(Received for publication, July 8, 1996, and in revised form, October 7, 1996)
From the Biochemistry Program, Department of Chemistry, College of Arts and Sciences, University of Massachusetts, Lowell, Massachusetts 01854
The incorporation of CN-hemin into three human
adult apohemoglobin species (apohemoglobin, -apohemoglobin, and
apohemoglobin modified at its
93 sulfhydryl with
p-hydroxymercuribenzoate) has been monitored at micromolar
concentrations in 0.05 M potassium phosphate buffer, pH
7.0, at 10 °C. In all cases, Soret spectral blue shifts accompanied
CN-protohemoglobin but not CN-deuterohemoglobin formation. This
finding in conjunction with isofocusing studies provided evidence of a
CN-protosemi-
-hemoglobin intermediate, the formation of which
appeared to be a direct consequence of CN-protohemin-
heme pocket
interactions. The kinetics of full reconstitution of
CN-protohemoglobin and CN-deuterohemoglobin revealed four distinct
phases that apparently correlated with heme insertion (Phase I), local
structural rearrangement (Phase II), global conformational response
(Phase III), and irreversible histidine iron bond formation (Phase IV).
These phases exhibited rates of 7.8-22 × 107
M
1 s
1, 0.19-0.23
s
1, 0.085-0.12 s
1, and 0.008-0.012 s
1, respectively. Partial (50%) reconstitution with
CN-protohemin, in contrast, revealed only three kinetic phases (with
Phase III missing) of heme incorporation into native and
p-hydroxymercuribenzoate-modified apohemoglobin.
Furthermore, the absence of Phase III slowed the rate of proximal bond
formation. These findings support the premise that irreversible
assembly of CN-protosemi-
-hemoglobin is deterred by the presence of
a heme-free
partner, the consequence of which may be that
intermolecular heme transfer is encouraged under conditions of heme
deficiency in vivo.
The structural, functional, and subunit assembly properties of
human hemoglobin have been intensely investigated (1, 2, 3, 4, 5). Yet the
precise nature and sequence of events that occur during hemoglobin
formation are still unknown. Not only is the mode(s) of combination of
mitochondrial Fe-protoporphyrin IX (heme) with cytoplasmic nascent and
polypeptide chains unknown, but the actual dimer precursor (or
precursors) remain undefined. Three distinct pathways of hemoglobin
tetramer assembly may be proposed, that of assembly through a
heme-containing heterodimer (
h
h), a
heme-globin pair (semihemoglobin;
h
o or
o
h), or a heme-free
dimer
(apohemoglobin;
o
o). The detection of
hemoglobin (
h) and
apohemoglobin (
o)
chains as well as semi-
-hemoglobin
(
h
o) and apohemoglobin
in vivo has served to strengthen the plausibility of these
three assembly mechanisms (6, 7).
Gibson and Antonini (8, 9) carried out pioneering studies that involved
the binding of a monomeric heme moiety to an apohemoglobin species
isolated from normal adult hemolysate (Hb A). Their rapid kinetic
investigations resulted in the development of a model which proposed
that hemoglobin formation occurred via a reversible intermediate
complex. Independent kinetic studies (10, 11, 12) have supported this
classical model, which postulated a two-step kinetic mechanism
involving a rapid second order heme insertion event followed by a
slower first order process attributed to structural rearrangement and
the irreversible formation of a histidine-iron bond. Experimental
variables that altered the heme insertion process (meso- and
deutero-derivatives of CN- or CO-heme) as well as those aimed at
modulating apohemoglobin structural response (pH changes; introduction
of polyanions) were explored, and no evidence of an ordered sequence
(either or
subunit) of heme binding was found.
