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Structure of isothiocyanate methemoglobin
J. Mol.
Biol.
(1981) ‘145, 815-824
Structure of Isothiocyanate Methemoglobin ZBIGNIEW
RICHARD
KORSXX
AND KEITH
MOFFAT
Section of Biochemistry Molecular and Cell Biology Clark Hall Cornell University Ithaca, N.Y. 14853, U.S.A. (Received 8 September 1980) The structure of the complex of the ambidentate ligand thiocyanate with horse methemoglobin was compared with that of acid methemoglobin and azide methemoglobin by X-ray difference Fourier techniques. Thiocyanate is co-ordinated to iron exclusively by its nitrogen atom, and thus forms the isothiocyanate complex. The ligand is bent as in the stereochemically very similar azide complex, and there is no evidence for strain in the ligand pocket of the globin, in contrast to earlier results on the cyanide complex. The higher spin of the isothiocyanate complex compared with azide is accompanied by only slight structural changes at the hemes, the most prominent of which is interpreted to be a slight lengthening of the iron-ligand nitrogen bond in the /3-heme.
1. Introduction Co-operative oxygen binding to hemoglobin is linked to quaternary structural exists as a ferrous, change (Monod et al., 1965; Perutz, 1970). Deoxyhemoglobin five-co-ordinate high-spin species in the T quaternary structure, and oxyhemoglobin is a ferrous, six-co-ordinate low-spin species in the R quaternary structure. The three-dimensional structure of oxyhemoglobin has not yet been determined, so that an understanding of the structural aspects of oxygen binding to hemoglobin can only be accomplished by extrapolation from results on other ligands. All six-coordinate hemoglobins, whether ferrous or ferric, high-spin or low-spin, normally exist in the R quaternary structure, though small but significant differences in tertiary structure have been found between methemoglobin (Ladner et al., 1977). carboxyhemoglobin (Heidner et al., 1976), cyanide MetHbt (Deatherage et al., 1976a), fluoride MetHb (Deatherage et al., 1976b), nitric oxide hemoglobin (Deatherage & Moffat, 1979) and azide MetHb (Deatherage et al., 1979). Perturbations in globin structure are larger for ligands such as CO and (more markedly) cyanide, which are strained from their normal linear, axial mode of binding to iron, and smaller or absent for bent ligands such as NO and azide (and by inference, 0,). These stereochemical differences are reflected in the kinetics of ligand t Abbreviation
used: MetHb, methemoglobin. 815
W2-2836/81/04081~10
002.00/O
0 1981 Academic Press Inc. (London) Ltd.
816
Z. R. KORSZUS
AND
K. MOFFAT
binding to ferrous (Moffat et al., 1979) and ferric (Moffat & Korszun, 1980) hemoglobin. Although heme stereochemistry depends on the spin state of the iron (Hoard, 1975), the structural differences between high-spin ligands such as fluoride and low-spin ligands such as azide are not pronounced; the stereochemical OI electronic basis for the control of spin state in hemoglobin is under active debate (see, for example, Perutz, 1979). Thiocyanate is a linear triatomic species similar to azide in bulk, but which forms higher spin complexes with hemoglobin than azide (Perutz et al., 1978; Messana et al.. 1978). Furthermore, it is an ambidentate ligand (Burmeister, 1966,1968), which exhibits stereochemical isomerism and may bind to iron via either its nitrogen or its sulfur atom. In model compounds, the mode of co-ordination of thiocyanate t,o metals depends on the metal, on the electronic nature ofthe other metal ligands. and on stereochemica,l interactions of the thiocyanate with these ligands and its environment (Burmeister, 1966,1968). It is therefore of interest to examine its complex with hemoglobin, seeking an explanation for its mode of co-ordination and for its spin state.
2. Experimental Procedures Horse MetHb prepared according to the procedure of Gibson et al. (1969) was crystallized by the scheme of Perutz (1968). Enough sodium thiocyanate was added to the supernatant above the crystals to bring it to 120 mM in thiocyanate. Under these conditions the crystals were saturated in thiocyanate, as evidenced by visible absorption spectroscopy. X-ray data were collected photographically to 26 a resolution using Supper precession cameras and CUKI+ radiation. Symmetry R-factors were calculated to be 5%“/0 on intensity. The data were scaled plane by plane to the native MetHb intensity data with a 10.25’$~~scaling R-factor: 6730 unique reflections were measured to be above 20 and were used throughout, Difference Fourier maps were calculated using observed structure amplitude differences and the refined MetHb phases (Ladner et al., 1977). Thiocyanate minus azide MetHb double difference Fouriers were calculated using coefficients observed as significant in both derivatives only; the azide MetHb data were those obtained by Deatherage et aZ. (1979). Derivative Fourier maps with coefficients 2IF(derivative)lIP(MetHb)[ were calculated for both the thiocyanate and azide derivatives, to estimate ligand positions. Again, only coefficients observed as significant in both the derivative and MetHb were used.
3. Results (a) Heme stereochemistry
Figure 1 shows sections of the thiocyanate minus MetHb difference map parallel to the hemes, sectioned at 1 A intervals along the normal to the mean heme planes from - 2 A (proximal) to + 4 A (distal), centered on the iron atoms. This Figure may be compared with Figure 2 of Moffat et al. (1979). which presents similar sections through the /3-heme for the NO, azide and cyanide derivatives. Large positive features representing the thiocyanate ligand are seen on the distal side of both hemes, lying over pyrrole II (using the pyrrole numbering convention of Heidner et al. (1976)). They are of equal magnitude, consistent with full occupancy. Large negative features surround them in both hemes, which may be diffraction ripples. In the s-heme, the propionic acid on pyrrole IV shifts from distal to proximal (as it does
3 .....
..:-.
%. K.
818
KORSZI’S
AND
K.
