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High resolution nuclear magnetic resonance spectra of hemoglobin
J. Mol. Biol. (1972) 70, 291-300
High Resolution Nuclear Magnetic Resonance Spectra of Hemoglobin I. The Cyanide Complexes of a and B Chains S. OC+AWA, R.
Murray
G. SHULMAN
AND
T.
YAMANE
Bell Laboratories Hill, N.J. 07974, U.S.A.
(Received 18 January
1972)
The high resolution nuclear magnetic resonance spectra of the isolated chains of human hemoglobin have been studied. Interest is mainly focussed on the paramagnetically shifted resonances in the cyanoferric state of these chains. Two resonance peaks of the heme methyls are easily observable, together with several single proton peaks in the downfield region. The strong peaks in the high-field region are due to two methyl resonances of some amino-acid residue near the periphery of the heme, shifted up-field by the pseudocontact interaction. Effects of pH and of a modification of the protein on those resonances are presented. The exchangeable NH protons of the ring and of the peptide of the proximal histidine have resonance positions very similar to those in cyanoferric myoglobin, although the methyl resonances of the heme group are at somewhat different positions.
1. Introduction Recently, the high resolution proton nuclear magnetic resonance spectra of heme proteins has been intensively studied (see Davis, Mock, Laman & Ho (1969) and Sheard, Yamane & Shulman (1970) for earlier references). An increase of resolution and interpretability ha8 been achieved because many of the proton resonances have been shifted by paramagnetic interactions away from the normal diamagnetic protein region. These paramagnetio shifts, coming from interactions with the unpaired electron of the heme iron, have been most strongly felt by protons near the iron. Hence, they have provided a means of monitoring this interesting region of the protein. With the great amount of information available from the n.m.r.t spectra of the paramagnetic low-spin ferric forms of myoglobin, particularly MbmCN (Sheard et al., 1970), it is now possible to discuss in detail the spectrum of hemoglobin cyanide Hb”ICN, and its constituent o?CN and /3mCN chains. Previous studies of HbmCN have shown that shifted resonances in the spectra can be resolved (Wiithrich, Shuhnan & Yamane, 1968) and are sensitive to modification8 in other regions of the protein. These modifications included mutant human hemoglobins (Davis et al., 1969), other mammalian hemoglobins (Yamane, Wiithrich, Shulman & Ogawa, 1970) and a preliminary account of the effects at one heme of oxygenating the neighbors (Shulman et al., 1969). In this series, we first discuss the n.m.r. spectra of the PCN and /3n1CN t Abbreviation
used: n.m.r., nuclear magnetic
resonance. 291
292
S. OGAWA,
R. G. SHULMAN
AND
T. YAMANE
chains. In the accompanying paper (Ogawa, Shulman, Fujiwara & Yamane, 1972), we show the effects of recombining these chains to form fully ligated hemoglobin. Finally, in the third paper (Ogawa t Shuhnan, 1972), we present the n.m.r. spectra of half ligated hemoglobins and their response to biochemical perturbations. These spectral changes are discussed in terms of a molecular mechanism for the co-operative oxygenation of hemoglobin.
2. Experimental Human hemoglobin A was prepared from freshly-drawn adult blood. The (Yand /? chains were separated by p-chloromercuribenzoate (PMB)t reaction and subsequent carboxymethyl cellulose chromatography (Bucci & Fronticelli, 1965). Regeneration of the SH group in the 01chain was carried out with O-015 a6-mercaptoethanol treatment on carboxymethyl cellulose for 30 minutes (Geracci, Parkhurst t Gibson, 1969).
FIQ. 1. n.m.r. Spectra of the isolated amCN and ,3mCNchaina in Da0 and HsO in 0.1 w-phosphate buffer at pH 643 and 20°C. t Abbreviation
used: PMB, p-&loromercuribenzoete.
n.m.r.
OF HEMOGLOBIN.
DSS
I.
CHAINS
293
.%N
r
II) = DSS
R%N
T
Fro. 2. (a) Temperature dependencea of the shifted resonance lines of the PCN oh&s st pH 6.6, 0.1 ax-phoephste buffer in D,O. The numbers in parentheses in the Figure are the ktensities in number of protons per heme. The letters b snd n correspond to relatively broad and narrow resonance lime as described in the text. (b) Temper&m-c dependences of the shifted resonance lines of the #mCN chains at pH 64, 0.1 x-phosphate buffer in D,O. The numbers in parentheses in the Figure are the intensities in number of protons per heme.
294
S. OGAWA,
R.
G. SHULMAN
AND
T. YAMANE
For the p chain, N-acetyl pennicillamine was used (Benesch, Benesch $ Enoki, 1968). Treatment of the p chains with mercaptoethanol on DEAE-cellulose at pH 8.0 was also tried, but the yield was better from the former method. The j3 SH chain obtained was further purified on carboxymethyl cellulose to eliminate oxidized material. Oxidation of the a: chain to the cyanoferric state was carried out at room temperature with 1-l to 1.2 molar equivalents of K,Fe(CN), in the presence of excess KCN in O-1 M-phosphate at pH ‘7.0. The j3 chain was oxidized at 36°C with l-2 equivalents of K,Fe(CN), and two of KCN, at pH 7-O. For n.m.r. measurements, the hemoglobin solutions were concentrated in a collodium bag to 5 to 10 x 10m3M in heme. The pH values in D,O buffer were expressed by the values read on the pH meter. A Varian high-resolution 220 MHz spectrometer was used for the n.m.r. experiments. The line positions were expressed by the separation in parts per million, from the position of 2,2-dimethyl-2-silapentane-5-sulfonate with negative sign for the downfield shifts. The spectra were obtained by accumulating the signal in a Fabritek 1062 computer for 0.5 to 6 hours, depending on the frequency range covered. The intensities were measured by taking ratios of the integrated areas under the peaks, after rough estimates were obtained by a comparison with the central diamagnetic proton resonances. The accuracy of the intensity measurements was limited by the ability to draw a base line. This introduced uncertainties up to fifteen per cent for overlapping lines, such as were found in the /3mCN spectra.
