SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - FUNCTIONAL AND STRUCTURAL ROLE OF INTERSUBUNIT 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, H-1-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. Although this hydrogen bond has been considered to fix the phenolic group of Tyr-145-beta in a pocket between the F and H helices, to strengthen the salt bridges formed by His-146-beta, and thereby to stabilize the T-quaternary structure, Hb Phe-145-beta exhibited only mild functional and structural alterations. This result led us to conclude that the van der Waals contacts between the benzene ring of Tyr-145-beta and the tyrosine pocket, rather than the hydrogen bond between Tyr-145-beta and Val-98-beta, make a major contribution to the stabilization of the T-quaternary structure. The present NMR spectra of deoxygenated Hb Phe-37-beta and Hb Phe-145-beta further showed that the exchangeable proton resonance observed at 6.4 ppm for deoxy Hb A originates from the intersubunit hydrogen bond between Asp-94-alpha and Trp-37-beta, although it has previously been assigned to the intrasubunit hydrogen bond between Val-98-beta and Try-145-beta.