(Received for publication, August 9, 1994; and in revised form, October 19, 1994)
From the
Heme iron out-of-plane displacement following ligand dissociation in hemoglobin, myoglobin, and the proximal cavity mutant H93G is shown to be as rapid as the heme iron out-of-plane vibrational period by sub-picosecond time-resolved resonance Raman spectroscopy. The results demonstrate that the effect of steric repulsion initiated by the spin change of the iron gives rise to heme doming independent of covalent attachment of the proximal ligand to the protein. It is concluded that the protein plays a passive role in the initial ultrafast heme iron motion toward the out-of-plane position observed in the deoxy structure of hemoglobin and myoglobin. The results suggest that the spin change of the heme iron is the primary cause of rapid heme doming and that steric repulsion of the proximal ligand with the heme plays a secondary role in forcing the iron out of the heme plane.
The study of cooperative ligand binding among the four subunits
of the protein hemoglobin has occupied a central role in the
understanding of allosteric transitions of enzymes (Monod et
al., 1965). The role of the heme iron out-of-plane displacement,
or doming, as the trigger for structural changes leading to the
cooperative transition in hemoglobin has been suggested based on an
observed correlation between the extent of iron out-of-plane motion and
the free energy of cooperativity (Perutz, 1979), as well as on quantum
chemical calculations (Olafson and Goddard, 1977) and molecular
dynamics simulations (Henry et al., 1985; Kuczera et
al., 1990; Gibson et al., 1992). Cooperative interactions
among the subunits of hemoglobin are induced by the breaking of a
chemical bond between the heme iron and a diatomic ligand that leads to
displacement of the heme iron by 0.4 Å out of the heme plane
(Baldwin and Chothia, 1979). To function as a trigger for the
cooperative transition, heme doming must be the first conformational
change to occur following ligand dissociation.
Both time-dependent
absorption (Sawicki and Gibson, 1976; Hofrichter et al., 1983;
Martin et al., 1983) and resonance Raman spectroscopies have
been used to evaluate the time scale of heme doming in hemoglobin
(Findsen et al., 1985; Franzen et al., 1994a). In the
present paper, we describe application of sub-picosecond time-resolved
resonance Raman as a probe of the dynamics of the heme iron. The
results presented here suggest that the heme-(histidine)-imidazole
complex undergoes an ultrafast reaction to which the protein matrix
reacts on longer time scales analogous to rapid formation of a dipolar
excited state in a polar solvent. We substantiate this view by
demonstrating ultra-rapid heme doming in the myoglobin mutant H93G(Im)
in which the proximal histidine is replaced by glycine, and exogenous
imidazole (Im) ()is not chemically bonded to the protein but
occupies the cavity created by the H93G mutation and is bonded to the
heme iron (Barrick, 1994).
The time-dependent resonance Raman signal was observed using
a two color experiment (Petrich et al., 1987). The pump
wavelength was 570 nm and had a duration of 100-150 fs as
determined from an autocorrelation measurement. The pump beam energy
was about 50 µJ, which was sufficient to photolyze 50% of the hemes
in the sample. The Raman beam at 435 nm, which is in resonance with the
Soret band of all species observed, was generated by using the
frequency-doubled output of a chain of three amplifiers using laser dye
LDS867. The frequency bandwidth of the Raman beam was fixed using a
bandpass filter with a 6-Å bandwidth after a cuvette containing
HO, in which a spectroscopic continuum was generated. The
LDS867 amplifiers were pumped by the output of a frequency-doubled
injection-seeded Nd:YAG (neodymium: yttrium-aluminum-garnet) laser at
30 Hz and functioned in a regime saturated in the femtosecond pulse
(Migus et al., 1980). This apparatus leads to a consistently
stable 435-nm beam over nearly an order of magnitude change in the
intensity of the input pulse from the continuum and permits signal
averaging for many hours with little drift. After frequency doubling to
435 nm, the bandwidth of the Raman probe pulse was 25
cm
, and the pulse duration was 700 fs. Raman probe
pulse energies were 500 nJ/pulse giving rise to no detectable
photodissociation of HbCO by the probe pulse alone. The pump and probe
pulses were made collinear using a dichroic mirror and had a
cylindrical form (100 µm
400 µm) at the sample. The
scattered light was collected in a 90 degree geometry and dispersed
onto a microchannel plate (EG&) using a 1 m Jobin-Yvon
monochromator.
The samples were the carbon monoxide (CO) complexes of HbCO, horse heart myoglobin (MbCO), and the H93G(Im)CO mutant of sperm whale myoglobin (200-400 µM in heme). The preparation of hemoglobin and horse heart myoglobin has been described elsewhere in detail (Franzen et al., 1994b). The H93G(Im) mutant of sperm whale myoglobin was prepared from an Escherichia coli strain containing the mutant plasmid grown in medium containing 10 mM imidazole. The protein purification procedure has been discussed in detail elsewhere (DePillis et al., 1994).