3. Results The n.m.r. spectra of the isolated a! and /3 chains in the cyanoferric state are shown in Figure 1. The magnetic field regions shown in Figure 1 are outside the ordinary n.m.r. absorption region in a diamagnetic system. The line positions of most of those resonances are temperature-dependent (see Fig. 2(a) and (b)), as expected in paramagnetically shifted systems (Shulman t Jaccarino, 1957). In the PCN chain, the two strong peaks at -22.7 and -16.6 p.p.m. at 20°C have intensities of 3 protons per heme and come from the ring methyls of the porphyrin. Four peaks of intensity one proton each are clustered around -14 p.p.m. They become completely separated into individual lines above 36°C. Two of the four stay narrow over the entire temperature range shown in Figure 2(a), while the other two become broader at the lower temperature. In the upfield region, there is a strong peak at 3.2 p.p,m. (see Fig. 3(a)), with an intensity of 8 to 10 protons per heme, which at the highest temperature begins to resolve. In the associated form of hemoglobin, this strong peak is resolved into two peaks under certain conditions, as discussed in the following paper (Ogawa et al., 1972). It appears likely that in this strong resonance two lines of intensity 3 protons and at least 2 single proton lines, which are resolved in the or& tetramer, are superimposed. Similar, but slightly different spectra are observed in the $WN chains. The two methyl lines in the downfield region are found at -21.7 and 15.7 p.p.m. (see Fig. l(c)). In the upfield region (see Fig. 3(c)), two single proton peaks are slightly upfield from the strong peak of ~6 protons at 4 p.p.m. From a comparison with the associated hemoglobin, where under certain circumstances it splits (see II), this strong peak consists most likely of two methyl resonances. The line widths of these resonances vary among the different peaks and are tempera-
n.m.r.
OF HEMOGLOBIN.
295
I. CHAINS
(a)
(b)
2w p.p.m.
B*CN
2L"4L"G wm
FIG. 3. Effeot of PMB on the n.m.r. speotra of the &WN and the @mCN ohains. After stoiohiometrio emounts of PMB were sdded to the cr’nCN chain solution, the pH and buffer were adjusted to pH 643 in 0.1 aa-phosphate buffer by gel filtration.
ture-dependent. Some semi-quantitative differences were as the following. At 4O”C, for example, the low-field peaks in the PCN spectra were about 40 Hz wide (full width at half height) and were approximately the mrne as the line widths of the analogous resonances in MIPCN. In contrast, the low-field resonances in the j3mCN spectra were about 20% wider. Most of those resonance lines are insensitive to pH in phosphate buffer in the physiologiaal pH region as shown in Figure 4. However, one line in the OPICN spectrum, with intensity of one proton, near - 13.6 p.p.m., titrateswith pH around pH 6.9
298
S. OGAWA,
R. G. SHULMAN
AND
T. YAMANE
There are some pH-sensitive peaks in the less-shifted field region near -11 p.p.m. but their origin is not understood. The presence of organic or inorganic phosphates in the cPCN solution did not affect the spectrum. In solutions of the /FCN chains, only the lowest field methyl resonance showed a pH dependence, which was small. In a stripped j? chain solution at pH 6.6, the lowest field methyl resonance was at a slightly lower field than at the same pH in phosphate buffer. Addition of 2,3diphosphoglycerate moved this methyl peak to a slightly higher field. These shifts were very small, of the order of O-3 p.p.m. No other lines in the downfield region were affected by the absence or presence of phosphates. Diphosphoglycerate caused similar small shifts in the upfield peaks.
z xI-
-2
I c 6
-x-x -x-x
b CH-3
” CH3 amCN(a)
and pmCN (x)
wx-x -5000
FIQ. 4. pH Dependenaes of the shifted rep88oxmncelines of the cPCN 0.1 i%x-phosphake buffer in D,O.
x- CH,
- CH3
and /3”‘CN chains at 2O”C,
The n.m.r. spectra of the chains in H,O rather than D,O show extra resonance lines from exchangeable protons similar to some reported for MbnICN (see Table 1). In the OrmcN spectrum, two extra peaks were observed below -13 p.p.m. labeled NHB and NRC. In the /FC!N chains, no additional resonances were definitely detected in H,O, although the low-field shoulder of the methyl resonance at -23 p.p.m. apparently increased. The measured positions are given in Table 1 where they are correlated with those previously reported in MbmCN (Sheard et al., 1970). As discussed below, the wrrelation was made on the basis of the pH dependence of the line widths, those in the cPCN and pmC?N chains being independent of pH between 6-6 and 85. The effects upon these resonances of a particular protein modification are presented
n.m.r.
OF HEMOGLOBIN.
I.
