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INTRODUCTION |
Thirty years ago, Perutz and Matthews (1) proposed that the
pathway for ligand entry and exit into mammalian hemoglobins and
myoglobins (Mb)1 involves
rotation of the distal histidine to form a short and direct channel
between the heme pocket and solvent. The experimental evidence in favor
of this histidine gate mechanism includes: (a) crystal
structures with large bound ligands that force upward and outward
rotation of the His64 side chain (2-4); (b)
significant increases in O2 association rate constants at
low pH, where the imidazole side chain becomes protonated and moves out
into the solvent (5, 6); (c) large increases in the rates of
ligand binding when the distal histidine is mutated to smaller apolar
residues (7-11); and (d) increases in the rate constants
for O2 association and dissociation when residue
Phe46(CD4) is replaced with Ala or Val, allowing greater
mobility and outward movement of the distal histidine (12).
Several groups have argued that the histidine gate may not be the
primary pathway for ligand movement into and out of myoglobin. Instead,
they have proposed that ligands may escape through the interior of
myoglobin by multiple transiently formed hydrophobic channels that
eventually lead to the solvent phase. In 1984, Tilton et al.
(13) suggested that the hydrophobic Xe binding cavities observed in
myoglobin crystal structures are important components of these escape
routes (orange spheres in Fig. 1). Molecular
dynamics simulations have provided additional support for indirect
pathways involving Xe sites (14, 15).
More recently, Huang and Boxer (16) suggested the existence of multiple
entry and exit routes based on changes in the kinetics of geminate and
overall O2 and CO rebinding to random point mutants of
myoglobin expressed in crude Escherichia coli lysates.
Significant effects were observed for amino acid replacements at
locations far removed from the distal and proximal histidines. Scott
and Gibson (17, 18) presented more direct kinetic evidence that dissociated ligands can access the Xe4 and Xe1 cavities. Filling these
sites with xenon gas eliminates the slow phases observed for geminate
rebinding on nanosecond and microsecond time scales but causes little
change in the total amount of geminate recombination. Three groups have
now reported low temperature crystal structures of wild-type and mutant
myoglobins in which electron density attributed to photodissociated CO
and O2 is present in the Xe1 or Xe4 sites (19-22). These
studies demonstrate that ligands do migrate into these internal
cavities and that population of the two Xe sites is associated with
protein relaxation that occurs after photolysis (19, 20).
These new crystallographic results prompted us to re-examine the
question of how ligands move into and out of myoglobin and whether or
not O2 can escape directly from the Xe cavities through the
interior of the protein. We have mapped entry and escape routes in
myoglobin by measuring geminate and overall O2 binding
parameters for 90 myoglobin mutants at 27 different positions in the
primary sequence. The locations of these residues are shown in Fig. 1, and the wild-type and mutant amino acids are listed in Tables I-V.
Replacements were made at virtually all positions in or near the heme
pocket and at locations near the Xe binding sites and along the escape
routes proposed by Tilton et al. (13, 14), Elber and Karplus
(15), and Huang and Boxer (16). Additional residues were chosen on the
basis of ligand movements observed in molecular dynamics simulations by
Gibson and co-workers over the past 6 years (17, 23-26). The remaining
mutants were made for other studies dealing with the fluorescence and
unfolding of apomyoglobin (residues 7, 8, 14, 79, and 131; see Refs. 18 and 27).
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MATERIALS AND METHODS |
Overall Association and Dissociation Rate Constants--
Oxygen
association time courses were measured by photolysis of
MbO2 samples using a 300-ns excitation pulse from a Phase-R model 2100B dye laser, and the results were analyzed using Equation 1
as described by Rohlfs et al. (8).
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(Eq. 1)
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O2 dissociation rate constants,
kO2, were
determined independently by analyzing stop-flow, rapid mixing time
courses in which bound oxygen was displaced by carbon monoxide. All
reaction conditions were 20 °C, 0.1 M potassium
phosphate, 0.3 mM EDTA, pH 7.0. Most of the association
rate constants for NO binding to various Mb mutants were taken from
Eich et al. (10, 11). The remainder were determined using
the same 300-ns dye laser photolysis apparatus used to measure
bimolecular O2 binding (23).
Measurement and Analysis of Geminate Recombination Time
Courses--
Time courses for geminate recombination were observed
after excitation with a 9-ns YAG laser. These internal, first order processes were recorded on multiple time scales ranging from 0-500 ns
to 0-5.0 µs (Fig. 2). The results were analyzed in terms of the
three-step, side path model shown in Scheme
1, which was first proposed by Chatfield
et al. (28) and then elaborated by Scott and Gibson
(17). The rate constants for intramolecular rebinding, escape, and
movement through the protein (k1 through
k4) were obtained by fitting sets of time
courses to this model. The algorithms for numerically integrating the
appropriate sets of rate equations, which account for recombination
during the excitation pulse and incorporate corrections for bimolecular
rebinding, are described in detail by Scott and Gibson (17). Sets of
fitted geminate rate constants are given in Tables I-V.
The geminate rebinding time courses can be fitted by the sum of two
exponential processes and an offset reflecting the amount of escape to
solvent. Any scheme that reduces to two exponential decays can
represent the data. The side path scheme was chosen by Scott and Gibson
(17) based on the effects of Xe binding and mutagenesis. The total
fraction of photolyzed ligands that rebind intramolecularly,
Fgeminate, can be measured directly or calculated as k1/(k1 + k2) based on Scheme 1. The fraction of ligands
that take the side path to state C and rebind more slowly, Fsecondary, can be calculated as
k3/(k1 + k2 + k3).
Determination of the Bimolecular Rate of Ligand Entry--
When
measuring the overall bimolecular rate of O2 binding to
deoxymyoglobin at room temperature, the concentrations of the intermediate B and C states are very small, and both
d[B]/dt and d[C]/dt are approximately zero.
Assuming a steady state approximation, the expression for the overall
association rate constant,
k'O2, is shown in
Equation 2.
