From the Department of Physiology and Biophysics,
Albert Einstein College of Medicine, Bronx, New York 10461 and the
§ CNR Center of Molecular Biology, c/o Department of
Biochemical Sciences, University "La Sapienza,"
00185 Rome, Italy
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ABSTRACT |
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The ferric form of the homodimeric
Scapharca hemoglobin undergoes a pH-dependent
spin transition of the heme iron. The transition can also be modulated
by the presence of salt. From our earlier studies it was shown that
three distinct species are populated in the pH range 6-9. At acidic
pH, a low-spin six-coordinate structure predominates. At neutral and at
alkaline pHs, in addition to a small population of a hexacoordinate
high-spin species, a pentacoordinate species is significantly
populated. Isotope difference spectra clearly show that the heme group
in the latter species has a hydroxide ligand and thereby is not
coordinated by the proximal histidine. The stretching frequency of the
Fe-OH moiety is 578 cm The homodimeric hemoglobin
(HbI)1 isolated from arcid
clam Scapharca inaequivalvis possesses unique structural
features (1-14). Although HbI has a low sequence homology with
tetrameric mammalian hemoglobins (Hb), it has a conserved globin fold.
It displays a unique structural basis for cooperative ligand binding in
that the two hemes face each other across the intersubunit contact and
are able to communicate directly through their propionate groups (2, 4,
15, 16). As a consequence of this unusual structural linkage,
cooperativity is accompanied by major tertiary changes in the heme
environment with only minor rearrangements of the quaternary structure,
in contrast to mammalian Hbs (2, 13). The subunit interface in HbI is
formed mostly by the helices E and F, which are solvent-exposed in
tetrameric mammalian Hbs.
Although a wealth of structural and functional information is available
on the ferrous deoxy and ligand-bound forms of HbI (2-14), little is
known about the oxidized form (1). The heme structure in the ferric
form undergoes a spin transition that is dependent on pH and salt
concentration (17, 18). From our earlier studies by optical and
resonance Raman spectroscopy, it was shown that a mixture of three
distinct species are populated in the pH range 6-9 (17, 18). The
formation of a six-coordinate low-spin heme is favored at acidic pH and
high ionic strength and is accompanied by the reversible dissociation
of the HbI dimer into monomers. This species is likely to be formed by
coordination of the distal histidine to the heme (in addition to the
proximal histidine) as shown by EPR studies (18). At neutral pH values, a dimeric pentacoordinate species appears and becomes the dominant form
at alkaline pH and low salt. A population of dimeric six-coordinate high-spin heme exists over the entire 6-9 pH-range and in both low and
high salt. This six-coordinate high-spin species is a typical aquomet
form (18) such as that commonly observed in mammalian myoglobins (Mb)
and hemoglobins (19, 20).
The occurrence of a low-spin bishistidine heme has been proposed to
exist in some invertebrate Hbs (21-23). However, conversion of a
hexacoordinate species into a pentacoordinate form, as seen in HbI, is
very rare in Mbs and Hbs, and the structure of the pentacoordinate
species in HbI is not known. The ferric Mb from the mollusk
Aplysia limacina is also reported to contain a
pentacoordinate species at acidic pH (5.9) in which the proximal
histidine is proposed to serve as the fifth ligand (24). The
observation of a strong pH-dependence of the ferric form of
Aplysia Mb was suggested to be the consequence, in part, of
the absence of a distal histidine (24). Scapharca HbI, on
the other hand, contains a distal histidine yet also shows a strong pH
dependence of the heme ligation structure. The optical spectrum and the
relative intensity of the Raman marker line ( Studies on the ferric form of Hbs (metHb) have significantly advanced
our understanding of the structural basis for the cooperative transition in Hb. Under physiological conditions, metHb is formed by
spontaneous autoxidation, and the redox equilibrium is shifted toward
the reduced form by the metHb reductase system. Recently it was shown
that the formation of metHb may result also from interaction with NO
in vivo (25). In addition, metHbs are extremely useful to
study because numerous ligands form stable ferric heme complexes that
have a wide range of electronic properties. Therefore, several studies
on metHbs have been carried out as a means of understanding the
mechanism of allosteric control (26-33).
In the present study, we have characterized the pentacoordinate form of
oxidized Scapharca HbI by resonance Raman spectroscopy and
identified the fifth ligand to the heme unambiguously as hydroxide rather than histidine. This finding, which provides direct evidence for
cleavage of the Fe-proximal His bond in ferric HbI, is discussed in the
framework of known manifestations of strain in that bond in ferrous HbI
and in liganded T-state human hemoglobin crystals (34).
