NMR study of human mutant hemoglobins synthesized in Escherichia coli. Consequences of tyrosine alpha 42 substitutions.
The hydroxyl group of Tyr alpha 42 in human hemoglobin forms a hydrogen bond with the carboxylate of Asp beta 99 which is considered to be one of the most important hydrogen bonds for stabilizing the "T-state." However, no spontaneous mutation at position 42 of the alpha subunit has been reported, and the role of the tyrosine has not been tested experimentally. Two artificial human mutant hemoglobins in which Tyr alpha 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 Tyr alpha 42----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 A. This may be attributed to the formation of a new hydrogen bond between His alpha 1(42) and Asp beta 2(99). These observations indicate that the hydrogen bond formed between Tyr alpha 42 and Asp beta 99 is required to convert unliganded Hb to the T-state.
Ishimori
K
K
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Morishima
I
I
Imai
K
K
Fushitani
K
K
Miyazaki
G
G
Shih
D
D
Tame
J
J
Pegnier
J
J
Nigai
K
K
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006455:Hemoglobins, Abnormal / Q000096:biosynthesis / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000235:genetics
D006455:Hemoglobins, Abnormal; D014443:Tyrosine; D010649:Phenylalanine; D006639:Histidine