CHAINS
297
in Figure 3(b) and (d). Reacting the amCN chains with stoichiometric amounts of PMB to form 01rMBmCN, slightly shifts the lower field methyl line and one or two single proton lines near -14 p.p.m., but does not affect the other lines. In the PmCN chain, there are two SH groups which can react with PMB. When both SH groups have reacted with PMB, the spectrum is very different from that of the SH chains, as can be seen by comparing Figure 3(c) and (d). An attempt to block only the fill2 SH group was made by reacting with iodoacetate (Neer, 1970). Although after the reaction nearly one SH group per heme was still titratable, the n.m.r. spectrum was quite complex and different from both the original SH chains and the PMB chains. It is worth pointing out that the oxygen binding properties of SH and PMB /l-chains are very different, especially in the oxygen-off rate, in contrast to the or-chains where PMB made very little difference (Brunori, Noble, Antonini & Wyman, 1966). Since the n.m.r. of the cyanoferric state monitors the ligated state of the protein around the heme, the large differences of the n.m.r. spectra of the /l-chains with and without PMB suggests that the off rate is affected by the differences in the ligated state. 4. Discussion The spectra presented here emphasize the paramagnetically shifted lines. Similar lines in MbmCN have been shown to be shifted from their diamagnetic positions by two well known mechanisms, i.e. contact and pseudocontact interactions (Shulman, Glarum & Karplus, 1971). The former arises because of spin-delocalimation through bonds, so that the unpaired electron spin actually comes into contact with the hydrogen nucleus, immersing it in a large magnetic field. This interaction is isotropic, so that it is not averaged to zero as the molecules tumble in solution and a net magnetic field is produced, shifting the proton nuclear resonance (Bloembergen, 1967). In contrast, the pseudocontact interaction operates via a dipolar interaction, which is transmitted through space and does not require electron delocalization (McComell & Robertson, 1958). When the electron has a different effective magnetic moment along different molecular directions (i.e. an anisotropio electron g-factor), then the dipolar field at a site in the molecule, averaged by molecular tumbling, does not vanish. Instead, depending upon the site’s co-ordinates with respect to the iron, there is either a net positive or negative magnetic field from this interaction. The pseudocontact interaction then is a function of position with respect to the unpaired spin, which is mainly on the iron, and does not depend upon a series of bonds to provide paths for electron delocalization. In this way, it shifts resonances from protein residues near the heme. The two peaks in both downfield regions (Fig. 1) with intensities of three protons each are assigned to the heme methyls. This assignment is made by analogy with Mbu’CN (Wiithrich, Shulman & Peisach, 1968) and the heme cyanides, where the heme methyl peaks are found in this region (Wiithrich, Shulman, Wyluda t Caughey, 1969) and by the impossibility that pseudocontact shifts alone could move two methyl resonances of the protein this far down in field. The other two methyl resonances are presumably hidden under the diamagnetic resonances above -9 p.p.m. The two methyl peaks near -16 and -22 p.p.m. can be assigned to the ring methyls of the PR and VL pyrrole in analogy to the MbmCN case,in which a theoretical analysis (Shulman et al., 1971) predicted larger spin densities in these pyrroles than in the other
298
S. OGAWA,
R. G. SHULMAN
AND
T. YAMANE
two. To distinguish on theoretical grounds between PR and VL, requires a more accurate knowledge of the electronic structure of the a: and fl chains than we have at present. The best argument, so far, for assigning these peaks was given by Davis et al. (1969) who suggested, by correlating the positions of the lowest field methyls resonance with amino-acid changes in different hemoglobins, that for the p-chain in the tetramer this resonance comes from the PR methyl group. Preliminary experiments on reconstituted Hb”‘CN where the PR and PL porphyrin methyl groups had been selectively deuterated show that the lowest field methyl resonance disappears, thereby showing it could not come from the VL methyl (Goncalves, Smith, Kenner, Yamane, Shulman $ Ogawa, manuscript submitted for publication). For assigning the single proton peaks around -15 to -10 p.p.m. in the JIICN and /3111CNchains, there are many candidates; the methylene groups of the propionates, the vinyl CH protons, proximal and distal histidine CH’s and protons of nearby amino-acid residues close to the heme plane, such as Phe CDI. One previous result shows that peaks from the vinyl CH protons fall in this region in both the heme cyanides (Wiithrich et al., 1969) and in MbI”CN (Shulman, Wiithrich, Yamane, Antonini & Brunori, 1969) and that their temperature-dependence is either very small or is in the reverse of the normal paramagnetic shifts, i.e. as the temperatureincreases the shifts also increase. It is shown in Figure 2 that the 01chains have a single proton peak near -3000 Hz with this unusual temperature-dependence, so that this peak can be assigned to a vinyl CH with some confidence. Finally, we consider the assignments of the resolved resonances at high fields. From their temperature-dependencea and by analogy with Mb”‘CN, the two methyl resonances at +3*3 p.p.m. come from amino-acid residues at the periphery of the heme plane, shifted upfield by pseudocontact interactions. Using Perutz’s (1969) co-ordinates for methemoglobin, a calculation was made in search of pseudocontactshifted resonances which might fall in this region. The results were, that methyl groups of FG3 Leu, F4 Leu and 68 Leu were the only possible candidates. Since the calculations are very sensitive to the directions of the principal axes of g tensor in the heme plane, an exact selection amongst these possibilities must await more definite electron paramagnetic resonance information. In addition to the upfield methyls, there are at least two more individual proton resonances in this region. In the PmCN chain, these two resonances are well resolved at still higher fields, while in the ocmCNisolated chains they apparently coincide with the superimposed methyl peaks near $3.3 p.p.m. We suggest this superposition partly because the intensity of this line in the #‘CN chain is definitely larger than that of two methyls but with more assurance because in the reconstituted ((YI*ICN /?nO& hemoglobins these additional peaks are clearly resolved. A vinyl CH, group is a possible assignment for these two proton peaks, because it is found in this region in the heme cyanide spectra, but it has not been identified in the MbII’CN spectra. In MblIICN three exchangeable proton peaks observable in H,O but not in D,O have been reported in the low-field region between -14 and -23 p.p.m. (Sheard et al., 1970). They have been assigned to the N,H and the peptide NH protons of the proximal histidine and N,H proton of the distal histidine, on the basis that no other exchangeable protons could be shifted by the contact and pseudocontact interactions into this low field region. For the c?‘ICN chains, two exchangeable peaks are readily observable at -21.9 p.p.m. and at -13.5 p.p.m. at 20°C. For the B1nCN chain, only one peak at -22.8 p.p.m. is definitely observed (see Table 1). The others
n.m.r.
OF HEMOGLOBIN.
I.
299
CHAINS
could be hidden under strong peaks or be too broad to be seen. There is also a possibility that the peptide NH is difficult to exchange with deuterium and therefore the peak is also present in D,O solution. A comparison of the shifted resonance spectra TABLE
Positions (in ppm.)
