeds of monocotyledonous (2,3) and dicotyledenous plants (3)(4)(5)(6). Nonsymbiotic Hbs of both monocots and dicots fall into a single coherent gene family, distinct from the family of genes that encode the symbiotic Hbs (6). They are functionally distinct from the familiar symbiotic plant leghemoglobins that are expressed by some plants in nitrogen-fixing symbiotic associations with the bacterium Rhizobium or the actinomycete Frankia. Distinguishing characteristics of nonsymbiotic Hbs (1,7,8) are extremely slow dissociation of bound oxygen and 6-coordinate, low spin ferrous (deoxy) species.
The barley nonsymbiotic Hb gene is induced under conditions of low oxygen pressure (2). Low oxygen tension, per se, is not responsible for the induction, because the gene can be induced in the presence of oxygen by respiratory inhibitors that interfere with mitochondrial ATP synthesis (9). Induction appears to be initiated by a decline in ATP within the cell, leading to a Ca 2ϩ -mediated signal transduction process (10). Studies using cultured maize cells transformed to express the sense and antisense gene (11) have demonstrated that cells containing the sense construct possessed higher levels of ATP and adenylates than wild type or antisense-transformed cells when grown under limiting oxygen, suggesting that Hb acts to maintain energy status under low oxygen tension (11). It has been postulated (12) that barley oxyHb, in conjunction with another protein, could function as an oxygenase, oxidizing NADH to maintain glycolytic ATP synthesis as an alternative pathway to ethanol or lactate formation.
The recombinant barley Hb studied here and native barley Hb are homodimers with a subunit molecular mass of 18.5 kDa, having one protoheme IX per subunit. Extraordinarily slow dissociation of bound oxygen (k off ϭ 0.027 s Ϫ1 ), corresponding to a t1 ⁄2 of ϳ25 s, taken together with an unremarkable combination rate constant, results in a very high affinity for oxygen (K D ϭ 3.8 nM (1)). The net rate of oxygen dissociation 2 (ϳ30 M min Ϫ1 ) is far too slow to support a metabolic function. The concentration of barley Hb within the aleurone or root cell is too small to store a metabolically significant amount of oxygen or facilitate oxygen movement, except perhaps within very limited structural domains. Ferrous deoxy barley Hb, in common with rice Hb (7), has an optical spectrum characteristic of a low spin 6-coordinate heme, indicating the presence of a ligand in the distal position (1,2). It has been suggested that an exogenous ligand, such as oxygen or carbon monoxide, can react with the heme iron only following prior dissociation of the bound endogenous ligand (1). In any hemeprotein, the heme pocket structure plays a crucial role in controlling protein function, such as heme ligand stability, and thus determines the class of a particular hemeprotein. Since barley Hb is a newly discovered hemeprotein, it is important to elucidate the structure of the heme pocket as a whole, particularly the nature of the heme axial ligands and the electronic environment of the heme crevice. Upon unveiling of the specific nonbonding interactions that prevail in the heme pocket, the origin of the remarkably slow oxygen dissociation rate may be elucidated.
The 6-coordinate nature of ferrous barley Hb is reminiscent of the structure of cytochromes. For this reason, it is of interest to identify the ligands to the heme iron. Here we present evidence that the histidine, placed by sequence alignment in the position proximal to the heme iron, ligates to the iron and has the character of an uncharged imidazole. We further show that a second histidine residue, placed by sequence alignment in the position distal to the heme iron, ligates to the heme. Furthermore, we show that the distal histidine interacts with exogenous iron-bound CO in a novel manner and may account for a remarkably high ligand stability associated with the slow oxygen dissociation rate of barley oxyHb.

EXPERIMENTAL PROCEDURES
Recombinant Barley Hb-Barley Hb was prepared as described previously (1). Briefly, the barley root Hb cDNA was cloned into pUC 19 plasmid (2). E. coli strain DH5-␣ was used as the host for the recombinant plasmid. The Hb was extracted and purified from the cells, and the most pure fractions of Hb were pooled and concentrated to a final volume of ϳ200 l in phosphate buffer, pH 7. The purified protein was stored at Ϫ80°C until used for spectroscopic measurements.