>lOFFA’I’
in the cyanide, azide and NO derivatives). A similar motion may occur in the j% heme, but this is much less pronounced than in the a-heme. or in the /3-heme of the NO and azide derivatives. Other features on the hemes are less readily interpreted. On the proximal side of the I-heme, a small positive feature is located between pyrroles II and III; on the proximal side of the fl-heme, a similar feature lies over pyrrole I, and may be associated with motion of that pyrrole to the proximal side. The prominent negative feature proximal to and in the center of the /3-heme plane is probably real, not a diffraction ripple (it does not appear in the rx-heme), and ma> represent a motion of the iron to distal. To determine the ligand stereochemistry, derivative Fouriers were calculated for both the thiocyanate and azide derivatives. Figure 2 shows a section perpendicular to the heme, passing through the iron atom and the peak ligand density, for the ‘r and fl-hemes of both derivatives. For thiocyanate. the peak ligand density due to L3 (the ligand atom most distant from the iron) lies 4.1 A from the center of the mean heme plane. Since the S-N distance in the linear thiocyanate is 2.78 A (Beard & Dailey. 1950), this is consistent with L, (the ligand atom bonded to the iron) lying on the
(a)
3
0
FIG. 2. Se&ions perpendicular to the a and /3-hemes of the thiocyanate MetHb derivative Fourier ((a) and (b)) and the azide MetHb derivative Fourier ((c) and (d)). (a) and (c) x-Heme: (b) and (d) fi-heme. The sections pass through the Fe-L,-L,-L3 plane; the outline of the ligand is shown.
STRUCTURE
OF
ISOTHIOCYANATE
MetHb
819
heme normal 2.1 A f 0.1 A from the iron, such that the angle Fe-L,--L, is 120” f 10”. In both hemes the thiocyanate lies nearly above pyrrole II, with the angle += 195+ 10” (Heidner et al., 1976). Non-bonded contacts between the ligand atoms and the heme plane are 2.8 A from L, to the pyrrole nitrogens, 2.7 A from L, and 3.4 A from L, to the mean heme plane. It is evident that steric effects which may influence ligation occur between the heme and positions L, and L, of the ligand; L, is at a comfortable van der Waals’ distance from the heme and does not sterically hinder ligation. The ligand geometry found in the thiocyanate derivative is closely similar to that of azide (Deatherage et al., 1979); the major difference is that little ligand density is found in azide MetHb at the L, position, which suggests some ligand disorder. In order to determine whether the thiocyanate is co-ordinated through its nitrogen or its sulfur atom (or a mixture of the two), a thiocyanate minus azide double difference Fourier was calculated; a positive feature was expected at the sulfur position. In both hemes a large positive feature was located 41 A from the center of the mean heme plane (the assumed location of L3 in thiocyanate). In the oc-heme,the double difference map was otherwise featureless from 2 A proximal to the heme to 2 A distal to the heme, including the assumed L, location. This result emphasizes the close similarity in the ligand stereochemistry of thiocyanate and azide. In the fi-heme, a negative feature extended along the heme normal from the iron position for 2 A on the distal side. These results suggested that thiocyanate was N-bonded in both hemes. Further support was obtained from examination of the electron density at the L, position in the azide difference Fourier and the L, position in the thiocyanate difference Fourier: the ratio of peak heights was 0.42 + 0.03, very close to the ratio of the atomic numbers of nitrogen to sulfur, O-44. Also as expected. the ratio of peak heights at the L, positions of both derivatives was close to 1.0. Taken together, these results show that in both hemes the thiocyanate ligand is coordinated exclusively via its nitrogen. That is, the isothiocyanate derivative of MetHb is formed.
al
SCN- Meliib
Fig. 3. Schematic representation of ~$3~ hemoglobin dimer, viewed down a pseudo-dyad axis. Arrows indicate motion of helices, on passing from acid MetHb to thiocyanate MetHb. Curved arrows indicate rotation of a helix as viewed from the amino terminus of the helix. All motions are small (see the text).
820
%. K.
KORSZUN
AND
K.
MOPl”AT
(b) Globin stereochemistry Figure 3 is a schematic representation of an alpZ dimer of IMetHb, viewed down a pseudo 2-fold axis, in which the arrows represent the motions found on going from MetHb to isothiocyanate MetHb. Overall, the tertiary structural differences between MetHb and thiocyanate MetHb are very small, even less than those found in azide MetHb (Deatherage et al., 1979); we estimate that no globin structural difference exceeds 0.2 A. In addition to the motions shown in Figure 3, the sidechain of LeuBlO(29)a is slightly displaced by non-bonded interactions. the (’ terminus of both chains becomes more disordered, and a very slight rearrangement of hydrophobic side-chains occurs. In contrast to the cyanide, azide, CO and NO derivatives, no motion of the distal histidine away from the ligand is found in isothiocyanate MetH b.
4. Discussion In considering the stereochemistry of thiocyanate binding to MetHb, two related results have to be accounted for: co-ordination via nitrogen rather than sulfur, and bent rather than linear co-ordination. In the structure of five-co-ordinate isothiocyanato FeTPP (unpublished results of A. Bloom & J. L. Hoard, quoted by Hoard (1975)) the ligand is N-bonded with an Fe-N bond length of 1.96 A and an Fe-N-C bond angle of nearly 180”. Unfortunately, no related six-co-ordinate structure has been determined. However, the structures of the corresponding five and six-co-ordinate azide FeTPP and pyridine azido FeTpp are known (unpublished results of K. Adams, IJ. G. Rasmussen & W. R. Scheidt, quoted by Hoard (1975)). In both, the Fe-N-N bond angle is about 125”, with closely similar Fe-N bond lengths of 1.91 and 1.93 A in the five and six-co-ordinate species. Addition of a sixth ligand trans to the azide therefore has relatively little effect on its mode of co-ordination, and the stereochemistry of azide binding to MetHb (Deatherage et al., 1979) is consistent with that observed in the six-co-ordinate model compound. If addition of a sixth ligand such as imidazole or pyridine trans to isothiocyanate also has little effect on its mode of co-ordination, then the six-co-ordinate isothiocyanate complex in model compounds will be linear, contrary to our observations on isothiocyanate MetHb. Determination of the crystal structures of suitable six-co-ordinate model complexes is clearly desirable. Structural and infrared absorption studies (reviewed by Burmeister, 1966,1968) also suggest that isothiocyanates are linear complexes. Ligation via nitrogen (preferred for first row transition metals such as iron) leads to metal-N-C bond angles near 180”, but via sulfur (preferred for late second and third row transition metals) leads to strongly bent ligation with bond angles near 115” (Lewis ef al.. 1961). DiSipio et al. (1966) and Hollebone (1971) have shown by molecular orbital calculations that the highest occupied molecular orbital of’ thiocyanate is 3dn centered on the sulfur atom, which would allow bent ligation via sulfur. An antibonding 4~0 orbital primarily residing on the nitrogen atom is thought to be available to strongly polarizing metal acceptors for linear ligation. The first unoccupied molecular orbital is 4pn centered on the nitrogen atom; promotion of
