(Received for publication, June 20, 1995; and in revised form, October 2, 1995)
From the
Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal
polarized absorption microspectrophotometry. The saturation curve,
characterized by a Hill coefficient n =
1.45 and an oxygen pressure at half saturation p
= 4.8 torr, at 15 °C, shows that HbI in the
crystalline state retains positive cooperativity in ligand binding.
This finding will permit the correlation of the oxygen-linked
conformational changes in the crystal with the expression of
cooperativity.
Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species.
The study of the homodimeric, cooperative hemoglobin from the
clam Scapharca inaequivalvis, HbI, ()has suggested
a mechanism of cooperativity in oxygen binding that is radically
different from that operative in HbA. The mechanism is based on the
comparison of the deoxy- and liganded HbI crystal structures,
determined in orthorhombic and monoclinic crystals, respectively, and
entails prominent tertiary changes in the heme environment but only
subtle quaternary
changes(1, 2, 3, 4) . Communication
between the heme groups via localized structural changes is made
possible by the unusual assembly of the globin chains. In HbI, the
heme-carrying E and F helices form the dimer interface (5) rather than being exposed to solvent as in HbA. Thereby,
the heme groups are brought into nearly direct contact through a
network of hydrogen bonds, which is modified upon
oxygenation(2, 3, 4, 5) .
In order to establish whether the crystal structures of HbI fully correlate with function in solution, we asked the question whether cooperative oxygen binding is retained by the protein in the crystalline state. In fact, it cannot be excluded, a priori, that crystallization conditions favor a protein conformation different from that prevailing in solution and that lattice interactions restrict its activity-related movements. The functional behavior has been commonly used as a stringent criterion to compare structural properties of proteins in solution and in the crystalline state(6, 7, 8, 9) .
The response
of crystals of human hemoglobin A (HbA) to oxygenation exemplifies the
delicate interplay between intramolecular rearrangements associated
with ligand binding and intermolecular interactions stabilizing the
crystal. Deoxy-HbA crystals, grown from high salt solutions, shatter in
the presence of oxygen(10, 11) , as lattice
interactions neither prevent nor accommodate the T R quaternary
transition(12, 13) . In contrast, deoxy-HbA crystals,
grown from polyethylene glycol solutions, are stable when exposed to
oxygen because the protein remains in the T quaternary state with
intact salt bridges(14, 15, 16) . A single
crystal polarized absorption microspectrophotometric study has shown
that oxygen binds noncooperatively to these crystals, saturating both
and
hemes (17, 18) . Similar studies have
been carried out on stable crystals of Hb Rothschild (19) and
des-Arg HbA(20) .
Single-crystal polarized absorption microspectrophotometry is here exploited to determine oxygen binding curves to HbI crystals grown in the deoxy state. To obtain a precise estimate of the fractional saturation of reduced hemes, it is critical to evaluate exactly the amount of HbI molecules present in the oxidized form. The spectrum of soluble HbI in the oxidized state is pH-dependent and reflects an equilibrium distribution between two high spin dimeric components and a low spin monomeric hemichrome(21) . Therefore, this equilibrium has been characterized in the crystalline state.
Single crystal polarized absorption spectra were recorded between 450 and 700 nm. The electric vector of the incident polarized light was parallel either to the a or to the b crystal axis. These axes coincide with the diagonals of the plate and are principal optical directions of the crystal. Polarized light absorption along these directions obeys the Beer-Lambert law. The optical theory for myoglobin and hemoglobin crystals has been previously reported(18, 24, 25) .
Oxygen binding curves were determined by recording polarized absorption spectra of HbI crystals equilibrated with either progressively increasing or progressively decreasing oxygen pressures, at 15 °C, as described previously(17, 18) . The time required to obtain a stable spectrum of oxygenated HbI crystals depends on crystal thickness and oxygen pressure. For oxygen pressures below 2 torr, equilibration times for HbI crystals suitable for microspectrophotometric studies are of the order of days, and oxygen electrode drift precludes measurements. Long equilibration times also cause the unavoidable formation of oxidized HbI. For these reasons, even at higher oxygen pressures, each crystal was used for no more than three measurements.
The pH-dependent absorption spectra of oxidized HbI in solution were analyzed with a singular value decomposition algorithm (Matlab, The Math Works Inc., Natick, MA) and fitted according to the equilibrium scheme given by Spagnuolo et al.(21) , which describes the pH-dependent interconversion of three components, a dimeric hexacoordinate, a dimeric pentacoordinate, and a monomeric hemichrome.