This was unexpected because detailed protein chemical studies (13, 14, 15),
which involved half-equivalency titration of apohemoglobin with
heme, had led to the conclusion that chains have a greater
preference than their
chain counterparts for heme. Furthermore,
Soret spectral kinetic studies focused on CO-heme (16) and CN-hemin
(17) binding to preassembled semi-hemoglobins (semi-hemoglobins
prepared in vitro by heme-chain transfer; Ref. 18) have
further documented differences in the heme affinity of
and
chains. In this report, the incorporation of CN-protohemin and
CN-deuterohemin into human apohemoglobins has been monitored, and
results indicate that a significant difference in binding of
and
subunits of apohemoglobin does exist, a finding consistent with
human hemoglobin assembly through a semi-
-hemoglobin intermediate in vitro and most probably in vivo.
Human adult hemoglobin and its isolated heme subunit
were prepared (19) and characterized as previously reported (20, 21).
Removal of heme was accomplished by treatment with acid acetone as
described (22) with modifications (23). Final solutions of the
hemoglobins were suspended in 0.05 M potassium phosphate buffer, pH 7.0, and concentrations were determined
(
280nm = 12.7 mM
1
cm
1, on a subunit basis) in a Cary 2200 spectrophotometer
(Varian Instruments). Nativeness of these species was confirmed by
carrying out a heme titration at a single wavelength. In addition,
reconstituted CN-protohemoglobin and CN-deuterohemoglobin were
chromatographed on Biogel P-6 (Bio-Rad) and subjected to spectral
measurements which confirmed maxima of 420 and 409 nm,
respectively.
Heme
titrations over a Soret spectral region of 400 to 450 nm were performed
in the following manner. Protohemin and deuterohemin (Porphyrin
Products Inc.) were dissolved in a minimal amount of 0.1 N
NaOH and distilled water, and their concentrations were determined
(390nm = 50 mM
1
cm
1 and
382nm = 57 mM
1 cm
1 for protohemin and
deuterohemin, respectively). These solutions were converted to the
cyanide derivative by adding excess KCN. Increments of the stock
CN-hemin solutions were added to both sample and reference cells, and
data acquisition was carried out by Lab Calc Software (Galactic).
Confirmation of apohemoglobin nativeness was obtained from isoelectric
focusing studies (Omega Horizontal Electrophoretic System; Isolab Inc.)
on an agarose gel between pH levels 6 and 8, with the gel surface
maintained at 10 °C. The samples were focused for 1 h at 1100 V
at the end of which the gel was fixed in a 10% trichloroacetic acid
solution followed by staining with a heme specific stain,
o-dianisidine. Titration of apohemoglobin with
p-hydroxymercuribenzoate (PMB; Sigma)1 was performed
according to the method of Boyer (24) and revealed that an end point
was achieved when half-equivalent amount of PMB was bound to the
apohemoglobin dimer. The concentration of PMB was determined
(
232nm = 16.9 mM
1
cm
1), and the titration was monitored at 255 nm.
Subsequent preparations of PMB-apohemoglobin were made by adding
half-equivalent amounts of a concentrated PMB solution to the
apohemoglobin sample. The integrity and stability of PMB-apohemoglobin
were evaluated in the UV region (see "Results and Discussion").
All kinetic
measurements were carried out in a Kinetic Instruments stopped flow
device online to OLIS 3820 data acquisition software. The pathlength of
the reaction cell was 20 mm, and the instrument had a dead time of 2 ms. All measurements were performed in 0.05 M potassium
phosphate buffer, pH 7.0 at 10 °C, by mixing equal volumes of the
respective CN-hemin and apohemoglobin in a 1:1 or 1:2 ratio. The
reaction of apohemoglobin was carried out only under equimolar
conditions. The time courses were monitored, at their respective
absorption maxima over a variety of time frames (0.02-300 s) to permit
the collection of a sufficient number of data points. Each time course
consisted of an average of three runs, and a minimum of three
independent trials was performed allowing the determination of standard
deviations. Standard fitting routines allowed the isolation of multiple
phases for each time course. Apparent first (and second order) rate
constants were derived from standard plots of log absorbance and
(1/[CN-hemin]) versus time, respectively. A slow reaction
(inaccessible by rapid kinetic technique) was observed when
CN-protohemin and apohemoglobin were in a 1:2 ratio and its baseline
was obtained in a Cary 2200 spectrophotometer after manual mixing of
the two reactant solutions.