STRUCTURE OF ISOTHIOCYANATE MetHb
821
electrons to this orbital could be a way of achieving bent ligation via nitrogen. Indeed, Beard & Dailey (1950) have demonstrated by rotational spectroscopy that in isothiocyanic acid the H-N-C moiety is bent, with a bond angle of about 140”. However, co-ordination ofthiocyanate to metals is strongly influenced by steric effects as well as by electronic effects (Basolo et al., 1964). For example, the structure of dimethyldiisothiocyanato(terpyridy1) tin (IV) determined by Naik & Scheidt (1973) showed that one of the isothiocyanate moieties formed a linear complex with tin (Sn-N-C bond angle of 177”), and the other formed a bent complex (Sn-N-C bond angle of 155”). They proposed that the bending was due to nonbonded interactions with a terpyridyl ring on a neighboring molecule in the crystal. It is not clear whether the bent isothiocyanate is bonding via a promoted 4pr orbital, or by a less effective overlap via the 4~0 orbital. More extreme steric hindrance may lead to interconversion of thiocyanate and isothiocyanate complexes (Burmeister, 1966). In MetHb, the ligand pocket is so constructed that linear, diatomic ligands such as cyanide (Deatherage et al., 1976a) and CO (Heidner et al., 1976) encounter appreciable steric hindrance and are displaced off the heme axis; by contrast, bent diatomic and triatomic ligands such as NO (Deatherage $ Moffat, 1979) and azide (Deatherage et al., 1979) encounter little or no steric hindrance. However, in contrast to the results on cyanide MetHb (Deatherage et aZ., 1976a), the ligand pockets in isothiocyanate MetHb show almost no evidence of strain relative to MetHb. In particular, there is no motion of the distal histidines, or of the E-helices. If the isothiocyanate is constrained to its bent configuration off the heme axis by sterie effects, then the stress associated with this strain must be extremely low. That is, it must require much less energy to bend the Fe-N-C bond than to displace the distal histidine or the E-helix of the globin. There thus appear to be two possible explanations for our results. If externally unconstrained six-co-ordinate iron isothiocyanates are linear, then the Fe-N-C bond must be very readily bent. This contrasts with cyanide and CO complexes, where theory suggests (Hoffmann et al., 1977) that the Fe-C--N (or 0) bond is relatively stiff, in accord with observation (Deatherage et al., 1976a, and references therein). Alternatively, if such isothiocyanates are bent, then the theoretical studies of DiSipio et al. (1966) and Hollebone (1971), and the experimental results on other model compounds which suggest linear co-ordination, may not be relevant to the present structure. Since oxygen binding to deoxyhemoglobin is accompanied by a change in spin from high-spin to low-spin, considerable attention has been paid to spin equilibria in hemoglobin, and the way in which they are coupled to heme stereochemistry and to the tertiary and quaternary structure of the globin. For many ligands, the spin equilibria are determined not merely by the nature of the ligands, but also by the quaternary structure and by temperature. Addition of the allosteric effector inositol hexaphosphate converts all liganded carp hemoglobins, and most human hemoglobins, from the R to the T state. For those derivatives of mixed spin in the R quaternary structure which are in a thermal spin equilibrium, this conversion is accompanied by an increase in spin, as judged by magnetic susceptibility and optical measurements (Perutz et al., 1978; Messana et al., 1978). For example, the
822
Z. R.
KORBZUN
AND
K.
MOFFA’I
high-spin fraction of carp azide MetHb increases from 099 to 0.50, and that of carp thiocyanate MetHb from 0.36 to 0.64, on addition of inositol hexaphosphate (Messana et al., 1978). These results show that either quaternary structure can accommodate either spin state. Magnetic susceptibility (George et al., 1964) and resonance Raman (Remba, 1980) studies on horse and human thiocyanate MetHb suggest that the high-spin fraction in this derivative is at least 0.60, while in the azide derivatives it is much less, 0 to O-05. The stereochemical manifestation of lowering the spin state has been noted by Hoard (1975) and Perutz et al. (1978) to be a shortening of the iron-pyrrole nitrogen bond lengths. If no radial contraction of the heme occurs? then lowering the spin must also produce a motion of the iron towards the mean plane of the pyrrole nitrogens. However, little or no change in position of the iron was observed (Deatherage et al., 19763,1979) on comparing the structures of acid MetHb (mixed, largely high-spin) with fluoride MetHb (high-spin) and azide MetHb (low-spin). Likewise, little or no change in iron position is seen here in comparing isothiocyanate MetHb (mixed, largely high-spin) with azide MetHb, despite the significant difference in spin state. This implies that iron motion is not a necessary consequence of change in spin state. Recently, Scheidt and collaborators (Scheidt et al., 1979) have found that in a six-co-ordinate high-spin ferric model compound, the iron is exactly centered in the plane of the pyrrole nitrogens (as it is in six-co-ordinate lowspin ferric and ferrous compounds), and radial expansion of the heme has occurred. That is, lowering the spin state and shortening the iron-pyrrole nitrogen bonds is accompanied in this example not by motion of the iron, but by radial contraction of the heme. They propose (Scheidt et al., 1979) that a similar structural change may occur on changing the spin state of hemoglobin. In acid MetHb, the iron atom is located 0.21 A out of the mean plane in the /3-heme, but only 0.07 A in the %-heme. Marker bands in resonance Raman spectra have also been identified