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(Eq. 2)
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In terms of Scheme 1, k'entry represents
the second order rate constant for formation of state B starting with
O2 in the solvent phase and deoxyMb. The values of
k'entry were calculated from
k'O2/Fgeminate,
and are listed in Tables I-V.
The values of k'entry are defined
experimentally. If a sequential binding scheme (Scheme
2) is used to analyze the geminate time
courses, the bimolecular rate constant for ligand entry into the
protein will still be defined as the ratio of the overall association
rate constant and the total fraction of internal rebinding.
The steady state expression for
k'O2 using this linear
scheme is still
k'entry·Fgeminate. In
this case, Fgeminate = k1k4/(k1k4 + k3k2 + k1k2), and the fitted
values of k1 through k4
will be significantly different from those obtained using a side path scheme. However, the computed value of Fgeminate
will be the same as that calculated from the side path parameters since
both schemes can accurately represent the observed data, and
Fgeminate is the experimentally defined total
amplitude of geminate rebinding (17). In the linear scheme,
k'entry represents the bimolecular rate of
formation of state C, and the fitted rate parameters for the formation
and decay of this secondary state will affect the overall association
rate constant (29, 30). In contrast, the rate parameters for the
formation and decay of C state in the side path scheme have no
influence on the steady state expressions for the overall association
and dissociation rate constants.
The overall association rate constant for NO binding,
k'NO, serves an independent check of the
calculated value of k'entry. Nitric oxide is the
most reactive of the physiological gases, has the lowest quantum yield
for complete photodissociation from the native myoglobin
(Q
0.01), and shows the largest amount of geminate
recombination, Fgeminate
0.99 (9, 29, 30). In
effect, every time a NO molecule enters the distal pocket, it binds to
the iron atom, and thus the rate-limiting step for the overall NO
binding reaction is ligand entry. Since O2 and NO have
effectively the same size and polarity, the calculated value of
k'entry for O2 binding should be
approximately equal to k'NO, which can be
measured independently in conventional photolysis experiments.
Myoglobin Mutants--
All mutants were constructed and
expressed using the sperm whale myoglobin gene first synthesized by
Springer and Sligar (31). The wild-type control protein has an extra
N-terminal Met residue and an Asp122 to Asn substitution.
This double mutant shows identical ligand binding properties to
authentic native SW Mb and recombinant proteins with and without the
D122N replacement and the N-terminal methionine residue (32). Most of
the mutations at residues between positions 60 and 75 were constructed
using cassette mutagenesis and a pUC19 expression vector (7, 33). The
other mutants were made using a variety of oligonucleotide-directed
techniques and a pEMBL19 expression vector (see Ref. 34). The basic
expression and purification procedure was developed by Springer and
Sligar (31) and used with minor modification (34).
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RESULTS |
Determination of the Individual Rate Parameters for O2
Binding--
Typical nanosecond time courses for intramolecular
O2 rebinding to wild-type and mutant myoglobins are shown
in Fig. 2. Geminate data for all 90 different mutants were analyzed in
terms of the side path scheme shown in Scheme 1, and the fitted
parameters, k1 to k4, are
listed in Tables I-IV. Overall
association and dissociation rate constants for each mutant were either
taken from the literature or determined in new experiments. The errors
in the latter rate parameters have been shown to be ~±10-20% based
on measurements with wild-type myoglobin controls (32). The errors for
the fitted geminate parameters are more difficult to define because
they are correlated with each other and the observed fraction of
recombination. Based on repeated experiments with wild-type
oxymyoglobin, the errors in k1,
k2, and Fgeminate were
estimated to be ~±20% (see also Ref. 25). The rate parameters for
the secondary rebinding process are less well defined than the
amplitude of this phase (17). Since the rate constant for ligand entry
was calculated from the ratio of
k'O2 and
Fgeminate, the estimated error for k'entry is ~±30% based on the errors of
these measured parameters.
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Table I
Oxygen geminate and bimolecular rate constants for mutants with
substitutions in the first shell of distal residues
The fraction of total geminate recombination,
Fgeminate, was calculated as
k1/(k1 + k2).
The fraction of slow, secondary geminate recombination,
Fsecondary, was calculated as
k3/(k1 + k2 + k3). The bimolecular rate constant for ligand entry,
k'entry, was calculated as
k'O2/Fgeminate. Values
of k1 through k4 were obtained
from fitting sets of geminate recombination time course and the values
of k'O2, kO2,
KO2, and k'NO were
measured independently or taken from the literature. The errors for
wild-type (WT) myoglobin were estimated from averages of multiple
geminate analyses ( 10) and previous measurements of the overall
binding parameters (32). ND, not determined; UD, undefined.
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The results are organized in terms of the three-dimensional position of
the mutated amino acid: Table I, the first shell of amino acids that
are directly adjacent to bound O2 (Leu29(B10),
Phe43(CD1), His64(E7), Val68(E11));
Table II, the second shell of amino acids
on the distal side of the heme group located toward the solvent
interface (Arg45(CD3), Phe46(CD4),
Leu61(E4), Gly65(E8), Val66(E9)),
Thr67(E10)) and toward the protein interior
(Ile28(B9), Leu32(B13), Ile107(G8),
Ile111(G12)); Table III,
residues on the proximal side of the heme group (Leu89(F4),
His97(FG3), Ile99(G1), Leu104(G5),
Phe138(H15)); and Table IV,
amino acids far removed from the heme pocket (Trp7(A5),
Gln8(A6), Trp14(A12), Met55(D5),
Ala71(E14), Leu72(E15), Lys79(EF3),
Met131(H8), Ala144(H21)). The effects of Xe on
wild-type, one distal, and two proximal pocket mutant myoglobins are
shown in Table V.
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Table II
Oxygen geminate and bimolecular rate constants for mutants with
substitutions in the second shell of distal residues
Parameters are defined as in Table I. WT, wild-type; ND, not
determined.
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Table III
Oxygen geminate and bimolecular rate constants for mutants with
substitutions on the proximal side of the heme
Parameters are defined as in Table I. WT, wild-type; ND, not
determined.