CHa C& NH* NHB NHC
1
of low jkld methyl and NH resonances
-22.7 -16.6 ? -21.9 -13.5
-21.7 -15.7 ? -22.8 ?
-27.6 -18.9 -23.8 -21.6 -14.1
of the chains and MbmCN is presented in Table 1. The exchangeable peaks NHC at -13.5 p.p.m. assigned to the peptide NH protons of the proximal histidine in &C!N have very similar line positions to those found in MIPCN. The peaks in the chains labeled NHB in Table 1 are correlated with the peak in MbnlCN assigned to the Nd-H proton of the proximal histidine, because all three of these lines are observed over the full range from pH 65 to 8.5. However, the peak NH* in MWWN broadened beyond detectability at pH <6*5. For this reason, and from its dependence upon the animal source of the myoglobin, it was assigned to the N,H proton of the distal histidine. The broadening was interpreted in terms of an increasing rate of exchange of the N,H proton with the solution, an exchange which is expected to be faster and more sensitive at this position than it would be for the proximal NdH proton. The fact that this resonance is not observed in the chain spectra suggests that the distal N,H proton exchanges more rapidly there than in Mb’nCN. The paramagnetic shift for the peptide NH should be almost completely from the pseudocontact term, which depends on the g-values, the amount of the unpaired spin on the iron atom and the distance from the iron. This proton is near the heme normal through the iron, hence, the pseudocontact term is not sensitive to the in-plane orientation. Since its distance from the iron is expected to be very similar in the two proteins, the closeness of the peak positions suggests that the other factors in the pseudocontact term are very similar. The similarity of the N,H proton positions in the chains and MbmCN also supports the suggestion that the factors responsible for the pseudocontact term are very similar in all three cases, because the major part of the N,H shift is also calculated to be pseudocontact in origin. In contrast to these similarities, the porphyrin ring methyl positions in the chains, while similar to each other, differ considerably from their positions in MbnlCN, as shown in Table 1. In the chains, the average contact shift of these two methyls is about ISo/o smaller than in MWWN. Since about 25% of the electron spin is delocalized into the porphyrin rr orbitals in MbuICN, the most direct interpretation of the PCN and j3mCN methyl positions is that there is only about 20% of the electron spin similarly delocalized in the chains. The small differences in the amount of spin left behind on the iron in the two cases of chains and MbmCN would not be quantitatively inconsistent with the similarities of the pseudocontact interactions observed in the exchangeable NH proton resonance.
300
S. OGAWA,
R. G. SHULMAN
AND
T. YAMANE
So far, we have discussed the paramaguetically shifted resonances iu terms of the two mechanisms, contact and pseudocontact interactions and tried in a tentative way to assign these resonances to specific protons. Those resonances are in their nature very sensitive to structural changes in the immediate vicinity of the heme group in the proteins. The simplest case is a slight change in the positions of some specific amino-acid residue, whose proton resonances are shifted mainly by pseudocontact interactions. A few tenths of an Angstrom unit change in the position of the protons relative to the heme iron can easily be detected as a movement of the resonance peak. In this case, only a limited number of the resonance peaks would be affected. Another type of spectral change is that all shifted resonances, including the porphyrin ring methyls, change their peak position due to some modification of the electronic state of the heme. This can come from slight modification of the axial ligands to the iron or of the coupling between the iron and porphyrin rr system. When there is a substantial conformational change around the heme, such a wide-spread spectral change can be expected. Various minor spectral changes are also possible by minor changes of the interaction between the heme and the protein. Many examples of those speotral changes, when the isolated chains are associated into +& tetramer, will be described in the following paper (Ogawa et CL, 1972). REFERENCES Benesch, R., Benesch, R. E. t Enoki, Y. (1968). Proc. Nat. Acud. Sci., Wa-sh. 61, 1102. Bloembergen, N. (1957). J. C&m. Phys. 27, 595. Brunori, M., Noble, R. W., Anton%, E. & Wyman, J. (1966). J. Mol. Biol. 241, 6238. Bucci, E. & Fronticelli, C. (1965). J. Bid. Chem. 240, 561. Davis, D. G., Mock, N. L., Laman, V. R. & Ho, C. (1969). J. Mol. BioZ. 40, 311. Geracci, G., Parkhurst, L. J. & Gibson, Q. H. (1969). J. Bio2. Chem. 244, 4664. McConnell, H. M. & Robertson, R. E. (1968). J. Chem. Phya. 29, 1361. New, E. J. (1970). J. BioZ. Chem. 245, 564. Ogawa, S. & Shuhnan, R. G. (1972). J. Mol. BioZ. 70, 315. Ogawa, S., Shulman, R. G., Fujiwara, M. & Yamane, T. (1972). J. Mol. BioZ. 70, 301. Perutz, M. F. (1969). Proc. Roy. Sot. B, 173, 113. Sheard, B., Yamane, T. & Shuhnan, R. G. (1970). J. Mol. BioZ. 53, 35. Shuhnan, R. G., Glarum, S. H. & Karplus, M. (1971). J. Mol. BioZ. 57, 93. Shulman, R. G. & Jaccarino. V. (1957). Phye. Rew. 108, 1219. Shulman, R. G., Ogawa, S., Wtithrich, K., Yamane, T., Peisach, J. & Blumberg, W. E. (1969). Science, 165, 251. Shulman, R. G., Wiithrich, K., Yamane, T., Antonini, E. & Bnmori, M. (1969). Proc. Nat. Acad. Sci., Woxh. 63, 623. Wiithrich, K., Shuhnan, R. G. & Peisach, J. (1968). PTOC.Nat. Acad. Sci., Wmh. 60, 373. Wiithrich, K., Shulman, R. G. & Yamane, T. (1968). Proc. Nat. Acad. Sci., Wash. 61, 1199. Wiithrich, K., Shulman, R. G., Wyluda, B. J. & Caughey, W. S. (1969). Proc. Nat. Acud. Sci., Wash. 62, 636. Yamane, T., Wiithrich, K., Shulman, R. G. & Ogawa, S. (1970). J. Mol. BioZ. 49, 197.