with heme expansion and contraction, and correlated with spin state (Spaulding et al., 1975; Spiro et al.. 1979: Remba, 1980). At very least, these results suggest that there is not a unique correlation between the position of the iron and its spin state; the globin may exert stress on the heme, and the heme may expand or contract radially, to varying extents in the 2 and /I-hemes and in different derivatives. In thiocyanate MetHb, qualitative differences between the ‘CX and /3-hemes exist. The /3-heme shows a negative feature which could be interpreted as motion of the iron to distal, relative to acid MetHb. This explanation is tenuous, however, since no corresponding positive feature is seen (although it, could be masked by the negative diffraction ripple surrounding the ligand), nor is there any displacement of the proximal histidine. As noted above, the double difference Fourier shows that the n-heme is closely similar to that ofazide MetHb, though in the,&heme there are some differences near the iron-ligand nitrogen bond. In human azide MetHb, the highspin fraction is believed to be 0.0 in the r-heme, and 0.10 in the /3-heme (Perutz et al., 1978). The overall high-spin fraction in thiocyanate MetHb is 0.60, but it is not known how this is distributed between the x and /3-hemes. The distribution most consistent with our crystallographic results places only a small high-spin fraction in the a-heme, say O-20, and a large fraction in the /I-heme, 1.00. This distribution
STRUCTURE
OF
ISOTHIOCYANATE
MetHb
823
would produce a lesser structural difference on comparing the a-hemes of azide and thiocyanate MetHb, and a larger structural difference in the /3-hemes. Although our data are not sufficiently precise to specify with confidence the structural differences at the hemes, (and in particular, to identify radial expansion or contraction) the most likely possibility is that the iron-ligand nitrogen bond (and possibly the ironproximal histidine bond) is longer in thiocyanate MetHb than in azide MetHb, especially in the /3-heme. The hemes are otherwise closely similar. Messana et al. (19’78) have suggested that in R state thiocyanate MetHb, the ligand is co-ordinated via the nitrogen; our results confirm this. They further suggest that on conversion to the T state, the ligand will adopt the other possible isomer and become co-ordinated via the sulfur, thus producing the increase in spin and other spectral changes they observe. It is equally possible that on conversion from the R to the T state, the ligand will remain co-ordinated via the nitrogen, but with an increase in the iron-ligand nitrogen bond length. The stereochemistry of the ligand pocket is quite different in the R and T states (Baldwin & Chothia, 1979), and it is possible that the strain imposed on the bulky thiocyanate ligand in the more constricted ligand pocket in the T state will lead to an increase in this bond length, perhaps via pivoting of the ligand about the carbon atom L,. Thanks are due to Dr D. M. Szebenyi for assistance in computing and to J. Wenban for his excellent artwork and photography. Supported by National Institutes of Health grant HL18309 (to K. M., who also is a National Institutes of Health Research Career Development Awardee)
REFERENCES Baldwin, J. M. & Chothia, C. (1979). J. Mol. Biol. 129, 175220. Basolo, F., Baddley, W. H. t Burmeister, J. L. (1964). Znorg. Chem. 3, 1202-1203. Beard, C. I. & Dailey, B. P. (1950). J. Chem. Phys. 18, 1437-1441. Burmeister, J. L. (1966). Co-ord. Chem. Revs, 1, 2OS221. Burmeister, J. L. (1968). Co-ord. Chem. Revs, 3, 225-245. Deatherage, J. F. & Moffat, K. (1979). J. Mol. Biol. 134, 401-417. Deatherage, J. F., Loe, R. S., Anderson, C. M. & Moffat, K. (1976a). J. Mol. Biol. 104, 687-706. Deatherage, J. F., Loe, R. S. & Moffat, K. (1976b). J. Mol. Biol. 104, 723-728. Deatherage, J. F., Obendorf, S. K. & Moffat, K. (1979). J. Mol. Biol. 134, 419429. DiSipio, L., Oleavi, L. & Michelis, G. (1966). Co-ord. Chem. Revs, 1, 7-12. George, P., Beetlestone, J. & Griffith, J. S. (1964). Revs, Mod. Phys. 36, 441-458. Gibson, Q. H. Parkhurst, L. J. & Geraci, G. (1969). J. Bill. Chem. 244, 4668-4676. Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976). J. Mol. Biol. 104, 707-722. Hoard, J. L. (1975). In Porphyrins and Metallqorphyrins (Smith; K. M., ed.), pp. 317-380, Elsevier, New York. Hoffman, R., Chen, M. M.-L. & Thorn, D. L. (1977). Znorg. Chem. 16, 503-511. Hollebone, B. R. (1971). J. Chem. Sot. ser. A, 19, 3021-3027. Ladner, R. C., Heidner, E. J & Perutz, M. F. (1977). J. Mol. Biol. 114, 385-414. Lewis, J., Nyholm, R. S. & Smith, P. W. (1961). J. Chem. Sot. 459Cb4599. Messana, C., Cerdonio, M., Shenkin, P., Noble, R. W., Fermi, G., Perutz, R. N. & Perutz, M. F. (1978). Biochemistry, 17, 3652-3662. Moffat, K. & Korszun, Z. R. (1980). In Interaction Between Iron and Proteins in Oxygen and Electron Transport (Ho et al., eds), Elsevier, New York, in the press.
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R.
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MOFFAT
Moffat, K., Deatherage, J. F. & Seybert, D. W. (1979). ScierLce, 266, 1035-l 042. Monod, J., Wyman, J. & Changeux, J.-P. (1965). J. Mol. Biol. 12, 8&l 18. Naik, D. V. & Scheidt, W. R. (1973). Znorg. Chm. 12, 272276. Perutz, M. F. (1968). J. Cryst. Growth, 2, 54-56. Perutz, M. F. (1970). Nature (London), 228, 726-734. Perutz, M. F. (1979). Annu. Rev. Biochem. 48, 327-386. Perutz, M. F., Sanders, J. K. M., Chenery, D. H.. Noble, R. W.. Pennelly, R. R.. Fung, L. W.-M., Ho, C., Giannini, I., Piirschke, D. & Winkler, H. (1978). Biochemistry, 17. 3640-3652. Remba, R. (1980). Ph.D. thesis, Cornell University. Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979). Biochemistry, 18, 354G3.552. Spaulding, L. D., Chang, C. C., Yu, N. T. & Felton, R. H. (1975). J. Amer. Chem. #ooc.97, 2517-2525. Spiro, T. G., Stong, J. D. & Stein, P. (1979). J. Amer. Chem. Sot. 101, 2648-2655.
Note added in proof: Scheidt and co-workers (Y. J. Lee, K. Hatano & W. R. Scheidt) have recently determined the crystal structure of pyridine isothiocyanato Fe (tetraphenylporphine), which may serve as a model compound for the heme stereochemistry ‘in isothiocyanate MetHb. They find that the ligand is N-bonded: the N-C-S moiety is linear (bond angle 177.0”), but the Fe-NC bond is appreciably bent, with a bond angle of 155.6”. Thus in both the model compound and hemoglobin, the ligand is bent. There are some weak steric constraints in the model, as the ligand sulfur is located 3.77 A from a solvent molecule: the greater bending in hemoglobin may arise from more severe steric constraints.
