Site-directed mutagenesis in hemoglobin: functional and structural role of inter- and intrasubunit hydrogen bonds as studied with 37 beta and 145 beta mutations.
In order to clarify the functional and structural role of intra- and intersubunit hydrogen bonds in human hemoglobin (Hb A), we prepared two artificial beta chain mutant hemoglobins by site-directed mutagenesis. The mutant Hb Phe-37 beta, in which Trp-37 beta is replaced by Phe to remove the intersubunit hydrogen bond between Asp-94 alpha and Trp-37 beta at the alpha 1-beta 2 interface in deoxy Hb A, showed a markedly increased oxygen affinity and almost completely diminished Bohr effect and cooperativity. However, 1H-NMR data indicated that the structure of deoxy Hb Phe-37 beta is rather similar to that of deoxy Hb A. The enhanced tetramer-to-dimer dissociation previously observed in Hb Hirose (Trp-37 beta----Ser) together with our observation of the effects of organic phosphate on the structure and function of Hb Phe-37 beta suggested that a large part of the abnormal properties of Hb Phe-37 beta observed for dilute solutions appears to result from partial dissociation into alpha beta dimers rather than direct destabilization of the T-quaternary structure in the deoxygenated state. Thus, the primary and direct role of the hydrogen bond between Asp-94 alpha and Trp-37 beta is to stabilize the tetrameric assembly, and thereby this hydrogen bond indirectly contributes to stabilization of the T-quaternary structure. The other mutant Hb Phe-145 beta has a Phe residue at the 145 beta site and lacks the intrasubunit hydrogen bond formed between Tyr-145 beta and the carbonyl group of Val-98 beta in deoxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)
Ishimori
K
K
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Imai
K
K
Miyazaki
G
G
Kitagawa
T
T
Wada
Y
Y
Morimoto
H
H
Morishima
I
I
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D006441:Hemoglobin A / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000235:genetics
D001224:Aspartic Acid; D014364:Tryptophan; D006441:Hemoglobin A; D010100:Oxygen