The pH-dependent
polarized absorption spectra of oxidized HbI in the crystal were fitted
to a linear combination of the basis spectra of the three oxidized
species in solution. The contributions of the different components to
the observed polarized spectra A were calculated
by the following expression:
where A, A
, and A
are the basis spectra in solution of the dimeric
hexacoordinate, dimeric pentacoordinate, and monomeric hemichrome,
respectively, and c
, c
, and c
are coefficients proportional to both the
fractional concentration of the various species and their extinction
coefficients along each crystal axis. c
A
represents a horizontal offset. The use of the
above expression is justified by the observation that the ratio of
absorbances at each wavelength in the two perpendicular directions of
polarization is almost constant, i.e. the electronic
transitions are almost perfectly x, y polarized(18, 24, 25) .
Figure 1: Single crystal polarized absorption spectra of HbI at different oxygen pressures. Crystals of HbI, suspended in a solution containing 3.5 M potassium phosphate, 1 mM EDTA, 0.35 mg/ml catalase, pH 8.5, at 15 °C, were equilibrated with humidified oxygen mixtures between 2 and 37 torr. Polarized absorption spectra were recorded with the electric vector parallel either to the a or to the b crystallographic axis. Due to the long time required to equilibrate a crystal at low oxygen pressures, one crystal was used for no more than three measurements. Spectra of different crystals were normalized to the reference crystal spectra (see Fig. 2). Oxygen pressure is plotted on a log scale such that distances are proportional to chemical potentials.
Figure 2: Single crystal polarized absorption spectra and polarization ratios of deoxy-HbI, oxy-HbI and oxidized HbI (reference spectra). The spectra of oxidized HbI were recorded on a crystal, oxidized with 2 mM ferricyanide and then washed with 3.5 M potassium phosphate, pH 8.5 (panel a); the spectra of deoxy-HbI were obtained by suspending the same crystal in 30 mM dithionite (panel b); the spectra of oxy-HbI were obtained by resuspending the crystal, after removal of dithionite, in a solution equilibrated at 760 torr of oxygen (panel c). The contribution of a small fraction of oxidized HbI to the oxy-HbI spectra, as evidenced by a shoulder at 602 nm, was subtracted, and the spectra were properly normalized as described previously(18) . For each set of spectra, the polarization ratio is plotted on the top portion of each panel. In panel d, the observed polarized spectrum, recorded with the electric vector parallel to the a crystal axis, at an oxygen pressure of 5.5 torr (-), is compared with that calculated from the fitting to a linear combination of the reference spectra(- - -), the fractional contribution of the spectra of deoxyHbI, oxyHbI, and oxidized HbI being 42.5, 50.1, and 7.4%, respectively. The fractional saturation with oxygen, defined as the ratio of oxygenated hemes over reduced plus oxygenated hemes, is 54.5%.
Hill plots of the data are shown in Fig. 3. The oxygen pressure required for half-saturation (p) is 4.9 ± 1.0 and 4.8 ± 0.8
torr, as determined from spectra recorded along the a and the b crystal axes, respectively. The Hill coefficient (n
) determined from the two sets of data is 1.43
± 0.07 and 1.46 ± 0.06, respectively. In solution, at
15.1 °C, in 0.1 M phosphate buffer, pH 7.8, the p
for HbI is 5.7 torr and the Hill coefficient is
1.44(26) .
Figure 3:
Hill plots of oxygen binding data for HbI
crystals. Crystals of HbI were suspended in 3.5 M potassium
phosphate, 1 mM EDTA, pH 8.5, at 15 °C. The Hill
coefficient n and the p
are
1.43 ± 0.07 and 4.9 ± 1.0 torr, respectively, from data
recorded along the a axis (open symbols, dashed
line), and 1.46 ± 0.06 and 4.8 ± 0.8 torr from data
recorded along the b axis (closed symbols, solid
line), either by increasing (
,
) or decreasing
(
,
) the oxygen pressure.
Polarized absorption spectra of hemoglobin
crystals provide information on the heme orientation in different
ligation states(24, 25) . The pertinent parameter is
the polarization ratio (PR), i.e. the ratio of the absorbances
in two perpendicular directions at wavelengths where the electronic
transitions are almost perfectly x, y polarized. A
good estimate of PR can be obtained by calculating the ratio of
absorbances at 556 nm for deoxy-HbI and at 542 nm for oxy-HbI. The PR
values (A/A
) are 1.79 and
1.81 for deoxy- and oxy-HbI, respectively (Table 1), indicating
that oxygen binding is not accompanied by a heme tilt. This result is
in agreement with the crystallographic finding that hemes have the same
orientation in deoxy-HbI (2) as in CO-HbI (1, 3) and oxy-HbI crystals(4) .