Biol.
(1981) ‘145, 815-824
Structure of Isothiocyanate Methemoglobin ZBIGNIEW
RICHARD
KORSXX
AND KEITH
MOFFAT
Section of Biochemistry Molecular and Cell Biology Clark Hall Cornell University Ithaca, N.Y. 14853, U.S.A. (Received 8 September 1980) The structure of the complex of the ambidentate ligand thiocyanate with horse methemoglobin was compared with that of acid methemoglobin and azide methemoglobin by X-ray difference Fourier techniques. Thiocyanate is co-ordinated to iron exclusively by its nitrogen atom, and thus forms the isothiocyanate complex. The ligand is bent as in the stereochemically very similar azide complex, and there is no evidence for strain in the ligand pocket of the globin, in contrast to earlier results on the cyanide complex. The higher spin of the isothiocyanate complex compared with azide is accompanied by only slight structural changes at the hemes, the most prominent of which is interpreted to be a slight lengthening of the iron-ligand nitrogen bond in the /3-heme.
1. Introduction Co-operative oxygen binding to hemoglobin is linked to quaternary structural exists as a ferrous, change (Monod et al., 1965; Perutz, 1970). Deoxyhemoglobin five-co-ordinate high-spin species in the T quaternary structure, and oxyhemoglobin is a ferrous, six-co-ordinate low-spin species in the R quaternary structure. The three-dimensional structure of oxyhemoglobin has not yet been determined, so that an understanding of the structural aspects of oxygen binding to hemoglobin can only be accomplished by extrapolation from results on other ligands. All six-coordinate hemoglobins, whether ferrous or ferric, high-spin or low-spin, normally exist in the R quaternary structure, though small but significant differences in tertiary structure have been found between methemoglobin (Ladner et al., 1977). carboxyhemoglobin (Heidner et al., 1976), cyanide MetHbt (Deatherage et al., 1976a), fluoride MetHb (Deatherage et al., 1976b), nitric oxide hemoglobin (Deatherage & Moffat, 1979) and azide MetHb (Deatherage et al., 1979). Perturbations in globin structure are larger for ligands such as CO and (more markedly) cyanide, which are strained from their normal linear, axial mode of binding to iron, and smaller or absent for bent ligands such as NO and azide (and by inference, 0,). These stereochemical differences are reflected in the kinetics of ligand t Abbreviation
used: MetHb, methemoglobin. 815
W2-2836/81/04081~10
002.00/O
0 1981 Academic Press Inc. (London) Ltd.
816
Z. R. KORSZUS
AND
K. MOFFAT
binding to ferrous (Moffat et al., 1979) and ferric (Moffat & Korszun, 1980) hemoglobin. Although heme stereochemistry depends on the spin state of the iron (Hoard, 1975), the structural differences between high-spin ligands such as fluoride and low-spin ligands such as azide are not pronounced; the stereochemical OI electronic basis for the control of spin state in hemoglobin is under active debate (see, for example, Perutz, 1979). Thiocyanate is a linear triatomic species similar to azide in bulk, but which forms higher spin complexes with hemoglobin than azide (Perutz et al., 1978; Messana et al.. 1978). Furthermore, it is an ambidentate ligand (Burmeister, 1966,1968), which exhibits stereochemical isomerism and may bind to iron via either its nitrogen or its sulfur atom. In model compounds, the mode of co-ordination of thiocyanate t,o metals depends on the metal, on the electronic nature ofthe other metal ligands. and on stereochemica,l interactions of the thiocyanate with these ligands and its environment (Burmeister, 1966,1968). It is therefore of interest to examine its complex with hemoglobin, seeking an explanation for its mode of co-ordination and for its spin state.
2. Experimental Procedures Horse MetHb prepared according to the procedure of Gibson et al. (1969) was crystallized by the scheme of Perutz (1968). Enough sodium thiocyanate was added to the supernatant above the crystals to bring it to 120 mM in thiocyanate. Under these conditions the crystals were saturated in thiocyanate, as evidenced by visible absorption spectroscopy. X-ray data were collected photographically to 26 a resolution using Supper precession cameras and CUKI+ radiation. Symmetry R-factors were calculated to be 5%“/0 on intensity. The data were scaled plane by plane to the native MetHb intensity data with a 10.25’$~~scaling R-factor: 6730 unique reflections were measured to be above 20 and were used throughout, Difference Fourier maps were calculated using observed structure amplitude differences and the refined MetHb phases (Ladner et al., 1977). Thiocyanate minus azide MetHb double difference Fouriers were calculated using coefficients observed as significant in both derivatives only; the azide MetHb data were those obtained by Deatherage et aZ. (1979). Derivative Fourier maps with coefficients 2IF(derivative)lIP(MetHb)[ were calculated for both the thiocyanate and azide derivatives, to estimate ligand positions. Again, only coefficients observed as significant in both the derivative and MetHb were used.
3. Results (a) Heme stereochemistry
Figure 1 shows sections of the thiocyanate minus MetHb difference map parallel to the hemes, sectioned at 1 A intervals along the normal to the mean heme planes from - 2 A (proximal) to + 4 A (distal), centered on the iron atoms. This Figure may be compared with Figure 2 of Moffat et al. (1979). which presents similar sections through the /3-heme for the NO, azide and cyanide derivatives. Large positive features representing the thiocyanate ligand are seen on the distal side of both hemes, lying over pyrrole II (using the pyrrole numbering convention of Heidner et al. (1976)). They are of equal magnitude, consistent with full occupancy. Large negative features surround them in both hemes, which may be diffraction ripples. In the s-heme, the propionic acid on pyrrole IV shifts from distal to proximal (as it does
3 .....
..:-.
%. K.
818
KORSZI’S
AND
K.
