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OCR for page 33
A SURVEY OF THE EVIDENCE FOR AND AGAINST
A CREVICE CONFIGURATION FOR THE HEME IN HEMOGLOBIN*
PHILIP GEORGE AND R. L. J. LYSTER;
I?'tro~nct~on. For many purposes the reactions of hemoglobin and methe-
moglobin can be quite adequately represented by the traditional formulae
Hb + O — HbO~
HA + OH- = HbOH
HA + CN- — ~bCN
However, in more detailed discussions of the factors which govern the reac-
tivity of the heme iron, a consideration of the way the heme is bound to the
protein becomes essential. The fact that heme is also the prosthetic group of
peroxidase and catalase, aIld of cytochrome c, too—although in a slightly
modified form emphasizes the dominant role of the heme-protein linkage
in determining specificity. This is further borne out by other considerations.
Hemoglobin among all the hemoproteins shows most clearly differences in
reactivity, depending not only on the species of origin, but also on its physical
state as this is altered from that of a fresh lysed-cell preparation to that of a
many-times recrystallized sample. Several as yet undefined changes in the
conformation of the molecule probably accompany this transition. Not only is
the primary reaction of the iron altered (for instance the oxygen amenity is
usually increased by dialysis, crystallization, etc.), but the secondary processes
that affect the primary reaction are also modified. Although the magnitude
of the Bohr effect is scarcely altered, heme-heme interaction is less, or is lost
entirely.) This is especially true of the reactions of recrystallized methemo-
globin; in some studies it has been found that n I, showing interaction
to be completely absent.'~3~4
Hence, the process of heme-heme interaction is to be regarded as resulting
from a far more critical and specific structural feature of the molecule. In the
case of muscle hemoglobin, myoglobin, it may be that the recrystallized con-
dition represents a final conformational state wh ich can only be further
changed by denaturation itself. The smaller Bohr effect observable in the
metmyog;lobin-fluoride reaction reappears unchanged with the hemoprotein
reconstituted from hemin and apomyoglobin.5
The binding of the heme to the globin has usually been discussed from
two points of view: either to suggest a mechanism for heme-heme inter-
action or the Bohr effect, or to account for the remarkable property of
~ This review has been based in part upon research, now in progress, that is sup-
ported by grants from the National Science Foundation ( G23 09 ) and the National
Institutes of Health (RG4850).
~r This paper was presented by Dr. George.
OCR for page 34
34
PART I. STRUCTURE OF HEMOGLOBIN
reversible oxygenation by considering specific bonding to a particular amino-
acid residue, notably histidine. However, some observations and quantitative
data bear on the more general question of the configuration of the heme, quite
apart from the chemical nature of the group or groups in the protein to
which the iron atom is attached, and quite apart from the role these may play
be
~ ~ ~ 1 — — _ _ ~ _ ~
in determining the interaction effects. This kind of evidence will first
reviewed under three headings that represent extreme structural types:
a) heme bonded only by its side-chains: no Ye-protein bond
b) a surface configuration: one Fe-protein bond
c) a crevice configuration: two Fe-protein bonds.
Then a comparison will be made between pH effects that can originate in
the ionization of a heme-linked group, and in the fission of a crevice bond.
In some respects these extreme types are not mutually exclusive, and in
particular certain structures may be regarded as intermediate between ~ and
c. For example, while there may be one actual iron-protein bond, another
part of the protein molecule may nevertheless shield the heme from direct
attack by ligand molecules, introducing steric hindrance as an important
factor in complex formation. Such a structure could also be considered as a
crevice configuration. However, for classifying the evidence, the extreme
structural types defined above provide ~ very convenient frame of reference.