The HbI from S. inaequivalvis was isolated and
purified as described elsewhere (5). The protein samples were oxidized
by addition of potassium nitrite to the oxygenated protein. The excess oxidant was removed by gel filtration through a Sephadex G-25 column
equilibrated with the desired buffer. The protein was stored in liquid
nitrogen until use.
The concentration of the protein samples used for the Raman
measurements was 35 µM in 40 mM buffer (MES,
pH 6.0; phosphate, pH 7.4; CHES, pH 9.0). To prepare the protein
samples in isotopic water, the following composition was used: 35 µM ferric HbI, 40 mM buffer, 80%
H218O or D2O (using 98%
H218O from Cambridge Isotope Laboratories,
Andover, MA; D2O from Aldrich, Milwaukee, WI), and 20%
H216O. Deoxy samples were prepared by adding an
aliquot of dithionite solution to an anaerobic HbI solution prepared in
buffers at the desired pH values. Absorption spectra were recorded
before and after the Raman measurements to ensure the stability of the
species studied. The samples were placed in a spinning cylindrical cell with a 2-mm light path. An incident laser frequency of 413.1 nm (Kr-ion
laser, Spectra Physics) was used, and 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) as
described elsewhere in detail (35). A holographic notch filter (Kaiser,
Ann Arbor, MI) was used to remove the laser scattering. Typically, five
30-s spectra were recorded and averaged after removal of cosmic ray
spikes by a standard software routine (CSMA, Princeton Instruments, NJ).
The resonance Raman spectra of ferric Scapharca HbI,
shown in Fig. 1, were measured at acid
(pH 6.0) and alkaline (pH 9.0) pHs in 40 mM buffer. At
alkaline pH, the pentacoordinate heme species is very prominent as
judged by its 1 and shifts to 553 cm
1 in H218O, as would be
expected for a Fe-OH unit. On the other hand, the ferrous form of the
protein shows substantial stability over a wide pH range. These
observations suggest that Scapharca hemoglobin has a unique
heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the
functional deoxy form of the protein but not in the ferric form.
INTRODUCTION
Top
Abstract
Introduction
References
3) of the
pentacoordinate species in Aplysia Mb, however, are very
different from the pentacoordinate species seen in Scapharca
HbI. Furthermore, as described above, the nature of the
pH-dependent transition in Scapharca HbI is very
unique as compared with the aquo-hydroxy transition in vertebrate Mbs
and Hbs. Hence, it is important to determine whether the unique optical
properties reflect some unanticipated axial ligation states in ferric
Scapharca HbI.
EXPERIMENTAL PROCEDURES
RESULTS
3 band at 1490 cm
1. The
spectrum shows also the presence of a six-coordinate low-spin (
3 = 1506 cm
1) species and a small
population of six-coordinate high-spin (
3 = 1480 cm
1) species. At acid pH, however, the low-spin species
is dominant.
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Fig. 1.
Resonance Raman spectra of the ferric form of
the Scapharca HbI in the high frequency region.
Spectra shown are in 100% H216O, pH 9.0 (a) and pH 6.0 (b). Laser excitation wavelength
was 413.1 nm, and power delivered at sample was 8 mW.
To identify the nature of the pentacoordinate species, the low
frequency region of the spectra were measured at alkaline pH as a
function of the isotopic composition of the water. The low frequency
region of the resonance Raman spectrum is very useful in identifying
metal-ligand vibrations as it can directly demonstrate the presence of
a particular ligand and the nature of its interactions in the heme
pocket (35). In particular, a line in the 490-560 cm1
region has been assigned to the Fe-OH stretching frequency in alkaline
ferric hemoglobin, myoglobin, and horseradish peroxidase (19, 20). The
position of this line is sensitive to the spin state of the metal and
the strength of hydrogen bonding to the hydroxide moiety of these
proteins. Fig. 2 shows the low frequency region of the resonance Raman spectra of ferric Scapharca
HbI in alkaline-buffered solutions of water of various isotopic
composition. The Raman line at 578 cm
1 in
H216O (Fig. 2, spectrum a) shifts to
553 cm
1 in H218O (spectrum
b), and yields a clear difference spectrum (spectrum d). However, the spectrum in D2O (spectrum
c) does not change appreciably compared with that in
H2O as also seen from the difference spectrum shown in
spectrum e. We assign the line with the oxygen isotope
sensitivity as the iron-hydroxide stretching mode
(
Fe-OH) arising from the Fe-OH moiety of the
five-coordinate heme in HbI. As expected for this assignment, the
feature at 578/553 cm
1 is absent at pH 6 (data not
shown), where the population of the five-coordinate heme is negligible.
