Multiple Active Site Conformers in the Carbon Monoxide Complexes of Trematode Hemoglobins*

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (∼60 cm–1) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm–1, for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm–1, respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm–1, respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.

It is now well recognized that hemoglobins (Hbs) 3 are widespread in all phyla. There has been a renewed interest in understanding the structure and function of Hbs in recent years (1)(2)(3)(4)(5), because these Hbs are located in diverse cellular compartments and possess atypical physico-chemical properties. Hbs have been discovered in about 33% of the animal species, and in addition to being present in vertebrates, they are found in the phyla that include nematode, mollusc, and annelid, to name but a few. Single subunit globins have been discovered lately in algae, nonsymbiotic plants, and a number of prokaryotes ranging from bacteria to cyanobacteria (see Refs. [2][3][4][5]. Additionally, flavoproteinbound chimeric Hbs have been found in bacteria (6) and yeast (7). In most cases, the nonvertebrate Hbs are proposed to have dissimilar functions from those in vertebrate Hbs; the latter class is established as consisting of O 2 carrier proteins. However, additional cellular activities have been proposed recently for vertebrate Hbs as well (8).
Although the nonvertebrate Hbs bind O 2 and other ligands just as the vertebrate Hbs, the kinetic and structural properties of the O 2 complexes (see for example Refs. 3-5 and 9 -15) differ significantly, suggesting functional diversity. However, identification of the physiological functions remains elusive. Diversity in ligand binding properties, despite sharing a common prosthetic group, can be attributed to subtle changes in the arrangement of the amino acid residues that either coordinate to the heme or form its immediate surroundings. Understanding of the interactions between heme-bound ligands in hemeproteins and the residues in its distal pocket has received a great deal of attention in recent years as it allows for the clarification of catalytic mechanisms in some proteins and the rationalization of ligand kinetic behaviors in others. Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions ( Fig. 1) (16 -17). In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is seen as displaced toward the globin CD region (18). In the present study, we report resonance Raman data on two closely related Hbs of the trematodes, P. epiclitum and G. crumenifer, for elucidation of the active site structure.
Resonance Raman spectroscopy is a very powerful method to study the nature of the interactions between the heme-bound ligands and their distal environment owing to the presence of Fe-ligand modes in the spectrum. The CO derivatives of heme proteins are especially useful in this regard because the Fe-CO ( Fe-CO ) and C-O ( C-O ) stretching vibrations are sensitive to the polarity of the heme pocket, and hence can be used to assess the electrostatic properties of the distal environment that play a crucial role in ligand association/dissociation events. We report here a combination of multiple protein active site conformers in the CO complex of the two trematode hemoglobins. We also discuss the implications of such protein conformers in modulating the ligand binding kinetics.

EXPERIMENTAL PROCEDURES
Biological Materials-P. epiclitum and G. crumenifer (Platyhelminthes, Trematoda, Paramphistomidae) parasitic in the rumen of the common * This work was supported by National Institutes of Health Grants GM54806 (to D. L. R.) and GM58890 (to J. M. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. 1  Indian water buffalo, Bubalis bubalis, was obtained from a local slaughterhouse in Aligarh, India. Trematodes were washed thoroughly with normal saline and incubated for 1 h at 37°C in 0.15 M NaCl, 8 mM glucose to make them shed their eggs and gut contents. Trematodes were stored at Ϫ80°C until use.
Purification of Trematode Hemoglobins and Sequence Determination-Purification and primary structure determination of the P. epiclitum Hb is described in Rashid et al. (17). Purification and protein sequencing of the G. crumenifer Hb was performed as described for P. epiclitum. Briefly, a single isoform was isolated by semipreparative isoelectrofocusing (IEF) under denaturing conditions. Peptides, generated by digestion with trypsin, endoproteinase Asp-N, CNBr cleavage, and N-terminal deblocking were purified by RP-HPLC on a Vydac C4 column (2.1 mm) developed with a 0.1% trifluoroacetic acid/acetonitrile gradient. Peptide sequencing was performed on an ABI 471-B sequencer operated in the pulsed liquid mode as recommended by the manufacturer (20 -21).