Figure 4:
Dependence on pH of the spectra of
oxidized HbI in the crystal and in solution. a, a single
crystal of oxidized HbI was suspended in a solution containing 3.5 M potassium phosphate at pH 6.3 (),
6.8(- - - -), 7.0(- - -), 7.5 (- -
-) and 8.5 (-). Polarized absorption spectra were
recorded along the a and the b crystal axes. Inset, the absorbance change at 600 nm along the a (
) and the b axis (
) was fitted to the
equation for the ionization of a group with pK
= 7.07 ± 0.13 and 7.02 ± 0.11,
respectively. b, absorption spectra of 1 mM oxidized
HbI in a solution containing 1 M potassium phosphate at pH 6.0
(
), 6.5(- - - -), 7.0(- - -),
7.5 (- - -), and 8.0 (-), at 15 °C.
Spectra were normalized by subtraction of a horizontal offset (see panel d). The optical path was 1 mm. c, fitting of
the oxidized HbI spectrum in the crystal at pH 7.5, recorded along the a axis (
), to a linear combination
(-) of the spectra of high spin hexacoordinate(-
- -), high spin pentacoordinate
(-
-
-), low spin hemichrome species (not
detectable), and an offset (- - -). The basis spectra
are taken from Spagnuolo et al.(21) . The residuals
between observed and calculated spectra are reported in the top part of
the panel. d, fitting of the spectrum of oxidized HbI in a
solution containing 1 M potassium phosphate, pH 7.5
(
), to a linear combination (-) of the
spectra of high spin hexacoordinate (- - -), high
spin pentacoordinate (-
-
-), low spin
hemichrome species (
) and an offset (- -
-)(21) . The residuals between observed and calculated
spectra are reported in the top part of the
panel.
Figure 5:
Dependence on pH and phosphate
concentration of the fraction of high spin aquomet, high spin
pentacoordinate, and low spin hemichrome for crystalline and soluble
oxidized HbI. The fractions of high spin hexacoordinate (,
), high spin pentacoordinate (
,
), and low spin
hemichrome (
,
) species were calculated by a linear
combination of the basis spectra as described under ``Materials
and Methods''. Panel a, crystalline HbI; open and closed symbols refer to data recorded along the a and b crystal axes, respectively. Panel b,
soluble HbI in 1 M phosphate buffer. Panel c, soluble
HbI in phosphate buffer, pH 7.5.
The calculated pK for the transition
between the two dimeric species is 7.07 ± 0.13 and 7.02 ±
0.11, as determined by polarized absorption spectra along the a and the b crystal axes, respectively (Fig. 4a, inset).
In solution, the hemichrome and the high spin hexacoordinate component are the predominant species under all the conditions investigated (Fig. 4d and Fig. 5, b and c). The amount of hemichrome decreases in the alkaline range with a concomitant increase of the pentacoordinate heme, whereas the amount of hexacoordinate heme is essentially constant over the pH range studied. In 3 M phosphate, namely at an ionic strength approaching that of the crystal suspending medium, the amount of hemichrome is significantly higher than in 1 M phosphate (Fig. 5c).
The polarization ratio for oxidized HbI crystals was calculated from the reference spectra at pH 8.5 (Fig. 2a) either at 602 nm or at 500 nm. At these wavelengths, which correspond to the absorbance maxima of the pentacoordinate and hexacoordinate high spin components, respectively, the heme behaves as a planar absorber(25) . The PR values obtained, 2.4 and 2.3, respectively, differ considerably from the value characteristic of the ferrous protein.
Allosteric transitions of oligomeric proteins within the crystal lattice are usually limited by intermolecular interactions that stabilize a particular conformational state. This is the case for human hemoglobin A, Hb Rothschild, and des-Arg Hb, crystallized from polyethylene glycol solutions, that remain in the T state upon oxygenation(14, 15, 16, 17, 18, 19, 20) . However, when trigonal crystals of Escherichia coli aspartate carbamoyltransferase in the T state are soaked in a solution containing the substrate L-aspartate and phosphate, a slow molecular transition to the R state takes place, suggesting a kinetic rather than a thermodynamic barrier between the two conformations(27) . Furthermore, in crystals of tetrameric L-lactate dehydrogenase from Bifidobacterium longum, grown from a solution where the R state conformation prevails, lattice forces favor a 1:1 mixture of T and R state molecules, the latter ones binding a substrate analog (28) .
Activity measurements have provided an independent
criterion to establish whether and to which extent lattice interactions
affect symmetry and regulation of some oligomeric proteins.
Glyceraldehyde-3-phosphate dehydrogenase from lobster muscle retains in
the crystalline state the negative cooperativity
(``half-of-the-sites reactivity'') exhibited in solution in
the reaction with the chromophoric acylating reagent
-(2-furyl)acryloylphosphate(29, 30, 31) .