No isotope sensitivity was observed for any other line in the spectra
at pH 6.
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It is important to note that the 25 cm1 isotope shift
(16O/18O) corresponds to a nearly ideal value
(23.8 cm
1 assuming that the two oscillating units are the
Fe and the OH) that would be expected from an "isolated" Fe-OH
harmonic oscillator. In the event the OH group had strong
hydrogen-bonding or strong non-bonding interactions, the expected
isotope shift would not be observed because of a deviation from a
perfect two-body oscillator (19). One intriguing observation is that
the Fe-OH moiety did not show any appreciable D2O effect.
We postulate that such a situation could arise if the Fe-O-H unit
exists in a significantly bent configuration so that the vibration of
the O-H unit is not coupled with that of the Fe-O unit. In such a case,
the Fe-O-H oscillator would show an isotope shift in
H218O but would not be responsive to
replacement of the hydrogen by deuterium (in D2O).
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DISCUSSION |
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In summary, the three heme species identified by optical and resonance Raman spectroscopy can be schematically represented as shown in Fig. 3. The pentacoordinate high-spin species (Fig. 3a) is unambiguously assigned in the present work as a hydroxide form. Therefore, the proximal histidine bond is no longer retained; instead, the hydroxyl group binds to the iron accounting for the fifth ligand. The species shown in Fig. 3, b and c are, respectively, the high- and low-spin hexacoordinated forms, both of which retain their proximal Fe-His bond, but have water and the distal histidine, respectively, as their sixth ligands. The identity of the high-spin hexacoordinate form (Fig. 3b) is based on the similarity of its optical and resonance Raman spectra to those of aquomet Mbs and Hbs. The assignment of the low-spin hexacoordinate form (Fig. 3c) relies on our previous EPR study (18) that determined g-values similar to those of the bishistidine heme complexes. Hence, it was inferred that the distal histidine binds the heme iron at acidic pH.
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In six-coordinate hydroxy complexes of hemeproteins with histidine or
imidazole as an axial ligand, the Fe-OH mode is located in the 450-560-cm
1 region (19, 20, 36). In the
five-coordinate form of HbI,
Fe-OH appears at a
significantly higher frequency. We attribute the high frequency to two
origins. First, the absence of a ligand trans to the hydroxide can
strengthen the Fe-OH bond in comparison with the case when the proximal
histidine is present. Second, the higher frequency of the line as
compared with the five-coordinate hydroxide species in the H175G mutant
of cytochrome c peroxidase (37), as well as it being
significantly narrower, indicates that there are no strong interactions
between it and any neighboring residues in its binding pocket. The
similarity of the very high frequency for
Fe-OH in the
hydroxide-bound heme in HbI to that in H93G Mb, also shown to be a
five-coordinate hydroxide
complex,2 suggest that the
hydroxide group has a hydrophobic environment and there is a partially
collapsed structure of the proximal cavity in HbI just as in H93G Mb.
Thus, on cleavage of its bonding to the heme iron, the proximal
histidine undergoes a substantial structural rearrangement resulting in
a partial collapse of the proximal cavity. In turn, the heme, which
remains inside the heme pocket because of stabilization by hydrophobic
interactions, has an altered orientation as indicated by the changes in
the polarized absorption spectra of ferric HbI crystals brought to
alkaline pH (38).
In contrast to ferric HbI, the ferrous form remains quite stable over a
wide pH range, as judged from the invariance of all its properties,
including the resonance Raman spectrum (data not shown). One specific
feature is of interest, namely the very low Fe-His stretching frequency
(203 cm1) of the deoxygenated protein, which indicates a
high degree of proximal strain (39). Despite the proximal strain, the
protein is able to bind oxygen cooperatively even if that leads to
movement of the iron into the heme plane presumably straining the
Fe-histidine bond even further. However, ligand-binding to the ferrous
protein is facilitated by movement of Phe-97 that swings out of contact with the heme and the proximal histidine on ligand binding in the
ferrous protein, hence performing a dual task of information transfer
to the contralateral subunit and partially reducing the extra strain on
the proximal histidine. We postulate that such structural
rearrangements characteristic of the deoxy
oxy transition do not
take place in the hydroxide form in the ferric protein. If binding
hydroxyl leaves the protein in its "T-like" state, Phe-97 does not
reorient and will cause steric crowding of the proximal histidine
destabilizing the Fe-His bond. The aftermath of the Fe-His bond rupture
is a partial collapse of the proximal cavity that retains the heme
group as a pentacoordinate hydroxide-bound species.