Raman Measurements-The Raman experiments were carried out with 413.1 nm excitation from a cw Kr-ion laser (Spectra Physics, Mountain View, CA). The instrumentation and measurement procedures have been described elsewhere (9). Deoxy (reduced) hemoglobin was prepared by the addition of a small aliquot of dithionite solution under anaerobic conditions. The CO complexes were prepared by the addition of CO ( 12 C 16 O or 13 C 18 O) to anaerobic solutions of dithionite-reduced protein (ϳ70 M) in 100 mM sodium phosphate buffer, pH 7.4 in tightly sealed Raman cells. All spectral measurements were made at pH 7.4 unless otherwise noted. The 13 C 18 O gas was obtained from ICON (Mt. Marion, NY) and 12 C 16 O was purchased from Matheson (Rutherford, NJ). The laser power was kept low at the sample (ϳ0.5 milliwatt) to minimize CO dissociation. Optical spectra were recorded before and after Raman measurements to ensure sample integrity.

RESULTS
Primary Structure of G. crumenifer Hemoglobin-When analyzed by IEF, under native and denaturing conditions, G. crumenifer Hb consists, as do P. epiclitum and Isoparorchis hypselobagri Hb, of different isoforms (17). It is unclear whether this heterogeneity is caused by the existence of genetically different isoforms or to post-translational modifications of a single Hb chain. A single isoform (glb1) was purified by semipreparative IEF and RP-HPLC (17).
The G. crumenifer globin primary structure was reconstructed from the sequence of relevant peptides generated by tryptic, endoprotease Asp-N and CNBr digestion (Fig. 1). The N terminus was inaccessible for Edman degradation suggesting that it is blocked. Deblocking by the Takara method resulted in the 6 terminal residues and proves that the N terminus is acetylated (22). All overlaps are presented, and the majority of the residues were sequenced twice. The total sequence contains 147 amino acids. The alignment with other trematode sequences is unam- biguous and confirms that no peptides are missing (Fig. 1). All four trematode sequences display an identity of 38 out of 147 residues (26.5%). Key positions of the globin structure at NA2, B6, B14, C2, CD1, F8, G5, and H8 are conserved. Deviations of the classical globin pattern are: Leu-A12, Tyr-B10, Tyr-C4, Tyr-E7, Tyr-F4, and Leu-H8. The presence of a Tyr at positions B10 and E7, a striking characteristic of the trematode Hbs, allows for the possible formation of two hydrogen bonds to the bound oxygen and therefore may be responsible for the high oxygen affinity because of a high k on and a low k off value (16,17,23). We therefore suggest that, to achieve a high oxygen affinity, all trematode Hbs might have a Tyr at the B10 and E7 positions. It should be noted that a Tyr at the B10 position is quite common in globins from various species (5, 25) (See Table 1).
