NMR Study of Human Mutant Hemoglobins Synthesized in Escherichia coli

The hydroxyl group of Tyr”42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp8@@ which is considered to be one of the most important hydrogen bonds for stablizing the “T-state.” However, no spontaneous mutation at position 42 of the a subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr”42 was replaced by phenylalanine or histidine were synthesized in Escherichia coli, and their proton NMR spectra were studied with particular attention to the hyperfine-shifted and hydrogen-bonded proton resonances. The site-directed mutagenesis of the Tyra42 + Phe removes the hydrogen bond described above and prevents transition to the T-state so that the mutant Hb is rather similar to the “R-state” even when deoxygenated. On the other hand, the mutation from tyrosine to histidine causes less drastic structural changes, and its quaternary and tertiary structures are almost the same as native deoxy-Hb


This may be attributed to the formation of a new hydrogen bond between
and Asp8+'@. These observations indicate that the hydrogen bond formed between Tyr"42 and Aspses is required to convert unliganded Hb to the T-state.
The expression of cloned genes in appropriate host cells has made it possible to study protein functions by site-directed mutagenesis (1)(2)(3)(4)(5)(6)(7). Studies of hemoglobin (Hb) by this method are of particular interest since it is the only allosteric protein whose structure has been solved to atomic resolution in both the T (tense) and R (relaxed) states (8,9). Subunit interactions, the binding of various allosteric effectors, and the interaction of oxygen with residues in the heme pocket can be studied in detail by introducing various mutations. The effects of such mutations on the electronic state of the heme and the features of ligand binding can be studied spectroscopically (10). Nagai and co-workers (6,7,11) had developed a cleavable fusion protein expression vector to produce a-or pglobin in Escherichia coli cells in amounts sufficient for biochemical and x-ray crystallograhic studies. We have used this method to synthesize two a subunit mutant Hbs in E. coli to investigate the effect of the hydrogen bonds located at the a& subunit interface on the tertiary and quaternary structures of Hb. The hydrogen bonds in subunit interfaces have been considered to play an important role in maintaining the quaternary structure of Hb. Ho and co-workers (12,13) reported some NMR spectra of natural mutant Hbs to assign the resonances of hydrogen-bonded residues and to discuss structure-function relationships in Hb. Of all the hydrogen bonds and salt bridges in the subunit interfaces, the hydrogen bond between T~r"1~' and Aspozg9 has been considered to serve as a key intersubunit interaction for the allosteric transition of Hb. Some natural mutant Hbs which lack this hydrogen bond such as Hb Yakima (Aspm9 + His) (14), Hb Kempsey (Aspm9 + Asn) (15), and Hb Radcliffe (Aspm9 4 Ala) (16) fail to assume a "T-state" conformation in the deoxygenated form, resulting in high oxygen affinities and low cooperativity. However, it has not yet been clarified how this intersubunit hydrogen bond causes these functional defects, because no naturally occurring a42 mutation has been discovered. Thus, we have designed new mutant Hbs (Hb Ya42F, Tyra42 + Phe and Hb Ya42H, Trya4' + His) to gain further insight into the role of this hydrogen bond in the tertiary and quaternary structures of Hb.

EXPERIMENTAL PROCEDURES
Protein preparation was essentially the same as described in the previous papers (6, 7,ll). The preparation of the a-globin expression vector will be published soon. 2 Proton NMR spectra at 300 MHz were recorded on a Nicolet NT-300 spectrometer equipped with a 1280 computer system. NMR spectra were obtained as reported by our previous papers (17,18).
' As discussed in previous papers (171, more than two quaternary structures may exist in the course of allosteric ligand binding to Hb. In this paper, the term "T-state" is used to describe the quaternary structure of fully deoxy-Hb A, and "R-state" is used to describe the quaternary structure of fully oxy-Hb A, respectively.

Synthesized Human Hemoglobins
Proton shifts were referenced with respect to the water signal, which is 4.8 ppm downfield from the proton resonance of 4,4-dimethyl-4silapentane-1-sulfonate at 23 "C. Oxygen equilibrium curves were determined with an automatic recording oxygenation apparatus (19). The spectrophotometer used was a Cary model 118C. The wavelength of detection light was 560 nm.