Resonance Raman Spectroscopy-The Raman instrumentation has been described in detail elsewhere (13). Briefly, the resonance Raman measurements were carried out with an excitation wavelength of 413.1 nm from a Kr-ion laser (Spectra Physics, Mountain View, CA). The sample cell was spun at 6000 rpm to avoid local heating. The Raman scattered light was dispersed through a polychromator (Spex, Metuchen, NJ) equipped with a 1200 grooves/mm grating and detected by a liquid nitrogen-cooled charge-coupled device camera (Princeton Instruments, Princeton, NJ). A holographic notch filter (Kaiser, Ann Arbor, MI) was used to remove the laser scattering. Typically, six 30-s spectra were recorded and averaged after the removal of cosmic ray spikes by a standard software routine (CSMA; Princeton Instruments, NJ). Frequency shifts in the Raman spectra were calibrated using acetone-CCl 4 or indene as a reference. The laser power was maintained at ϳ1 mW to minimize CO dissociation from HbCO samples. In photolysis experiments with HbCO samples, partial CO photodissociation was achieved with 400 mW of laser power.
The concentration of the protein samples used for the Raman measurements was typically 30 -100 M in 100 mM buffer (sodium acetate, pH 5; sodium phosphate, pH 7.4; CAPS, pH 10.5). For the ferrous samples, ferric barley Hb was reduced by the addition of a freshly prepared anaerobic solution of dithionite to the degassed protein solution. HbCO was prepared by exposing dithionite-reduced samples to either 12 C 16 O or 13 C 18 O in tightly sealed Raman cells. 13 C 18 O gas was a product of ICON (Mount Marion, NY). Absorption spectra were recorded before and after the Raman measurements to verify the stability of the species studied.
Electron Paramagnetic Resonance-EPR spectra were obtained at 6 K using a Varian E112 spectrometer equipped with a Systron-Donner frequency counter and a PC-based data acquisition program. The samples of ferric barley Hb were dissolved in 20 mM Hepes, pH 7.5, for the EPR measurements. The spectrum was recorded at a microwave frequency of 9.29 GHz, a microwave power of 10 mW, a modulation frequency of 100 kHz, and a modulation amplitude of 5 G.

High Frequency Resonance Raman Spectra of Fe(III) and
Fe(II) Barley Hb-The high frequency region (1300 -1700 cm Ϫ1 ) of the resonance Raman spectra of hemeproteins is comprised of porphyrin in-plane vibrational modes that are sensitive to the electron density in the porphyrin macrocycle and also to the oxidation, coordination, and spin state of the central iron atom (13,14). The resonance Raman spectra of the ferric and ferrous protein (at pH 7.4) in the high frequency region are shown in Fig. 1.
The ferric form (Fig. 1a) displays a frequency of the electron density marker, 4 , at 1374 cm Ϫ1 , a frequency characteristic of the Fe(III) state. The location of 3 (at 1505 cm Ϫ1 ) and 10 (at 1635 cm Ϫ1 ) in the spectrum indicates that the protein contains a 6-coordinate low spin ferric heme. However, the observation of a weak 3 line at 1474 cm Ϫ1 suggests that a minor population of a 6-coordinate high spin complex is also present. The spectrum of the ferric complex did not show any pH dependence in the pH range 5.0 -10.5.
The spectrum of ferrous barley Hb at pH 7.4 ( Fig. 1b) is dominated by a 6-coordinate low spin heme ( 4 ϭ 1361 cm Ϫ1 ; 3 ϭ 1493 cm Ϫ1 ) in addition to a population of a 5-coordinate high spin heme ( 3 ϭ 1470 cm Ϫ1 ). The population of the 5-coordinate heme is much smaller than that of the 6-coordinate heme, although the intensity of the 1470 cm Ϫ1 line is higher than the 1493 cm Ϫ1 line. This results from the fact that the intrinsic intensity of 3 (1470 cm Ϫ1 ) is very high for the 5-coordinate species compared with that of the 6-coordinate species and thus makes it difficult to assess the relative populations by inspection of the resonance Raman spectrum. The optical absorption spectrum (1) confirms that the 6-coordinate form dominates at a neutral pH. The population of the ferrous high spin heme observed at both neutral and acidic pH values becomes immeasurably small at an alkaline pH value (pH 10.5). Ferrous mammalian Hbs, on the other hand, remain in a 5-coordinate high spin form over a wide pH range.
