(Received for publication, September 12, 1995)
From the
Hemoglobin C (Glu
Lys) shares with
hemoglobin S (Glu
Val) the site of mutation,
but with different consequences: deoxyHbS forms polymers, whereas
oxyHbC readily forms crystals. The molecular mechanism for this
property of oxyHbC is unknown. Since no detailed oxyHbC crystal
structural information exists, spectroscopic probing is used in this
study to investigate possible solution-phase conformational changes in
HbC compared with HbA. Intrinsic fluorescence combined with UV
resonance Raman data demonstrate a weakening of the
Trp
-Ser
hydrogen bond that
most likely leads to a displacement of the A helix away from the E
helix.
The 6 hemoglobin variants, in particular, sickle cell
hemoglobin (HbS) and HbC, are aggregating hemoglobins and, for decades,
have been of interest to structural biologists, pathophysiologists, and
clinicians. HbC is the second most commonly encountered abnormal
hemoglobin in the United States and, next to HbS and HbE, the third
most prevalent hemoglobin variant worldwide(1) . HbC
(Glu
Lys) shares with HbS (Glu
Val) the site of mutation at position
6, but the
consequences of the specific substitutions are very different. In red
blood cells, deoxygenated HbS forms polymers, whereas HbC forms
intraerythrocytic crystals in the oxygenated state as demonstrated by
absorption spectroscopy as well as by their tendency to melt upon
deoxygenation(2) . However, to date, no structural basis for
the abnormal properties of these mutant hemoglobins has been defined (3) .
In this study, we focus upon conformational changes in
HbC as a means of addressing the remaining questions: is the localized
6 mutation alone responsible for the tendency of HbC to
crystallize, or does this mutation induce extended tertiary and/or
quaternary conformational changes necessary for crystallization? Hence,
to investigate possible dynamic distal conformational changes in HbC
compared with HbA, several optical spectroscopies were sequentially
used in this study to probe conformational differences in liganded HbC.
Resonance Raman spectroscopic
measurements were obtained using the following system. The second
harmonic (532 nm, 20 Hz) of a neodymium:YAG laser (Continuum NY-81C)
passed through a hydrogen cell (200 p.s.i.) was used to generate the
blue (435.71 nm) pulse (1 ml, 7-ns pulses). A single laser pulse
photolyzes the sample and probes the Raman spectrum of the transients
within
7 ns. Scattered light was collected with a Nikon F/1.4
50-mm camera lens and focused with an f-matching lens onto a 150-µm
entrance slit of an ISA HR640 monochrometer. A notch filter was used to
reduce the Raleigh scattering, and a depolarizer was used to reduce
grating biases. The detector was a Princeton Instruments 1024 element
intensified diode array (IRY 1024 S/B) connected to a Princeton
Instruments ST120 detector controller interfaced to a personal
computer, where the spectra were stored and later analyzed with LabCalc
software (Galactic Industries Corp.).
Front-face fluorescence (5, 6, 7) has been used to probe changes in
both the environment of tryptophans and heme-Trp distances in
hemoglobins(9) . The first positive findings were obtained
comparing the intrinsic fluorescence intensities of the oxy and deoxy
forms of HbA and HbC as shown in Fig. 1. 296 nm excitation was
used to eliminate any resonance energy transfer from Tyr
Trp(9) . A significant difference (p = 0.0035)
in the fluorescence emission intensity of oxyHbC and oxyHbA is
observed. This is not the case for the deoxy derivatives. A
significant difference (p = 0.00008) in the percent
change in the fluorescence intensity of HbC compared with HbA upon
deoxygenation is also shown in Fig. 1, but is accounted for by
the initial intensity difference between the liganded species.