Resonance Raman Spectra of the Deoxy Complexes: Detection of Fe-His Mode-The low frequency region of resonance Raman spectra of hemeproteins is comprised of several vibrational modes of the heme, including modes of the peripheral groups and those involving the axial ligands (26 -29). Enhancement of the vibrational modes involving the axial ligand bound to the central metal atom arises from electronic coupling of the ligand orbitals to the heme orbitals. Assignment of a ligand vibrational mode is extremely useful as it directly identifies a particular ligand and the nature of its interactions with amino acid residues in the heme pocket. The low frequency region of the resonance Raman spectrum of P. epiclitum deoxyHb (Fig. 2) shows several in-plane skeletal modes ( 8 ϭ 353, 7 ϭ 678, 15 ϭ 754 cm Ϫ1 ) similar to those observed in deoxyMb (30), although a significant frequency shift in 8 is observed (in deoxyMb, 8 is assigned at 342 cm Ϫ1 ). Bending modes sensitive to the conformation of propionate (␦ C␤CcCd ϭ 379 cm Ϫ1 ) and vinyl (␦ C␤CaCb ϭ 410 and 423 cm Ϫ1 ) groups of the protoheme are also assigned. The low-frequency modes in G. crumenifer deoxyHb appear at slightly different but comparable frequencies (Fig. 2, 8 ϭ 347, 7 ϭ 674, 15 ϭ 750, propionate bending ␦ C␤CcCd ϭ 375 cm Ϫ1 , vinyl bending ␦ C␤CaCb ϭ 413 and 421 cm Ϫ1 ); however, the intensity pattern of the bands differs significantly from that in P. epiclitum deoxyHb. Whereas it is unclear what the origin of the differences is, it is likely that the two deoxyHbs may have a significant difference in their heme peripheral interactions, although they share a substantial amino acid sequence identity (ϳ60%) (Fig. 1). However, some of these differences could result from our observation, from the high frequency resonance Raman spectra, that G. crumenifer deoxyHb is in a five-coordinate/ six-coordinate mixture whereas P. epiclitum is purely five-coordinate (data not shown).
The Fe-histidine (proximal) stretching mode, Fe-His , which is typically seen at ϳ220 Ϯ 20 cm Ϫ1 in five-coordinate Fe(II)-hemeproteins reflects out-of-plane characteristics of the heme iron as well as the strain experienced by the proximal histidine, both of which are related to the ligand binding propensity of the heme iron. Additionally, hydrogen bonding to the proximal histidine can also influence the value of the Fe-His mode. The Fe-His line in deoxyMb and human deoxyHb occurs at 220 and 215 cm Ϫ1 , respectively (27,28,31). The Fe-His modes in P. epiclitum and G. crumenifer deoxyHbs are assigned at 206 and 207 cm Ϫ1 , respectively ( Fig. 2), indicating conformational similarity of proximal histidine bonding to the heme in the two Hbs despite differences in their heme peripheral interactions as noted above.
Assignment of Fe-CO , C-O , and ␦ Fe-C-O in the CO Complex of P. epiclitum Hb-The Fe-CO stretching mode ( Fe-CO ) in the CO complexes of hemeproteins is sensitive to interactions of the bound CO with neighboring residues, thus offering a very useful tool to probe the active site conformation of hemeproteins in great detail. In the low frequency region of the resonance Raman spectrum of the CO complex of P. epiclitum Hb, three Fe-CO frequencies are detected at 539, 496, and 479 cm Ϫ1 (Fig. 3, bottom and Table 2). Assignment of these frequencies as Fe-CO stretching modes is confirmed by isotope ( 13 C 18 O) replacement experiments, where the corresponding lines appear at 519, 484, and 464 cm Ϫ1 , close to the values expected for a two body harmonic oscillator between iron and CO (calculated values of ⌬ Fe-CO of the two CO isotopes are 17.6, 16.2, and 15.6, respectively). As discussed below we label these species as the C, O, and N conformers, respectively. It may be noted here that observation of as many as three Fe-CO stretching modes widely separated in frequency and coexisting in a hemeprotein is uncommon, although two Fe-CO have been observed recently in a number of nonvertebrate hemoglobins (5,10,11,(32)(33)(34)(35). Recently, in human cytoglobin three closely lying Fe-CO modes (518, 510, and 492 cm Ϫ1 ) have been reported (36). Additionally, although a very low Fe-CO fre-   (7) 6 0 Bacteria (98) 85 0 a Alignment and database available from M. Marden, unpublished data. APRIL 28, 2006 • VOLUME 281 • NUMBER 17 quency (479 cm Ϫ1 ) has not been reported in a hemoglobin, such low frequencies have been detected in other types of heme proteins, mutants, and model complexes (37)(38)(39).