Soret absorption spectra of hemoglobins, which are sensitive not
only to the type and state of ligand on the central iron of heme but
also the heme environment of the protein itself, have been used to
study heme binding kinetics (8, 9, 10, 11, 12, 16, 17). In addition, an increased
preference of the over the
subunit for heme is postulated to be
the basis for the occurrence of semi-
-hemoglobins. The present study
represents a novel method of monitoring for the existence of this
kinetic intermediate during reconstitution of hemoglobin. Static and
kinetic Soret spectral changes of apohemoglobin model systems that
occur upon incorporation of two distinct heme moieties, the native
CN-protohemin and its less hydrophobic derivative, CN-deuterohemin,
have been monitored.
The
change in absorption spectra of apohemoglobin upon binding of
CN-protohemin (Fig. 1, top panel) and
CN-deuterohemin (Fig. 1, bottom panel) was followed in the
Soret region between 400 and 450 nm. Titration curves (Fig. 1,
insets) of absorbance changes at 420 and 409 nm for
CN-protohemin and CN-deuterohemin, respectively, clearly indicate an
end point corresponding to one heme bound per monomer subunit. Under
the standard experimental conditions of 0.05 M potassium
phosphate buffer, pH 7, at 10 °C incremental addition of
CN-protohemin to apohemoglobin (5 µM) resulted in a
significant blue spectral shift (5 ± 0.5 nm) until
half-saturation (one heme/apohemoglobin dimer) was reached (Fig. 1,
top panel), and then no further spectral shift was observed
with additional CN-protohemin.
Wavelength dependence has been observed by Kawamura-Konishi and Suzuki
(25) upon addition of a caffeine adduct of hemin to apohemoglobin and
was reported to be a consequence of caffeine-heme binding to the subunit of apohemoglobin. In addition, the blue shift seen here is
consistent with CN-protosemi-
-hemoglobin formation, because the
Soret spectra of this semihemoglobin is more blue-shifted than that of
its semi-
-hemoglobin counterpart in both CO-protoheme (16, 18) and
more importantly the CN-protohemin form (17).
CN-deuterohemin incorporation into apohemoglobin, on the other hand,
revealed a titration whose spectra were wavelength-independent over the
region of study from 400 to 450 nm. The CN-deuterohemin lacks the
vinyl groups in positions 2 and 4 of protohemin, and this would be
expected to alter heme-protein contacts and consequently spectral
properties (see below). Furthermore, electrophoretic studies from the
laboratories of Winterhalter et al. (13) and Cassoly and
Banerjee (18) indicate that CN-deuterohemoglobin reconstitution does
not exhibit chain differences. Random heme binding could obscure
any spectral shifts and would also preclude preferential formation of a
CN-deuterosemi-
-hemoglobin.
Isofocusing studies (Fig. 2) reveal that a
semihemoglobin intermediate is present only during CN-protohemin
incorporation into apohemoglobin. These results are in general
agreement with earlier zonal electrophoresis studies (13). Furthermore,
the cathodic heme-containing component observed here has been
previously identified as semihemoglobin (16, 18). CN-deuterohemin
incorporation into apohemoglobin reveals no semihemoglobin formation; a
fact that corresponded well with the lack of detectable wavelength dependence (Fig. 1, bottom panel) during titration.
Static Titrations of
The heme-protein
interactions of the subunit were probed independently of its
coupling to its
chain partner. Soret spectral monitoring of
-apohemoglobin upon the addition of increments of CN-protohemin
(Fig. 3, top panel) and CN-deuterohemin
(Fig. 3, bottom panel) was carried out under conditions of
0.05 M potassium phosphate buffer, pH 7.0, at 10 °C.
Titration curves (Fig. 3, insets) revealed one heme bound
per
-apohemoglobin monomer. Only CN-protohemin was able to induce a
blue spectral shift from 422.5 to 420.5 (±0.5) nm in
-apohemoglobin. This strongly implies that interaction of
CN-protohemin with the unique environment of the
subunit results
in an alteration in the heme chromophore spectrum. The decreased
magnitude of this change compared with that seen for apohemoglobin
(Fig. 1, top panel) also may indicate that this chromophoric
perturbation is augmented by the presence of a
chain partner.