No Iron-Protein Bonds. This structure would require firm bonding of
the heme group to the protein by the side-chains of the heme. Only the pro-
pionic acid groups need be considered, since the very ready formation of
hematoheme with hemoglobin excludes the possibility of linkage by the
reaction of the vinyl groups, as in cytochrome c.6 Esterification or amide
formation with groups on the protein is unlikely in view of the very rapid
removal of the heme that occurs in strong acid. A more likely possibility is
the formation of salt bridges between the negatively-charged carboxyl groups
and positively-charged groups on the protein. One consequence of this structure
would be weaker binding of heme in solutions acid to the pK of the carboxyl
groups; and this would accour~t for the easy removal of heme in solutions at
pH 3.7 However, this reaction probably involves several groups other than
those directly linked to the heme, in view of Zaiser and Steinhardt's finding
that in this pH region, 36 extra basic groups per molecular weight of 68,000
become titratable.S
Were it not for the
tact that both heme and hemin exist as polymeric
species in aqueous solution, a comparison of their absorption spectra with
those of hemoglobin and methemoglobin would provide valuable evidence
as to the plausibility of this structure. The differences (which are especially
marked between hemin and me/hemoglobin, and which permit the spectro-
photometric titration of apohemoglobin with heme and hematin) could how-
ever be attributed entirely to the monomeric state of the prosthetic group
ashen it is attached to the protein. It is interesting that, in certain solvents,
OCR for page 35
CREVICE CONFIGURATION GEORGE AND LYSTER 35
especially alcohol, hemin gives an absorption spectrum that resembles methe-
moglobin very closely.9
The difficulty of accounting for the specificity of hemoglobin that this
configuration would entail might be overcome by supposing that the iron
atom becomes simultaneously attached to the protein when a ligand is bound.
One Iron-Protein Bond. In this structure the iron in hemoglobin is
regarded as forming a classical octahedral complex with four bonds in a
plane to the pyrrole N-atoms, a fifth bond to the protein on one side of the
porphyrin ring, and a sixth bond on the other side to a coordinated water
molecule:
id N
e.g., Globin Fe—HERO
/ I
At N
The magnetic susceptibility data,10 which establish that hemoglobin and
methemoglobin can form octahedral complexes, taken in conjunction with
the recent theoretical developments in ligand field theory which account for
the stability of transition metal compounds in general,~~~-~~3 make the in-
clusion of a water molecule to complete the octahedral coordination shell an
extremely reasonable assumption.
This hydrate structure was first discussed in some detail by Hauro~vitz in
1935,14 and, seeking direct experimental confirmation, he later extended
Zeynek's observations on the drying of hemoglobin.45~~6~i' He showed that
with oxyhemoglobin scarcely any change in the absorption spectrum occurs,
and ever at very low pressures (0.1 mm Hg) the oxygen cannot be removed.
In contrast, unoxygenated hemoglobin on drying is converted into a com-
pound named anhydro-hemoglobin characterized by a hemochromogen-like
spectrum. Although not stated explicitly, it appears from the description of
the experiments that anhydro-hemoglobin is unable to combine with oxygen
unless re-hydrated. Haurowitz pointed out that the formation of anhydro-
hemoglobin may be due either to an intramolecular change, with the iron
atom forming a bond to nitrogen within the same molecule, or to an inter-
molecular reaction with the iron exchanging the water for a N-atom on a
neighboring molecule. The latter polymerization mechanism was preferred.
In the presence of a large excess of glucose no hemochromogen-like spectrum
was observed in accord with the expectation that, through its multiple polar
groups, the glucose would become attached to the surface of the hemoglobin
and thus prevent the mutual association forming the polymeric nemochrom-
ogen. Keilin and Hartree extended these studies by showing that, on drying,
methemoglobin underwent a similar change giving the corresponding para-
hematin while the fluoride, abide and cyanide complexes could be dried
without change, thus resembling oxyLemcglobin.:S
OCR for page 36
36
PART I. STRUCTURE OF HEMOGLOBIN
These observations appear to demonstrate the presence of a water molecule
attached to the iron in both hemoglobin and me/hemoglobin; but this con-
clusion would only be valid if it could be shown that an alteration in the
number of solvent water molecules, hydrating the protein as a whole, played
no part in bringing about the change in structure around the iron atom. If
salvation effects were significant, then on the basis of these observations alone
no choice could be made between the hydrate structure, a structure in which
another unidentified labile group is attached to the iron, and a structure in
which the sixth coordination position is unoccupied. At present there is some
evidence to suggest that the water does play an essential role beside that of a
potential ligand for the iron. lIaurowitz noted that by changing the partial
pressure of the water the conversion of hemoglobin into anhydro-hemoglobin
could be repeatedly shifted from left to right
Hb.H2O ~ ~ anhydro-Hb + H2O
while anhydro-hemoglobin apparently only forms oxyhemoglobin when both
oxygen and water are present. Now it is understandable that dried oxyhemo-
g~obin resists deoxygenation, since calculation shows that its formation is
probably accompanied by a very favorable free energy change. But, unless
water also enters into the reaction in the capacity of an unspecific solvating
agent, it is difficult to understand why oxygen should not be able to react
with anhydro-hemoglobin just as water is presumed to do according to the
hydrate hypothesis. Further experiments on anhydro-hemoglobin with oxygen
and other ligands, such as CO and NO for which the iron has a greater
affinity, are really needed to bring out more clearly the role of the water.