Figure 1:
Comparison of the oxy
and deoxy intrinsic fluorescence intensities of HbC and HbA. The
hemoglobin solutions were 1 g % (0.62 mM heme) in 0.1 M Hepes buffer, pH 6.85. The excitation wavelength was 296 nm,
selectively exciting tryptophans(8) . On the x axis, %
refers to the percent change in relative intensity upon the oxy
deoxy transition, which is calculated by intensity
- intensity
. The inset shows the
difference spectra. Curve A is the difference spectrum
resulting from subtracting the oxyHbA emission spectrum from the oxyHbC
emission spectrum. This significant spectrum is consistent with the
statistical evaluation that the intrinsic fluorescence intensity of
oxyHbC is significantly different from that of oxyHbA. Curve B (the difference spectrum resulting from subtracting the deoxyHbA
emission spectrum from the deoxyHbC emission spectrum) is not a
significant difference spectrum, supporting the observation that the
intrinsic fluorescence intensities of deoxyHbA and deoxyHbC do not
differ.
This
difference in the intrinsic fluorescence can arise from any of the Trp
residues (Trp, Trp
, and
Trp
) within Hb. It is also possible that this
difference may arise from differences in orientation and efficiency of
transfer from tryptophan(s) to the heme. Hence, to further localize the
origin of the increase in fluorescence, we used conformationally
sensitive vibrational spectra of tryptophans and tyrosines using UV
resonance Raman spectroscopy(10, 11, 12) ,
building on the findings of Spiro and
co-workers(10, 13) . These authors showed that the
distinct low frequency shoulder of the Trp(W3) UV resonance Raman band
at
1550 cm
arises from Trp
,
whereas the main band at
1555 cm
is derived
from the composite contribution of Trp
. Fig. 2shows a comparison of the UV resonance Raman spectra of
the liganded (CO) forms of HbA compared with HbC. All differences can
be attributed to the Trp(W3)
band since no
other spectral differences are observed.
Figure 2: UV resonance Raman spectra of HbCCO compared with HbACO. The dashed line is HbC, and the solid line is HbA. For a description of the instrument, see ''Experimental Procedures.``
Visible resonance Raman
spectroscopy is used to probe the functionally important heme
environment(14, 15, 16) . This method probes
heme vibrational modes, many of which are highly sensitive to the local
tertiary structure. It is used here to resolve any differences, between
HbC and HbA, in the heme and local heme environment that could
contribute to the observed fluorescence alteration. Comparisons between
the equilibrium deoxy forms and the photoproducts of ligand-bound forms
reflect both ligand binding and quaternary structure-induced changes in
the heme environment. The low frequency region contains the
iron-proximal histidine stretching mode, which is highly sensitive to
changes in the functionally important proximal heme pocket. Other bands
in the low frequency region reflect the environment of the vinyl and
propionate groups. The high frequency region contains modes that are
sensitive to the iron displacement and the electron density of
the porphyrin ring(14, 15) .
Fig. 3(a and b) shows that the high and low frequency regions of the resonance Raman spectra of both the equilibrium deoxy form and the 10-ns transient photoproduct of HbA and HbC are identical within our spectral resolution. Identical proximal and distal heme pocket environments would be consistent with the recent report by Shapiro et al.(17) that there are no differences between HbA and HbC with respect to CO geminate recombination kinetics (10-ns resolution). Geminate rebinding has been shown to be sensitive to both proximal (18) and distal (19) perturbations. Considering all of the above, a difference in heme orientation between HbC and HbA is an unlikely mechanism to explain the increased fluorescence.
Figure 3:
The high (v,
reporting the porphyrin breathing motion) (a) and low (v
, reporting the Fe-His stretch mode) (b) frequency 10-ns transient resonance Raman spectra of HbACO
and HbCCO (1 mM heme) and the equilibrium deoxy forms. The
buffer used was 0.05 M sodium phosphate buffer, pH 7.25.
Excitation was at 435.7 nm. For a description of the instrument, see
''Experimental Procedures.`` The purified proteins were
stored under liquid nitrogen. Storage did not change the absorption
spectra or scattered light intensities. To prepare the deoxy
derivatives, the sample was degassed with vacuum/nitrogen cycles. HbCO
was formed by saturating the protein solutions with gaseous CO. The
ligation state of the hemoglobin, sample integrity, and stability were
verified by absorption spectroscopy obtained before and after the
resonance Raman experiments for each
sample.
