The Heme Environment in Barley Hemoglobin*

Tapan Kanti DasDagger , H. Caroline LeeDagger , Stephen M. G. Duff§, Robert D. Hill§, Jack PeisachDagger , Denis L. RousseauDagger , Beatrice A. WittenbergDagger , and Jonathan B. WittenbergDagger

From the Dagger  Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 and § Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada

    ABSTRACT
Top
Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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 µM, 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 µmol/kg wet tissue weight) in roots, stems, or germinating seeds of monocotyledonous (2, 3) and dicotyledenous plants (3-6). 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 Ca2+-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 (koff = 0.027 s-1), corresponding to a t1/2 of ~25 s, taken together with an unremarkable combination rate constant, results in a very high affinity for oxygen (KD = 3.8 nM (1)). The net rate of oxygen dissociation2 (~30 µM min-1) 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-alpha 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 ~200 µl in phosphate buffer, pH 7. The purified protein was stored at -80 °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-CCl4 or indene as a reference. The laser power was maintained at ~1 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 µM 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 12C16O or 13C18O in tightly sealed Raman cells. 13C18O 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.

    RESULTS AND DISCUSSION

High Frequency Resonance Raman Spectra of Fe(III) and Fe(II) Barley Hb-- The high frequency region (1300-1700 cm-1) 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.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Resonance Raman spectra of (a) ferric and (b) ferrous barley Hb at pH 7.4 in the high frequency region. The laser power at the sample was ~5 mW.

The ferric form (Fig. 1a) displays a frequency of the electron density marker, nu 4, at 1374 cm-1, a frequency characteristic of the Fe(III) state. The location of nu 3 (at 1505 cm-1) and nu 10 (at 1635 cm-1) in the spectrum indicates that the protein contains a 6-coordinate low spin ferric heme. However, the observation of a weak nu 3 line at 1474 cm-1 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 (nu 4 = 1361 cm-1; nu 3 = 1493 cm-1) in addition to a population of a 5-coordinate high spin heme (nu 3 = 1470 cm-1). The population of the 5-coordinate heme is much smaller than that of the 6-coordinate heme, although the intensity of the 1470 cm-1 line is higher than the 1493 cm-1 line. This results from the fact that the intrinsic intensity of nu 3 (1470 cm-1) 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 deoxy-type 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-1 is assigned to the Fe-His (proximal) stretching mode (nu Fe-His). This assignment is supported by the appearance of this line at 219 cm-1, 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 (nu 4 = 1356 cm-1; nu 3 = 1470 cm-1). 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).


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Resonance Raman spectra of ferrous barley Hb in the low frequency region at pH 7.5. a, ferrous (deoxy); b, CO derivative; c, photolyzed CO derivative. The three spectra are normalized with respect to the porphyrin mode nu 7 (at 676 cm-1). The intensity in spectrum a is amplified by a factor of 2 for the sake of clarity. The laser powers used were (a) 5 mW, (b) 1 mW, and (c) 400 mW.

It is to be noted that in peroxidases, the nu Fe-His mode is detected at a significantly higher frequency (>240 cm-1) (15-18) compared to that in globins (200-230 cm-1) (13, 18, 19). Whereas the origin of the anomalous frequency of nu Fe-His in peroxidases is not well understood, it is likely that the high frequency of the nu 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 nu 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, 5.63, and 1.99. The g values for the low spin signal are 3.02, 2.22, and 1.48, values that are virtually identical to those of bovine liver cytochrome b5 (3.03, 2.23, 1.43; Ref. 20) for which the x-ray crystal data (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 b5 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 = 3.32 was resolved for the ferric cyanide complex of barley Hb. This value is similar to gmax 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.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3.   EPR spectrum of ferric barley Hb in 20 mM Hepes, pH 7.5. The spectrum was recorded at a microwave frequency of 9.29 GHz, a microwave power of 10 mW, a modulation frequency of 100 kHz, a modulation amplitude of 5 G, and a temperature of 6 K.