Multiple Fe-CO Conformers in Trematode Hbs
Two Fe-C-O bending modes (␦ Fe-C-O ) are assigned at 586 and 578 cm Ϫ1 for the 12 C 16 O complex, that shift to 564 and 555 cm Ϫ1 , respectively with 13 C 18 O (Fig. 3). It may be noted that the width of the difference bands in the ␦ Fe-C-O frequency region of the isotope difference spectrum suggests the presence of more than one mode in this region. Therefore, two closely lying bending modes are assigned in the spectra. The ␦ Fe-C-O mode at 586 cm Ϫ1 most likely corresponds to the FeCO conformer (C) with Fe-CO at 539 cm Ϫ1 , in analogy to the frequencies in Ascaris suum Hb ( Fe-CO at 543 cm Ϫ1 , and ␦ Fe-C-O at 588 cm Ϫ1 ) (10). The second bending mode (578 cm Ϫ1 ) may correspond to the FeCO conformer (O) with Fe-CO at 496 cm Ϫ1 on the basis of the empirical relation that the stretching and the bending frequencies bear a linear relationship as will be discussed in the next section. It may be noted though that the second bending mode observed here at 578 cm Ϫ1 is more intense than the analogous bending modes (for a FeCO conformer with Fe-CO in the 490 -500 cm Ϫ1 region) in other heme proteins.
The resonance Raman spectra of the C-O stretching modes ( C-O ) of P. epiclitum Hb (Fig. 3, top) shows one prominent C-O mode at 1927 cm Ϫ1 (1839 cm Ϫ1 with 13 C 18 O). We assign this C-O band as being associated with the FeCO conformer (C) that has a Fe-CO mode at 539 cm Ϫ1 . A second difference feature, albeit very weak, is seen at 1956/1874 cm Ϫ1 , which may be a candidate for C-O corresponding to the FeCO conformer (O) with Fe-CO at 496 cm Ϫ1 .
Assignment of Fe-CO , C-O , and ␦ Fe-C-O in the CO Complex of G. crumenifer Hb-Three Fe-CO frequencies are detected at 535, 492, and 476 cm Ϫ1 (Fig. 4, bottom) in the low frequency region of the resonance Raman spectrum of the CO complex of G. crumenifer Hb and they are also designated as the C, O, and N conformers. Assignment of these frequencies as Fe-CO stretching modes was confirmed by isotope ( 13 C 18 O) substitution measurements, in which the corresponding modes are detected at 514, 478, and 462 cm Ϫ1 , close to the values expected for a two-body harmonic oscillator between iron and CO (cal-     (Fig. 4). Assignment of the bending modes is made on the same basis as that presented above in the case of P. epiclitum Hb. The correspondence of the bending and stretching modes, based on the correlation described under "Discussion," are assigned as follows: the ␦ Fe-C-O mode at 582 cm Ϫ1 to the Fe-CO at 535 cm Ϫ1 (C conformer); the ␦ Fe-C-O mode at 575 cm Ϫ1 to the Fe-CO at 492 cm Ϫ1 (O conformer). The C-O frequency in G. crumenifer Hb (Fig. 4,  top) is assigned at 1929 cm Ϫ1 (1846 cm Ϫ1 with 13 C 18 O). We attribute this C-O to the FeCO conformer with Fe-CO at 535 cm Ϫ1 (C conformer). A spectrum was measured from a sample at low pH to determine if there was any pH dependence. In Fig. 4 (bottom) the spectrum in the low frequency region of the CO complex of G. crumenifer Hb at low pH (pH 5) is shown. No major changes are observed in Fe-CO as well as in other modes. All other CO spectra reported here are of samples at pH 7.4.