Six residues of the subunit and eight residues of the
subunit
have been shown by Perutz and Fermi to interact with the vinyl groups
of the heme moiety and have shown considerable structural homology (4,
26). Residues at helical positions G5 and G8 are invariant in both the
and
chains of human hemoglobin. This is of interest because the
G-helical regions of both chains have been implicated in apohemoglobin
dimer stability (27, 28). Furthermore, the G5 residue is aromatic in
nature, and as such, this phenylalanyl residue has the potential to
noncovalently interact with the heme moiety; an interaction that would
be expected to contribute to changes in Soret spectral behavior.
Interpretation of Soret spectral shifts for -apohemoglobin is
precluded by the fact that this species exists as a dimer (29, 30, 31, 32). An
alternate approach would be to modify the
subunit of apohemoglobin,
a challenging endeavor because the heme-free protein is rather
unsuitable for extensive protein chemical manipulation. Nonetheless,
modification of apohemoglobin has been reported, and fortuitously, the
most successful was that of the site-specific modification of the
reactive
93 (F9) cysteine residue (15, 27). Furthermore, a reagent
attached to this
93 (due to the residue being adjacent to the
proximal histidine
92) would be expected to be a "reporter
group" of the
chain heme insertion event.
The sulfhydryl
reagent, PMB, has been shown to bind rapidly and specifically to the
93 (F9) cysteine residue of apohemoglobin at one PMB bound per
apohemoglobin dimer. Investigation of the UV spectral region (240-290
nm) upon addition of PMB to apohemoglobin (Fig. 4,
left panel) revealed two significant spectral changes. One
corresponds to the formation of a mercaptide bond in the 250-260 nm
region (24), and the other (in the 280 nm region) may correlate with
changes in the
chain heme environment (see kinetic studies below).
Under conditions of 0.05 M potassium phosphate buffer, pH
7.0 at 10 °C, spectral scans of the UV region demonstrated stability
of the PMB-apohemoglobin species (Fig. 4, center panel) over
the time frame required for the present investigations. CN-protohemin (Fig. 4, right panel) and CN-deuterohemin (not shown)
titration of this PMB modified protein showed Soret spectral
changes identical to those seen for native apohemoglobin (Fig.
1). Thus, this sulfhydryl modification showed no apparent affect on the
static spectral properties of CN-hemin apohemoglobin. However,
significant differences in the binding kinetics of CN-hemin to native
and modified apohemoglobin may be discernible (see "Kinetics of
CN-Hemin Binding to PMB-Apohemoglobin").
Kinetics of CN-Hemin Binding to Apohemoglobin
This present
kinetic investigation was aimed at evaluating the heme incorporation
process in vitro and attempting to extrapolate these
findings to the in vivo event. Our current studies of static titrations have demonstrated that the CN-protohemin-protein binding involves a spectrally definable intermediate (presumably
semi--hemoglobin) that is not seen in CN-deuterohemin-protein
association and that this intermediate is most discernible up to
half-saturation (one heme per apohemoglobin dimer). Taking this into
account CN-protohemin and CN-deuterohemin were mixed in a 1:1 and 1:2
ratio with apohemoglobin in 0.05 M potassium phosphate
buffer, pH 7.0, at 10 °C, and the change in Soret absorbance (at 420 and 409 nm, respectively) was followed in a stopped flow device. All
four reactions were multiphasic, and the resultant rate plots are
presented (Fig. 5). The kinetics of full reconstitution
of CN-protohemin and CN-deuterohemin are displayed in rows
1 and 3, respectively, whereas those that promote partial (50%) reconstitution are in rows 2 and
4, respectively.