Fe-CO Stretching Frequency-- The Fe-CO stretching mode (nu 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-1, respectively, are observed at pH 7.4 (Fig. 4, spectrum a). Assignment of these two frequencies is confirmed by isotope replacement with 13C18O, in which the corresponding frequencies appear at 518 and 486 cm-1, respectively (Fig. 4, spectrum b). The isotope shift of the 493 cm-1 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-1 band on isotope replacement (12C16O/13C18O) is difficult because of the overlap of nu Fe-CO with a porphyrin peak at ~490 cm-1. The Fe-C-O bending mode (delta Fe-C-O) associated with the 534 cm-1 species is assigned to the band at 586 cm-1 that shifts to 568 cm-1 in 13C18O.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 4.   Resonance Raman spectra of barley HbCO in the low frequency region as a function of the pH and the isotopic composition of CO. a, pH 7.4, 12C16O; b, pH 7.4, 13C18O; c, pH 5, 12C16O; d, pH 5.0, 13C18O; e, pH 10.5, 12C16O. The laser power at the sample was ~1 mW.

It is to be noted that at pH 7.4 (Fig. 4, spectrum a), the intensity of the nu Fe-CO band at 534 cm-1 is much stronger than that at 493 cm-1. However, upon lowering the pH to 5.0, the intensities of the bands are reversed (Fig. 4, spectrum c); the nu Fe-CO line at 493 cm-1 becomes dominant. Frequency shifts of both of these lines by isotope replacement of CO at the lower pH value confirm their assignment as nu 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 nu Fe-CO line at 493 cm-1 is observed (Fig. 4, spectrum e).

Fe-CO stretching frequencies at ~495 cm-1 have been observed in the A0 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 (27-29). However, a value as high as 534 cm-1 for nu 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 nu Fe-CO at ~520 cm-1 (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 nu Fe-CO modes in the 505-510 cm-1 range (28, 35-37) at neutral pH value (A1 state), whereas horseradish peroxidase and cytochrome c peroxidase have nu Fe-CO in the range of 530-540 cm-1 (13, 15, 16, 30, 31).

The origin of such high frequencies for nu 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 nu Fe-CO must arise from distal interactions. We propose that the origin of the high frequency of nu Fe-CO in barley Hb is due to a strong interaction of CO with a positively polarized (delta +) residue on the distal side of the heme. It has been suggested that electrostatic interactions with CO can modulate nu Fe-CO significantly. Specifically, a positively charged environment tends to increase the frequency of nu Fe-CO (37-40). The amino acid sequence homology (with mammalian globins) of barley Hb suggests that His-61 is located distal (E7) to the heme. It also suggests that, there are no other positively charged residues present in the distal heme pocket (Phe at B10, Phe at CD1, Val at E11). It is likely that the imidazole of this histidine residue interacts with CO. Evidence of a hydrogen bonding interaction comes from the observation of an isotope effect on the nu Fe-CO line shown in Fig. 5. The nu Fe-CO line at 534 cm-1 in H2O (spectrum b) shifts to 535 cm-1 in D2O (spectrum a) with a concomitant decrease in the intensity of the delta Fe-CO located at 586 cm-1. The difference spectrum (H2O-D2O) shows the effect of deuterium substitution more clearly (spectrum c), indicating the presence of a proton in close interaction with CO. 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 Nepsilon H to CO is shorter than that in mammalian Mb. Close proximity of the polar Nepsilon H group to CO should increase the nu Fe-CO frequency because it was shown that such an effect is distance dependent; the shorter the distance, the higher the frequency of nu 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.


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Resonance Raman spectra of barley HbCO in the low frequency region at pH 7.4 as a function of isotopic composition of water. a, D2O; b, H2O; c, difference spectrum (H2O-D2O). The laser power at the sample was ~1 mW.