High Resolution Nuclear Magnetic Resonance Spectra of Hemoglobin I. The Cyanide Complexes of a and B Chains S. OC+AWA, R.
Murray
G. SHULMAN
AND
T.
YAMANE
Bell Laboratories Hill, N.J. 07974, U.S.A.
(Received 18 January
1972)
The high resolution nuclear magnetic resonance spectra of the isolated chains of human hemoglobin have been studied. Interest is mainly focussed on the paramagnetically shifted resonances in the cyanoferric state of these chains. Two resonance peaks of the heme methyls are easily observable, together with several single proton peaks in the downfield region. The strong peaks in the high-field region are due to two methyl resonances of some amino-acid residue near the periphery of the heme, shifted up-field by the pseudocontact interaction. Effects of pH and of a modification of the protein on those resonances are presented. The exchangeable NH protons of the ring and of the peptide of the proximal histidine have resonance positions very similar to those in cyanoferric myoglobin, although the methyl resonances of the heme group are at somewhat different positions.
1. Introduction Recently, the high resolution proton nuclear magnetic resonance spectra of heme proteins has been intensively studied (see Davis, Mock, Laman & Ho (1969) and Sheard, Yamane & Shulman (1970) for earlier references). An increase of resolution and interpretability ha8 been achieved because many of the proton resonances have been shifted by paramagnetic interactions away from the normal diamagnetic protein region. These paramagnetio shifts, coming from interactions with the unpaired electron of the heme iron, have been most strongly felt by protons near the iron. Hence, they have provided a means of monitoring this interesting region of the protein. With the great amount of information available from the n.m.r.t spectra of the paramagnetic low-spin ferric forms of myoglobin, particularly MbmCN (Sheard et al., 1970), it is now possible to discuss in detail the spectrum of hemoglobin cyanide Hb”ICN, and its constituent o?CN and /3mCN chains. Previous studies of HbmCN have shown that shifted resonances in the spectra can be resolved (Wiithrich, Shuhnan & Yamane, 1968) and are sensitive to modification8 in other regions of the protein. These modifications included mutant human hemoglobins (Davis et al., 1969), other mammalian hemoglobins (Yamane, Wiithrich, Shulman & Ogawa, 1970) and a preliminary account of the effects at one heme of oxygenating the neighbors (Shulman et al., 1969). In this series, we first discuss the n.m.r. spectra of the PCN and /3n1CN t Abbreviation
used: n.m.r., nuclear magnetic
resonance. 291
292
S. OGAWA,
R. G. SHULMAN
AND
T. YAMANE
chains. In the accompanying paper (Ogawa, Shulman, Fujiwara & Yamane, 1972), we show the effects of recombining these chains to form fully ligated hemoglobin. Finally, in the third paper (Ogawa t Shuhnan, 1972), we present the n.m.r. spectra of half ligated hemoglobins and their response to biochemical perturbations. These spectral changes are discussed in terms of a molecular mechanism for the co-operative oxygenation of hemoglobin.
2. Experimental Human hemoglobin A was prepared from freshly-drawn adult blood. The (Yand /? chains were separated by p-chloromercuribenzoate (PMB)t reaction and subsequent carboxymethyl cellulose chromatography (Bucci & Fronticelli, 1965). Regeneration of the SH group in the 01chain was carried out with O-015 a6-mercaptoethanol treatment on carboxymethyl cellulose for 30 minutes (Geracci, Parkhurst t Gibson, 1969).
FIQ. 1. n.m.r. Spectra of the isolated amCN and ,3mCNchaina in Da0 and HsO in 0.1 w-phosphate buffer at pH 643 and 20°C. t Abbreviation
used: PMB, p-&loromercuribenzoete.
n.m.r.
OF HEMOGLOBIN.
DSS
I.
CHAINS
293
.%N
r
II) = DSS
R%N
T
Fro. 2. (a) Temperature dependencea of the shifted resonance lines of the PCN oh&s st pH 6.6, 0.1 ax-phoephste buffer in D,O. The numbers in parentheses in the Figure are the ktensities in number of protons per heme. The letters b snd n correspond to relatively broad and narrow resonance lime as described in the text. (b) Temper&m-c dependences of the shifted resonance lines of the #mCN chains at pH 64, 0.1 x-phosphate buffer in D,O. The numbers in parentheses in the Figure are the intensities in number of protons per heme.
294
S. OGAWA,
R.
G. SHULMAN
AND
T. YAMANE
For the p chain, N-acetyl pennicillamine was used (Benesch, Benesch $ Enoki, 1968). Treatment of the p chains with mercaptoethanol on DEAE-cellulose at pH 8.0 was also tried, but the yield was better from the former method. The j3 SH chain obtained was further purified on carboxymethyl cellulose to eliminate oxidized material. Oxidation of the a: chain to the cyanoferric state was carried out at room temperature with 1-l to 1.2 molar equivalents of K,Fe(CN), in the presence of excess KCN in O-1 M-phosphate at pH ‘7.0. The j3 chain was oxidized at 36°C with l-2 equivalents of K,Fe(CN), and two of KCN, at pH 7-O. For n.m.r. measurements, the hemoglobin solutions were concentrated in a collodium bag to 5 to 10 x 10m3M in heme. The pH values in D,O buffer were expressed by the values read on the pH meter. A Varian high-resolution 220 MHz spectrometer was used for the n.m.r. experiments. The line positions were expressed by the separation in parts per million, from the position of 2,2-dimethyl-2-silapentane-5-sulfonate with negative sign for the downfield shifts. The spectra were obtained by accumulating the signal in a Fabritek 1062 computer for 0.5 to 6 hours, depending on the frequency range covered. The intensities were measured by taking ratios of the integrated areas under the peaks, after rough estimates were obtained by a comparison with the central diamagnetic proton resonances. The accuracy of the intensity measurements was limited by the ability to draw a base line. This introduced uncertainties up to fifteen per cent for overlapping lines, such as were found in the /3mCN spectra.
