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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, tooalthough 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.

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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,

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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 FeHERO / 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

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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 possibilitynamely, 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

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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

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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

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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 ~

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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

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CREVICE CONFIGURATIONGEORGE 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

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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

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CREVICE CONFIGURATIONGEORGE 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-

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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~/KaK<.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.

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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

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~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. REFEREl\-i CES Hi. 1. 3. Altschul, A. M. and lIogness, T. R.: The hemoglobin-oxygen equilibrium, J. Biol. Chem. 129: 315, 1939. Scheler, W., and Jung, F`.: Hamoglobinstudien. IV. Uber die Temperaturabhang;ig- keit der Reaktionsgleichgewichte einiger Methamoglobinverbindungen, Biochem. Z., 325: 515, 1954. Havemann, R., and Hirse, H.: Dissoziation und Hydrolyse den Methamoglobin- rhodanids und Methamoglobincyanats, Z. Physikal. Chem., 204: 68, 1955. 4. George, P., and Hanania, G.: A spectrophotometric study of ionizations in met- haemoglobin. Biochem. J., 55: 236, 1953. 5. 6. 7. 8. Raiser, E. M., and Steinhardt, J.: Kinetic evidence on the mechanism of the acid denaturation of horse CO hemoglobin, J. Am. Chem. Soc., 73: 5568, 1951. S. Adler, A. D., and George, P.: Unpublished observations. 10. Pauling, L., and Coryell, C. D.: The magnetic properties and structure of hemo- globin' oxyhemoglobin, and carbonmonoxyhemoglobin, Proc. Nat. Acad. Sci., 22: 210, 1936. Orgel, L. E.: The en ects of crystal fields on the properties of transition-metal ions, J. Chem. Soc., 4756, 1952. 1~. Griffith, l. S.: On the stabilities of transition metal complexes, J. Inorg. and Nucl. Chem. 2: 1, 1956. 13. George, P.: Crystal field theory of complex formation in ions in the first transition Series-- Experimental thermodynamic data, Rec. Trav. Chim. des Pays-gas, 75: 131, 1956. 14. Haurowitz, F`.: Ober Globin und seine hamaffine Gruppe, Z. Physiol. Chem., 232: 146, 1935. 15. V. Zeynek, R.: Nowing Lekarske Poznan, 3S: 10, 1926. 16. Haurowitz, F`.: The Bond between Haem and Globin, in Haemoglobin: a sym- posium based on the Barcroft Memorial Conference, Butterworths, London, 1949. 17. Haurowitz, F.: Hemoglobin, anhydrohemoglobin, and oxyhemoglobin, J. Biol. Chem., 193: 443, 1951. 18. Keilin, D., and Hartree, E. F`.: Effect of drying upon the absorption spectra of haernoglobin and its derivatives, Nature, 170: 161, 1952. 1C'. Keilin, D., and Hartree, E. F.: Effect of low temperature on the absorption spectra of haemoproteins, Nature, 161: 254, 1949. 20. George, P., and Hanania, G.: The ionization of acidic metmyoglobin, Biochem. J., 65: 756, 1957. George, P., and O'Hagan, J.: Unpublished observations. Davenport, H. E.: Reductive cleavage of cytochrome c, Nature, 169: 75, 1952. Lewis, U. J.: Acid cleavage of heme proteins, J. Biol. Chem. 206: 109, 1954. -

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CREVICE CONFIGURATION GEORGE AND LYSTER 47 21. St. George, R. C. C., and Pauling, L.: The combining power of hemoglobin for alkyl isocyanides, and the nature of the heme-heme interactions in hemoglobin, Science, 114: 629, 1951. Keilin, D.: Position of haems in the haemoglobin molecule, Nature, 171: 922, 1953. Of myoglobin and the 22. 23. Kendrew, J. C., and Parrish, R. G.: Imidazole complexes position of the haem group, Nature, 175: 206, 1955. 24. George, P., and Irvine, D. H.: Electron-transfer reactions between metallic ions, co-ordination complexes, and haemoglobin, J. Chem. Soc., 587, 1954. 25. George, P.: The chemical nature of the second hydrogen peroxide compound formed by cytochrome c peroxidase and horseradish peroxidase, Biochem. J. 54: 267, 1953. . Lein, A., and Pauling, L.: The combining power of myoglobin for alkyl iso- cyanides and the structure of the myoglobin molecule, Proc. Natl. Acad. Sci., 42: 51, 1956. Bjerrum, J.: Metal ammine formation in aqueous solutions, P. Haase, Copenhagen, 1941. 28. Bruehlman, R. J., and Verhoek, F. H.: The basic strengths of amines as measured by the stabilities of their complexes with silver ions, J. Am. Chem. Soc., 70: 1401, 1948. 29. Eyfe, W. S.: Complex-ion formation. Part III. The entropies of reaction of the silver and hydrogen ions with some aliphatic amines, J. Chem. Soc. 1347, 1955. Roughton, F. J. W., Otis, A. B., and Lyster, R. L. J.: The determination of the individual equilibrium constants of the four intermediate reactions between oxygen and sheep haemoglobin, Proc. Roy. Soc., Series B. 144: 29, 1955. 31. George, P.: Discussions of the Faraday Society, 20: 291, The physical chemist.y of enzymes, 1955. 32. Roughton, F~. J. W., and George, P.: Discussions of the Faraday Society, 20: 288, The physical chemistry of enzymes, 1955. 33. Conant, J. B.: The oxidation of hemoglobin and other respiratory pigments, The Harvey Lectures, 28: 159, 1933. 34. Wyman, J.: The heat of oxygenation of hemoglobin, J. Biol. Chem., 127: 581, 1939. 3 5. Coryell, C. D., and Pauling, L.: A structural interpretation of the acidity of groups associated with the hemes of hemoglobin and hemoglobin derivatives, J. Biol. Chem., 132: 769, 1940. 36. Wyman, J., and Ingalls, E. N.: Interrelationships in the reactions of horse hemo- globin, J. Biol. Chem., 139: 877, 1941. Paul, NV., and Roughton, F. J. W.: The equilibrium between oxygen and sheep haemoglobin at very low percentage saturations, J. Physiol., 111: 23, 1951. Gibson, Q. H., and Roughton, F. J. W.: The determination of the velocity con- stants of the four successive reactions of carbon monoxide with sheep haemo- globin, Proc. Roy. Soc., Series B. 146: 206, 1957. Corwin, A. lI., and Reyes, Z.: Preparation and properties of imidazole ferro- and ferriprotoporphyrin complexes, J. Am. Chem. Soc., 78: 2437, 1956. 40. George, P., and Hanania, G.: Thermodynamic data for myoglobin, haemoglobin and cytochrome c reactions, and the position of the haem groups, Nature, 175: 1034, 1955. 41. Anson, M. L., and Mirsky, A. E.: The equilibrium between active native trypsin and inactive denatured trypsin, J. Gen. Physiol., 17: 393, 1934. George, P., and Hanania, G.: Effect of haem-linked and other ionizations on haemoprotein reactions, Nature, 174: 33, 1954 43. George, P., and Hanania, G.: The haem-linked ionizing group in myoglobin, Dis- 37.