Observation of a pentacoordinate ferric heme in native myoglobins or
hemoglobins is uncommon (19, 34, 40). A pentacoordinate species
observed in the Aplysia Mb at acidic pH values has a much broader and less intense 3 line at 1495 cm
1, which is suggested to arise because of the
destabilization of water binding to the heme and not to the breakage of
the proximal histidine (24). In the proximal-histidine mutant of sperm
whale Mb (H93G) on the other hand, the
3 line appears at
1490 cm
1. In the absence of the proximal histidine, H93G
stabilizes the heme as a pentacoordinate hydroxide-bound
species2. Horseradish peroxidase stabilizes a
pentacoordinate heme but without cleaving the proximal bond. Similarly,
in freshly prepared native cytochrome c peroxidase, a
five-coordinate heme is the stable form, coordinated by the proximal
histidine (41). In a mutant (H175G) of cytochrome c
peroxidase, where the proximal histidine was mutated to a
non-coordinating ligand, the heme showed a mixture of aquomet complexes
including a pentacoordinate species that was suggested to be a
heme-hydroxide complex (37).
The cleavage of the Fe-proximal His bond in alkaline ferric HbI is
particularly intriguing in light of recent crystallographic observations on human cyano-met T-state ferric HbA (34). Quite unexpectedly, the analysis of the heme geometry and coordination state
revealed that the distance between the iron and the nitrogen of the
proximal histidine in the cyanide-bound -chains is much too long to
allow for covalent bond formation (2.9-3.1 Å). Thus, the
-chains
in ferric, cyanide-bound T-state HbA is another example of a
pentacoordinate species in which the proximal bond is broken because of
the presence of a strong iron ligand on the distal side. Yet, in the
chains, cyanide binding leaves the proximal bond unaltered,
indicating that its effective strength does not depend solely on the
presence of a strong distal ligand. Moreover, in contrast to the
behavior of the hydroxide adduct, in the cyano complex of HbI, there is
no evidence for rupture of the proximal histidine bond (12). In this
connection, it should be noted that a number of spectroscopic markers
point to a strained, distorted coordination of the heme iron in ferrous
HbI, e.g. the broad anisotropic EPR signal of the
Co-porphyrin protein (42), the small hyperfine shift of the
N
proton resonance (43) and the unusually low frequency
of the Fe-His mode (39). Interestingly, these features are shared by
the
chains in the ferrous HbA tetramer (44, 45). Thus, the heme
coordination geometry and the properties of the exogenous ligand both
determine whether or not the proximal histidine bond will be ruptured.
In several ferrous heme proteins, rupture of the proximal histidine
bond has been observed when NO adducts are formed (13, 46-51). In
deoxy hemoglobin, a five-coordinate NO complex forms in the -chains
in the presence of inositol hexaphosphate, and in myoglobin, the
five-coordinate NO complex forms at low pH (pH ~4). The
five-coordinate form of ferrous guanylate cyclase, generated by the
coordination of NO, has been postulated as the active structure that
catalyzes the formation of cyclic GMP (47, 49-51). Thus, determination
of the factors that lead to the rupture of the proximal histidine bond
are essential for the full understanding of the functional forms of
these proteins.
In conclusion, the present data on HbI and the comparison with liganded
T-state HbA suggest that pentacoordinate derivatives which lack the
proximal iron-histidine bond do not occur solely in selected hemoglobin
and myoglobin mutants but may have a general significance in the native
proteins. The biochemical consequences of this unique heme coordination
on the kinetics and thermodynamics of anionic ligand binding to ferric
hemoproteins remain to be determined and will be the object of future investigations.
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FOOTNOTES |
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* This work was supported in part by Research Grants GM54806 and GM54812 from the National Institutes of Health (to D. L. R.), and the grant "Structural Biology" from Ministero dell'Universita della Ricerca Scientifica e Tecnologica of Italy (to E. C.).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: Dept. of Physiology and Biophysics, Albert Einstein college of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4264; Fax: 718-430-4230; E-mail: rousseau{at}aecom.yu.edu.
The abbreviations used are: HbI, homodimeric hemoglobin; HbA, human adult hemoglobin; Hb, hemoglobin; Mb, myoglobin; metHb, ferric hemoglobin; MES, 4-morpholineethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.
2 T. K. Das, S. Franzen, A. Pond, J. H. Dawson, and D. L. Rousseau, unpublished results.
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REFERENCES |
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