Fe-His Stretching Frequency-The low frequency region of resonance Raman spectra of hemeproteins is comprised of several in-plane and out-of-plane vibrational modes of the heme, including heme propionate modes and ligand vibrational modes (13,14). Enhancement of the axial ligand (bound to the central metal atom) vibrational modes arises from electronic coupling of the orbitals of the ligand to the metalloporphyrin electronic orbitals. Assignment of a ligand vibrational mode is extremely useful because it directly identifies a particular ligand and the nature of its interactions with amino acid residues in the heme pocket. As noted above, the ferrous barley Hb contains both a low spin 6-coordinate form and a smaller population of a deoxytype 5-coordinate high spin species. The low frequency region of the resonance Raman spectra of the ferrous species is shown in Fig. 2 (spectrum a). The line at 219 cm Ϫ1 is assigned to the Fe-His (proximal) stretching mode ( Fe-His ). This assignment is supported by the appearance of this line at 219 cm Ϫ1 , generated by photodissociation of the CO derivative (Fig. 2, spectrum c) that was completely absent from the spectrum of the CO derivative (Fig. 2, spectrum b). The high frequency region of the photolyzed species (data not shown) has the characteristics of a typical 5-coordinate high spin heme ( 4 ϭ 1356 cm Ϫ1 ; 3 ϭ 1470 cm Ϫ1 ). Observation of a line attributed to the Fe-His stretching frequency suggests that histidine is the proximal ligand to the heme in barley Hb. As expected, this line is not observed in the resonance Raman spectrum of the 6-coordinate species of ferrous barley Hb at high pH values (data not shown).
It is to be noted that in peroxidases, the Fe-His mode is detected at a significantly higher frequency (Ͼ240 cm Ϫ1 ) (15)(16)(17)(18) compared to that in globins (200 -230 cm Ϫ1 ) (13,18,19). Whereas the origin of the anomalous frequency of Fe-His in peroxidases is not well understood, it is likely that the high frequency of the Fe-His mode in peroxidases is due in part to the imidazolate character of the proximal histidine (13,18). Consideration of the above facts suggests that barley Hb in which the frequency of the Fe-His mode is similar to that of mammalian Hbs and Mbs has an uncharged proximal imidazole (histidine) and not an imidazolate.
EPR Spectrum of Ferric Barley Hb-The EPR spectrum of ferric barley Hb at pH 7.5 shows the presence of both high and low spin signals (Fig. 3). The high spin signal is slightly rhombic, with g values of 6.04, 5.  (21) and solution NMR studies (22) have shown a bis-histidyl imidazole-ligated heme structure. The EPR spectrum also resembles that of the bis-imidazole model heme complexes (23). Furthermore, the g tensor anisotropy of low spin barley Hb does not resemble that of the alkaline form of cytochrome b 5 and the high pH complex of bis-imidazole heme that contain axial imidazolate ligands (20). Thus, the possibility that one or both of the axial ligands of barley Hb is imidazolate is excluded. In addition, a signal at g ϭ 3.32 was resolved for the ferric cyanide complex of barley Hb. This value is similar to g max for cyanide complexes of globins (24) but larger than that for peroxidases (25) that have an imidazolate axial ligand (26). This further supports the assignment of a neutral histidyl imidazole as the proximal ligand and is consistent with the resonance Raman observation of a Mb-like (proximal imidazole and not imidazolate) Fe-His stretching frequency.
Fe-CO Stretching Frequency-The Fe-CO stretching mode ( Fe-CO ) has been identified in CO complexes of hemeproteins. Its frequency is sensitive to interactions of bound CO with neighboring residues. Fig. 4 shows the spectra of the CO derivative of barley Hb as a function of pH and isotopic composition of the bound CO. Interestingly, two Fe-CO stretching frequencies at 534 and 493 cm Ϫ1 , respectively, are observed at pH 7.4 (Fig. 4, spectrum a). Assignment of these two frequencies is confirmed by isotope replacement with 13 C 18 O, in which the corresponding frequencies appear at 518 and 486 cm Ϫ1 , respectively (Fig. 4, spectrum b). The isotope shift of the 493 cm Ϫ1 line is seen more clearly in the comparison of spectra c and d obtained at pH 5.0. However, determination of the exact magnitude of the frequency shift of the 493 cm Ϫ1 band on isotope replacement ( 12 C 16 O/ 13 C 18 O) is difficult because of the overlap of Fe-CO with a porphyrin peak at ϳ490 cm Ϫ1 . The Fe-C-O bending mode (␦ Fe-C-O ) associated with the 534 cm Ϫ1 species is assigned to the band at 586 cm Ϫ1 that shifts to 568 cm Ϫ1 in 13 C 18 O.