>lOFFA’I’
in the cyanide, azide and NO derivatives). A similar motion may occur in the j% heme, but this is much less pronounced than in the a-heme. or in the /3-heme of the NO and azide derivatives. Other features on the hemes are less readily interpreted. On the proximal side of the I-heme, a small positive feature is located between pyrroles II and III; on the proximal side of the fl-heme, a similar feature lies over pyrrole I, and may be associated with motion of that pyrrole to the proximal side. The prominent negative feature proximal to and in the center of the /3-heme plane is probably real, not a diffraction ripple (it does not appear in the rx-heme), and ma> represent a motion of the iron to distal. To determine the ligand stereochemistry, derivative Fouriers were calculated for both the thiocyanate and azide derivatives. Figure 2 shows a section perpendicular to the heme, passing through the iron atom and the peak ligand density, for the ‘r and fl-hemes of both derivatives. For thiocyanate. the peak ligand density due to L3 (the ligand atom most distant from the iron) lies 4.1 A from the center of the mean heme plane. Since the S-N distance in the linear thiocyanate is 2.78 A (Beard & Dailey. 1950), this is consistent with L, (the ligand atom bonded to the iron) lying on the
(a)
3
0
FIG. 2. Se&ions perpendicular to the a and /3-hemes of the thiocyanate MetHb derivative Fourier ((a) and (b)) and the azide MetHb derivative Fourier ((c) and (d)). (a) and (c) x-Heme: (b) and (d) fi-heme. The sections pass through the Fe-L,-L,-L3 plane; the outline of the ligand is shown.
STRUCTURE
OF
ISOTHIOCYANATE
MetHb
819
heme normal 2.1 A f 0.1 A from the iron, such that the angle Fe-L,--L, is 120” f 10”. In both hemes the thiocyanate lies nearly above pyrrole II, with the angle += 195+ 10” (Heidner et al., 1976). Non-bonded contacts between the ligand atoms and the heme plane are 2.8 A from L, to the pyrrole nitrogens, 2.7 A from L, and 3.4 A from L, to the mean heme plane. It is evident that steric effects which may influence ligation occur between the heme and positions L, and L, of the ligand; L, is at a comfortable van der Waals’ distance from the heme and does not sterically hinder ligation. The ligand geometry found in the thiocyanate derivative is closely similar to that of azide (Deatherage et al., 1979); the major difference is that little ligand density is found in azide MetHb at the L, position, which suggests some ligand disorder. In order to determine whether the thiocyanate is co-ordinated through its nitrogen or its sulfur atom (or a mixture of the two), a thiocyanate minus azide double difference Fourier was calculated; a positive feature was expected at the sulfur position. In both hemes a large positive feature was located 41 A from the center of the mean heme plane (the assumed location of L3 in thiocyanate). In the oc-heme,the double difference map was otherwise featureless from 2 A proximal to the heme to 2 A distal to the heme, including the assumed L, location. This result emphasizes the close similarity in the ligand stereochemistry of thiocyanate and azide. In the fi-heme, a negative feature extended along the heme normal from the iron position for 2 A on the distal side. These results suggested that thiocyanate was N-bonded in both hemes. Further support was obtained from examination of the electron density at the L, position in the azide difference Fourier and the L, position in the thiocyanate difference Fourier: the ratio of peak heights was 0.42 + 0.03, very close to the ratio of the atomic numbers of nitrogen to sulfur, O-44. Also as expected. the ratio of peak heights at the L, positions of both derivatives was close to 1.0. Taken together, these results show that in both hemes the thiocyanate ligand is coordinated exclusively via its nitrogen. That is, the isothiocyanate derivative of MetHb is formed.
al
SCN- Meliib
Fig. 3. Schematic representation of ~$3~ hemoglobin dimer, viewed down a pseudo-dyad axis. Arrows indicate motion of helices, on passing from acid MetHb to thiocyanate MetHb. Curved arrows indicate rotation of a helix as viewed from the amino terminus of the helix. All motions are small (see the text).
820
%. K.
KORSZUN
AND
K.
MOPl”AT
(b) Globin stereochemistry Figure 3 is a schematic representation of an alpZ dimer of IMetHb, viewed down a pseudo 2-fold axis, in which the arrows represent the motions found on going from MetHb to isothiocyanate MetHb. Overall, the tertiary structural differences between MetHb and thiocyanate MetHb are very small, even less than those found in azide MetHb (Deatherage et al., 1979); we estimate that no globin structural difference exceeds 0.2 A. In addition to the motions shown in Figure 3, the sidechain of LeuBlO(29)a is slightly displaced by non-bonded interactions. the (’ terminus of both chains becomes more disordered, and a very slight rearrangement of hydrophobic side-chains occurs. In contrast to the cyanide, azide, CO and NO derivatives, no motion of the distal histidine away from the ligand is found in isothiocyanate MetH b.
4. Discussion In considering the stereochemistry of thiocyanate binding to MetHb, two related results have to be accounted for: co-ordination via nitrogen rather than sulfur, and bent rather than linear co-ordination. In the structure of five-co-ordinate isothiocyanato FeTPP (unpublished results of A. Bloom & J. L. Hoard, quoted by Hoard (1975)) the ligand is N-bonded with an Fe-N bond length of 1.96 A and an Fe-N-C bond angle of nearly 180”. Unfortunately, no related six-co-ordinate structure has been determined. However, the structures of the corresponding five and six-co-ordinate azide FeTPP and pyridine azido FeTpp are known (unpublished results of K. Adams, IJ. G. Rasmussen & W. R. Scheidt, quoted by Hoard (1975)). In both, the Fe-N-N bond angle is about 125”, with closely similar Fe-N bond lengths of 1.91 and 1.93 A in the five and six-co-ordinate species. Addition of a sixth ligand trans to the azide therefore has relatively little effect on its mode of co-ordination, and the stereochemistry of azide binding to MetHb (Deatherage et al., 1979) is consistent with that observed in the six-co-ordinate model compound. If addition of a sixth ligand such as imidazole or pyridine trans to isothiocyanate also has little effect on its mode of co-ordination, then the six-co-ordinate isothiocyanate complex in model compounds will be linear, contrary to our observations on isothiocyanate MetHb. Determination of the crystal structures of suitable six-co-ordinate model complexes is clearly desirable. Structural and infrared absorption studies (reviewed by Burmeister, 1966,1968) also suggest that isothiocyanates are linear complexes. Ligation via nitrogen (preferred for first row transition metals such as iron) leads to metal-N-C bond angles near 180”, but via sulfur (preferred for late second and third row transition metals) leads to strongly bent ligation with bond angles near 115” (Lewis ef al.. 1961). DiSipio et al. (1966) and Hollebone (1971) have shown by molecular orbital calculations that the highest occupied molecular orbital of’ thiocyanate is 3dn centered on the sulfur atom, which would allow bent ligation via sulfur. An antibonding 4~0 orbital primarily residing on the nitrogen atom is thought to be available to strongly polarizing metal acceptors for linear ligation. The first unoccupied molecular orbital is 4pn centered on the nitrogen atom; promotion of