There is another aspect of these reactions that has received less attention.
If it is accepted that the heme is joined to globin by one strong bond, then
the fact that anhydro-hemoglobin has a hemochromogen structure suggests
very strongly that the bond is to a N-atom. The other possibility—namely,
that a strong bond to some other atom breaks and two new F`e-N bonds
form appears far less likely in view of the ready reversal of the equilibrium
with changes in water vapor pressure.
An important feature of the hydrate structure is that it leads directly to a
simple and reasonable mechanism for the conversion of acidic methemoglobin
to the alkaline form. As the following equations show, the ionization of
proton from the coordinated water molecule is equivalent to its replacement
by a hydroxyl ion.
Fe+H.,O = FeOH + H+
:lTe ~ H.',O + OH - ~eOH + H ,O
Keilin and Hartree observed that in freezing to liquid-air temperatures,
alkaline methemoglobin reverts to the acidic form. They lateriS considered
this to be direct evidence for the hydrate structure, but an effect of this kind
would be shown by any endothermic ionization, as demonstrated by Keilin
and Hartree in their earlier experiments. Hence it cannot serve to identify
OCR for page 37
CREVICE CONFIGURATION GEORGE AND LYSTER 37
the chemical nature of the ionizing group. That the methemoglobin and
rretmyoglobin ionizations are endothermic was later established by George
and Hanania, the values of AH being 5.8 + 0.7 and 3.9 A- 0.5 kcals/mole
respectively.4~~0
A suggestion that has received considerable attention is that while the
heme groups are attached by only one iron-protein bond, the groups are
sufficiently deeply buried in the body of the protein that combination of a
limed with the iron requires a dilation of the nrotein.-i This the of steric
~ ~ - 1~ r- - ------ - ---- -a r -
~ ' ~ ~ ~ · . · ' 1 ~ T' '1 · ~ 1 . ~ . . ~ -
h~ndrance has been criticized bY merlin on the grounds that the iron atoms
of hemoglobin are known to bind firmly some rather large molecules;~'' for
example, nitrosobenzene appears to be bound about as strongly as carbon
rr~onoxide. Moreover with 4-methyl imidazole, Kendrew and Parrish found
such a small change in the unit cell of metmyoglobin that they suggested that
the heme group is more likely to be located on the surface.''3 Certain oxidizing
and reducing agents have also been instanced as large molecules having
apparently easy access to the heme group. But close approach to the metal
atom itself is not essential for electron transfer, as is shown in reactions in-
volving the octahedral coordination con~plexes of iron, ruthenium, and
osmium with dipyridyl and phenanthroline.'4 A similar example is found
in the reaction of ferrocytochrome c and the second intermediate compound
of cytochromec peroxidase.-, All these reactions are very fast indeed, their
velocity constants being greater than 10;3 1. mole-i sec.-i
Evidence of a more direct kind for this type of steric hindrance divas sought
by St. George and Pauling by studying the binding of a series of aliphatic
isocyanides with ferroheme and hemoglobin.'') Their results, together with
those of Lein and Pauling's later Fort on myoglobin,~6 are given in Table I.
