Received for publication, October 11, 2000, and in revised form, November 20, 2000
Sequence alignment of Hmp with other hemoglobins suggests that the
proximal ligand to the heme is histidine, and the distal residues at
the E7 and B10 positions are glutamine and tyrosine, respectively (2).
Although the exact arrangement of these distal residues surrounding the
ligand binding site in Hmp is not clear, it is plausible that the
distal pocket is large and flexible (2, 21) based on the crystal
structure of Alcaligenes (Ralstonia) Hb (the only
available crystal structure for flavohemoglobins). We postulate that
the large and flexible distal pocket evolved to accommodate both
O2 and NO molecules simultaneously for performing O2/NO chemistry. Hmp binds O2 strongly with a
Kd of ~11.5 nM (20) (as compared with
857 nM for sperm whale myoglobin). Replacement of the B10
Tyr with Phe increases the O2 dissociation constant
~80-fold (20), demonstrating the importance of B10 Tyr in stabilizing
the heme-bound O2. A similar role of the B10 Tyr residue
has been found in several nonvertebrate hemoglobins, such as those from
Mycobacterium tuberculosis (22), Ascaris suum
(23-25), Chlamydomonas eugametos (26), and
Cyanobacterium synechocystis (27). In this work, resonance
Raman spectroscopy was employed to reveal the structural features of
Hmp that underlie its chemical reactivity.
Hmp was cloned, expressed, and purified to near homogeneity as
described elsewhere (28). The protein was buffered at the desired pH
with 50 mM Tris, pH 7.5. For all of the experiments reported here, the protein concentration was 40 µM. The Raman measurements were made with previously described instrumentation (22).
The output at 406.7 and 413.1 nm from a krypton ion laser (Spectra
Physics), for the measurements of the ferric and the ferrous
derivatives, respectively, were focused to a ~30-µm spot (laser
power ~ 2 milliwatts) on a rotating cell to prevent photodamage to the sample. The acquisition time was about 20-30 min for each spectrum. The scattered light was collected at right angles to the
incident beam and focused on the entrance slit of a 1.25-m polychromator (Spex) where it was dispersed and then detected by a
charge-coupled device camera (Princeton Instruments).
Resonance Raman spectroscopy has been demonstrated to be extremely
informative in probing active site structures of heme proteins (22, 29,
30). In the low frequency region of the spectrum between 200 and 800 cm
1, the specific axial ligands coordinated to the
prosthetic heme group can be identified by detecting iron-ligand
stretching modes. In the high frequency region of the spectrum between
1300 to 1700 cm
1, the oxidation state, spin state, and
the axial coordination state of the iron at the center of the heme can
be characterized. In particular, the
2 mode, in the
region between 1550 and 1600 cm
1, is sensitive to the
iron spin state. The line in the 1475-1520 cm
1 region,
assigned as the
3 mode, is sensitive to both the axial coordination and spin state of the iron. The strong line between 1350 and 1400 cm
1, assigned as the
4 mode, is
sensitive to the
-electron density of the porphyrin macrocycle and
therefore the oxidation state of the iron. The frequency and intensity
of these Raman lines are further modulated by the protein environment
surrounding the heme and, therefore, provide useful structural
information on heme proteins.
The high frequency resonance Raman spectrum of the ferrous deoxy form
of Hmp is shown in Fig. 1. The electron
density marker line,
4, is located at 1353 cm
1 and the spin/coordination sensitive line,
3, is located at 1470 cm
1. The spectrum is
characteristic of a ferrous five-coordinate heme protein. In the low
frequency region (Fig. 2), a very strong line at 244 cm
1 is present that we attribute to an
iron-histidine stretching mode. The presence of this mode is consistent
with the assignment of the ferrous form being five-coordinate high-spin
with histidine as the proximal ligand. In general, the Fe-His
stretching frequency of globins, which have a neutral histidine as the
proximal ligand, is in the range of 200-230 cm
1
(31-34). On the other hand, that of the peroxidases, which have an
imidazolate ligand, is much higher in the range of 240-260 cm
1 (32, 35, 36). Thus, the stretching frequency of 244 cm
1 suggests that the proximal ligand of Hmp has
imidazolate character. This conclusion is consistent with the
crystallographic data of another flavohemoglobin from
Alcaligenes eutrophus, which has high sequence homology with
Hmp. This hemoglobin has a Fe-His-Glu grouping on the proximal side of
the heme (2), that resembles the catalytic triad (Fe-His-Asp) observed
in cytochrome c peroxidase (CCP) (37) as illustrated in Fig.
3.
The heme iron of the ferric protein is five-coordinate high-spin at
neutral pH, as indicated by the
3 and
2
lines located at 1491 and 1570 cm
1, respectively, in the
resonance Raman spectrum (Fig. 1). These data suggests that the distal
binding site of heme is unoccupied. The distal binding site in most
ferric globins is occupied by a water molecule at neutral pH, and as a
result, the electronic configuration of the heme iron is normally a
six-coordinate high-spin and low-spin mixture (38). The absence of a
water bound to the heme in the distal site is consistent with the
presence of a CCP-like proximal imidazolate ligand for the heme. It has
been shown in peroxidases that the imidazolate character of the
proximal histidine leads to a strong Fe-His bond. The strong Fe-His
bond forces the iron to move out of the porphyrin plane and thereby
prevents the coordination of weak distal ligands to the heme (39-41).