The H/D isotope effect observed here is not a simple mass effect because in that case, a D2O-induced shift to lower frequency would be observed. Whereas the specific mechanism of the increase in the Fe-CO stretching frequency in D2O 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 CO···D-N assembly becomes more rigid than the CO···H-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 nu 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 (nu Fe-CO at 493 cm-1) becomes the major population at the expense of the second conformer (534 cm-1) (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 nu 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 nu Fe-CO line at 534 cm-1 and the delta Fe-C-O at 586 cm-1 are completely lost from the spectrum of barley HbCO, resulting in the sole appearance of nu Fe-CO at 493 cm-1 (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 (>440-fold) in the oxygen dissociation rate (koff) 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 pi -electron back-donation from the dpi (dxz, dyz) of Fe to the empty pi * 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 (nu C-O) were detected in barley Hb: 1) a strong line at 1924 cm-1, and 2) a weak line at ~1960 cm-1 (Fig. 6, spectrum a). With 13C18O, a line at 1836 cm-1 presumably corresponding to the mode at 1924 cm-1 was detected. Although the isotopically shifted line corresponding to the line at ~1960 cm-1 could not be detected (Fig. 6, spectra b and c), probably due to the higher noise level in the 13C18O spectrum, we assign the ~1960 cm-1 line to the nu C-O of a minor population of the CO conformer in which the Fe-CO stretching mode is located at 493 cm-1. Whereas the nu C-O line at ~1960 cm-1 is similar to the nu C-O line in mammalian Mbs and Hbs with an open conformation (1965 cm-1), the frequency of the nu C-O line at 1924 cm-1 in barley Hb is significantly lower than that in any state (A1,2, ~1946 cm-1; A3, ~1932 cm-1) of the closed conformation in mammalian Mbs (37). Some vertebrate Hbs (lamprey Hb, rabbit Hb) however, have a CO conformer with low frequency of the nu C-O line (1927 cm-1 for lamprey Hb; 1928 cm-1 for rabbit Hb) (50). It was proposed that the low frequency of the nu 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 nu C-O against nu 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 nu Fe-CO mode at 534 cm-1 (nu C-O at 1924 cm-1) 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 nu C-O and the high frequency of nu Fe-CO result from a direct interaction of distal residues with the bound CO.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6.   Resonance Raman spectra of barley HbCO in the CO frequency region at pH 7.4 as a function of isotopic composition of CO. a, 12C16O; b, 13C18O; c, difference spectrum (12C16O-13C18O). The laser power at the sample was ~1 mW.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   Correlation between Fe-CO (nu Fe-CO) and C-O (nu C-O) stretching frequencies for various hemeproteins that have histidine as the proximal ligand. open circle , stretching frequencies of globins; , those of peroxidases. black-square (indicated by vertical arrows), the frequencies of CO barley Hb.

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-1, respectively) are proposed to arise from an electrostatic effect of the strong positive polarity of a short hydrogen bond between Nepsilon 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.

    ACKNOWLEDGEMENT

We thank Doug Durnin for skilled technical assistance.

    FOOTNOTES

* This work was supported by Natural Sciences and Engineering Research Council of Canada Grants OGP4689 and STR0149182 (to R. D. H.) and by National Institutes of Health Grants GM54806 and GM54812 (to D. L. R.) and GM40168 and RR02538 (to J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 718-430-4264; Fax: 718-430-4230; E-mail: rousseau{at}aecom.yu.edu.

The abbreviations used are: Hb, hemoglobin; HbCO, carbonmonoxy hemoglobin; Mb, myoglobin; EPR, electron paramagnetic resonance; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

2 The net rate of oxygen dissociation is calculated by multiplying koff by the concentration of Hb (~20 µM) in barley aleurone tissue.