TABLE I
LOGARITHM OF EQUII IBRIUM CONSTANTS FOR THE COMBINATION OF
ALKY! ~ SOCYANIDES WITH HEME AND HEMoPRoTErss AT PH 6.8 AND 25 C.-1 -I;
iso- tert.-
ethyl n-propyl propyl butyl
Ferroheme 9.43 9.14 9.04
Horse Myoglobin 5.30 5.04 4.15 3.00
Bovine Hemoglobin 3.9 6 — 3.48 1.70
Human Hemoglobin 4.11 3.52 1.80
The most interesting feature is the sharp drop in the affinity of hemoglobin
for the tertiary butyl isocyanide, which is not reflected in the results with
ferroheme. This sharp drop was ascribed to steric hindrance. If this inter-
pretation is correct, the problem now arises whether steric hindrance affects
the binding of only those ligands equal (or larger) in size to tertiary butyl
isocyanide, or whether it is always present as a factor in determining ligand
affinities, however small the ligand. If the latter alternative is adopted, it
becomes necessary to postulate for the iso-propyl isocyanide some sort of
OCR for page 38
38
PART I. STRUCTURE OF HEMOGLOBINT
steric assistance, such as a complementary hollow in the appropriate part of
the protein near the heme group. This difficulty is further emphasised by a
consideration of Lein and Pauling's data for myoglobin and approximate
estimates of the maximum lengths of the ligand molecules, based on the ap-
piopriate covalent radii, which are given in Table II. In its fully extended
TABLE II
APPROXIMATE MAXIMUM LENGTHS IN
A NTGSTROM U NITS OF AL KYL I SOCYAN IDES
ethyl 7.7 iso-propyl 7.7
n-propyl 9.0 ter;.-butyl 7.7
form, n-propyl isocyanide would project furthest of all from the plane of
the heme group, yet its affinity is only half that of ethyl isocyanide. On the
other hand, in its coiled configuration, it would project to the same extent
as tertiary butyl isocyanide, but it would appear necessary to postulate an
additional complementary hollow to contain the extra methyl group.
The difficulties involved in comparing a series of primary, secondary and
tertiary alkyl ligands may be illustrated by considering Table III. These
TABLE III
BINDING OF BASES By' SILVER IONS AT 25 C. - ~ - - S- - S3
log K /\G AH /\S
Amine: Ag ratio 1 :1 (kcals/mole) (kcals/mole) (e.u.)
Ammonia 3.32 —4.52 —6.9 —8.0
Ethylamine 3.37 —4.6 —6.73 —7.15
Iso-propylamine 3.3 8 —4.61 —6.73 —7.1
Benzylamine 3.29 —4.49 —6.73 —7.5
Amine: Ag ratio 2 : ~ ~
Ammonia 7.23 —9.86 —13.8 —13.3
Methylamine 6.79 —9.26 —11.5 —7.5
Ethylamine 7.30 —9.96 —13.5 —11.8
Iso-propylamine 7.24 —9.87 —13.5 —12.0
Benzylamine 7.14 —9.74 —13.5 —12.5
Dimethylamine 5.37 —7.32 —9.7 —8.0
Diethylamine 6.38 —8.70 —10.65 —6.5
results are from the work of Bj errum, 'I Bruehlman and Verhoek,9S and
Fyfe,99 on the binding of amines by silver ions. Silver can bind either one
or two molecules of base; in either case, if the base is a primary amine, the
thermodynamic data are almost identical for all the amines listed. However,
for the formation of the 2:1 complete with secondary amines, there is a
difference of a factor of ten between diethvlamine and dimethylamine for the
affinity, though the acid dissociation constants of these two amines differ by
less than a factor of two. Thermodynamically, ~ H for diethylamine is
more favorable to the extent of 0.S kcals/mole, and AS also more favorable
to the extent of 1.5 e.u. Similar thermodynamic data for the reaction of
OCR for page 39
CREVICE CONFIGURATION GEORGE ANTD LO STER 39
hemoglobin and myoglobin with the isocyanides would be of the greatest
interest.
The apparent existence of steric interaction of tertiary butyl isocyanide
with the protein in hemoglobin led St. George and Pauling to propose that
the phenomenon of heme-heme interaction also had a steric origin.') How-
ever, it appears from their curves of percentage formation versus ligand con-
centration that heme-heme interaction almost disappears in the on crucial case
of tertiary brutal isocvanide: it is difficult to see who a large litany should
. . ~ .
, , , . j ~ c,
Produce the hemoglobin to show less heme-heme interaction than a small
ligand like oxygen.
Characteristic of this hypothesis that heme-heme interaction has a steric
origin is the trend in thermodynamic data to be expected from such a
mechanism. If the first ligand molecule to combine reduced the steric hind-
rance for the second, and so on, it would be predicted that in comparing data
for the first and last oxygen molecules the heats of reaction should show a
trend to a more favorable value, since more er~ergy would be required to
dilate the molecule for the entry of the first; whereas the entropy change
should show a trend to a less favorable value, because the entry of the last
oxygen molecule would be accompanied by a smaller increase in the freedom
of the molecule in the neighborhood of the heme. Experimentally, exactly the
opposite trend is shown in the only system for which direct experimental
results are available. The data of Roughton, Otis and Lyster for the reaction
of oxygen and sheep hemoglobin at pH 9.1 are given in Table INT.:~° The first
TABLE IV
THERMCDYNTAMIC D ETA FOR THE OXYGENATION OF
SHEEP HEMOGLOBIN AT pH 9.1 AND 25 C.:~°
1st Oxygen
2nd Oxygen
3 rd Oxygen
4th Oxygen
(I./^'nole)
1.1 X 104
5.S X 104
1.2 ~ 10;'
3.9 X 106
(locals /mole)
—12.1 + 0.8
2.5
—4.2 + 3.7
—5.1 + 3.3
AS
(e.~.)