The ferric protein in most peroxidases thus favors a five-coordinate
structure. When the Asp that forms a hydrogen bond with the proximal
His in CCP is mutated, the ferrous state exhibits a much lower Fe-His stretching frequency, and the ferric state is converted from a five-coordinate to a six-coordinate structure (39). Because Hmp has a
similar hydrogen bonding network on the proximal side of the heme as
peroxidases, and it contains a strong Fe-His bond as indicated by the
high Fe-His stretching frequency, the five-coordinate structure of the
ferric state is attributed to the same origin.
In the high frequency region of the resonance Raman spectrum of the
CO-coordinated protein,
4 is located at 1373 cm
1 and
3 and
2 are located
at 1498 and 1582 cm
1, respectively (Fig. 1). The spectrum
is characteristic of a ferrous six-coordinate heme protein. In the low
frequency region, two isotope sensitive lines, located at 494 and 535 cm
1, were observed that are shifted to 478 and 516 cm
1, respectively, in the 13C18O
derivative (Fig. 2). These two Fe-CO modes are associated with two
C-O stretching modes at 1907 and 1960 cm
1, respectively,
in the 12C16O derivative that are shifted to
1825 and 1868 cm
1, respectively, in the
13C18O derivative (Fig.
4). There is a well established inverse
correlation curve linking the Fe-CO stretching frequencies with the
associated C-O stretching frequencies (42-44). This correlation is
attributed to back-donation of Fe2+ d
electrons to the CO
* orbitals. When CO is coordinated to a ferrous
heme iron, the
bond formed by donation of electrons from CO to the
iron greatly increases the electron density on the iron. A partial
double bond resonance (i.e. "back bonding" from iron
d
electrons to the CO
* orbitals) is used to decrease the rich electron density added to the iron to stabilize the L-M-CO complex (M and L stand for the ferrous heme iron and the proximal heme
ligand, respectively) as is shown in Reaction 2.
It is widely accepted that the O-O bond activation in peroxidases
is facilitated by a "push-pull" (37, 48-51) mechanism that is
illustrated in the middle panel of Fig. 3. In this model,
the presence of a strong hydrogen bond between the proximal histidine and an aspartate leads to partially anionic character in the proximal histidine that supplies a better electronic push for the heterolytic cleavage of the O-O bond (48-51). On the other hand, the pull effect on the distal side of the heme is created by an arginine to polarize the O-O bond and a histidine functioning as an acid/base catalyst in
proton transfer during the catalytic cycle. Although recent new
evidence suggests that the push effect from the proximal side of the
heme is not required for some peroxidase activity (50-54), the
structural features of Hmp revealed by resonance Raman spectroscopy suggest that the push-pull mechanism may underlie the enzymatic reactivity of Hmp.
The distal structural information of Hmp is provided by the resonance
Raman spectra of the CO complex. Two distinct structures are evident
from the two sets of the Fe-CO and C-O stretching modes. One set of
frequencies is characteristic of heme proteins in which there are no
polar interactions between the CO and the distal residues (an open
structure) whereas the other set corresponds to a structure in which
there are strong H-bonding interactions (a closed structure). The
frequencies of the closed structure are similar to those of peroxidases
and the inverse correlation between
Fe-CO and
C-O indicates that the distal pocket of Hmp is as polar
as peroxidases. The distal environment in Hmp thus may provide a strong
electronic pull for the O-O bond activation just like that in
peroxidases. The most likely residues offering the pull effect in the
distal pocket are the B10 Tyr and the E7 Gln as may be seen in Fig. 3.
It has been shown that the reaction rate of oxy-Hmp with NO decreases
by a factor of ~30 when the B10 Tyr is mutated to Phe (20),
demonstrating the importance of this residue in modulating the reaction.
The catalytic role of B10 Tyr in nonvertebrate hemoglobins is
intriguing. For example, in a bacterial hemoglobin (HbN) from M. tuberculosis that belongs to the truncated hemoglobin family (1),
the B10 Tyr interacts strongly with the heme-bound O2 and
OH ligands, and it causes the CO adduct to exhibit both an open and a
closed conformation (22). As observed in Hmp, the closed conformation
has spectroscopic properties similar to those of peroxidases as may be
seen from the Fe-CO versus C-O correlation curve in Fig.
5. When the B10 Tyr is mutated to Phe, only the open conformation is
observed, and the O2 off-rate increases by a factor of 150 (22). Similarly interactions between the B10 Tyr and the heme-bound
ligands have been identified in A. suum (23-25), C. eugametos (1, 26), and C. synechocystis (27) hemoglobins based on both spectroscopic and crystallographic
studies. Like Hmp, it was proposed that HbN and Ascaris Hb may
perform NO/O2 chemistry physiologically (22, 56). A Tyr at
the B10 position thus may play an essential role in this chemistry.