The three Fe-CO modes, the C-O mode as well as the two ␦ Fe-C-O modes in G. crumenifer are very similar to those detected in P. epiclitum Hb. It may also be noted that the other low frequency region modes as well as their intensity patterns in the spectrum of G. crumenifer HbCO are very similar to those seen in P. epiclitum HbCO. Although the CO complexes of the two Hbs are very similar, the deoxyHb species are markedly different in their heme peripheral modes (cf. Fig. 2). Thus, despite a difference in conformation of the deoxy forms, upon ligand binding the active site conformation in the CO complexes becomes very similar.
Resonance Raman Spectra of the Oxy Complexes-To test for the presence of multiple conformations in the oxy complexes of these two Hbs we recorded the spectra of the O 2 -bound proteins. In both cases the high frequency resonance Raman spectra and the optical absorption spectra confirmed that the oxy complex was formed. In the G. crumenifer Hb a mode was detected at 573 cm Ϫ1 with 16 O 2 in the resonance Raman spectrum, which shifted to 548 cm Ϫ1 with 18 O 2 (see supplemental data, Fig. S1). No additional oxygen isotope-sensitive lines from alternate conformations were detected in the spectrum. This line is assigned as the Fe-O 2 stretching mode. In the P. epiclitum Hb no oxygen-isotope sensitive line was found (see supplemental data, Fig. S2). Thus, for this complex the Fe-O 2 stretching mode is too weak to be detected. The Fe-O 2 stretching mode of many oxy complexes is weak so the absence any contribution from the P. epiclitum Hb is not unprecedented.

Low Frequency of Fe-His Stretching Mode-
The frequency of the Fe-His stretching modes in the P. epiclitum and G. crumenifer Hbs (206 and 207 cm Ϫ1 , respectively) are both very low compared with most other Hbs in which the mode is typically in the 220 -230 cm Ϫ1 region. The frequency of the Fe-His stretching mode in globins is sensitive to both proximal steric strain and the electronic coupling between the orbitals of the histidine and those of the heme iron. Both of these factors depend on the iron out-of-plane displacement, the hydrogen bonding between the imidazole N ␦ with proximal side residues, the tilting of the imidazole with respect to the heme and the orientation of the histidine with respect to the iron-pyrrole nitrogen bonds (32, 40 -43).
The P. epiclitum hemoglobin crystal structure shows typical hydrogen bonding of the imidazole N ␦ and a staggered orientation of the proximal imidazole with respect to the heme pyrrole nitrogens resulting in a planar heme macrocycle (18). In addition, the tilt of the imidazole ring, within its plane, with respect to the heme plane is typical of that in other hemoglobins. However, the plane of the imidazole ring is highly tilted with respect to the heme plane as may be seen for the two crystalline forms shown in Fig. 5. In one form (Structure A) the angle is ϳ75°a nd in the other (Structure B) it is ϳ60°. We propose that this tilted heme imidazole plane weakens the electronic coupling between the imidazole orbitals and the iron -orbitals resulting in a weaker bond and the associated lowered Fe-His stretching frequencies for these two Hbs.
Correlation among the Iron Carbonyl Modes-It is well established that there is an inverse correlation between the frequencies of the Fe-CO and the C-O modes shown in Fig. 6A. The two sets of modes assigned to the C and O structures of P. epiclitum lie on the inverse correlation curve and the one set of frequencies from the C conformer of G. cru-   Table 3.