—22.2 +
3
—4.4 + 8
+9.2 + 12
- 13.0 + 11
molecule to combine has a large and favorable heat of reaction and art un-
favorable entropy change, while the affinity of the last molecule to combine
is high despite a low heat of reaction, because of a large and favorable entropy
change.3i This trend is in direct conflict with the mechanism for heme-
heme interaction suggested by St. George and Pauling. However, the only
data as yet available for the carbon monoxide reaction indicate no such trend.3>
The entropy changes for the first and last carbon monoxide molecules are
very similar, and the heme-heme interaction originates entirely in the change
in ASH from 7.1 kcals/mole to 10.5 kcals/mole, in the direction opposite
to that for oxygen. It would thus appear that heme-heme interaction may
not only be a oronertv of the hemoglobin molecule, but also have a different
1 1 ~
OCR for page 40
40
PART I. STRUCTURE OF HEMOGLOBIN
thermodynamic origin, depending on the chemical nature of the ligand. If
this is borne out by future quantitative measurements and for two ligands
like oxygen and carbon monoxide that have such similar thermodynamic
properties there are such enormous differences in ~ H and ~ S then it
may be necessary to question whether in fact simple steric effects like those
considered so far play any significant part in this intricate phenomenon.
Two Iron-Protein Bonds. Conant, in his Harvey Lecture of 1933, first
considered various structural possibilities in which iron was bound to the
protein on both sides of the heme group.:33 Wyman suggested that the imi-
dazole residues of histidine, one of these possibilities, were likely to be the
groups concerned, on the basis of his finding that the heat of ionization for
both the heme-linked acid groups of hemoglobin was 6.5 kcals/mole;34 this
value is characteristic for histidine. Assuming that imidazoles are involved,
Coryell and Pauling have given a detailed picture of the reactions in terms of
resonating structures.3;, The dissociation constants of these heme-linked ion-
izing groups are listed in Table V.
TABLE V
HEME-~NKED IONIZATIONS OF
HORSE HEMOGLOBIN AT 25 C. 30
Pi:,. pK~
l\Iethemoglobin 5.75 6.68
Oxyhemoglobin 5.75 6.68
Hemoglobin 5.25 7.93
Figure 1 shows one of the possible resonating structures proposed by Coryell
and Pauling; they assign pK, to the glyoxalinium nitrogen of the distal group
and pi to the imino nitrogen of the proximal group. The structure shown
differs from Conant's original suggestion in having a water molecule co-
ordinated to the iron atom; this removes the objection that hemoglobin does
Proximal
HN~1 Fe+(H20)
G Iobin
Distal
N~NH
FIG. 1. Diagram of a possible res-
onating structure proposed by Coryell
and Pauling.
not have an absorption spectrum like histidine hemochromogen. A weak link
between the iron atom and the distal group, however, is postulated. George
and Hanania found spectroscopic evidence for ionizations with similar pK',
in methemoglobin solutions of low ionic strength.4 These ionizations became
undetectable at higher ionic strengths; and from this effect of neutral salt, it
was possible to argue that while pK~ could be attributed to such a group as
that shown in figure 1, pK~ could not, if their pK~ is the same as Wyman's
OCR for page 41
CREVICE CONFIGURATION—GEORGE AND LYSTER
phi. This difficulty persists in any structure where a proton acceptor is in-
volved in bonding to the iron.
Apart from this difficulty, the identification of pK~ and pK2 with imidazole
groups may be criticized in two ways. First, there seems to be no obvious
reason why the heat of ionization of the imino group of the proximal ring in
figure 1 should have the same heat of ionization as that found in free his-
tidine. Secondly, the recent studies by Roughton and co-workers have shown
that the individual equilibrium constants of the oxygenation reactions 3' and
the velocity constants for the combination of CO 35 are not uniformly affected
by pH changes. In view of this, it should not be assumed too readily that
Wyman's value for the average heat of ionization is actually that of each
heme-linked acid group.