It is to be noted that at pH 7.4 (Fig. 4, spectrum a), the intensity of the Fe-CO band at 534 cm Ϫ1 is much stronger than that at 493 cm Ϫ1 . However, upon lowering the pH to 5.0, the intensities of the bands are reversed (Fig. 4, spectrum c); the Fe-CO line at 493 cm Ϫ1 becomes dominant. Frequency shifts of both of these lines by isotope replacement of CO at the lower pH value confirm their assignment as Fe-CO modes (Fig. 4, spectrum d) that arise from two different conformations of the protein that have different electronic environments around the Fe-CO moiety. More importantly, the transition between these two conformers is linked to a proton-induced phenomenon in the heme pocket. At a very alkaline pH value, pH 10.5, only the Fe-CO line at 493 cm Ϫ1 is observed (Fig. 4, spectrum e). Fe-CO stretching frequencies at ϳ495 cm Ϫ1 have been observed in the A 0 state of Hbs and Mbs at acidic pH values and in many other hemeproteins as well and are believed to arise from an open heme pocket in which CO has very little interaction with the surrounding amino acid residues (27)(28)(29). However, a value as high as 534 cm Ϫ1 for Fe-CO is unprecedented in globins and in other hemeproteins, with the exception of some peroxidases (13,15,16,30,31). The terminal oxidases also exhibit a high frequency of Fe-CO at ϳ520 cm Ϫ1 (32-34). However, the high frequency of the Fe-CO modes in oxidases is believed to result from an interaction with the nearby copper atom. Mammalian Hbs and Mbs containing a distal histidine, on the other hand, show Fe-CO modes in the 505-510 cm Ϫ1 range (28, 35-37) at neutral pH value (A 1 state), whereas horseradish peroxidase and cytochrome c peroxidase have Fe-CO in the range of 530 -540 cm Ϫ1 (13,15,16,30,31).
The origin of such high frequencies for Fe-CO has been debated extensively. In peroxidases, it is attributed to an increase in the Fe-CO bond order caused by the imidazolate character of the proximal histidine ligand and hydrogen bonding interactions of a distal residue with CO. Because the proximal histidine of barley Hb does not have imidazolate character, the high frequency for Fe-CO must arise from distal interactions. We propose that the origin of the high frequency of Fe-CO in barley Hb is due to a strong interaction of CO with a positively polarized (␦ ϩ ) residue on the distal side of the heme. It has been suggested that electrostatic interactions with CO can modulate Fe-CO significantly. Specifically, a positively charged environ- A similar isotope effect has been reported to occur in the CO complex of sperm whale Mb, in which it was suggested that a hydrogen bond exists between the distal histidine and the iron-bound CO (41). It is well known that such hydrogen bonding occurs between the distal histidine and the bound oxygen in mammalian oxyMb (42,43). In barley Hb, however, we propose that the hydrogen bonding distance of the histidine N ⑀ H to CO is shorter than that in mammalian Mb. Close proximity of the polar N ⑀ H group to CO should increase the Fe-CO frequency because it was shown that such an effect is distance dependent; the shorter the distance, the higher the frequency of Fe-CO (38). The fact that the distal histidine of barley Hb can bind to the heme iron in both the ferric and ferrous states (in the absence of exogenous ligands) is consistent with the distal histidine residing closer to the heme than seen in mammalian Mbs and Hbs.
The H/D isotope effect observed here is not a simple mass effect because in that case, a D 2 O-induced shift to lower frequency would be observed. Whereas the specific mechanism of the increase in the Fe-CO stretching frequency in D 2 O remains to be solved, it has been postulated that the hydrogen bond strength between the FeCO and the histidine N-H is changed upon the H/D exchange due to a decrease in the zero point energy of the N-D bond (41,44,45) relative to the N-H bond in an anharmonic potential well. The N-D bond is more stabilized due to a retardation of the vibrational amplitude of one of its bending modes that primarily represents the wagging motion of the bridging deuterium. As a result, the CO⅐⅐⅐D-N assembly becomes more rigid than the CO⅐⅐⅐H-N moiety; thus, the stretching vibrational frequency of the Fe-CO mode is increased.
The above results, which suggest that the conformer associated with the high frequency of Fe-CO involves an interaction with the distal histidine, are further supported by observations at acidic pH values. At pH 5.0, the presumptive open conformer ( Fe-CO at 493 cm Ϫ1 ) becomes the major population at the expense of the second conformer (534 cm Ϫ1 ) (Fig. 4). This transition is consistent with protonation-induced changes in the distal histidine that result in its moving out of the heme pocket, leaving it open. Similar transitions in Fe-CO have been seen in sperm whale Mb at acidic pH values and have been ascribed to an open-closed transition of the heme pocket supported by the crystal structure of the acid form of Mb (37, 46,47). At an alkaline pH value (pH 10.5), the Fe-CO line at 534 cm Ϫ1 and the ␦ Fe-C-O at 586 cm Ϫ1 are completely lost from the spectrum of barley HbCO, resulting in the sole appearance of Fe-CO at 493 cm Ϫ1 (Fig. 4). This indicates an open conformation of the heme pocket in which the hydrogen bonding network becomes very weak or is completely abolished at strongly alkaline pH values.