In summary, based on the increase in the intrinsic
fluorescence of oxygenated HbC and the lack of differences in the heme
environment as demonstrated by the visible resonance Raman spectroscopy
and CO geminate rebinding kinetics(17) , it is likely that
differences exist between oxyHbA and oxyHbC in the microenvironment of
at least one tryptophan. Trp, a possible candidate
since it contributes to the intrinsic fluorescence of Hb using 296 nm
excitation(3, 4, 5, 6, 7, 20) ,
can be eliminated since the UV resonance Raman spectroscopy (Fig. 2) shows no HbA/HbC difference involving this residue. In
contrast, a substantial intensity difference is apparent at the peak
assigned to either Trp
or Trp
(which are not distinguishable by this technique). Given that the
6 mutation is on the same helix as Trp
, we
conclude that Trp
is the source of the change in
signal in both the fluorescence and UV resonance Raman spectra.
Trp is an A helix residue that lies in the crevice
formed by the A and E helices and normally forms a hydrogen bond with
Ser
of the E helix in both the deoxy and oxy
structures(13) . Based on the analysis of Rodgers and
Spiro(13) , the UV resonance Raman data presented here are
interpreted as a weakening of this hydrogen bond in oxyHbC. In
addition, the increased fluorescence intensity pattern for oxyHbC is
consistent with Trp being farther away from the heme, with the A helix
looser or more distant. Finally, as reported in the transient resonance
Raman studies, there are no detectable differences in the heme
environment between HbA and HbC in either the deoxy T state or liganded
R state as reflected in the transient photoproduct spectrum. Therefore,
the proximal heme pocket of the liganded R structure does not appear to
be altered as a result of the Lys
substitution within
the 10-ns timeframe. The Raman data presented here, the observation
that HbC and HbA are functionally identical, and the findings by
Shapiro et al.(17) indicating no difference in the CO
geminate recombination of HbC compared with HbA indicate that both the
proximal and distal heme pocket architectures are indistinguishable in
these two proteins. Hence, the perturbation of the the A helix
alteration is not transmitted to the critical heme interactions with
the E and F helices.
We conclude that the weakened hydrogen bond
between the A and E helices, reflected by UV resonance Raman
spectroscopy, involving Trp of the A helix and
Ser
of the E helix and induced by the Lys
replacement, in turn induces a swinging away of the A helix from
the E helix, with the E helix remaining unperturbed (Fig. 4).
The absence of comparable differences for the deoxy derivatives could
be due to the known increase in the tightness of packing between the EF
corner and the N terminus in the deoxy T state, which could be
sufficient to maintain the A helix in its standard configuration
relative to the E helix.
Figure 4:
Illustration of the A, E, and F helices of
the oxyhemoglobin -chain modified from the data base of Shaanan (21) . Our results indicate that the Glu
Lys substitution of HbC results in a weakened hydrogen bond
(Trp
-Ser
) between the A and E
helices, with a likely swinging away of the A helix from the E helix
(direction indicated by the arrows). The view of the A, E, and
F helices is similar to that presented by Rodgers and Spiro (13) used to illustrate the
-chain helical movements of
the HbA nanosecond transient intermediate.
This model is adopted as the simplest
explanation that accounts for our observations and incorporates
physical and chemical intuition. Other possible alternative mechanisms
to explain the observed fluorescence difference are unlikely in light
of the following. 1) A different heme orientation is ruled out by the
lack of differences in the visible resonance Raman spectra and by the
work of others(17) . 2) Resonance energy transfer from Tyr
Trp is ruled out by the use of 296 nm excitation, which
selectively excites Trp(8) . 3) Differences in Trp
are ruled out as the origin of the fluorescence change by the UV
resonance Raman data. Although the resonance Raman cross-section
depends on the absorption spectra while fluorescence quantum yield
mechanisms depend upon resonance energy transfer rate efficiency, this
study exemplifies the strength by which these two spectroscopic
approaches may serve to complement each other in the search for
assignments.
Finally, the altered packing of the A helix in oxyHbC
becomes a likely focus for mechanistic studies dealing with the
increased tendency of oxyHbC to crystallize. Moreover, these results
suggest that sickle cell hemoglobin (HbS, Glu
Val) should be revisited in terms of delocalized conformational changes
inducing deoxyHbS polymerization, particularly considering earlier
reports implying nonpolymerization-related functional differences
between tetrameric HbS and
HbA(22, 23, 24, 25) .