The recent work of Corwin and Reyes, however, provides circumstantial
evidence in favor of the imidazole hypothesis.;3'3 These workers prepared imi-
dazole ferrohemochromogen and found that, in the solid state, it was able to
combine reversibly with oxygen. l his property was not shared by the similar
pyridine ferrohemo<:hromogen. III this renotinn In 1~re~lim~hl~r rlicnln~ e~
., ~ . . .- .
--I --a A--- ret ~~: ---or-
ar ~m~ctazote group bound directly to tile iron atom, as in the formulations
given by (:onant. Such displacement reactions, regardless of the chemical
nature of the groups involved, are characteristic of all structures in which
the iron is joined to the protein by two bonds, and will show certain general
features to which we now turn.
Crevices in General. The general features of reactions involving crevice
structures will be considered from two points of view; first, the trend in
thermodynamic data to be expected from such a reaction, and secondly, the
effect such ~ structure has on the pH dependence of the affinity for a ligand.
In considering thermodynamic data a useful comparison with cytochrome c
can be made, since a crevice configuration has beers fully established for this
hemoprotein. George and Hanania have compared the thermodynamic data
for binding of cyanide ions by ferricytochrome c ant by ferrimyoglobin ;~0
the figures are given in Table VI. Though the affinities are quite similar, the
T H ERA ODY N.N M IC D ATA
Ferricytochrome c
Ferrimyoglobin
difference
TABLE VI
FOR BINDING OF CYANIDE AT 25 C.~O
/\C~° WHO
( locals /mole ) ~ - ~
—8.3
—11.4
+3.1
( kcals / mole )
+1.1
—18.6
+19.7
AS°
(e.u.)
+31.3
—24
—55.3
heats and entropies of reaction differ in a very significant fashion. While
the heat change is favorable for ferrimyoglobin complex formation, it is ad-
verse for the ferricytochrome c reaction; exactly the reverse holds for the
entropy change, so that, but for the favorable entropy change ferricytochrome ~
cyanide would not in fact be formed. The adverse AH for ferricytochromec
OCR for page 42
PART I. STRUCTURE OF HEMOGLOBIN
can be attributed to energy lost in breaking the Fe-N bond and other links
that may be concerned in forming the crevice in the parent compound. The
favorable entropy change may be attributed to the freeing of the polypeptide
chain carrying this basic group. This freeing may be compared to a process
of partial denaturation. The reversible heat denaturation of proteins is char-
acterized by unfavorable heat changes and large and favorable entropy
changes; for example, Anson and Mirsky found for trypsin that the heat of
reversible thermal denaturation was + 68 kcals/mole, and the entropy
change +213 e.u.4i
From the figures in Table VI, it seems reasonable to conclude that, if the
above interpretation is correct, crevice opening plays little or no role in the
formation of the ferrimyoglobin complex. No thermodynamic data are avail-
able for the corresponding reaction with hemoglobin, but it is known that
the corresponding data for the ionizations of ferrihemoglobin and ferrimyo-
giobin, and for the formation of their fluoride complexes, are very similar
indeed. Thus it seems that in these particular recrystallized preparations the
reactions of the ferric form of myoglobin and hemoglobin involve no crevice
structures. Furthermore it may be noted that with the ferrihemoglobin no
heme-heme interaction divas observed.
The effect of a crevice structure on the pH dependence of the affinity for
a ligand can be readily deduced. It is natural to assume that the crevice-
J
forming group is basic since this property is characteristic of all ligands that
bind to metal atoms. Hence when the crevice bond Fe-Y is broken as a
consequence of the iron atom combining with a ligand L, the group Y will
associate with a proton in solutions where the pH is less than the pK of the
group YH+, and remain in its conjugate basic form Y in solutions where
the pH is less than this pK value.
i.e., pH ~ pi-:
pH > pi-:
Fe Y + L + H ~: FeL YH +
Fat e Y + L ~ > FeL Y
These are generalized equations. With ferro- and ferrihemoglobin, the iron
atom probably carries a net charge of zero and +1 respectively (the other
two ionic charges being balanced by negative charges on two of the pyrrole
N-atoms of the porphyrin ) and the ligands are usually neutral molecules
and anionic species respectively. The crevice-forming group has a charge of
zero arbitrarily assigned to it: similar equations can be written with the group
as Y-, giving the conjugate acid YH. Since combination with the ligand
breaks the crevice bond, it is convenient to consider a hydrate structure of
. . . . .... .