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Table IV
Oxygen geminate and bimolecular rate constants for mutants with
substitutions distant from the heme
Parameters are defined as in Table I. WT, wild-type.
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Table V
Oxygen geminate and bimolecular rate constants for the addition of Xe
(12 atm) to selected mutants
Parameters are defined as in Table I. WT, wild-type.
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Plots of k'entry versus
k'NO show a roughly linear correlation (Fig. 3).
In general, the rate constant for O2 entry should be
greater than the measured association rate constant for NO binding
because the calculated value of k'entry takes
into account all ligand movements into the protein, regardless of
eventual location, orientation, and escape. The value of
k'NO reflects only those movements that lead to
bond formation with the iron atom and concomitant absorbance changes.
Equality between k'NO and
k'entry also assumes
Fgeminate = 1.0 for NO binding to all of the
proteins examined, which may not be the case for those mutants with
very high quantum yields (i.e. F43V, F46A, H64A, and V68I).
In our view, the correspondence between k'entry
and k'NO is remarkably good, considering the
errors and difficulties in the measurements, particularly with those
MbO2 mutants that autooxidize rapidly. The results in Fig.
3 serve to validate experimentally the use of
k'entry in mapping the location of pathways for
ligand entry.
Primary Site for Non-covalent Binding, State B--
A structural
interpretation of the side path scheme of Scott and Gibson (17, 18) is
shown in Fig. 4. This mechanism attempts to take into account the
effects of added Xe gas, all of the mutagenesis data in Tables I-V,
and the previous ideas of Olson, Phillips, Gibson, and co-workers (9,
23, 35). In the wild-type protein, transient outward movements of the
distal histidine allow distal pocket water molecules to leave the
protein and apolar ligands to enter through the His64(E7)
gate. Dioxygen molecules are then captured in the space above pyrrole
rings B and C that is circumscribed by Leu29,
Leu32, Val68, and Ile107. Ligands
in this location are designated as being in state "B," which is the
location of the initial nanosecond photoproduct. This state has been
well defined by mutagenesis experiments, IR spectroscopy, and molecular
dynamics simulations (for a review, see Ref. 35 and references therein)
and by time-resolved x-ray crystallography (19, 20, 22, 36, 37).
Ligands can then move further into the protein, bind to the heme iron
atom, or exit the protein back through the histidine gate. The observed movement depends on the accessibility of internal cavities, the reactivity of the iron atom, and the position of the His64
side chain.
Secondary Sites (State C) on the Distal Side of the Heme--
The
most obvious path for secondary ligand movement is into the Xe4 cavity.
This site is continuous with the primary photodissociated site, making
its exact boundaries difficult to define (orange sphere,
Fig. 1; upper gray sphere,
Fig. 4; inner cavity, Fig. 9). As described by Scott and
Gibson (17, 18), the geminate rebinding of O2 to wild-type
and 24 different myoglobin mutants is biphasic. The slower secondary
phase accounts, on average, for about 25% of the observed absorbance
change (Tables I and V; Ref. 17). When these proteins are equilibrated
with 12 atm of Xe, the secondary phase is much reduced, and the primary
phase becomes larger and significantly more rapid (Fig. 7C).
In most cases, the total fraction of geminate recombination does not
change significantly with Xe addition, and the overall second order
rate constant for O2 binding,
k'O2, is little affected
(Table V; Ref. 17). All these observations suggest that ligands do not
enter or leave the protein through the Xe pockets, which instead appear to be on side paths (Scheme 1 and Fig. 4).

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Fig. 1.
RibbonsTM drawings of the
structure of sperm whale metmyoglobin containing bound Xe (13).
The backbone is drawn as silver ribbons,
and the amino acid side chains drawn as red
sticks mark the 27 native residues that were mutated. The
locations of bound Xe are represented by orange
spheres, labeled sites 1-4. The helices are
labeled in yellow capital letters
(A-H). The heme group is shown in blue.
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Building on the initial work of Scott and Gibson (17), we have now
increased and decreased the size of most of the residues adjacent to
the Xe4 pocket, including Ile28, Leu32,
Ile107, and Ile111. Decreasing the size of
residues 28, 32, and 107 to Ala increases the amplitude of the slow,
secondary phase of geminate recombination, whereas increasing the size
to Trp almost completely abolishes it (Fig.
2C, Tables II and V). The
effects of these mutations on the total amount of geminate
recombination were more variable. For example, the I107W and I28W
mutations cause Fgeminate to increase from 0.47 to 0.67 and 0.79, respectively, whereas the I107A and I28A
substitutions cause only slight decreases to 0.38. Mutation of
Ile111, which is located at the interior edge of the Xe4
pocket, had little effect on any of the kinetic parameters for
O2 binding (Table II).

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Fig. 2.
Time courses for geminate O2
rebinding to sperm whale myoglobin mutants at pH 7.0, 20 °C.
Panel A, effects of mutations at or near the
distal histidine gate (positions Phe43(CD1),
Phe46(CD4), and His64(E7)). Panel B,
effects of mutations at the Val68(E11) and
Leu29(B10) positions. Panel C, effects of
mutations at Ile107(G8) and the addition of 7 atm of
Xe.
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Little or no effect on the total amount of recombination is observed
for amino acid replacements at positions farther away from the iron
atom, which include residues in the middle of the D helix
(Met55), at the end of the E helix (Ala71,
Leu72), at the EF (Lys79) and FG
(His97) corners, and in the A (Trp7,
Gln8, Trp14) and H helices (Met131,
Ala144) (Table IV). Huang and Boxer (16) reported that the
A144V mutation had significant effects on CO and O2
recombination in crude lysates of E. coli containing
recombinant myoglobin. In contrast, the purified A144V mutant has
geminate recombination properties very similar to the wild-type protein
(Table IV). The Q8V, M131L, and A144V mutations do cause 2-, 3-, and
1.5-fold, respectively, increases in the O2 dissociation
rate constant and therefore measurable decreases in oxygen affinity.
The origin of these latter effects is unclear and may somehow be
related to the overall stability of the globin molecule. However, these
mutations cause little change in the rate constants for ligand entry
and exit.