Recently, a hemoglobin, DHP, with peroxidase activity, was discovered
from in a marine worm, Amphitrite ornate (57, 58). This
hemoglobin catalyzes the oxidative dehalogenation of polyhalogenated phenols in the presence of H2O2 at a rate at
least 10 times faster than that of all known halohydrolases of
bacterial origin. The catalytic mechanism involves the binding of
H2O2 to the heme iron in the distal site and
the heterolytic cleavage of the O-O bond, as that observed in
peroxidases. Surprisingly, the structure of DHP has a classic globin
fold with only small variations on the distal and proximal heme side
(57). Two structural features of DHP are important for its efficient
peroxidase activity. First, the Fe-His bond is stronger than in
typical globins as evidenced by a high Fe-His stretching frequency at
233 cm
1 (53), presumed to originate from a strong H-bond
between the proximal histidine and a peptide carbonyl oxygen. Second,
the distal histidine is one Å further away from the heme iron than in
typical globins and readily swings out of the heme pocket enabling the
substrate to enter the distal pocket and undergo the oxidation reaction
(57). Although the physiological function of Hmp is distinct from that
of DHP, the presence of a strong proximal Fe-His bond, and the ability
to convert between an open and a closed structure in Hmp suggests that
both features may be critical for globins to carry out enzymatic functions.
An intriguing structural feature of Hmp that is distinct from other
globins is the orientation of the proximal histidine (assuming that the
structure of Hmp is similar to the flavohemoglobin from A. eutrophus) as illustrated in Fig. 7.
In most globins, including DHP, the imidazole plane of the proximal
histidine is almost coincident with the heme normal (i.e. a
tilt angle of 0°), in contrast to a large tilt angle of ~10° in
CCP. The tilt of the imidazole ring appears to be a consequence of the
strong hydrogen bond between its N
and a carboxylate
side chain of an aspartate (Asp-235) located in a nearby helix that
packs against the proximal helix, based on the fact that this tilt
angle becomes zero when Asp-235 is mutated to an alanine or a glutamate
(37). In Hmp, this tilt angle is even larger (~25°). We attribute
it to the strong hydrogen bonding between the N
and the
carboxylate side chain of a glutamate in another helix. The tilt of the
imidazole ring could generate strain in the molecule and affect the
covalent bonding between the proximal histidine and the heme iron,
which consequently influences the reactivity of the distal ligands.
The rotation angle of the imidazole ring along the Fe-His bond
is also very interesting. It is rotated by ~60°, 90°, and 180° for DHP, Hmp, and CCP, respectively, with respect to that in Mb (Fig.
7). In DHP, this rotation is made possible by the replacement of the
last helix turn of the proximal helix with a short 310 helix (57). It allows the proximal histidine to form a hydrogen bond
with a peptide carbonyl that is six residues away in sequence. The
resulting hydrogen bond is stronger than that in Mb as reflected by the
Fe-His stretching frequency at 233 cm
1 versus
220 cm
1 in Mb (57). In Hmp, the 90° rotation of the
proximal histidine is permitted by the shift of the F helix away from
the center of the heme (see the projection view in Fig. 7). It allows
the proximal histidine to form a hydrogen bond with a carboxylate side
chain of a glutamate in another helix (2). This hydrogen bond strength
is comparable with that in CCP as indicated by the Fe-His stretching
frequency at 244 cm-
1 versus 248 cm
-1 in CCP (35). In CCP, the proximal helix
is rotated 90O and shifted away from the center of the heme
with respect to that in Mb (37). This allows the proximal histidine to
form a hydrogen bond with a neighboring aspartate. It is noteworthy that the orientation of the proximal helix in DHP and Hmp lies somewhat
between that in Mb and CCP, which may have implications for the
evolution of hemoglobin from an oxygen activator to an oxygen carrier
(59).
It is well established that hemoglobin is an oxygen carrier, and the
heme pocket structure of hemoglobin is optimized to bind and release
oxygen reversibly. In contrast, in peroxidases the residues lining the
heme pocket are tailored to activate heme-bound oxygen species. The
discovery of the dioxygenase activity of Hmp, the nitric
oxide-activated deoxygenase activity of Ascaris Hb, and the
peroxidase activity of DHP from A. ornata, clearly
demonstrate that globins share a wide spectrum of catalytic activities
with peroxidases. The results from this work suggest that Hmp has a peroxidase-like active site structure that may promote
O2/NO chemistry instead of oxygen delivery. This finding
presents a paradigm shift in our understanding of hemoglobin structure
and function. Hmp, along with Ascaris Hb, DHP, and HbN from M. tuberculosis offer excellent examples that appropriate
modification of the distal residues and subtle control of the proximal
ligand in hemoglobins can alter their catalytic reactivity drastically.
The essential role of the Tyr at the B10 position on the enzymatic
activities is now becoming clear. The understanding of the structural
and functional relationships in these hemeproteins will help us to reconcile the old view of an oxygen transport and storage function for
globins and a wide range of new enzymatic functions in invertebrate hemoglobins that were discovered in recent years.
We thank Dr. Denis L. Rousseau for
many valuable discussions.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M009280200
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