The initial part of all four time courses is dominated by a second
order process that is designated Phase I. This is the heme insertion
event, and studies with an array of monomeric heme derivatives (8, 9, 10, 11, 12,
16, 17, 25) have yielded rates in the order of 107
M1 s
1. As expected the rates of
CN-protohemin insertion (Table I) for both full and
half-saturation (10 and 14 × 10
7
M
1 s
1, respectively) were
1.3-2-fold faster than the rate of entry of the less hydrophobic
CN-deuterohemin derivative (full and half-saturation yielded rates of
7.8 and 7.1 × 107 M
1
s
1, respectively). Interestingly enough, the formation of
CN-protosemihemoglobin was 1.4 times more rapid than that of the
CN-protohemoglobin. This faster rate could result from an increased
accessibility of the
chain for heme and is consistent with the
finding that the
subunit structure is more rigid (less
accommodating; Ref. 29) possibly due to the presence of its D-helix
(33). All subsequent phases (Phases II-IV) were found to be first
order in nature and almost certainly attributed to structural changes in the apohemoglobin molecule. Phase II exhibited a rate of 0.21 s
1, which was invariant with the type of CN-hemin
derivative or the degree of reconstitution achieved. Phase III (0.085 s
1) was approximately 2.5-fold slower than Phase II for
all reactions except that it was apparently missing in the formation of
CN-protosemi-
-hemoglobin (Fig. 5, row 2,
III). This suggests that the absence of this phase is
related to lack of CN-hemin insertion into the
chain partner. The
final phase of the reaction (Phase IV) displayed a rate of 0.013 s
1 (6.5-fold slower than Phase III) except in the case of
CN-protosemi-
-hemoglobin (Fig. 5, row 2, IV)
where the rate obtained was 0.008 s
1. This slower rate
for Phase IV is of interest because recent studies have assigned this
rate of reaction to the formation of the bond between the central iron
of heme and the proximal histidine (F-8) in myoglobin (34, 35).
|
Thus, it appears that binding half-saturating amounts of CN-protohemin
to apohemoglobin results in a process that allows faster heme
insertion, that lacks one of two discernible first order structure
rearrangement components, and that possibly results in a 1.5-fold
decrease in the rate of iron-histidine bond formation. This bond
formation ensures irreversible heme incorporation and prevents the
possibility of heme exchange between the subunits of apohemoglobin
(36, 37, 38). The 1.5-fold decrease in this rate of bond formation for
CN-proto semi--hemoglobin would therefore allow more time for such
a heme transfer (from
to
) to occur.
CN-protohemin (Fig. 6,
row 1) and CN-deuterohemin (Fig. 6, row 2) were
mixed in a l:l ratio with -apohemoglobin in 0.05 M potassium phosphate buffer, pH 7.0, at 10 °C, and both
reactions yielded four independent kinetic phases (Table I). The rate
of CN-protohemin insertion (Phase I) into this monomeric apohemoglobin was 1.4-fold faster than that for CN-deuterohemin entry yielding values of 12 and 8.4 × 107
M
1 s
1, respectively. Phase II
exhibited a rate (0.20 s
1) similar to that seen for
apohemoglobin in the case of both CN-hemins, presumably indicative of
a similar event in the presence and the absence of a partner chain. The
additional structural event (designated as Phase III) was present for
the incorporation of both CN-protohemin and CN-deuterohemin into
-apohemoglobin yielding values of 0.087 and 0.099 s
1, respectively. These rates are comparable with those
seen for full incorporation of CN-hemin into apohemoglobin (Fig. 5,
rows 1 and 3, III). The final Phase IV
exhibited rates (0.012 s
1) comparable with those of the
fully reconstituted parent hemoglobins.
This current investigation is of considerable interest because it
allows comparison with the earlier study of Leutzinger and Beychok (11)
in which these workers demonstrated that the kinetics of CN-protohemin
incorporation into -apohemoglobin is multiphasic. Their
three mixed phases can be readily correlated with the four phases seen
here for CN-protohemin and CN-deuterohemin incorporation. Their Soret
spectral, fluorescence quenching, and far UV circular dichroism studies
revealed a process in which heme entry (Phase I) was followed by
structural rearrangements local (Phase II) and global (Phase III).