RESULTS AND DISCUSSION
In Fig. 1 are shown the 'H NMR spectra of the oxygenated mutant Hbs. For native Hb A (truce C), the ring currentshifted proton peak at -7.2 ppm, which was assigned to the yl-methyl resonance of Vala1' and Vala1', has been shown to serve as a marker for the tertiary structure in the heme vicinity (20). The exchangeable peak at 5.9 ppm in the downfield region arises from the hydrogen bond between and Asna2'oz, which is used as an indicator of the R-state quaternary structure (21). The corresponding signals for Hb Ya42F and Hb Ya42H (truces A and B ) were observed at the same positions, and only small spectral changes were found in the region from -5 to -6 ppm, showing that the tertiary and quaternary structures of the oxygenated mutants are little perturbed by the amino acid substitutions.   completely disappears in going from native Hb A to Hb Ya42F. This exchangeable proton resonance is also absent in the NMR spectra of deoxy-Hb Yakima and Hb Kempsey (13) in which aspartic acid p99 is replaced by histidine and asparagine, respectively. It is worth noting that the R-state marker around 6 ppm is missing, suggesting that the Hb Ya42F could experience some quaternary structural changes upon deoxygenation without undergoing the normal R "-$ T transition.
In the hyperfine-shifted region (truce A ) , the proximal histidyl NIH resonances of Hb Ya42F were observed with a substantial downfield bias as compared with native Hb A, and their resonance positions appear close to those for the isolated chains as found for deoxy-Hb Kempsey (22). This downfield bias of the NH contact shift implies that a strain imposed on the bond between the heme iron and the proximal His is released in favor of electron spin delocalization from the iron to the imidazole (22, 23). The structural differences between Hb Ya42F and native Hb A are also manifested by changes in the 5-25-ppm region of the NMR spectra. The hyperfineshifted resonance pattern of Hb Ya42F looks similar to the reported spectrum of deo~y-des-His~'~~-Arg"'~~-Hb, which is considered to be in the R-state (22). It should be noted that the NMR resonances arising from the unmodified p subunit are also altered by the amino acid substitution in the a subunit. Therefore, we can conclude that the tertiary structures of both subunits of the Hb Ya42F become "R-like." The observations described above indicate that the formation of the hydrogen bond between Tyr"14* and Aspi0zg9 plays a key role not only in stabilizing the "T" quaternary structure but also in increasing the strain between the heme iron and the proximal histidine in both subunits, which is characteristic of the normal deoxy tertiary structure.
Hb Ya42H, on the other hand, exhibits an NMR spectrum substantially different from that of Hb Ya42F (Fig. 2). In spite of substitution of His for Tyr, a small exchangeable proton resonance was observed around 9 ppm where the Tstate marker resonance is expected to appear for native deoxy-Hb A. Sequence analysis clearly showed that the amino acid residue at position 42 of the a subunit is His.3 This exchangeable broad signal may arise from the Hisa4' imidazoyl NH' group which is capable of forming a new hydrogen bond to carboxylate (-COO-) of Asppgg.
The positions of two histidyl NIH signals in the hyperfine-

Synthesized Human Hemoglobins
shifted region are not significantly perturbed by the amino acid substitution of His for Tyr, except for the slight signal broadening of the a subunit NIH signal. The heme methyl signal around 18 ppm from the p subunit is also unperturbed, while the heme methyl signals from the a subunits are significantly shifted or broadened. Thus, although the substitution for the tyrosine induces structural changes in the heme cavity of the a subunit, the tertiary structure of the Hb Ya42H, which is manifested by the resonance positions of the proximal histidine NIH and heme methyl groups, is almost the same as that of native deoxy-Hb A. This suggests that the possible new hydrogen bond between His"14' and AspPzg9 can fix the aC helix and the PG helix to rearrange the tertiary structure of both subunits to retain the T-state structure.
It is now clearly shown that the hydrogen bond between Tyral4' and Asppzgg plays a key role in maintaining the T-state in the deoxygenated form. Complete removal of this hydrogen bond (Hb Ya42F, Tyra4' + Phe) causes significant structural changes in the deoxygenated state. The 'H NMR spectrum of this mutant reveals that its tertiary and quaternary structures are rather similar to the R-state even in the deoxy form. We have also studied the oxygen equilibrium curves for these mutant Hbs. As shown in Table I, Hb Ya42F exhibited extremely high oxygen affinity and a hyperbolic oxygen equilibrium curve (nmax = 12), as is the case for the natural mutants having a substitution for aspartic acid p99 (24,25). The fact that the replacement of tyrosine at a42 and aspartic acid at p99 affects the oxygen binding properties in the same manner clearly lent credence to the hypothesis that the intersubunit hydrogen bond between Tyf4' and Aspog9 stabilizes the low oxygen affinity form, the T-state. This is also consistent with the view that the structure of the mutant remains in the R-state during oxy-and deoxygenation, although the R-state NMR markers arising from the intersubunit hydrogen bond between Aspag4 and Asno'" were not observed.
It is of great interest that the oxygen binding property of Hb Ya42H, in which a hydrogen bond is possibly formed between Hi~"1~' and AspPzg9, is different from those of Hb Ya42F and Aspmg mutants in that Hb Ya42H exhibited a lower oxygen affinity (P50 = 1.4) and more significant cooperativity (nmax = 2.0). These reduced functional defects in Hb Ya42H are also consistent with its structural characteristics ( d e supra) determined by the present NMR study.
In summary, mutations of a particular amino acid residue forming a hydrogen bond at the subunit interfaces have shown that this bond is crucial in balancing the stabilities of the Tand R-states of Hb. Since discovery of new natural mutant Hbs is not assured and actually occurring natural mutations do not necessarily satisfy the interests of researchers, intensive site-directed mutagenesis studies of Hb will be requisite for further studies of the structure-function relationships in Hb. Medicale.