Secondary Sites (State C) on the Proximal Side of the
Heme--
Scott and Gibson (17) postulated that photodissociated
O2 migrates into or near the high affinity Xe1 binding site
because even low pressures of Xe have a significant effect on the time course of geminate recombination. More recently, Ostermann et al. (19) and Chu et al. (20) observed photodissociated
CO in this pocket using time-resolved x-ray crystallographic
techniques, and Vojtechovsky et al. (21) reported
O2 in this pocket when native crystals were equilibrated
with 100 atm of pure oxygen.
The Xe1 pocket is located underneath the porphyrin ring in a cavity
circumscribed by Leu89, Phe138, and
Leu104. Ala replacements at positions 104 and 138 increase
and Trp replacements decrease, respectively, the fractional amount of
slow secondary phases. However, these mutations have little effect on
the total amount of geminate recombination, the fitted rate parameter
for ligand escape, and the calculated rate constant for ligand entry (Tables IV and V). Surprisingly, the amplitude of the secondary geminate phase decreases when Leu89 is replaced with either
glycine or tryptophan. This apparent anomaly was resolved when Liong
et al. (38, 39) found that the L89G mutation allows several
water molecules to enter the Xe1 pocket through the "hole" created
by this substitution. Thus, both mutations cause the Xe1 cavity to be
filled, with water in the case of Gly89 myoglobin and an
indole ring in the Trp89 mutant.
The effects of mutations at Ile99 are more complex and
difficult to interpret because there are significant changes in the
conformation of the porphyrin ring (Table III; Refs. 38 and 39).
Decreasing the size of residue 99 to an alanine causes an increase, not
a decrease, in the extent of geminate rebinding. Similar results are
observed for the isosteric L99N mutation, and both substitutions cause
2-fold increases in overall O2 affinity, suggesting changes in reactivity of the iron atom due to puckering of the porphyrin ring
(38, 39).
The Distal Histidine Gate--
The most compelling evidence in
favor of ligands entering myoglobin through the distal histidine gate
is shown in Fig. 3C. Most
mutations that cause k'entry to be
100 µM
1
s
1 involve insertions of smaller residues at
position 64, and there is a inverse correlation between the size of
residue 64 and both the calculated value of
k'entry and the measured value of
k'NO (Table I). Karplus, Elber, and others have
argued that these mutations "poke" a hole in an inner tube, allow
gas to enter and escape rapidly, and do not identify the location of
the naturally occurring "valve" for ligand entry (15). This
criticism is valid but may be addressed by examining the effects of the
H64W mutation. If ligand entry does occur primarily through the distal
histidine gate, blocking this route with a large Trp residue should
decrease the bimolecular rate constant for ligand entry.

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Fig. 3.
Correlations between the calculated values of
k'entry and the experimental values of
k'NO. As described in the text,
k'entry was calculated as
k'O2/Fgeminate
and k'NO was measured independently in
microsecond laser photolysis experiments. Panel A shows all
available data and indicates an ~1.5:1 correspondence between
k'entry and k'NO. The
data in the lower range of rate constants are shown in panel
B. The data for mutations at and near the distal histidine are
shown in panel C. The latter results show that there is
strong inverse correspondence between the size of the residue at
position 64 and k'entry.
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The calculated value of k'entry for the
Trp64 mutant is 9 µM
1
s
1, which is substantially smaller than that
for either the Phe64 mutant (130 µM
1
s
1) or the wild-type His64
protein (34 µM
1
s
1). Thus, the large indole side chain does
appear to "plug," although not completely, the valve for ligand
entry. This conclusion is supported by the laser photolysis results
shown in Fig. 2A. When the distal histidine is replaced with
alanine, the rate constant for O2 entry increases
~20-fold (Table I), and bimolecular rebinding can be seen in the
2-µs geminate time courses measured at 1 atm O2 (Fig.
2A). The H64A mutation also decreases the fraction of true
geminate rebinding to almost zero, presumably because ligands can
escape directly to solvent. The H64W mutation produces the opposite
effect, an increase in Fgeminate to ~0.7.
The effects of the F46A, F46V, and F43V mutations provide additional
support for the histidine gate hypothesis. These replacements cause
large increases in k'entry and decreases in the
extent of geminate recombination (Figs. 2A and
3C). The distal histidine in the crystal structures of these
mutants is highly disordered (12, 40). In the case of F46V MbCO, the
imidazole side chain is rotated upward and outward, creating a direct
channel from the bound ligand to the solvent interface. This
conformation accounts for the marked increase in the rate and fraction
of ligand escape from the mutant protein (12, 41). Unlike the H64W
mutation, tryptophan replacements at positions 46 and 43 do not appear
to block ligand entry. The F46W mutation causes no change in
k'entry, and the F43W mutation results in an
increase in this rate constant (Tables I and II). In the latter case,
the large indole ring appears to push against the distal histidine
causing it to be more disordered (40).
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DISCUSSION |
Maps for Ligand Movement in Myoglobin--
We have created
three-dimensional maps to summarize the effects of mutagenesis on the
individual rate parameters for geminate and overall bimolecular
O2 binding (Fig. 5). The design of these maps follows the
presentation made by Huang and Boxer (16) for the effects of point
mutations on CO and O2 rebinding to recombinant myoglobin
in bacterial lysates. In their work, an automated procedure was used to
screen the effects of large numbers of random single mutations. In our
study, mutations from small to large residues were made rationally, the
proteins were expressed and purified, and complete sets of overall and
geminate O2 binding rate parameters were determined. With
one or two exceptions, there is good agreement between the two
approaches in terms of identifying amino acid side chains that regulate
the kinetics of ligand binding.
Secondary sites for photodissociated ligands were defined by
highlighting those positions which, when mutated from a large to a
small amino acid, cause marked increases in the fraction of slow
geminate rebinding, Fsecondary. The results of
this analysis are shown in the upper two
panels in Fig. 5. Positions where mutations cause large,
moderate, and very little change in Fsecondary
are marked in green, yellow, and white
or gray, respectively. All the mutations that affect the
slow phase of geminate recombination cluster around the Xe4 and Xe1
sites. These results support the low temperature, time-resolved x-ray
crystallographic results (19, 20, 22). Small amounts of
photodissociated ligands are able to move into these cavities under
physiological conditions.