3. Results The n.m.r. spectra of the isolated a! and /3 chains in the cyanoferric state are shown in Figure 1. The magnetic field regions shown in Figure 1 are outside the ordinary n.m.r. absorption region in a diamagnetic system. The line positions of most of those resonances are temperature-dependent (see Fig. 2(a) and (b)), as expected in paramagnetically shifted systems (Shulman t Jaccarino, 1957). In the PCN chain, the two strong peaks at -22.7 and -16.6 p.p.m. at 20°C have intensities of 3 protons per heme and come from the ring methyls of the porphyrin. Four peaks of intensity one proton each are clustered around -14 p.p.m. They become completely separated into individual lines above 36°C. Two of the four stay narrow over the entire temperature range shown in Figure 2(a), while the other two become broader at the lower temperature. In the upfield region, there is a strong peak at 3.2 p.p,m. (see Fig. 3(a)), with an intensity of 8 to 10 protons per heme, which at the highest temperature begins to resolve. In the associated form of hemoglobin, this strong peak is resolved into two peaks under certain conditions, as discussed in the following paper (Ogawa et al., 1972). It appears likely that in this strong resonance two lines of intensity 3 protons and at least 2 single proton lines, which are resolved in the or& tetramer, are superimposed. Similar, but slightly different spectra are observed in the $WN chains. The two methyl lines in the downfield region are found at -21.7 and 15.7 p.p.m. (see Fig. l(c)). In the upfield region (see Fig. 3(c)), two single proton peaks are slightly upfield from the strong peak of ~6 protons at 4 p.p.m. From a comparison with the associated hemoglobin, where under certain circumstances it splits (see II), this strong peak consists most likely of two methyl resonances. The line widths of these resonances vary among the different peaks and are tempera-
n.m.r.
OF HEMOGLOBIN.
295
I. CHAINS
(a)
(b)
2w p.p.m.
B*CN
2L"4L"G wm
FIG. 3. Effeot of PMB on the n.m.r. speotra of the &WN and the @mCN ohains. After stoiohiometrio emounts of PMB were sdded to the cr’nCN chain solution, the pH and buffer were adjusted to pH 643 in 0.1 aa-phosphate buffer by gel filtration.
ture-dependent. Some semi-quantitative differences were as the following. At 4O”C, for example, the low-field peaks in the PCN spectra were about 40 Hz wide (full width at half height) and were approximately the mrne as the line widths of the analogous resonances in MIPCN. In contrast, the low-field resonances in the j3mCN spectra were about 20% wider. Most of those resonance lines are insensitive to pH in phosphate buffer in the physiologiaal pH region as shown in Figure 4. However, one line in the OPICN spectrum, with intensity of one proton, near - 13.6 p.p.m., titrateswith pH around pH 6.9
298
S. OGAWA,
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There are some pH-sensitive peaks in the less-shifted field region near -11 p.p.m. but their origin is not understood. The presence of organic or inorganic phosphates in the cPCN solution did not affect the spectrum. In solutions of the /FCN chains, only the lowest field methyl resonance showed a pH dependence, which was small. In a stripped j? chain solution at pH 6.6, the lowest field methyl resonance was at a slightly lower field than at the same pH in phosphate buffer. Addition of 2,3diphosphoglycerate moved this methyl peak to a slightly higher field. These shifts were very small, of the order of O-3 p.p.m. No other lines in the downfield region were affected by the absence or presence of phosphates. Diphosphoglycerate caused similar small shifts in the upfield peaks.
z xI-
-2
I c 6
-x-x -x-x
b CH-3
” CH3 amCN(a)
and pmCN (x)
wx-x -5000
FIQ. 4. pH Dependenaes of the shifted rep88oxmncelines of the cPCN 0.1 i%x-phosphake buffer in D,O.
x- CH,
- CH3
and /3”‘CN chains at 2O”C,
The n.m.r. spectra of the chains in H,O rather than D,O show extra resonance lines from exchangeable protons similar to some reported for MbnICN (see Table 1). In the OrmcN spectrum, two extra peaks were observed below -13 p.p.m. labeled NHB and NRC. In the /FC!N chains, no additional resonances were definitely detected in H,O, although the low-field shoulder of the methyl resonance at -23 p.p.m. apparently increased. The measured positions are given in Table 1 where they are correlated with those previously reported in MbmCN (Sheard et al., 1970). As discussed below, the wrrelation was made on the basis of the pH dependence of the line widths, those in the cPCN and pmC?N chains being independent of pH between 6-6 and 85. The effects upon these resonances of a particular protein modification are presented
n.m.r.
OF HEMOGLOBIN.