Although these workers postulated that the His (F8)-iron bond formation
preceded these structural changes, recent evidence (34, 35) suggests
that this step (Phase IV) occurs later in the overall heme
incorporation process. Taken together these investigations suggest that
the heme pocket of
-apohemoglobin is quite accessible and can
readily accommodate both CN-protohemin and CN-deuterohemin, that this
-apohemoglobin monomer is capable of undergoing structural
rearrangements comparable with those observed during full
reconstitution of its apohemoglobin parent, and that if these
structural adjustments are permitted allow a stable linkage between its
proximal histidine and the heme iron to be formed at a normal rate. If,
however, as may be the case during half-saturation of apohemoglobin
with CN-protohemin (see above), conformational restraints (presumably
due to
coupling) are present, then this rate of bond
formation is diminished.
The static
Soret absorption changes accompanying titration with CN-protohemin
were identical for apohemoglobin (Fig. 1, top panel) and
PMB-apohemoglobin (Fig. 4, right panel), and the overall kinetic profile of CN-hemin binding to PMB-apohemoglobin (Fig. 7) was comparable with that of apohemoglobin (Fig. 5)
but not the rates. All four time courses (full reconstitution, Fig. 7, rows 1 and 3; partial (50%) reconstitution, Fig.
7, rows 2 and 4 for CN-protohemin and
CN-deuterohemin, respectively) reveal heme insertion rates (Phase I)
1.7-fold more rapid for CN-protohemin and 1.5-fold more rapid for
CN-deuterohemin than seen for unmodified apohemoglobin. Furthermore,
the difference between the heme insertion rate of CN-protohemin and
CN-deuterohemin increased to 1.8-fold, whereas the rate of formation
of CN-protohemin-semi--hemoglobin (Fig. 7, row 2)
actually decreased when compared with that of the fully reconstituted
species (Fig. 7, row 1); a finding heretofore only seen with
CN-deuterohemin insertion. Phase II exhibited a rate of 0.23 s
1 for all four time courses and is remarkably similar to
that seen for both apohemoglobin and
-apohemoglobin.
Phase III (0.12 s
1) was 1.9-fold slower than Phase II for
reactions involving full reconstitution and either missing or much
slower (2.5-fold) for half-reconstitution with CN-protohemin and
CN-deuterohemin, respectively. Phase IV displayed rates of 0.008 (15-fold slower than Phase III) and 0.011 s
1 (10-fold
slower than Phase III) for CN-protohemin and CN-deuterohemin binding
irrespective of the degree of reconstitution. In fact, full
reconstitution of PMB-apohemoglobin displayed a Phase IV rate 1.4-fold
slower than that for reconstitution of unmodified apohemoglobin. Thus,
even the presence of Phase III could not restore proximal bond
formation to its original rate when PMB is bound.
These present studies of CN-hemin incorporation into PMB-apohemoglobin
showed that 93 (F9) sulfhydryl modification not only accelerated but
also accentuated the difference in the rate of heme insertion (Phase I)
of CN-protohemin and CN-deuterohemin. It appears that even though
subunit accessibility has been enhanced, the vinyl groups continue to
play a key role in the kinetics of CN-hemin binding. Phase II
consistently reflected local protein-heme interactions, the majority of
which, interestingly enough, are on the proximal (F8) side of the
and
chains (26). Although the rate of Phase III of
PMB-apohemoglobin increased during complete reconstitution, it
continued to be absent during partial reconstitution of this modified
protein. This demonstrated that PMB alone is able to alter
coupling (PMB has been reported to affect dimer coupling in hemoglobin;
Ref. 39) but not enough to allow the
subunit to respond as it would
if decoupled (Fig. 6) from its
partner. This absence of Phase III
inevitably resulted in a slowed rate of proximal bond formation for the
subunit. This finding is not a direct consequence of the presence
of PMB because the residue adjacent to the proximal (F8) histidine, is
a sulfhydryl, only in the case of the
subunit.