the hemoprote~n In equ~l~0r~um with the crevice configuration,
, ~
Fe(H~O) Y
~
Fe Y + H,O
OCR for page 43
CREVICE CONFIGURATION—GEORGE AND LYSTER 43
and the formation of the complex via the reaction of the ligand L with the
hydrate structure,
-- KI, A__
F`e ~ HERO ~ Y + L ~ ~ lTeL Y + H '0
where the equilibrium constants are KCr and KI. respectively. The equilib-
rium reaction giving the complex is then directly comparable with the formu-
lation usually employed when no crevice is being considered,
i.e., Fe(H.,O) + L ~ ~ FeL + H O
The solution of the appropriate equations based upon the above mechanism
gives the follow-in" expression for the observed equilibrium constant, evalu-
ated as
[complex]
Free hemoprotein] x ~ligand]
(H+ + KHY)
Kobs = Kin x ~ H + + Ker. KITN- ~
This may be compared with the corresponding expression reflecting the in-
uence of a heme-linked ionization on the affinity of the iron for a ligand,
Kobs — KL x ~ H + Kp)
Denoting the heme-linked group by H ~ X, then Kit, refers to the equilibrium
constant for complex formation with the group in its conjugate acid form
i.e., H +X.F~e ~ H..0 ~ + L
(Kr)
H ~ X.FeL + HERO
(Kp)
and Kr and Kit, are the ionization constants of the group in the reactant and
product respectively. The two expressions are identical in so far as they give
the same variation of Kobs with pH
(H+ + Ka)
i . e., }(obs tax
where Ka and Kb are simply the experimentally determined constants. Ac-
cording to the crevice mechanism Ka would be identified with KAY, and Kb
with Ken Kuy; whereas according to the heme-linked ionization mechanism
Ka would be identified with Kp, and Kb with Kr A consideration of the
relative magnitudes of Kit and Kb shows that it is nevertheless possible in
certain circumstances to distinguish between the two mechanisms.
For a significant fraction of the hemoprotein to be present in the crevice
configuration the equilibrium constant KCr must be greater than unity. This
implies that only when Kb > Ka is the crevice mechanism applicable: those
cases for which Kb < Ka must be attributed to the ionization of a heme-
OCR for page 44
44
PART I. STRUCTURE OF HEMOGLOBIN
linked group. If the condition Kb > Ka holds then when data are only avail-
able for one single reaction it is impossible to distinguish between the two
mechanisms. However, provided that data are available for several reactions
of the same hemoprotein, then, according to the crevice mechanism, Ka (ider~-
tified with Kim) should to a first appro~cimatior~ be independent of the nature
of the ligand, whereas according to the heme-linked ionization mechanism
Kit (identified with Kp) would be expected to vary from one ligand to an-
other, since the ionizing group H+X is understood to be linked in some way
to the iron atom, i.e. H+~.FeL. According to both mechanisms Kb would
be independent of the ligand, since it is identified respectively with K`r Kim
and Kr, the ionization constant of the group in the parent hemoprotein. Since
an ionizing group forming a crevice in this way results in the affinity of the
iron for a ligand being dependent on pH it could still be described as a heme-
linked group. The ionization is entirely normal in the complex and is affected
in the parent hemoprotein only by virtue of its conjugate base forming a co-
ordination bond to the iron above a threshold pH value (given by KCr.KH~-~.
Nevertheless it is better to regard such a group as a special case because of
the inter-relationship imposed on K;, and Kb.
Turning now to hemoglobin, and the data in Table V, it is at once clear
that pK~ cannot be explained by the crevice mechanism. This is the ionization
responsible for the main Bohr effect, and as can be seen, the pK charge on
oxygenation is in the wrong direction. However, it is clear that SKI, from
the scant: available data, does meet the required condition, Kb > A. Never-
theless, if a crevice is formed, it is a weak one with KCr ~ 3; this is readily
calculated from the change in pK since K~/Ka—K<.r. This particular ion-
ization is the one responsible for the reverse Bohr effect in acid solutions,
and as yet there is no good evidence for a similar group in myoglobin.4:3 The
other criterion can be illustrated by considering the pK values~for myoglobin
derivatives in Table VII. Although Kit, ~ Kit, there is a different pK value
for each complex, which thus again rules out the possibility of a crevice
mechanism.