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CHAINS
297
in Figure 3(b) and (d). Reacting the amCN chains with stoichiometric amounts of PMB to form 01rMBmCN, slightly shifts the lower field methyl line and one or two single proton lines near -14 p.p.m., but does not affect the other lines. In the PmCN chain, there are two SH groups which can react with PMB. When both SH groups have reacted with PMB, the spectrum is very different from that of the SH chains, as can be seen by comparing Figure 3(c) and (d). An attempt to block only the fill2 SH group was made by reacting with iodoacetate (Neer, 1970). Although after the reaction nearly one SH group per heme was still titratable, the n.m.r. spectrum was quite complex and different from both the original SH chains and the PMB chains. It is worth pointing out that the oxygen binding properties of SH and PMB /l-chains are very different, especially in the oxygen-off rate, in contrast to the or-chains where PMB made very little difference (Brunori, Noble, Antonini & Wyman, 1966). Since the n.m.r. of the cyanoferric state monitors the ligated state of the protein around the heme, the large differences of the n.m.r. spectra of the /l-chains with and without PMB suggests that the off rate is affected by the differences in the ligated state. 4. Discussion The spectra presented here emphasize the paramagnetically shifted lines. Similar lines in MbmCN have been shown to be shifted from their diamagnetic positions by two well known mechanisms, i.e. contact and pseudocontact interactions (Shulman, Glarum & Karplus, 1971). The former arises because of spin-delocalimation through bonds, so that the unpaired electron spin actually comes into contact with the hydrogen nucleus, immersing it in a large magnetic field. This interaction is isotropic, so that it is not averaged to zero as the molecules tumble in solution and a net magnetic field is produced, shifting the proton nuclear resonance (Bloembergen, 1967). In contrast, the pseudocontact interaction operates via a dipolar interaction, which is transmitted through space and does not require electron delocalization (McComell & Robertson, 1958). When the electron has a different effective magnetic moment along different molecular directions (i.e. an anisotropio electron g-factor), then the dipolar field at a site in the molecule, averaged by molecular tumbling, does not vanish. Instead, depending upon the site’s co-ordinates with respect to the iron, there is either a net positive or negative magnetic field from this interaction. The pseudocontact interaction then is a function of position with respect to the unpaired spin, which is mainly on the iron, and does not depend upon a series of bonds to provide paths for electron delocalization. In this way, it shifts resonances from protein residues near the heme. The two peaks in both downfield regions (Fig. 1) with intensities of three protons each are assigned to the heme methyls. This assignment is made by analogy with Mbu’CN (Wiithrich, Shulman & Peisach, 1968) and the heme cyanides, where the heme methyl peaks are found in this region (Wiithrich, Shulman, Wyluda t Caughey, 1969) and by the impossibility that pseudocontact shifts alone could move two methyl resonances of the protein this far down in field. The other two methyl resonances are presumably hidden under the diamagnetic resonances above -9 p.p.m. The two methyl peaks near -16 and -22 p.p.m. can be assigned to the ring methyls of the PR and VL pyrrole in analogy to the MbmCN case,in which a theoretical analysis (Shulman et al., 1971) predicted larger spin densities in these pyrroles than in the other
298
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T. YAMANE
two. To distinguish on theoretical grounds between PR and VL, requires a more accurate knowledge of the electronic structure of the a: and fl chains than we have at present. The best argument, so far, for assigning these peaks was given by Davis et al. (1969) who suggested, by correlating the positions of the lowest field methyls resonance with amino-acid changes in different hemoglobins, that for the p-chain in the tetramer this resonance comes from the PR methyl group. Preliminary experiments on reconstituted Hb”‘CN where the PR and PL porphyrin methyl groups had been selectively deuterated show that the lowest field methyl resonance disappears, thereby showing it could not come from the VL methyl (Goncalves, Smith, Kenner, Yamane, Shulman $ Ogawa, manuscript submitted for publication). For assigning the single proton peaks around -15 to -10 p.p.m. in the JIICN and /3111CNchains, there are many candidates; the methylene groups of the propionates, the vinyl CH protons, proximal and distal histidine CH’s and protons of nearby amino-acid residues close to the heme plane, such as Phe CDI. One previous result shows that peaks from the vinyl CH protons fall in this region in both the heme cyanides (Wiithrich et al., 1969) and in MbI”CN (Shulman, Wiithrich, Yamane, Antonini & Brunori, 1969) and that their temperature-dependence is either very small or is in the reverse of the normal paramagnetic shifts, i.e. as the temperatureincreases the shifts also increase. It is shown in Figure 2 that the 01chains have a single proton peak near -3000 Hz with this unusual temperature-dependence, so that this peak can be assigned to a vinyl CH with some confidence. Finally, we consider the assignments of the resolved resonances at high fields. From their temperature-dependencea and by analogy with Mb”‘CN, the two methyl resonances at +3*3 p.p.m. come from amino-acid residues at the periphery of the heme plane, shifted upfield by pseudocontact interactions. Using Perutz’s (1969) co-ordinates for methemoglobin, a calculation was made in search of pseudocontactshifted resonances which might fall in this region. The results were, that methyl groups of FG3 Leu, F4 Leu and 68 Leu were the only possible candidates. Since the calculations are very sensitive to the directions of the principal axes of g tensor in the heme plane, an exact selection amongst these possibilities must await more definite electron paramagnetic resonance information. In addition to the upfield methyls, there are at least two more individual proton resonances in this region. In the PmCN chain, these two resonances are well resolved at still higher fields, while in the ocmCNisolated chains they apparently coincide with the superimposed methyl peaks near $3.3 p.p.m. We suggest this superposition partly because the intensity of this line in the #‘CN chain is definitely larger than that of two methyls but with more assurance because in the reconstituted ((YI*ICN /?nO& hemoglobins these additional peaks are clearly resolved. A vinyl CH, group is a possible assignment for these two proton peaks, because it is found in this region in the heme cyanide spectra, but it has not been identified in the MbII’CN spectra. In MblIICN three exchangeable proton peaks observable in H,O but not in D,O have been reported in the low-field region between -14 and -23 p.p.m. (Sheard et al., 1970). They have been assigned to the N,H and the peptide NH protons of the proximal histidine and N,H proton of the distal histidine, on the basis that no other exchangeable protons could be shifted by the contact and pseudocontact interactions into this low field region. For the c?‘ICN chains, two exchangeable peaks are readily observable at -21.9 p.p.m. and at -13.5 p.p.m. at 20°C. For the B1nCN chain, only one peak at -22.8 p.p.m. is definitely observed (see Table 1). The others
n.m.r.
OF HEMOGLOBIN.
I.
299
CHAINS
could be hidden under strong peaks or be too broad to be seen. There is also a possibility that the peptide NH is difficult to exchange with deuterium and therefore the peak is also present in D,O solution. A comparison of the shifted resonance spectra TABLE
Positions (in ppm.)
CHa C& NH* NHB NHC
1
of low jkld methyl and NH resonances
-22.7 -16.6 ? -21.9 -13.5
-21.7 -15.7 ? -22.8 ?