Ligands in state C are almost certainly not in one specific location
but dispersed throughout the region marked by the green and
yellow residues and the two orange
spheres representing the Xe4(distal) and Xe1(proximal)
cavities (Fig. 5, upper panels). Residues further
removed from the heme group, in the area between the A, G, and H
helices, including Xe sites 2 and 3, do not appear to be accessible to
dissociated ligands. The latter region of the protein remains intact in
the molten globule intermediate that is observed during either acid,
urea, or guanidinium chloride-induced denaturation of apomyoglobin
(42-46). Thus, this portion of the intact myoglobin structure may be
too rigid to allow rapid penetration by ligand molecules.
Determinants of the Amount of Internal Rebinding--
In an
attempt to define the pathways for ligand escape, we have mapped the
effects of mutations from large to small amino acids on the total
fraction of geminate recombination, Fgeminate. The results are shown in the middle panels of
Fig. 5. Positions where mutations cause large, intermediate, small, and
no changes in Fgeminate are marked in
red, orange, yellow, and
white or gray, respectively. In this map, fewer
interior residues are marked in color, and only one proximal residue,
Ile99, is highlighted (yellow). The positions
showing the largest effects occur at His64
(red), Val68 (red), Ile28
(orange), Leu29 (orange),
Phe43 (orange), and Phe46
(orange). These positions are at the solvent interface and
in or near the major non-covalent binding site (state B) for
dissociated or entering ligands.
The middle panels in Fig. 5 suggest that only a
few ligands exit myoglobin by migrating from one Xe site to another and
then out through the interior of the protein. If those pathways were dominant, Ala substitutions at interior positions near Xe sites 1, 2, and 3 should have decreased to a greater extent the total amount of
geminate recombination by allowing more rapid escape, and Trp mutations
should have prevented escape, causing larger increases in
Fgeminate. These types of changes are observed
to a greater extent for mutations in the regions near His64
or the major non-covalent binding site (state B).
Pathways for Ligand Entry and Exit and Non-covalent
Binding--
The effects of changing from a large (Phe, Trp) to a
small amino acid (Ala, Val) on the rates of entry and exit are plotted versus position along the primary sequence in Fig.
6A. If the position being examined is located along a
pathway for ligand movement into the protein, such mutations should
increase both k'entry and
kescape significantly. The largest effects are
observed for mutations at positions 28, 29, 32, 43, 46, 64, 68, and 107 (Fig. 6A). In general, the variation in
kescape is much smaller,
5-fold, than the
200-fold range of values for k'entry. The
effects of changing the native residue to a small amino acid differ
significantly, depending on whether the residue is internal or located
near the solvent interface. The external F43V, F46A, and H64A mutations cause both k'entry and
kescape to increase significantly (Tables I and
II), demonstrating that the naturally occurring residues at these
positions must form significant parts of the barrier to ligand movement
into and out of myoglobin.
Alanine replacements at the internal Ile28,
Leu29, Leu32, Val68, and
Ile107 positions cause either no effect or only small
increases (
2-fold) in the rates of ligand entry and escape (Tables I
and II). In contrast, tryptophan mutations at these positions inhibit
both the rate and extent of non-covalent ligand binding, as seen by decreases in k'entry, increases in
kescape, and concomitant decreases in the
equilibrium constant for non-covalent binding (entry) into the protein
(i.e. Kentry = k'entry/kescape; Tables I
and II; Fig. 6B). These results confirm experimentally that
residues 28, 29, 32, 68, and 107 surround the primary non-covalent
binding site (state B in Fig. 4, see also
Fig. 9).

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Fig. 4.
Stereochemical interpretation of the side
path scheme for geminate recombination and bimolecular binding of
O2 from solvent. Upper panel,
photodissociated ligands first move into the empty space toward the
back of the distal pocket (state B). Some of the molecules move further
into the protein toward the Xe4 and perhaps Xe1 binding sites (states
C). O2 moves back from these more remote positions into
state B and either rebinds to form MbO2 (state A) or
escapes when the His64 gate is open. Lower
panel, this reaction scheme corresponds to Scheme 1 in the text,
where k1 = kbond,
k2 = kescape, and
k3 and k4 equal the rates
of movement into the secondary sites and back to the primary site or
state B.
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In order to emphasize the kinetic barrier, the ratios
k'entry(small)/k'entry(large)
and
kescape(small)/kescape(large)
were multiplied together, which is equivalent to adding the logarithmic values for the filled and empty bars
shown in Fig. 6A. The resultant product was used to generate
the three-dimensional map shown in the bottom
panels of Fig. 5. Those
residues that show large, intermediate, small, and no effects on the
rates of both entry and escape are marked in red
(Phe43, His64, and Val68),
orange (Phe46), yellow
(Leu29 and Leu32), and white or
gray, respectively. This map argues strongly that ligands
enter and exit myoglobin primarily through the channel created by
outward movement of the distal histidine. Mutations in the interior of
the protein have little effect on the rate constants for ligand entry
and escape, even though these replacements often have significant
effects on the rates and amplitudes of geminate recombination. The
latter changes in internal rebinding were seen for many of the interior
random mutants screened by Huang and Boxer (16). Small numbers of
ligands do reach these positions; however, the majority of them appear
to return to the distal pocket and escape through the histidine
gate.

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Fig. 5.
Maps of the effects of mutagenesis on the
fraction of secondary geminate rebinding, the total fraction of
geminate recombination, and the rate constants for ligand entry and
escape. The side view (left panels) and top view
(right panels) correspond to those shown in Fig. 1, where
the Xe pockets and helices are labeled. Top panels, map of
amino acids, which, when mutated from small to large residues, cause
marked decreases in the extent of secondary geminate rebinding.