Fig. 1shows three significant changes in the
equilibrium HbCO vibrational spectrum upon photolysis. The iron-His F8
out-of-plane vibration, (Fe-His), appears at
225
cm
(shifted relative to the
212 cm
in equilibrium deoxy Hb) along with an unassigned mode at
304
cm
. The iron-ligand stretching vibration,
(Fe-CO), at
505 cm
decreases in intensity
upon photolysis due to the departure of the CO ligand (Tsubaki et
al., 1982). A considerable amount of experimental effort has been
devoted to assigning
(Fe-His) and showing that this mode is the
signature of a domed heme iron, thus correlating the resonance Raman
signal with a change in structure (Kitagawa et al., 1979).
From the time-resolved RR difference spectra shown in Fig. 1,
one can follow the breaking of the iron-ligand bond by observing the
reduction in scattering intensity of the Fe-CO stretch
(Fe-CO) at
505 cm
. This serves as both an internal
calibration for the extent of photolysis and a clock for structural
changes. The
(Fe-His) vibration and
304 cm
mode appear together in less than 1 ps following photolysis with
a rise in intensity, which tracks the bond-breaking event. The mode
(Fe-His) appears with a band shift of 12 ± 3
cm
to higher energy in photolyzed HbCO with constant
frequency out to 60 ps (Findsen et al., 1985; Franzen et
al., 1994b).
Figure 1: Time-resolved resonance Raman spectra for HbCO. Equilibrium HbCO Raman spectra were produced using probe pulses, which arrive at the sample 5 ps prior to the pump pulse. Difference Raman spectra consist of subtraction of the HbCO spectrum from a Raman spectrum in which the 435-nm probe arrives at the sample after the 575-nm pump.
Evidence for ultrarapid heme doming can be found
in the time-resolved RR spectra of MbCO and in H93G(Im)CO as shown in Fig. 2. At a delay of 1 ps, there is a clear increase in
intensity of (Fe-His) at
220 cm
and of an
unassigned mode at
300 cm
(as seen in the
hemoglobin spectrum in Fig. 1), indicating that the same modes
appear in myoglobin and hemoglobin on the picosecond time scale. Both
the equilibrium MbCO and H93G(Im)CO difference RR spectra are very
similar between 1 and 60 ps within the spectral resolution of the
700-fs Raman pulse. The intensity of the iron-histidine out-of-plane
mode is larger in H93G(Im) myoglobin than in horse heart. (
)This is observed in the continuous wave Raman spectrum as
well, and this intensity difference can be understood in terms of the
larger intensity of the
(Fe-Im) mode in deoxy H93G(Im) when
compared with
(Fe-His) in wild type deoxy Mb (see below). The
iron-histidine out-of-plane mode is more symmetrical in deoxy H93G(Im)
than in deoxy wild type horse heart or sperm whale myoglobin;
however, the level of resolution required to observe the
line-shape difference is much higher than that attainable in a
sub-picosecond time-resolved resonance Raman experiment.
Figure 2: A comparison of equilibrium Raman spectra and Raman difference spectra at 1 and 60 ps is shown for MbCO and H93G(Im)CO. The spectra are overlaid to show their overall similarity. The difference in the intensity of the lowest frequency peaks is due to the differences in the deoxy resonance Raman spectra of the respective species.
One
significant difference between myoglobin and hemoglobin is the
frequency of the (Fe-His) mode relative to the equilibrium deoxy
spectrum. The approximate 12-cm
shift that is found
in hemoglobin appears to be entirely absent in myoglobin. The error in
determining the peak position is inherently larger in myoglobin due to
the mode at
250 cm
. This mode, which appears in
the MbCO but not the HbCO RR spectra, is thought to be a heme pyrrole
out-of-plane tilt (Choi and Spiro, 1983). Regardless of origin, this
mode is observed in MbCO but not in deoxy Mb or any Hb RR spectrum
(Tsubaki et al., 1982). This accounts for the negative feature
that appears in the RR difference spectrum of photolyzed MbCO and
H93G(Im)CO (Fig. 2) but not HbCO (Fig. 1).
The stability of the iron-histidine (Fe-N) bond is
important both for the biological relevance of flash photolysis as a
technique and the properties of ligand binding in the physiological
function of myoglobin and hemoglobin. The observation of the
(Fe-His) out-of-plane mode at the earliest times in both myoglobin
and hemoglobin demonstrates that the Fe-N
bond is not broken on
the femtosecond time scale. The fact that the iron-histidine bond
remains intact on the picosecond time scale is important for the
validity of conclusions about ligand rebinding on a fast time scale
based on flash photolysis experiments. NO rebinding occurs on the 10-ps
time scale in hemoglobin, and the most rapid phase of the CO and
O
ligand recombination reactions could also be affected by
proximal ligand dynamics if photolysis occurred. In the H93G mutant of
myoglobin, the proximal imidazole is no longer covalently attached to
the protein, and even in this cavity mutant, the iron-histidine bond
appears to remain intact on the picosecond time scale. The results in Fig. 1and Fig. 2demonstrate that there is no detectable
proximal ligand photolysis on the time scale relevant to any of the
ligand binding processes studied.