-27.6 -18.9 -23.8 -21.6 -14.1
of the chains and MbmCN is presented in Table 1. The exchangeable peaks NHC at -13.5 p.p.m. assigned to the peptide NH protons of the proximal histidine in &C!N have very similar line positions to those found in MIPCN. The peaks in the chains labeled NHB in Table 1 are correlated with the peak in MbnlCN assigned to the Nd-H proton of the proximal histidine, because all three of these lines are observed over the full range from pH 65 to 8.5. However, the peak NH* in MWWN broadened beyond detectability at pH <6*5. For this reason, and from its dependence upon the animal source of the myoglobin, it was assigned to the N,H proton of the distal histidine. The broadening was interpreted in terms of an increasing rate of exchange of the N,H proton with the solution, an exchange which is expected to be faster and more sensitive at this position than it would be for the proximal NdH proton. The fact that this resonance is not observed in the chain spectra suggests that the distal N,H proton exchanges more rapidly there than in Mb’nCN. The paramagnetic shift for the peptide NH should be almost completely from the pseudocontact term, which depends on the g-values, the amount of the unpaired spin on the iron atom and the distance from the iron. This proton is near the heme normal through the iron, hence, the pseudocontact term is not sensitive to the in-plane orientation. Since its distance from the iron is expected to be very similar in the two proteins, the closeness of the peak positions suggests that the other factors in the pseudocontact term are very similar. The similarity of the N,H proton positions in the chains and MbmCN also supports the suggestion that the factors responsible for the pseudocontact term are very similar in all three cases, because the major part of the N,H shift is also calculated to be pseudocontact in origin. In contrast to these similarities, the porphyrin ring methyl positions in the chains, while similar to each other, differ considerably from their positions in MbnlCN, as shown in Table 1. In the chains, the average contact shift of these two methyls is about ISo/o smaller than in MWWN. Since about 25% of the electron spin is delocalized into the porphyrin rr orbitals in MbuICN, the most direct interpretation of the PCN and j3mCN methyl positions is that there is only about 20% of the electron spin similarly delocalized in the chains. The small differences in the amount of spin left behind on the iron in the two cases of chains and MbmCN would not be quantitatively inconsistent with the similarities of the pseudocontact interactions observed in the exchangeable NH proton resonance.
300
S. OGAWA,
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T. YAMANE
So far, we have discussed the paramaguetically shifted resonances iu terms of the two mechanisms, contact and pseudocontact interactions and tried in a tentative way to assign these resonances to specific protons. Those resonances are in their nature very sensitive to structural changes in the immediate vicinity of the heme group in the proteins. The simplest case is a slight change in the positions of some specific amino-acid residue, whose proton resonances are shifted mainly by pseudocontact interactions. A few tenths of an Angstrom unit change in the position of the protons relative to the heme iron can easily be detected as a movement of the resonance peak. In this case, only a limited number of the resonance peaks would be affected. Another type of spectral change is that all shifted resonances, including the porphyrin ring methyls, change their peak position due to some modification of the electronic state of the heme. This can come from slight modification of the axial ligands to the iron or of the coupling between the iron and porphyrin rr system. When there is a substantial conformational change around the heme, such a wide-spread spectral change can be expected. Various minor spectral changes are also possible by minor changes of the interaction between the heme and the protein. Many examples of those speotral changes, when the isolated chains are associated into +& tetramer, will be described in the following paper (Ogawa et CL, 1972). REFERENCES Benesch, R., Benesch, R. E. t Enoki, Y. (1968). Proc. Nat. Acud. Sci., Wa-sh. 61, 1102. Bloembergen, N. (1957). J. C&m. Phys. 27, 595. Brunori, M., Noble, R. W., Anton%, E. & Wyman, J. (1966). J. Mol. Biol. 241, 6238. Bucci, E. & Fronticelli, C. (1965). J. Bid. Chem. 240, 561. Davis, D. G., Mock, N. L., Laman, V. R. & Ho, C. (1969). J. Mol. BioZ. 40, 311. Geracci, G., Parkhurst, L. J. & Gibson, Q. H. (1969). J. Bio2. Chem. 244, 4664. McConnell, H. M. & Robertson, R. E. (1968). J. Chem. Phya. 29, 1361. New, E. J. (1970). J. BioZ. Chem. 245, 564. Ogawa, S. & Shuhnan, R. G. (1972). J. Mol. BioZ. 70, 315. Ogawa, S., Shulman, R. G., Fujiwara, M. & Yamane, T. (1972). J. Mol. BioZ. 70, 301. Perutz, M. F. (1969). Proc. Roy. Sot. B, 173, 113. Sheard, B., Yamane, T. & Shuhnan, R. G. (1970). J. Mol. BioZ. 53, 35. Shuhnan, R. G., Glarum, S. H. & Karplus, M. (1971). J. Mol. BioZ. 57, 93. Shulman, R. G. & Jaccarino. V. (1957). Phye. Rew. 108, 1219. Shulman, R. G., Ogawa, S., Wtithrich, K., Yamane, T., Peisach, J. & Blumberg, W. E. (1969). Science, 165, 251. Shulman, R. G., Wiithrich, K., Yamane, T., Antonini, E. & Bnmori, M. (1969). Proc. Nat. Acad. Sci., Woxh. 63, 623. Wiithrich, K., Shuhnan, R. G. & Peisach, J. (1968). PTOC.Nat. Acad. Sci., Wmh. 60, 373. Wiithrich, K., Shulman, R. G. & Yamane, T. (1968). Proc. Nat. Acad. Sci., Wash. 61, 1199. Wiithrich, K., Shulman, R. G., Wyluda, B. J. & Caughey, W. S. (1969). Proc. Nat. Acud. Sci., Wash. 62, 636. Yamane, T., Wiithrich, K., Shulman, R. G. & Ogawa, S. (1970). J. Mol. BioZ. 49, 197.