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Site-directed mutagenesis in hemoglobin: Functional and structural roles of inter- and intrasubunit hydrogen bonds
B035 SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN: FUNCTIONAL AND STRUCTURAL ROLES OF INTEW m,a
.
AND INTRASUBW
HYDROGEN BONDS
M. Hashimoto,a K. Imajb G. Miyazaki,c Y. Wada,d
T. Kitagawap H. Morimoto,c I. Morishimaa aDivision of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan, bDepartment of Physicochemical Physiology, Medical School, Osaka University, Suita 565, Japan, CDepartment of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka 560, Japan, dOsaka Medical Center and Research Institute for Maternal and Child Health, Izumi, 590-02, Japan, Research Institutes, eInstitute of Molecular Science, Okazaki National Okazaki 444, Japan In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin, we prepared several artificial 01or /3 chain mutant hemoglobins by site-directed mutagenesis. The mutation sites we have examined were Thr-38a [ll, Tyr-42a 121, Tyr140a [l], Trp-37p [31, and Tyr-145p 131. The mutant Hb Phe-42a, in which Tyr-42a is replaced by Phe to remove the intersubunit hydrogen bond between Tyr-42a and Asp-99P at the al-P2 interface in deoxyHb A, showed increased oxygen affinity and completely diminished cooperativity. v-7 ‘l’al_S5., ;-;:;i;h ls..ks CL^ :...;J_..,._- L...x I 1 ..‘.-*..r.n T%.. yt; ..;.; T-*_o..;scc._, ~3 UIL, ,lJUl “g”“* k,“lAd “.dCI”b4_. _. .a Asp-99P in the deoxygenated state, exhibited the nearly normal oxygen binding property, indicating that the hydrogen bond between Tyr-42a and Asp-99P is a key interaction to stabilize the deoxygenated state and the interaction by the hydrogen bond between Thr-38a and Asp-99p is not so important. On the other hand, the substitution of Trp-37P (Trp + Phe), which corresponds to Thr in the a subunit, caused the dissociation of tetramer to dimer, resulting in high oxygen affinity and no cooperativity. The penultimate Tyr at 140a and 145p is hydrogen bonded to the carbonyl group in Val-97a and Val-980, respectively. These intrasubunit hydrogen bonds have been also considered to be important for stabilizing the deoxygenated state. Hb Phe-140a, in which the hydrogen bond between Tyr-14Oa and Val-97a is missing, exhibited high oxygen affinity and reduced cooperativity. However, normal oxygen affinity and slightly decreased cooperativity were observed for the corresponding 0 mutant, Hb Phe-145/3. Considering these results, we suppose that the contribution of the intrasubunit hydrogen bond in the /3 subunit to the structure and function of hemoglobin is quite different from that in the UIsubunit. 1. M. Hashimoto, K. Ishimori, K. Imai, G. Miyazaki, Kitagawa, H. Morimoto, I. Morishima, to be submitted 2. K. Imai, K. Fushitani, G. Miyazaki, K. Ishimori, T. Wada, H. Morimoto, I. Morishima, D. Shih, J. Tame, 218,769 (1991) 3. K. Ishimori, K. Imai, G. Miyazaki, T. Kitagawa, Morimoto, I. Morishima, Biochemistry, 31, 3256 (1992)
Y. Wada,
T.
Kitagawa, Y. J. Mol. Biol., Y. Wada,
H.
.
AND INTRASUBW
HYDROGEN BONDS
M. Hashimoto,a K. Imajb G. Miyazaki,c Y. Wada,d
T. Kitagawap H. Morimoto,c I. Morishimaa aDivision of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan, bDepartment of Physicochemical Physiology, Medical School, Osaka University, Suita 565, Japan, CDepartment of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka 560, Japan, dOsaka Medical Center and Research Institute for Maternal and Child Health, Izumi, 590-02, Japan, Research Institutes, eInstitute of Molecular Science, Okazaki National Okazaki 444, Japan In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin, we prepared several artificial 01or /3 chain mutant hemoglobins by site-directed mutagenesis. The mutation sites we have examined were Thr-38a [ll, Tyr-42a 121, Tyr140a [l], Trp-37p [31, and Tyr-145p 131. The mutant Hb Phe-42a, in which Tyr-42a is replaced by Phe to remove the intersubunit hydrogen bond between Tyr-42a and Asp-99P at the al-P2 interface in deoxyHb A, showed increased oxygen affinity and completely diminished cooperativity. v-7 ‘l’al_S5., ;-;:;i;h ls..ks CL^ :...;J_..,._- L...x I 1 ..‘.-*..r.n T%.. yt; ..;.; T-*_o..;scc._, ~3 UIL, ,lJUl “g”“* k,“lAd “.dCI”b4_. _. .a Asp-99P in the deoxygenated state, exhibited the nearly normal oxygen binding property, indicating that the hydrogen bond between Tyr-42a and Asp-99P is a key interaction to stabilize the deoxygenated state and the interaction by the hydrogen bond between Thr-38a and Asp-99p is not so important. On the other hand, the substitution of Trp-37P (Trp + Phe), which corresponds to Thr in the a subunit, caused the dissociation of tetramer to dimer, resulting in high oxygen affinity and no cooperativity. The penultimate Tyr at 140a and 145p is hydrogen bonded to the carbonyl group in Val-97a and Val-980, respectively. These intrasubunit hydrogen bonds have been also considered to be important for stabilizing the deoxygenated state. Hb Phe-140a, in which the hydrogen bond between Tyr-14Oa and Val-97a is missing, exhibited high oxygen affinity and reduced cooperativity. However, normal oxygen affinity and slightly decreased cooperativity were observed for the corresponding 0 mutant, Hb Phe-145/3. Considering these results, we suppose that the contribution of the intrasubunit hydrogen bond in the /3 subunit to the structure and function of hemoglobin is quite different from that in the UIsubunit. 1. M. Hashimoto, K. Ishimori, K. Imai, G. Miyazaki, Kitagawa, H. Morimoto, I. Morishima, to be submitted 2. K. Imai, K. Fushitani, G. Miyazaki, K. Ishimori, T. Wada, H. Morimoto, I. Morishima, D. Shih, J. Tame, 218,769 (1991) 3. K. Ishimori, K. Imai, G. Miyazaki, T. Kitagawa, Morimoto, I. Morishima, Biochemistry, 31, 3256 (1992)
Y. Wada,
T.
Kitagawa, Y. J. Mol. Biol., Y. Wada,
H.