Phe43, Phe46, and His64 side chains
were removed since mutating these residues to alanine or valine causes
the total fraction of geminate recombination to decrease to 0.15,
making it difficult to define the secondary phase. Arg45
was removed from the side view to allow visualization of other residues
in the CD corner and D helix but was placed in the top view. Positions
where mutations cause 100% changes in the fraction of secondary
phase are green. Positions where mutations cause <100% but
50% changes in Fsecondary are
yellow. Positions where mutations cause less than 50%
changes in Fsecondary are marked in
white, if a complete size series (Ala or Val to Phe or Trp)
was examined, or in dark gray, if only a limited
set of mutations was examined. The Xe binding sites are
orange spheres. Middle panels, map of
mutations that affect the total fraction of geminate recombination,
Fgeminate. Positions were marked in
red when mutation from a small to large or wild-type residue
causes 100% changes in the fraction of total geminate recombination.
Positions are marked in orange when mutations cause changes
in Fgeminate <100% but 67%; and positions
are marked in yellow where mutations cause changes in
Fgeminate <67% but 33%. The remaining
residues are highlighted in white or dark
gray as in the upper panels.
Bottom panels, map of amino acids that affect the rate
constants for ligand entry and exit. Residues were marked in
red (Phe43, His64, and
Val68) when the product
k'entry(small)/k'entry(large) × kescape(small)/kescape(large)
was >30 and as high as 500. Phe46 was marked in orange and
has a value of
k'entry(small)/k'entry(large) × kescape(small)/kescape(large)
between 30 and 10. Residues were marked in yellow
(Leu29 and Leu32) when their product ratios
were between 10 and 3. The remaining residues show little or no effects
and are marked in white or gray as described for
the top and middle panels.
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The Problem of Water Barriers--
Most of the distal histidine
mutants also alter the apparent equilibrium constant for the formation
of state B. In wild-type deoxymyoglobin, a water molecule is present in
the distal pocket and stabilized by hydrogen bonding to the distal
histidine. This water molecule inhibits the entry of all ligands,
regardless of the pathway taken (47). Replacement of the distal
histidine with apolar amino acid acids results in the loss of internal
water. This effect accounts in part for the large increases in
k'entry and Kentry
observed for the formation of state B in Gly64,
Ala64, Val64, Leu64, and
Phe64 mutants (9, 35, 48).
Perhaps the most remarkable result in Tables I-V is the relative
invariance of the fitted rate constant for ligand escape. The value of
kescape ranges from 5 to
30
µs
1, with an average value of ~10
µs
1. In contrast, the rate constant for
internal bond formation, kbond, ranges from
0.05 to >200 µs
1, and the bimolecular
rate constant for ligand entry, k'entry, ranges
from 0.2 to 540 µM
1
s
1. The large variance in
k'entry is clearly a reflection of the importance of the size of the internal pocket available for capturing entering ligand molecules. The rate of entry can be increased if
internal water is lost by replacing His64 with an apolar
amino acid or decreased if the size of the distal pocket is reduced
further by insertion of large aromatic residues at positions 28, 29, 32, 68, and 107. The invariance of kescape may
be the result of a more general "ice-like" water barrier to ligand
escape from the protein. Clathrate structures are found on the surface
of all proteins and may limit outward movement of both the distal
histidine and the ligand molecules.
Multiple Pathways--
An alternative interpretation of the
invariance of kescape is that there are multiple
pathways with roughly equal resistance to ligand escape (15, 16). For
this situation, it would be difficult to slow the observed rate of
escape by a single point mutation. For example, if there are four
pathways with equal rates of ligand movement, blocking one of them
would only decrease the net rate of entry or exit by ~25%. This
argument could account for the lack of effect of interior tryptophan
substitutions near the Xe4 and Xe1 sites. However, if one of these
pathways were opened up by an alanine substitution, the values of
k'entry and kescape
should increase markedly. Elber and Karplus (15) and Huang and Boxer
(16) used the latter argument to rationalize the results observed for
the H64G, H64A, and H64V mutations. They argued that the 10-fold
increases in k'entry for these replacements are
due to creation of a "hole," which allows rapid ligand entry and
escape. However, if the multiple pathway interpretation were true, then
valine and alanine substitutions in the internal escape pathways should
also cause large increases in k'entry and
kescape. In contrast to these predictions, large
effects are not observed for Ala mutations located in the interior of
the distal and proximal heme pockets.
Some escape from the Xe sites cannot be ruled out. An upper limit for
the bimolecular rate of entry through alternative pathways can be
estimated from the calculated value of k'entry
for the H64W mutant, which is 4-fold lower than that for wild-type
myoglobin. Thus, as much as ~25% of the ligands could be entering
the protein by routes through the protein interior. However, adding Xe
to wild-type myoglobin has little or no effect on
k'entry and Fgeminate (Fig. 2C and Table V). Small increases in
k'entry are observed when alanine replacements
are made at positions located in the Xe4 and Xe1 cavities. For
example, the I28A, L104A, and F138A substitutions cause
k'entry to increase ~40-50%. However,
these changes are only slightly greater than the estimated error for these parameters, ±30% (see Fig. 6),
and indicate that the amount of ligand entry through these alternative
pathways is small even when the Xe cavities are made larger.

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Fig. 6.
Effects of mutagenesis on the rate constants
for ligand entry and exit as a function of residue position.
Panel A, the logarithm of the ratio of
k'entry for the mutant with the smallest amino
acid divided by k'entry for the mutant with the
largest amino acid was calculated for each position along the primary
sequence. In cases where the wild-type protein showed the smallest or
largest rate constant, the ratio was calculated using
k'entry for wild-type myoglobin in the
denominator. All the parameters are listed individually in Tables
I-IV. The filled bars represent the ratios for
k'entry and the open bars,
kescape, which is equal to the fitted value of
k2 for the side path scheme in Scheme 1 and Fig.