menifer HbCO also falls on the curve. (No C-O mode was detected for the O conformer in G. crumenifer.) Although the C-O frequencies for the C conformer are much lower than that in Mb ( C-O at ϳ1945 cm Ϫ1 ), the fact that they and the frequencies for the O conformer in P. epiclitum HbCO lie in the correlation line indicates that the increase in Fe-CO bond order (because of -electron back donation from d of Fe to the empty * orbitals of CO) is compensated by a decrease in the C-O bond order, as has been observed in other systems (5,10,38,44). Fig. 6B shows a plot of the ␦ Fe-C-O mode against the Fe-CO mode for hemeproteins with both histidine and cysteine as axial ligands. The frequencies are listed in Table 3. A linear correlation is seen between the bending and stretching modes. Resolving and assigning the Fe-C-O bending mode is not straightforward when there are multiple CO conformers present (35,45) or when there are spectroscopic complexities such as Fermi resonance coupling of the bending mode (e.g. wild-type myoglobin shows a doublet at 576 and 585 cm Ϫ1 ; Ref. 46). Finally, linking a ␦ Fe-C-O mode to the associated Fe-CO mode (i.e. of the same CO conformer) requires assumptions in a multiconformer system. Fig. 6B provides a framework that will facilitate assignment of Fe-C-O bending modes in a complex system provided the bending modes are sufficiently resolved in the spectra. If the linear correlation holds for very low Fe-CO stretching frequency ranges (such as below 480 cm Ϫ1 ) one would expect a low frequency of the Fe-C-O bending mode as well. The model complex data supports this trend (Table 3); however, more data covering the low frequency ranges would be helpful for further confirmation of the correlation. The positive correlation is likely a consequence of the strengthening of the Fe-C bond. As the bending mode is primarily a motion of the carbon, when the Fe-C bond order increases, the carbon motion is more restricted so the bending frequency also increases.
Structural Similarity between P. epiclitum and G. crumenifer Hbs-Both P. epiclitum and G. crumenifer Hbs display three Fe-CO modes in their CO complex. The three Fe-CO modes represent three different conformations of the protein that have different electronic environments around the Fe-CO moiety. These conformers may participate in a kinetic equilibrium thus exerting direct influence on ligand association/dissociation events. A similar mechanism was proposed for A. suum Hb (10). The simultaneous presence of two CO conformers has been observed in several nonvertebrate Hbs (4, 5, 10 -12, 32-35, 47) as well as in other heme proteins (48 -52); and such CO conformers have been suggested to correspond to multiple conformers of the protein active site. The three FeCO conformers observed in P. epiclitum are very similar to those in G. crumenifer. This is consistent with significant amino acid sequence similarity that the two Hbs share (Fig.  1), including the residues that are likely to form the active site in these proteins. The x-ray crystallographic structure of P. epiclitum Hb has been solved (18), and it displays the classical globin fold on the basis of an eighthelix comparison with sperm whale Mb. However, several differences are observed, most notably in and around the active site that contains two tyrosine residues at the E7 and B10 helical positions in P. epiclitum Hb as compared with His and Leu, respectively, in sperm whale Mb. The crystal structure data match well with the predictions from the sequence alignment, although it may be noted that the E7 Tyr is significantly displaced from the exogenous ligand heme binding site. When the E7 residue is not such a bulky group (e.g. Tyr), which is indeed true in numerous globins, its side chain usually is accommodated in the ligand binding site. Displacement of the second tyrosine side chain in the trematode Hbs likely occurs because it is difficult for two bulky aromatic side chains to point toward the heme-bound ligand (for hydrogen bond formation). We postulate that G. crumenifer Hb shares its overall three-dimensional structure with that of P. epiclitum Hb, on the basis of sequence identity (ϳ60%), and the similar active site conformations in the CO complex of the two Hbs. The three CO conformers observed here are named as the C (closed) conformer (539/535 cm Ϫ1 in P. epiclitum/G. crumenifer), the O (open) conformer (496/492 cm Ϫ1 ), and N (negative environment) conformer (479/476 cm Ϫ1 ). The basis for these assignments is discussed below.
