CONTRIBUTIONS OF ASPARAGINE AT ALPHA-97 TO THE COOPERATIVE OXYGENATION PROCESS OF HEMOGLOBIN

According to the X-ray crystallographic results from human deoxyhemogl obin, beta 99Asp at the alpha(1) beta(2) interface forms hydrogen bend s with alpha 42Tyr and alpha 97Asn. To clarify the structural and func tional roles of the hydrogen bond between alpha 97Asn and beta 99Asp, we have engineered a recombinant hemoglobin in which alpha 97Asn is re placed by Ala, and have investigated its oxygen-binding properties, an d have used proton nuclear magnetic resonance spectroscopy to determin e the structural consequences of the mutation. Recombinant Hb (alpha 9 7Asn-->Ala) shows a milder alteration of functional properties compare d to the severely impaired beta 99 mutants of the human abnormal hemog lobins, The addition of inositol hexaphosphate, an allosteric effector , causes recovery of the functional properties of recombinant Hb (alph a 97Asn-->Ala) almost to the level of human normal adult hemoglobin wi thout this allosteric effector. r l-lb (alpha 97Asn-->Ala) shows very similar tertiary structure around the heme pockets and quaternary stru cture in the alpha(1) beta(2) interface compared to those of human nor mal adult hemoglobin. The proton nuclear magnetic resonance spectrum o f the deoxy form of this recombinant hemoglobin shows the existence of an altered hydrogen bond which is believed to be between alpha 42Tyr and beta 99Asp at the alpha(1) beta(2) interface. Thus, the present re sults suggest that the intersubunit hydrogen bond between alpha 97Asn and beta 99Asp at the alpha(1) beta(2) interface is not as crucial as the one between alpha 42Tyr and beta 99Asp in the deoxy quaternary str ucture. Preliminary molecular dynamics simulations have been used to c alculate the contributions of specific interactions of several amino a cid residues in r Hb (alpha 97Asn-->Ala) to the free energy of coopera tivity of this recombinant hemoglobin. The results of these calculatio ns are consistent with the experimental results.