4. Panel B, equilibrium constants for the formation of state
B were calculated as Kentry = k'entry/kescape. Then, as
in panel A, the logarithm of the ratio of
Kentry(small)/Kentry(large)
was calculated and plotted versus sequence position. The
large increases in the latter ratio for Ala mutations at
positions 28, 29, 32, 64, 68, and 107 define the position of ligands in
the major non-covalent binding site (state B in Fig. 4).
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In some cases, tryptophan replacements at or near the Xe4 and Xe1
pockets do cause large decreases in k'entry and
increases in Fgeminate, which might indicate the
closing of a pathway for ligand entry and escape (Fig. 2, B
and C). Correlations between these parameters and the fitted
rate constants for bond formation and ligand escape are shown in Fig.
7 for mutations at internal positions
near the Xe cavities. As the size of the amino acid side chain
decreases from Trp to Ala, the rate of ligand entry increases, the
total fraction of geminate recombination decreases and the amplitude of
the slow geminate phase increases (Fig. 7A). If these
changes were reflecting an opening up of interior pathways, the
decrease in Fgeminate should be due to an
increase in the rate of escape with no change in the rate of internal
bond formation. However, the opposite effect is observed (Fig. 7,
B and C); kbond decreases
markedly as k'entry increases and
kescape is either unchanged or decreases
slightly.

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Fig. 7.
Correlations of fitted geminate rebinding
parameters with the calculated rate constant for ligand entry into
mutants with replacements in or near the Xe4 and Xe1 binding
sites. The parameters were taken from Tables I and II for apolar
mutations at positions Ile28, Leu32,
Val68, Leu89, Leu104,
Ile107, Ile111, and Phe138.
Panel A, correlations between log
(k'entry) and Fgeminate
and Fslow. Fslow is
defined as the absolute amplitude of the slow geminate rebinding phase
and is given by Fgeminate × Fsecondary. The solid line
for Fgeminate is a fit to a polynomial of degree
2 with r = 0.897; the solid line
for Fslow is a fit to power function with
r = 0.61. Panel B, correlations between log
(k'entry) and log(kbond).
The solid line represents a linear fit with
r = 0.862. Panel C, correlations between log
(k'entry) and
log(kescape). The solid
line represents a linear fit with r = 0.198.
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Free Energy Diagrams--
The origin of the strong inverse
correlation between kbond and
k'entry can be seen clearly by constructing free
energy diagrams for O2 binding (Fig.
8). The inner and outer barrier heights
and the wells for covalent (state A) and non-covalent (state B) ligand binding were calculated using the analyses described originally by
Carver et al. (9) and reviewed by Olson and Phillips (35). The most extreme case is seen for the V68W mutant, in which the tryptophan side chain fills the back portion of the distal pocket. This
replacement causes large and roughly equal increases in the absolute
free energy of state B and the outer barrier to ligand entry (Fig.
8A). Thus, there is a direct correlation between the internal target size and the rate and extent of ligand entry into myoglobin. The absolute free energy of the inner barrier for bond formation is unaffected by the decrease in pocket volume. As a result,
G
B
A for
internal rebinding in V68W myoglobin is very low, and
kbond is markedly increased with respect to that
for the wild-type protein. In contrast,
G
B
X
for escape is still large, and the apparent rate constant for escape is
unchanged or increases slightly. The net result is that
photodissociated ligands almost always rebind and the total fraction of
geminate recombination approaches 1 (Fig. 2B).

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Fig. 8.
Free energy barrier diagrams for
O2 binding to wild-type, V68W, F46A, and L29W sperm whale
myoglobin. The free energy wells for states A and B and the
absolute barrier heights were calculated as described by Carver
et al. (9) and Olson and Phillips (35). The solid
lines describe the barriers and wells for wild-type
myoglobin, and the dashed lines represent the
free energy diagrams for the mutants.
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Similar, but less pronounced changes in the free energy diagram occur
for I28W, I32W, and I107W mutations. In our view, the increases
observed in the fractional amount of geminate recombination for these
tryptophan mutations are due primarily to a decrease in the internal
volume for dissociated ligands and not to blocking alternative pathways
for entry and escape. Carver et al. (9) showed that
increasing the size of the ligand molecule from O2 to
methyl and ethyl isocyanide also causes dramatic increases in
Fgeminate and kbond,
little change in kescape, and marked
decreases in k'entry and
Kentry. The free energy diagram for ethyl
isocyanide binding to wild-type myoglobin looks very similar to that
for O2 binding to the V68W mutant shown in Fig.
8A. Thus, the ratio of the size of the primary
non-covalent binding pocket (state B) to that of the ligand is a
major determinant of the rate constant for ligand entry.
The free energy diagram for O2 binding to F46A myoglobin
contrasts with that for the V68W mutant (Fig. 8B). This
replacement causes a selective decrease in the absolute free energy of
the outer barrier, with little change in the free energy of state B and
the inner barrier to bond formation. The decrease in the barrier to
entry and escape is almost certainly a result of the greater
flexibility of the distal histidine side chain in this and the F46V
mutant (12).
Distal Pocket Size and the Rates of Ligand Entry and
Escape--
Fig. 8 (A and B) emphasizes the
complex nature of the kinetic barrier to ligand entry and exit. The
height of the barrier includes an entropic contribution due to
sequestering the ligand in a small volume and a steric contribution
requiring movement of the distal histidine and disruption of water
structure at the solvent interface near the heme propionates. These
effects also suggest multiple positions for ligands in state B. A
three-dimensional representation of the spaces available to ligands in
the distal pocket of myoglobin is shown in Fig.
9 along with the key amino acid side
chains that determine the size and shape of the cavity. Some entering
or dissociated O2 molecules are located directly above the
iron atom in an orientation perpendicular to the heme and poised for
rapid bond formation. Some are oriented with the dioxygen bond parallel
to the plane of porphyrin ring in a position that facilitates escape
through the histidine gate. Others are more randomly positioned in the
interior of the distal pocket and perhaps even in the Xe4 cavity.
Interconversion between these positions occurs rapidly on nanosecond
time scales at room temperature. As a result, the observed ns time
courses for geminate rebinding of O2 show simple one or two
exponential behavior at room temperature. These results contrast with
the complex behavior observed for O2 and CO rebinding at
low temperatures (49, 50) or for NO rebinding on picosecond time scales
(9, 23, 24, 30, 51).