The similarity of the
time-resolved resonance Raman signal in photolyzed Mb*CO and
H93G(Im)*CO is not entirely expected. There are large structural
differences between the position of imidazole ring in the ligated H93G
mutant and in ligated wild type myoglobin based on a comparison of the
aquo-met myoglobin crystal structures (Barrick, 1994). The angle
between the imidazole ring and the N(pyrrole)-Fe-N(pyrrole) line on the heme is nearly
0° in wild type ligated myoglobin and is 40-45° in the
aquo-met form of the H93G mutant. The difference in angle of the
imidazole obtained in the aquo-met structure is preserved in the
cyano-met form based heme-methyl NMR hyperfine shift patterns. ()The available evidence from NMR studies of the CO complex
suggests that the imidazole ring is similarly rotated. (
)Given these observations, a strong hydrogen bond formed
between the imidazole nitrogen and OH of serine 92 seen in the aquo-met
x-ray crystal structure may be the origin of the rotation angle of the
imidazole in H93G(Im). These considerations lead us to suggest the
hydrogen bond and hence the rotation angle of the imidazole are
preserved in the deoxy structure as well.
As a consequence of the
greater freedom for the rotated histidine to approach the heme, the
Fe-N bond is 1.85 Å in H93G, as opposed to 2.17 Å in
the aquo-met Mb form. The origin of the longer bond length in wild type
myoglobin is likely to be steric repulsion of the histidine by the
pyrrole nitrogens of the heme. If this difference in bond length is
preserved in the deoxy, Mb should result in an altered
(Fe-Im)
frequency when compared with
(Fe-His) in wild type. The similarity
of the frequency of the
(Fe-His) mode in the RR spectrum of the
photolyzed species suggests that the iron-histidine bond lengths are
not significantly different in Mb*CO and H93G(Im)*CO. However, there is
at present no structural evidence from NMR or x-ray diffraction on
deoxy species to substantiate this suggestion. In principle, resonance
Raman should be sensitive to small changes in rotation angle and bond
length; however, detailed analysis of the structural implications of
small changes in linewidth and frequency require high spectral
resolution continuous wave resonance Raman experiments. Preliminary
continuous wave resonance Raman spectra show that the frequency of
(Fe-Im) in H93G(Im) is at 226 cm
,
which is shifted <2 cm
from 224
cm
, the weighted average of the two components (80%
at 220 cm
and 20% at 240 cm
),
which comprise the asymmetric
(Fe-His) mode in wild type horse
heart (or sperm whale) myoglobin (Bangcharoenpaurpong et al.,
1984). Based on the continuous wave resonance Raman spectra, the
intensity of the
(Fe-Im) mode in H93G(Im) is greater by nearly a
factor of two than
(Fe-His) in wild type Mb,
which
explains the difference in intensity of this mode in the difference
Raman spectrum shown in Fig. 2.
The results in Fig. 1and Fig. 2confirm the hypothesis of significant
sub-picosecond heme iron doming based on the appearance of a deoxy-like
species in the time-resolved absorption spectrum within 300 fs
following photo-dissociation in HbCO and MbCO (Petrich et al.,
1988). The 300-fs iron doming time corresponds to one-half period of
the 50 cm Fe-heme out-of-plane vibration frequency
in deoxy myoglobin (Zhu et al., 1994). Our data show the
separation of time scale between heme doming and the subsequent large
conformational changes, which can be viewed as a rapid chemical
reaction followed by slower solvent (i.e. protein) relaxation.
The fact that ultrafast heme doming is observed even in a
heme-imidazole complex in the H93G(Im) mutant proves that the forces
responsible for this reaction do not arise from the protein matrix that
holds the heme-imidazole complex in place. Nor do the steric repulsive
forces between the proximal ligand (histidine in wild type) and the
heme appear to be responsible, as previously suggested, based on
molecular orbital calculations (Olafson and Goddard, 1977). Rather,
these results suggest that the spin change of the heme iron forces the
iron out of the heme plane. The lack of a frequency shift in
(Fe-His) further suggests that the motion of the accompanying
proximal histidine occurs on a similarly rapid time scale, thereby
allowing the protein structure on the proximal side to evolve rapidly
toward the deoxy configuration without dissipation of the energy of
heme doming. The time-resolved resonance Raman data, together with
other experiments (Franzen et al., 1994b), suggest that a
significant part of F-helix motion associated with the iron
out-of-plane displacement occurs on the picosecond time scale in
myoglobin. In hemoglobin, a similar set of changes occurs within each
subunit as tertiary structure evolves following ligand dissociation. In
this cooperative protein, the alterations in tertiary structure that
capture the energy of heme doming, such as the initial phase of F-helix
motion, are crucial to the formation of intersubunit interactions. The
specificity of these motions allows contact to be made on the surface
of each subunit on the microsecond time scale, which gives rise to the
R-T conformational switch. The rapid protein response to the
out-of-plane motion of the heme iron is responsible for the
communication of the rupture of the iron-ligand bond over a distance of
>25 Å, which leads to cooperative interactions in hemoglobin.