The C Conformer-The active site conformer with the highest frequency of Fe-CO (and low C-O ) is designated as the C conformer. This conformer represents a rigid (closed) structure in which we attribute strong ligand stabilization by hydrogen bonding to the B10 Tyr. Such conformers with strong hydrogen bonding stabilization to the hemebound ligand have also been observed in other nonvertebrate Hbs. For example, in A. suum Hb, the C conformer is stabilized by the B10 Tyr as well as E7 Gln (10). In barley Hb such conformers are stabilized primarily by the E7 His (11). In the two Hbs from Mycobacterium tuberculosis the conformer is stabilized by the B10 Tyr and the CD-1 Tyr (5,12,14,15,34). Corresponding to the high Fe-CO frequencies in P. epiclitum and G. crumenifer Hbs, the C-O modes are assigned at 1927 and 1929 cm Ϫ1 , respectively, for the C conformer and lie on the inverse correlation curve (Fig. 6A).
The O Conformer-The conformer with intermediate frequency for Fe-CO (at 496 and 492 cm Ϫ1 , respectively, in P. epiclitum and G. crumenifer) is designated as the O conformer. This conformer represents an open structure that should allow much faster ligand escape than the C conformer. In the O conformer, we postulate that the B10 Tyr has moved away from the CO such that the CO no longer experiences positive polarity from it or any other residue. Such an open conforma-  (54 -56). These open conformers in Mb have Fe-CO frequencies in the 490 cm Ϫ1 region. Open conformers also naturally occur in some nonvertebrate Hbs, such as in barley (11), M. tuberculosis (5,12), and in human cytoglobin (36), and may remain in equilibrium with other CO conformers as demonstrated by the pH dependence in barley Hb (11). Such conformational changes have also been associated with mechanisms of intersubunit communication (15).
The N Conformer-The conformer with the lowest frequency for Fe-CO in P. epiclitum and G. crumenifer (at 479 and 476 cm Ϫ1 , respectively) is designated as the N conformer. A conformer with such a low frequency is rare and has not been reported in wild-type Hbs or Mbs, although such low frequencies have been detected in mutant Mbs and model complexes (38,39). The only reported example of such a low Fe-CO frequency in a native hemeprotein is at 473 cm Ϫ1 in guanylate cyclase (37). On the basis of the hypotheses that an interaction between a negative charge (or polarity) with the heme-bound CO would increase the C-O bond order, and that the Fe-CO bond order would concomitantly decrease because of the loss of strong Fe3 CO -electron back donation, the N conformer may be envisaged as one in which a negative dipole in the active site interacts with CO, and the structure is pushed toward Fe-C'O ␦ϩ configuration rather than a FeϭCϭO ␦configuration resulting from a positive distal environment and strong back bonding (5,10,38,39,57). Empirical calculations using point negative charges predict that such a bond order relation would hold, and the Fe-CO bond order is expected to decrease because of the negative charge (44). This effect was confirmed in a Mb mutant (His(E7)Val/Val(E11)Thr) in which the Fe-CO stretching mode shifted from ϳ510 cm Ϫ1 in the wild-type protein to 478 cm Ϫ1 in the mutant (45,47). This was attributed to the interaction of the CO with the hydroxyl oxygen atom of the threonine. Thus, the N conformer likely results from an interaction between the CO and a negatively charged or non-polar group.
Structural Origin of the Conformer Differences-To determine the origin of the conformer differences we first examined the original crystal structure of P. epiclitum Hb (18). In the distal pocket the only residue that could interact with the heme ligand is the B10 Tyr. However, recently Milani et al. (58) reported that there is a great deal of plasticity in the structure of P. epiclitum Hb. Of particular interest was their observation that the Fe-O distance of the B10 Tyr 32 was 5.53 Å in one structure and 3.37 Å in another as shown in Fig. 5. This flexibility of the Tyr suggests that through changes in its position several different types of interaction could occur as illustrated in Fig. 7 and thus account for the three different CO forms. When B10 Tyr 32 is closest to the ligand, its oxygen atom may directly interact with the oxygen atom of the CO resulting in weak back bonding and a correspondingly low Fe-CO stretching mode (the N conformer). When the distance between B10 and the CO is more distant, the interaction will depend on the orientation of the proton on the Tyr hydroxyl group. When it is rotated away from the CO an open structure (O) is formed whereas when it is pointed toward the CO the positive interaction results in the closed structure (C) and the very strong back-bonding. The three different forms then are a consequence of the two different reported structures and two different rotational conformers of the Tyr hydroxyl group.