    REFERENCES
Top
Abstract
Introduction
References

  1. Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752[Abstract/Free Full Text]
  2. Taylor, E. R., Nie, X. Z., MaGregor, A. W., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-852[Medline] [Order article via Infotrieve]
  3. Appleby, C. A. (1992) Sci. Prog. 76, 365-398
  4. Bogusz, D., Llewellyn, D. J., Craig, S., Dennis, E. S., Appleby, C. A., and Peacock, W. J. (1990) Plant Cell 2, 633-641[Abstract/Free Full Text]
  5. Appleby, C. A. (1984) Annu. Rev. Plant. Physiol. 35, 443-478[CrossRef]
  6. Andersson, C. R., Jensen, E. O., Llewellyn, D. J., Dennis, E. S., and Peacock, W. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5682-5687[Abstract/Free Full Text]
  7. Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. (Bethesda) 115, 1259-1266[Abstract/Free Full Text]
  8. Trevaskis, B., Watts, R. A., Andersson, C. R., Llewellyn, D. J., Hargrove, M. S., Olson, J. S., Dennis, E. S., and Peacock, W. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12230-12234[Abstract/Free Full Text]
  9. Nie, X. Z., and Hill, R. D. (1997) Plant Physiol. (Bethesda) 114, 835-840[Abstract/Free Full Text]
  10. Nie, X. Z. (1997) Regulation and Function of Hemoglobin in Barley Aleurone Tissue.Ph.D. thesis, University of Manitoba
  11. Sowa, A. W., Duff, S. M. G., Guy, P. A., and Hill, R. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10317-10321[Abstract/Free Full Text]
  12. Hill, R. D. (1998) Can. J. Bot. 76, 707-712[CrossRef]
  13. Wang, J., Caughey, W. S., and Rousseau, D. L. (1996) in Methods in Nitric Oxide Research (Feelish, M., and Stamler, J. S., eds), pp. 427-454, John Wiley & Sons Ltd., New York
  14. Hu, S., Smith, K. M., and Spiro, T. G. (1996) J. Am. Chem. Soc. 118, 12638-12646[CrossRef]
  15. Dasgupta, S., Rousseau, D. L., Anni, H., and Yonetani, T. (1989) J. Biol. Chem. 264, 654-662[Abstract/Free Full Text]
  16. Uno, T., Nishimura, Y., Tsuboi, M., Makino, R., Iizuka, T., and Ishimura, Y. (1987) J. Biol. Chem. 262, 4549-4556[Abstract/Free Full Text]
  17. Mylrajan, M., Valli, K., Wariishi, H., Gold, M. H., and Loehr, T. M. (1990) Biochemistry 29, 9617-9623[Medline] [Order article via Infotrieve]
  18. Kitagawa, T. (1988) in Biological Application of Raman Spectroscopy, Vol. 3, Resonance Raman Spectra of Heme and Metalloproteins (Spiro, T. G., ed), pp. 97-131, John Wiley & Sons, New York
  19. Rousseau, D. L., and Friedman, J. M. (1988) in Biological Application of Raman Spectroscopy, Vol. 3, Resonance Raman Spectra of Heme and Metalloproteins (Spiro, T. G., ed), pp. 133-215, John Wiley & Sons, New York
  20. Bois-Poltoratsky, R., and Ehrenberg, A. (1967) Eur. J. Biochem. 2, 361-365[Medline] [Order article via Infotrieve]
  21. Durley, R. C. E., and Mathews, F. S. (1994) Protein Data Bank in Brookhaven National Laboratory, pdb: 1CYO, compound: cytochrome b5(oxidized), source: bovine(Bos Taurus) liver
  22. Muskett, F. W., Kelly, G. P., and Whitford, D. (1996) J. Mol. Biol. 258, 172-189[CrossRef][Medline] [Order article via Infotrieve]
  23. Blumberg, W. E., and Peisach, J. (1971) in Probes of Structure and Function of Macromolecules and Membranes, Vol. II, Probes of Enzymes and Hemoproteins (Chance, B., Yonetani, T., and Mildvan, A. S., eds), pp. 215-228, Academic Press, New York
  24. Hori, H. (1971) Biochim. Biophys. Acta 251, 227-235[Medline] [Order article via Infotrieve]
  25. Peisach, J., Blumberg, W. E., Wittenberg, B. A., and Wittenberg, J. B. (1968) J. Biol. Chem. 243, 1871-1880[Abstract/Free Full Text]