As discussed above, the location of the distal histidine in close proximity to the diatomic ligand-binding site of the heme iron should play a significant role in controlling the ligand stability. This, in fact, is manifested in the observation of a dramatic decrease (Ͼ440-fold) in the oxygen dissociation rate (k off ) relative to sperm whale Mb (1,48). Such a low dissociation rate can be explained by the extra stability of the bound ligand due to a strong hydrogen bonding interaction with the distal histidine.
Correlation between Fe-CO and C-O Stretching Frequencies-It is well established that the Fe-CO and C-O stretching modes follow an inverse correlation due to -electron backdonation from the d (d xz , d yz ) of Fe to the empty * orbitals of carbon monoxide, which results in an increase of the Fe-CO bond order and a concomitant decrease in the C-O bond order (13,16,36,49). The correlation between these two frequencies depends on the nature of the proximal ligand, because the electron density in the Fe-proximal ligand bond affects the Fe-CO bond order. Back-bonding to CO is controlled by many factors such as steric crowding of the bound CO, polarity of the neighboring environment, and hydrogen bonding of the CO oxygen to an adjacent proton of a distal residue. At pH 7.4, two C-O stretching frequencies ( C-O ) were detected in barley Hb: 1) a strong line at 1924 cm Ϫ1 , and 2) a weak line at ϳ1960 cm Ϫ1 (Fig. 6, spectrum a). With 13 C 18 O, a line at 1836 cm Ϫ1 presumably corresponding to the mode at 1924 cm Ϫ1 was detected. Although the isotopically shifted line corresponding to the line at ϳ1960 cm Ϫ1 could not be detected (Fig. 6, spectra b and c), probably due to the higher noise level in the 13 (50). It was proposed that the low frequency of the C-O line in rabbit Hb is caused by the proximity of the CD6 leucine to the distal histidine (50). To determine whether the electronic interactions in barley Hb correspond to those in other Hbs and Mbs, we plotted C-O against Fe-CO (Fig.  7). Both frequencies fall on a correlation line that is characteristic for hemeproteins that contain histidine as the proximal ligand, showing that the bond order of Fe-CO is inversely related to that of C-O. The Fe-CO mode at 534 cm Ϫ1 ( C-O at 1924 cm Ϫ1 ) falls toward the left end of the correlation line, close to the points for the peroxidases. However, as already discussed, the frequencies of the Fe-CO and C-O modes in barley Hb result from distal interactions, not the imidazolate character of the proximal ligand. Thus, for barley Hb, the low frequency of C-O and the high frequency of Fe-CO result from a direct interaction of distal residues with the bound CO.
Dihistidyl Heme in Nonsymbiotic Plant Hbs-There are three histidine residues in the amino acid sequence deduced from the nucleotide sequence of barley Hb (2). Sequence alignment (2) suggests that His-105 and His-70 are the proximal and distal ligands, respectively, to the heme iron atom of barley Hb. Evidence from several spectroscopies, as discussed above, indicates that the distal histidine ligates to the heme iron in both the ferrous and ferric forms of the protein. Growing evidence suggests that all nonsymbiotic plant Hbs may have dihistidyl ligation of the heme iron. Sequence alignment places histidine in the distal heme pocket of soybean (6), rice Hb1 and Hb2 (7), Trema (4,51), Arabidopsis AHB1 (8), and barley (1,6) Hbs. Optical spectra of ferrous deoxy rice Hb1 (7), Arabidopsis AHB1 (8), and barley (1) Hbs indicate that these proteins are in 6-coordinate low spin form, as expected for ferrous dihistidyl structures. This was confirmed for rice Hb1 by mutation of the distal histidine (7).
Conclusions-Barley Hb has a bis-histidine coordinated heme in which both the imidazoles are uncharged. Among the distal histidine-containing globins, barley appears to have a unique heme environment judged from Fe-CO stretching vibrational frequencies in the CO complex. Unusual Fe-CO and C-O stretching frequencies (at 534 and 1924 cm Ϫ1 , respectively) are proposed to arise from an electrostatic effect of the strong positive polarity of a short hydrogen bond between N ⑀ H of the distal histidine and CO and are confirmed by isotopic substitution studies. At low pH values, the distal histidine is believed to move out of the heme pocket due to protonation, leaving an open heme pocket, just as in mammalian Mbs. Existence of strong hydrogen bonding between the bound CO and the distal histidine implies that a similar situation may exist in the oxy complex of barley Hb. Such strong hydrogen bonding is expected to impart a great stability to the bound ligand that is in fact manifested by the observation of a very low oxygen dissociation rate.