The tryptophan synthase
complex from Salmonella typhimurium maintains the reciprocal regulation of
ligand binding and catalytic activity of the
and
sites
mediated by intersubunit interactions(32, 33) . On the
other hand, the asymmetric environment of the two chemically equivalent
subunits of aspartate aminotransferase from chicken heart mithochondria
within triclinic crystals causes a kinetic asymmetry in the reaction
with natural substrates that has no counterpart in
solution(34) .
Suspensions of cross-linked microcrystals of
rabbit muscle phosphorylase a showed homotropic cooperativity
in the reaction involving a fixed concentration of glucose 1-phosphate
and variable concentrations of maltoheptose (Hill coefficient in the
crystal n = 1.08 versus 1.17 in
solution) but not in other reactions(35) . HbI represents a
most favorable study case as it fully retains positive homotropic
ligand binding cooperativity in crystals of the quality used for x-ray
studies. The structure of liganded HbI presently available has been
determined using crystals belonging to the monoclinic C2 space group (1, 2, 3, 4) , whereas the structure
of deoxy-HbI has been determined using crystals belonging to the
orthorhombic C222
space group(2, 3) . The
observed conformational differences between the liganded and the
unliganded states of HbI involve small changes in the relative position
of the two subunits (3.3° rotation and 0.4 Å translation)
with striking changes in the heme environment. These include sinking of
the heme groups 0.6 Å deeper into the subunits due to the
extrusion of Phe-97 from the proximal side of the heme pocket into the
subunit interface. The movement of one heme alters the hydrogen bond
network that links the heme propionates to F helix residues (i.e. Lys-96 and Asn-100) of the other subunit, thus increasing its
oxygen affinity. It will be of interest to compare these conformational
differences with those occurring in orthorhombic crystals grown in the
deoxy state and exposed to increasing oxygen pressure, in order to
verify whether some of them may simply be due to different lattice
contacts.
The oxygen affinity measured along the two orthogonal axes is the same. Since the contribution of the two hemes to polarized light absorption is different along the two crystal axes (Table 1), this result indicates that, within experimental error, the two hemes possess the same oxygen affinity and, hence, that the monoliganded intermediates cannot be distinguished.
The comparison of the polarization ratios of liganded and unliganded HbI provides information on a possible reorientation of the heme upon oxygenation. The similarity of the PR values (1.79-1.81) indicates that ligand binding is not accompanied by a heme tilt, in agreement with the existing crystallographic evidence(1, 2, 3, 4) . The observed PR value for deoxy-HbI (1.79) is lower than that (2.0) calculated from the x-ray coordinates of the deoxy molecule, assuming a rigid heme plane. In all myoglobin and hemoglobin crystals, the observed PR value is consistently lower than the calculated value, due to fluctuations of the heme that produce an apparent out-of-plane (z-polarized) component in the heme absorption(36) .
For oxidized HbI
crystals, the PR value (2.3-2.4) observed at pH 8.5 differs from
that characteristic of reduced HbI crystals and can be attributed to a
different heme orientation. Although the available data do not allow
one to estimate the extent of rotation, even a tilt of a few degrees
would be expected to involve appreciable conformational changes in the
surrounding globin, due to the tight packing of the heme inside the
protein. Indeed, in the crystal, oxidation of reduced HbI induces the
development of fine cracks, which rapidly disappear, while, in
solution, the transition from reduced to oxidized HbI causes quaternary
changes that ultimately lead to dissociation of the dimer into the
monomeric, low spin hemichrome (M). The dissociation
equilibrium in solution involves also two spectroscopically distinct
dimeric species, a hexacoordinate aquomet (D) and
a pentacoordinate (D
) component, according to the
scheme: M &rlarr2; D
&rlarr2; D
. As described by Spagnuolo et al.(21) , the amount of D
is essentially
constant between pH 6.5 and 8.0 and over the ionic strength range
0.01-0.1 M. The monomeric hemichrome is favored by acid
pH and higher ionic strength, while the pentacoordinate species
prevails at alkaline pH and at low ionic strength. The monomerization
process occurs in solution even under solvent conditions approaching
those of the crystal mother liquor. In contrast, in the crystal the
dimeric pentacoordinate form is stabilized over the whole pH range
studied, whereas the monomeric hemichrome formation is absent due to
lattice forces that drastically shift the equilibrium toward the right.
Thus, HbI crystals provide an interesting example of the balance
between lattice energies and energies involved in tertiary and
quaternary rearrangements of multisubunit proteins. Lattice
interactions that do not restrict the large tertiary and subtle
quaternary conformational changes associated with cooperative oxygen
binding are strong enough to impair the quaternary transition that
would lead to dissociation of the oxidized protein into monomers.