  26. de Ropp, J. S., Thanabal, V., and La Mar, G. N. (1985) J. Am. Chem. Soc. 107, 8268-8270
  27. Mourant, J. R., Braunstein, D. P., Chu, K., Frauenfelder, H., Nienhaus, G. U., Ormos, P., and Young, R. D. (1993) Biophys. J. 65, 1496-1507[Abstract]
  28. Sage, J. T., Morikis, D., and Champion, P. M. (1991) Biochemistry 30, 1227-1237[Medline] [Order article via Infotrieve]
  29. Ramsden, J., and Spiro, T. G. (1989) Biochemistry 28, 3125-3128[Medline] [Order article via Infotrieve]
  30. Smulevich, G., Evangelista-Kirkup, R., English, A., and Spiro, T. G. (1986) Biochemistry 25, 4426-4430[Medline] [Order article via Infotrieve]
  31. Evangelista-Kirkup, R., Smulevich, G., and Spiro, T. G. (1986) Biochemistry 25, 4420-4425[Medline] [Order article via Infotrieve]
  32. Wang, J., Takahashi, S., Hosler, J. P., Mitchell, D. M., Ferguson-Miller, S., Gennis, R. B., and Rousseau, D. L. (1995) Biochemistry 34, 9819-9825[Medline] [Order article via Infotrieve]
  33. Wang, J., Ching, Y.-C., Rousseau, D. L., Hill, J. J., Rumbley, J., and Gennis, R. B. (1993) J. Am. Chem. Soc. 115, 3390-3391
  34. Argade, P. V., Ching, Y.-C., and Rousseau, D. L. (1984) Science 225, 329-331[Medline] [Order article via Infotrieve]
  35. Hirota, S., Ogura, T., and Kitagawa, T. (1995) J. Am. Chem. Soc. 117, 821-822
  36. Yu, N.-T., and Kerr, E. A. (1988) in Biological Application of Raman Spectroscopy, Vol. 3, Resonance Raman Spectra of Heme and Metalloproteins (Spiro, T. G., ed), pp. 39-95, John Wiley & Sons, New York
  37. Ray, G. B., Li, X.-Y., Ibers, J. A., Sessler, J. L., and Spiro, T. G. (1994) J. Am. Chem. Soc. 116, 162-176
  38. Kushkuley, B., and Stavrov, S. S. (1996) Biophys. J. 70, 1214-1229[Abstract]
  39. Ling, J., Li, T., Olson, J. S., and Bocian, D. F. (1994) Biochim. Biophys. Acta 1188, 417-421[Medline] [Order article via Infotrieve]
  40. Park, K. D., Guo, K., Adebodun, F., Chiu, M. L., Sligar, S. G., and Oldfield, E. (1991) Biochemistry 30, 2333-2347[Medline] [Order article via Infotrieve]
  41. Unno, M., Christian, J. F., Olson, J. S., Sage, J. T., and Champion, P. M. (1998) J. Am. Chem. Soc. 120, 2670-2671[CrossRef]
  42. Phillips, S. E., and Schoenborn, B. P. (1981) Nature 292, 81-82[Medline] [Order article via Infotrieve]
  43. Kitagawa, T., Ondrias, M. R., Rousseau, D. L., Ikeda-Saito, M., and Yonetani, T. (1982) Nature 298, 869-871[Medline] [Order article via Infotrieve]
  44. Scheiner, S., and Cuma, M. (1996) J. Am. Chem. Soc. 118, 1511-1521[CrossRef]
  45. Bell, R. L., and Truong, T. N. (1994) J. Chem. Phys. 101, 10442-10451[CrossRef]
  46. Morikis, D., Champion, P. M., Springer, B. A., and Sligar, S. G. (1989) Biochemistry 28, 4791-4800[Medline] [Order article via Infotrieve]
  47. Yang, F., and Phillips, G. N., Jr. (1996) J. Mol. Biol. 256, 762-774[CrossRef][Medline] [Order article via Infotrieve]
  48. Springer, B. A., Egeberg, K. D., Sligar, S. G., Rohlfs, R. J., Mathews, A. J., and Olson, J. S. (1989) J. Biol. Chem. 264, 3057-3060[Abstract/Free Full Text]
  49. Li, X.-Y., and Spiro, T. G. (1988) J. Am. Chem. Soc. 110, 6024-6033
  50. Caughey, W. S., Houtchens, R. A., Lanir, A., Maxwell, J. C., and Charache, S. (1978) in Biochemical and Clinical Aspects of Hemoglobin Abnormalities (Caughey, W. S., ed), pp. 29-53, Academic Press, New York
  51. Bogusz, D., Appleby, C. A., Landsmann, J., Dennis, E. S., Trinick, M. J., and Peacock, W. J. (1988) Nature 331, 178-180[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.