TABLE VII
ACIDITY OF THE IIEME LINKED IONIZATION
FOR VARIOUS MYOGLOBIN DERIVATIVES4:]
pie: 1'(°~)
Acidic Ferrimyoglobin 6.1 20
Ferrimyoglobin Fluoride 6.5 20
F`errimyoglobin Cyanide 7.1 20
lTerromyoglobin 6.6 30
Oxymyoglobin 6.3 27
The One-Equivalent 7.5 20
Higher Oxidation State
of Ferrimyoglobin*
* The most satisfactory structure to account for the chemical reactions of this compound is
that of a complex; "ferry!" ion.
OCR for page 45
CREVICE CONFIGURATION GEORGE AND LYSTER 45
The criteria discussed above are of a general nature, and cannot alone
lead to any identification of the chemical groups involved. For this, accurate
thermodynamic data would be very helpful. Nevertheless a difficulty arises
in the wide variability in the hemoglobin from different species, the vari-
ability being manifested not only in the aidinitv for oven, but also in the
-- -d ~ ~ D---
magnitude and pH range of the Bohr effect.45~40 To -~ BIAS Air Ai ~ ;~^
. . . . . . .
11 LllC ~l ~1 Ly ~1 1~VUl~lUl~
oxygenation requires a bond from the iron to a particular group, such as imi-
dazole, then the variability suggests that the configuration of adjacent parts
of the protein has a considerable influence on its reactions. In this case, the
thermodynamic functions for its ionization will not be the same for the group
in free solution, and it 'will be necessary to call upon other criteria for its
. . ,~ .
1C .elltl~CatlOIl.
Conclusions. At present, x-ray analysis, amino-acid sequence studies, and
direct physical and chemical methods for determining protein structure have
not yet progressed far enough to establish how the heme is bound to globin.
Hence this can only be discussed in the light of the very extensive but more
indirect chemical and physicochemical evidence. Nevertheless, the discussion
of such evidence has a value in itself. In a more general manner it prepares
the way for the further problems that will arise when the chemical structure
is fully known, namely the correlation between chemical reactivity and specific
structural elements.
The crevice configuration that has been examined is just such a structural
element that could have a profound influence on the reactivity of the iron. On
the other hand, it is quite possible that a crevice, if it were a shallow one,
would have no effect on the reactivity, and so be indistinguishable in this
respect from a surface configuration.
Depending on the type of crevice, it is not entirely certain whether the
direct methods could substantiate its presence or absence in aqueous solution.
If the iron is joined to the protein by bonds on both sides of the heme, no
doubt would arise. However, if there is only one Fe-protein bond and part
of the protein is merely in the vicinity of the heme on the other side, then it
is quite conceivable that the extent of this shielding could differ in the
crystalline state and in aqueous solution. That some kind of conformational
changes in the protein occur in solution is suggested by experiments of many
lacings, and there is little doubt that, with accompanying changes in solution
and neutral salt interaction, they are an extremely important factor in de-
termining the full biochemical reactivity of the heme. The precise nature,
however, of such changes remains obscure.
Unlike cytochrome c, where the hemochromogen-like spectrum of its ferrous
form provides direct evidence for a crevice configuration,46 all the evidence
with regard to hemoglobin is indirect. It is certain that a hemochromogen
structure is not involved, and very unlikely that the iron is joined to the
globin by bonds on both sides of the porphyrin ring. Whether or not part of
OCR for page 46
~6
PART I. STRUCTURE OF HEMOGLOBIN
the protein shields the heme that is otherwise held by one iron-protein bond
(leaving aside side-chain interactions) is more difficult to decide. The entree
of large ligands might be sterically hindered: the evidence is somewhat con-
flicting. There is little reason though to believe that the combination of small
ligands is subject to any steric hindrance. This structure with one iron-protein
bond is the most satisfactory, and although there is no unequivocal experi-
mental evidence that the sixth coordination position is occupied by a water
molecule in the absence of an added ligand, theoretical considerations support
this as a very reasonable assumption.
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OCR for page 47
CREVICE CONFIGURATION GEORGE AND LYSTER 47
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Representative terms from entire chapter:
iron atom