In conclusionCN-hemin incorporation, although not seen
under normal physiological conditions, may nonetheless allow insight into the probable sequence of events leading up to hemoglobin tetramer
formation in vivo. CN-deuterohemin appears to randomly bind
to the
and
chains of apohemoglobin and as such does not promote
either Soret spectral shifts or anomalous kinetic behavior when
incorporated into the apohemoglobin models employed here. Furthermore, absence of the vinyl groups may impair the heme insertion (Phase I) process (possibly due to heme orientation stereospecificity factors; Ref. 40) but does not impede structural rearrangements (Phases
II and III) nor timely proximal histidine-iron bond formation (Phase
IV).
The presence of the porphyrin 2,4 vinyl groups, on the other hand, has
interesting consequences. CN-protohemin promoted Soret spectral shifts
upon binding in all apohemoglobin models. The invariant G5
phenylalanine of the subunit appears to be a likely candidate for
involvement in this spectral shift, especially because the G5 residue
of its
chain partner is not reported to interact with the vinyl
groups (4, 26). Furthermore, the magnitude of Soret spectral shift, the
presence of Phase III, and the rate of Phase IV were all governed by
whether the
apohemoglobin was free as a monomer or sequestered in
an apohemoglobin dimer, implying that
interplay is primarily
responsible. The G-helical segments are reported to be essential for
coupling of apohemoglobin, and thus it would appear that amino
acid residues in this region account for static enhancements and
kinetic restraints imposed on the preassembled
subunit. Although
the B-helical region may be important (26, 41, 42), focusing on the FG
and G regions, where the majority of interface contacts in both
apohemoglobin and hemoglobin reside, may be informative. In this
region, four
chain residues (FG5, G4, G5 and G8) account for 11 out
of 13 vinyl contacts, whereas three
chain residues (FG5, G4, and
G8) are responsible for 5 out of 14 vinyl contacts. Furthermore, these same residues not only interact with the heme moiety but also contribute one
1
1 and six
1
2 interface contacts in hemoglobin.
A possible scenario, consistent with the kinetics of
PMB-apohemoglobin, would be that the FG5 residue is involved in Phase III (the bulky PMB could enhance this residue's overall interaction; Table I). Movement of this FG5 valyl residue could reorient the G-helix, strengthen the 1
1 contact, and
prime the
1
2 region for tetramer assembly
(a process encouraged by heme binding to the
subunit). Histidine
bond formation would be inevitable. At half-saturating amounts during
semi-
-hemoglobin formation, however, the momentum of these
structural movements is lost, and the rate of irreversible proximal
bond formation is impaired.
In vivo heme and globin production are delicately balanced
processes so that ample quantities of both are available for hemoglobin formation in the typical precursor red blood cell (3); yet the exact
manner in which four nascent globin chains and four Fe-protoporphyrin-IX groups combine to form the heme-containing tetramer is still a mystery. As with all complex biochemical phenomena, isolation of given reactions have aided in understanding this process.
Previous studies have attempted to fine tune aspects of assembly
through kinetic investigations of association of heme-containing and
subunits (20, 43, 44); however, another plausible pathway of
assembly may be that of combination of heme-containing and heme-free
partner chains. If this is indeed the case then the heme-containing
partner must be the
subunit. Evidence for this is overwhelming and
consistent with findings that heme is readily inserted (possibly
cotranslationally; Ref. 45) into
o (1, 11), that both
o and
h are found in vivo (6,
7), and that
h chains are present only in the
case of severe
-thalassemia (HbH disease; Ref. 3). This stable
viable
h species may then combine with its heme-free
ribosomal bound
partner to form a stable semi-
-hemoglobin. The
results presented here suggest that its preassembled counterpart may
also be converted into hemoglobin by either acceptance of a heme moiety
from another semihemoglobin precursor or by binding heme directly. The
existence of the former pathway is intriguing, not only because both
semi-
-hemoglobin and apohemoglobin have been found in
vivo but also because it implies that the
chain plays a key
role in heme currency exchange.
We thank Fumin Chiu and Adrianna Morris for critical reading of this manuscript.
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