STRUCTURE OF ISOTHIOCYANATE MetHb
821
electrons to this orbital could be a way of achieving bent ligation via nitrogen. Indeed, Beard & Dailey (1950) have demonstrated by rotational spectroscopy that in isothiocyanic acid the H-N-C moiety is bent, with a bond angle of about 140”. However, co-ordination ofthiocyanate to metals is strongly influenced by steric effects as well as by electronic effects (Basolo et al., 1964). For example, the structure of dimethyldiisothiocyanato(terpyridy1) tin (IV) determined by Naik & Scheidt (1973) showed that one of the isothiocyanate moieties formed a linear complex with tin (Sn-N-C bond angle of 177”), and the other formed a bent complex (Sn-N-C bond angle of 155”). They proposed that the bending was due to nonbonded interactions with a terpyridyl ring on a neighboring molecule in the crystal. It is not clear whether the bent isothiocyanate is bonding via a promoted 4pr orbital, or by a less effective overlap via the 4~0 orbital. More extreme steric hindrance may lead to interconversion of thiocyanate and isothiocyanate complexes (Burmeister, 1966). In MetHb, the ligand pocket is so constructed that linear, diatomic ligands such as cyanide (Deatherage et al., 1976a) and CO (Heidner et al., 1976) encounter appreciable steric hindrance and are displaced off the heme axis; by contrast, bent diatomic and triatomic ligands such as NO (Deatherage $ Moffat, 1979) and azide (Deatherage et al., 1979) encounter little or no steric hindrance. However, in contrast to the results on cyanide MetHb (Deatherage et aZ., 1976a), the ligand pockets in isothiocyanate MetHb show almost no evidence of strain relative to MetHb. In particular, there is no motion of the distal histidines, or of the E-helices. If the isothiocyanate is constrained to its bent configuration off the heme axis by sterie effects, then the stress associated with this strain must be extremely low. That is, it must require much less energy to bend the Fe-N-C bond than to displace the distal histidine or the E-helix of the globin. There thus appear to be two possible explanations for our results. If externally unconstrained six-co-ordinate iron isothiocyanates are linear, then the Fe-N-C bond must be very readily bent. This contrasts with cyanide and CO complexes, where theory suggests (Hoffmann et al., 1977) that the Fe-C--N (or 0) bond is relatively stiff, in accord with observation (Deatherage et al., 1976a, and references therein). Alternatively, if such isothiocyanates are bent, then the theoretical studies of DiSipio et al. (1966) and Hollebone (1971), and the experimental results on other model compounds which suggest linear co-ordination, may not be relevant to the present structure. Since oxygen binding to deoxyhemoglobin is accompanied by a change in spin from high-spin to low-spin, considerable attention has been paid to spin equilibria in hemoglobin, and the way in which they are coupled to heme stereochemistry and to the tertiary and quaternary structure of the globin. For many ligands, the spin equilibria are determined not merely by the nature of the ligands, but also by the quaternary structure and by temperature. Addition of the allosteric effector inositol hexaphosphate converts all liganded carp hemoglobins, and most human hemoglobins, from the R to the T state. For those derivatives of mixed spin in the R quaternary structure which are in a thermal spin equilibrium, this conversion is accompanied by an increase in spin, as judged by magnetic susceptibility and optical measurements (Perutz et al., 1978; Messana et al., 1978). For example, the
822
Z. R.
KORBZUN
AND
K.
MOFFA’I
high-spin fraction of carp azide MetHb increases from 099 to 0.50, and that of carp thiocyanate MetHb from 0.36 to 0.64, on addition of inositol hexaphosphate (Messana et al., 1978). These results show that either quaternary structure can accommodate either spin state. Magnetic susceptibility (George et al., 1964) and resonance Raman (Remba, 1980) studies on horse and human thiocyanate MetHb suggest that the high-spin fraction in this derivative is at least 0.60, while in the azide derivatives it is much less, 0 to O-05. The stereochemical manifestation of lowering the spin state has been noted by Hoard (1975) and Perutz et al. (1978) to be a shortening of the iron-pyrrole nitrogen bond lengths. If no radial contraction of the heme occurs? then lowering the spin must also produce a motion of the iron towards the mean plane of the pyrrole nitrogens. However, little or no change in position of the iron was observed (Deatherage et al., 19763,1979) on comparing the structures of acid MetHb (mixed, largely high-spin) with fluoride MetHb (high-spin) and azide MetHb (low-spin). Likewise, little or no change in iron position is seen here in comparing isothiocyanate MetHb (mixed, largely high-spin) with azide MetHb, despite the significant difference in spin state. This implies that iron motion is not a necessary consequence of change in spin state. Recently, Scheidt and collaborators (Scheidt et al., 1979) have found that in a six-co-ordinate high-spin ferric model compound, the iron is exactly centered in the plane of the pyrrole nitrogens (as it is in six-co-ordinate lowspin ferric and ferrous compounds), and radial expansion of the heme has occurred. That is, lowering the spin state and shortening the iron-pyrrole nitrogen bonds is accompanied in this example not by motion of the iron, but by radial contraction of the heme. They propose (Scheidt et al., 1979) that a similar structural change may occur on changing the spin state of hemoglobin. In acid MetHb, the iron atom is located 0.21 A out of the mean plane in the /3-heme, but only 0.07 A in the %-heme. Marker bands in resonance Raman spectra have