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Fig. 9.
Space-filling model of the heme pocket of
wild-type sperm whale oxymyoglobin. The structure was taken from
PDB file 2MGM (48). Key amino acids in the distal pocket are shown as
stick models and labeled in black. The heme group and the
proximal histidine (His93) are also presented as stick
models. The rest of the protein is drawn as a space-filling model in
yellow. The entire D helix and the CD corner were cut away
to show the interior of the active site. The view is looking across the
heme group toward the E helix and is rotated 180° from the
orientation shown in Figs. 1 (left), 4, and 5 (left column) to allow better visualization of
state B, the Xe1 and Xe4 cavities, and their interconnections.
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Tryptophan insertions at positions 28, 32, 68, and 107 sequester
dissociated ligands closer to the iron atom. In most cases, confining
the ligand near the heme group increases both
kbond and kescape, and
reduces the number of states required to fit the observed time courses
from two to one (Tables I and II; Fig. 2, B and
C). These effects are due to the increased probability of
finding the ligand above the iron atom or poised to leave the pocket
when the interior cavities are reduced in size and no longer available
to the dissociated ligand (Fig. 9). The same interpretation explains
why these Trp mutations cause even larger decreases in the rate
constant for ligand entry.
In the case of non-covalent binding, the ligand must enter the distal
pocket when the histidine gate is open and then remain inside long
enough to be captured when the gate closes. If the cavities in the back
of the distal pocket are open, entering ligands will spend more time
moving around inside the protein before migrating back out into
solvent. If these cavities are filled with large aromatic amino acid
side chains, the entering ligands will be reflected back out at a
higher frequency and have a much lower probability of being caught
inside when the histidine gate closes. As a result, the net rate for
ligand entry, k'entry, is much smaller when the
distal pocket is reduced in size by Trp insertions.
Ligand Escape from Mutant Myoglobins--
In contrast to the
native protein, direct escape from the Xe4 and Xe1 pockets may be the
dominant pathway in some mutants. The energy diagram for the L29W
mutant (Fig. 8C) shows that both the absolute value of the
internal barrier to bond formation and the bound state are increased
dramatically by direct steric hindrance with the large indole side
chain (52). Thus, photodissociated ligands are prevented from rebinding
to the iron atom and probably also from escaping via the distal
histidine gate due to the presence of the large indole side chain
hanging directly over the center of the heme group. The amount of
geminate recombination is effectively zero (Fig. 2B).
Similarly, entering ligands are unable to reach the interior portion of
the distal pocket (state B in Fig. 9) and are reflected back out into
solvent at a high frequency, causing dramatic decreases in
k'entry, k'NO, and
k'O2 (Table I).
Ostermann et al. (19) used crystals of L29W myoglobin to
visualize photodissociated CO non-covalently bound in both the Xe4 and
Xe1 sites, under conditions where complete escape from the protein was
prevented by freezing. The large indole ring completely prevented
rebinding to the iron atom under these conditions, maximizing the
amount of electron density associated with photodissociated ligands.
Large decreases in the extent of geminate recombination also occur when
Leu29 is mutated to Tyr (53-55), and Brunori et
al. (22) used the triple L29Y/H64Q/T67R mutant to determine the
crystal structure of photodissociated CO in the Xe4 pocket at low temperatures.
In an attempt to simulate escape from the Xe4 site, we placed ligand
molecules in this cavity and then used the LES method (15) and the MOIL
set of programs (56, 57) to calculate ligand trajectories. Simulations
were carried out in wild-type myoglobin for up to 100 ps. If no
external water molecules were placed around the protein, ligands
streamed out of the structure through gaps between the B and G helices
near His24, Arg118, and His119, as
was observed by Elber and Karplus (15). When crystallographic waters
were incorporated into the structure, particularly those waters
associated with His24, Arg118, and
His119, or a box of water was built around the protein, no
ligands migrated out of the Xe4 pocket on the time scales that were
examined (
100 ps). When external water was present, the entire
protein structure was more compact, inhibiting ligand movement in any
direction on picosecond time scales. These theoretical results
emphasize the importance of surface water as a barrier to ligand
escape, regardless of the pathway taken.
Conclusions--
The side path scheme in Fig. 4 provides a simple
and quantitative interpretation of the effects of mutagenesis and Xe
addition on geminate and overall rate constants for O2
binding to sperm whale myoglobin. In effect, the globin acts as a
baseball glove to "catch" and then trap incoming ligand molecules
long enough to allow bond formation with the iron atom. Opening of the
glove occurs by upward and outward movements of the distal histidine, and the ligands are trapped in the interior "webbing" of the distal pocket. Following this analogy, it is more difficult to catch in-coming
ligands if the pocket of the glove is already filled with water
molecules or if it is reduced in size with tryptophan substitutions
since many more of the entering ligands will bounce back out into
solvent. Thus, the rate of ligand capture correlates directly with the
size and depth of the internal pocket, whereas the rate of escape from
this non-covalent binding site depends more on the frequency of gate opening.
The cavities in the interior of myoglobin are also important for ligand
release. These spaces allow dissociated O2 molecules to
remain unattached to the iron atom long enough for net escape through
the distal histidine gate. A three-dimensional picture of the
non-covalent binding site (state B) is shown in Fig. 9. Filling the
interior distal cavity with Trp residues at positions 28, 32, 68, or
107 causes 3-30-fold decreases in the overall rate constant for oxygen
dissociation due to sequestering the ligand near the iron atom, which
selectively enhances the rate of internal rebinding
(kO2 and
kbond values in Tables I and II). Without compensating mutations, such decreases would impair the physiological function of myoglobin, which is to release oxygen rapidly for mitochondrial respiration during muscle contraction when there is no
blood flow and then to take up it quickly during muscle relaxation.
Consequently, the cavity in the interior of the distal pocket is
conserved in all mammalian myoglobins and is also present in the
and
subunits of vertebrate hemoglobins.