Relation of the Conformers to Ligand Kinetics-Our data confirm the presence of multiple conformers of the distal pocket for the CO-bound complexes of these two trematode Hbs. It has been shown that the nature of the residues in the distal pocket significantly affects the ligand kinetics but quite unexpectedly there is no correlation between the Fe-C-O vibrational frequencies and the ligand affinity (47). On the other hand there is a rough correlation between the frequency of the Fe-CO stretching mode and the CO dissociation rate constant. The lack of a correlation between the affinity and the mode frequency has been attributed to the need, upon ligand entry, to displace a water molecule from the distal pocket that has a stabilization which depends on the electrostatic properties of the distal residues, whereas the CO dissociation rate depends only on its direct stabilization (47). In a case such as that presented here, the ligand binding properties are further dependent on the equilibrium among the three conformers.
The combination of stabilizing and destabilizing interactions in trematode Hbs is different from the multiple conformations observed in other hemoglobins., For example in A. suum Hb, E7 Gln and B10 Tyr both provide hydrogen bonding stabilization to the bound ligand (10, 32, 59 -63). In trematode Hbs, while B10 Tyr provides hydrogen-bonding stabilization to the bound carbon monoxide, interaction with a negative polarity of the B10 Tyr lone pair in the alternate conformation causes a destabilizing effect. This effect may be manifested in the oxy complex as well. However, the presence of only one Fe-O 2 mode in the G. crumenifer Hb at a high frequency (573 cm Ϫ1 ) suggests that the O 2 is bound in the conformation in which the Tyr 32 is at the longer distance from the heme group. It is unlikely that the oxygen would bind to the heme in the alternate conformation, the N conformer, as in that case the negatively charged environment resulting from the nearby Tyr would destabilize the structure. There is a substantial increase of ligand off-rates in the oxy complex of trematode Hbs in comparison to A. suum Hb (see Table 4) despite the fact that the Fe-O 2 stretching mode in the A. suum Hb is at 570 cm Ϫ1 (10). However, the higher frequency (573 cm Ϫ1 ) may signify a weaker interaction in the G. crumenifer Hb with the bound oxygen. Whereas in A. suum Hb a dual hydrogen bonding from both E7Gln and B10 Tyr provides strong stabilization of the oxy complex, the single B10  Tyr in the trematode Hbs may not stabilize the bound oxygen as much, resulting in the higher off-rates. It is difficult to pinpoint the exact contribution of the three active site conformers toward ligand kinetics. However, a qualitative assessment can be made by assuming a kinetic equilibrium between the conformers for the CO complex. A similar mechanism was proposed in the case of A. suum Hb (10). If a rapid equilibrium between the conformers is assumed, the most destabilizing conformer (N conformer) will determine the ligand dissociation rate as shown in Reaction 1.
͓͑Hb-CO͒ c 7 ͑Hb-CO) O ͔ 7 ͓͑Hb-CO͒ N ͔ 3 Hb ϩ CO REACTION 1 The brackets represent the two different reported crystal structures (18,58). The O and the C conformers, differing only by a rotation of the Tyr hydroxyl group, are both in the conformation in which the Tyr is far from the heme iron (5.53 Å). The N conformer is in the configuration in which the Tyr is close to the heme iron (3.37 Å). This model means that the most stabilized conformer (C conformer) has minimal effect. Such a proposal is consistent with the nanosecond photolysis data that show a minimal effect of the initial distribution of stabilized conformers on rebinding kinetics in mutant Mbs and nonvertebrate Hbs. 4 Instead, a rapid relaxation of the B10 residue subsequent to photodissociation dictates rebinding kinetics. This model accounts for the high CO dissociation rates listed in Table 4.