also been identified with heme expansion and contraction, and correlated with spin state (Spaulding et al., 1975; Spiro et al.. 1979: Remba, 1980). At very least, these results suggest that there is not a unique correlation between the position of the iron and its spin state; the globin may exert stress on the heme, and the heme may expand or contract radially, to varying extents in the 2 and /I-hemes and in different derivatives. In thiocyanate MetHb, qualitative differences between the ‘CX and /3-hemes exist. The /3-heme shows a negative feature which could be interpreted as motion of the iron to distal, relative to acid MetHb. This explanation is tenuous, however, since no corresponding positive feature is seen (although it, could be masked by the negative diffraction ripple surrounding the ligand), nor is there any displacement of the proximal histidine. As noted above, the double difference Fourier shows that the n-heme is closely similar to that ofazide MetHb, though in the,&heme there are some differences near the iron-ligand nitrogen bond. In human azide MetHb, the highspin fraction is believed to be 0.0 in the r-heme, and 0.10 in the /3-heme (Perutz et al., 1978). The overall high-spin fraction in thiocyanate MetHb is 0.60, but it is not known how this is distributed between the x and /3-hemes. The distribution most consistent with our crystallographic results places only a small high-spin fraction in the a-heme, say O-20, and a large fraction in the /I-heme, 1.00. This distribution
STRUCTURE
OF
ISOTHIOCYANATE
MetHb
823
would produce a lesser structural difference on comparing the a-hemes of azide and thiocyanate MetHb, and a larger structural difference in the /3-hemes. Although our data are not sufficiently precise to specify with confidence the structural differences at the hemes, (and in particular, to identify radial expansion or contraction) the most likely possibility is that the iron-ligand nitrogen bond (and possibly the ironproximal histidine bond) is longer in thiocyanate MetHb than in azide MetHb, especially in the /3-heme. The hemes are otherwise closely similar. Messana et al. (19’78) have suggested that in R state thiocyanate MetHb, the ligand is co-ordinated via the nitrogen; our results confirm this. They further suggest that on conversion to the T state, the ligand will adopt the other possible isomer and become co-ordinated via the sulfur, thus producing the increase in spin and other spectral changes they observe. It is equally possible that on conversion from the R to the T state, the ligand will remain co-ordinated via the nitrogen, but with an increase in the iron-ligand nitrogen bond length. The stereochemistry of the ligand pocket is quite different in the R and T states (Baldwin & Chothia, 1979), and it is possible that the strain imposed on the bulky thiocyanate ligand in the more constricted ligand pocket in the T state will lead to an increase in this bond length, perhaps via pivoting of the ligand about the carbon atom L,. Thanks are due to Dr D. M. Szebenyi for assistance in computing and to J. Wenban for his excellent artwork and photography. Supported by National Institutes of Health grant HL18309 (to K. M., who also is a National Institutes of Health Research Career Development Awardee)
REFERENCES Baldwin, J. M. & Chothia, C. (1979). J. Mol. Biol. 129, 175220. Basolo, F., Baddley, W. H. t Burmeister, J. L. (1964). Znorg. Chem. 3, 1202-1203. Beard, C. I. & Dailey, B. P. (1950). J. Chem. Phys. 18, 1437-1441. Burmeister, J. L. (1966). Co-ord. Chem. Revs, 1, 2OS221. Burmeister, J. L. (1968). Co-ord. Chem. Revs, 3, 225-245. Deatherage, J. F. & Moffat, K. (1979). J. Mol. Biol. 134, 401-417. Deatherage, J. F., Loe, R. S., Anderson, C. M. & Moffat, K. (1976a). J. Mol. Biol. 104, 687-706. Deatherage, J. F., Loe, R. S. & Moffat, K. (1976b). J. Mol. Biol. 104, 723-728. Deatherage, J. F., Obendorf, S. K. & Moffat, K. (1979). J. Mol. Biol. 134, 419429. DiSipio, L., Oleavi, L. & Michelis, G. (1966). Co-ord. Chem. Revs, 1, 7-12. George, P., Beetlestone, J. & Griffith, J. S. (1964). Revs, Mod. Phys. 36, 441-458. Gibson, Q. H. Parkhurst, L. J. & Geraci, G. (1969). J. Bill. Chem. 244, 4668-4676. Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976). J. Mol. Biol. 104, 707-722. Hoard, J. L. (1975). In Porphyrins and Metallqorphyrins (Smith; K. M., ed.), pp. 317-380, Elsevier, New York. Hoffman, R., Chen, M. M.-L. & Thorn, D. L. (1977). Znorg. Chem. 16, 503-511. Hollebone, B. R. (1971). J. Chem. Sot. ser. A, 19, 3021-3027. Ladner, R. C., Heidner, E. J & Perutz, M. F. (1977). J. Mol. Biol. 114, 385-414. Lewis, J., Nyholm, R. S. & Smith, P. W. (1961). J. Chem. Sot. 459Cb4599. Messana, C., Cerdonio, M., Shenkin, P., Noble, R. W., Fermi, G., Perutz, R. N. & Perutz, M. F. (1978). Biochemistry, 17, 3652-3662. Moffat, K. & Korszun, Z. R. (1980). In Interaction Between Iron and Proteins in Oxygen and Electron Transport (Ho et al., eds), Elsevier, New York, in the press.
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2.
R.
KORSZUN
AND
K.
MOFFAT
Moffat, K., Deatherage, J. F. & Seybert, D. W. (1979). ScierLce, 266, 1035-l 042. Monod, J., Wyman, J. & Changeux, J.-P. (1965). J. Mol. Biol. 12, 8&l 18. Naik, D. V. & Scheidt, W. R. (1973). Znorg. Chm. 12, 272276. Perutz, M. F. (1968). J. Cryst. Growth, 2, 54-56. Perutz, M. F. (1970). Nature (London), 228, 726-734. Perutz, M. F. (1979). Annu. Rev. Biochem. 48, 327-386. Perutz, M. F., Sanders, J. K. M., Chenery, D. H.. Noble, R. W.. Pennelly, R. R.. Fung, L. W.-M., Ho, C., Giannini, I., Piirschke, D. & Winkler, H. (1978). Biochemistry, 17. 3640-3652. Remba, R. (1980). Ph.D. thesis, Cornell University. Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979). Biochemistry, 18, 354G3.552. Spaulding, L. D., Chang, C. C., Yu, N. T. & Felton, R. H. (1975). J. Amer. Chem. #ooc.97, 2517-2525. Spiro, T. G., Stong, J. D. & Stein, P. (1979). J. Amer. Chem. Sot. 101, 2648-2655.
Note added in proof: Scheidt and co-workers (Y. J. Lee, K. Hatano & W. R. Scheidt) have recently determined the crystal structure of pyridine isothiocyanato Fe (tetraphenylporphine), which may serve as a model compound for the heme stereochemistry ‘in isothiocyanate MetHb. They find that the ligand is N-bonded: the N-C-S moiety is linear (bond angle 177.0”), but the Fe-NC bond is appreciably bent, with a bond angle of 155.6”. Thus in both the model compound and hemoglobin, the ligand is bent. There are some weak steric constraints in the model, as the ligand sulfur is located 3.77 A from a solvent molecule: the greater bending in hemoglobin may arise from more severe steric constraints.