Proton NMR investigation of the influence of subunit assembly on the low-spin in equilibrium high-spin equilibrium of met-azido hemoglobin A.

The 1H nuclear magnetic resonance signals for the side-chain labile protons of the proximal His-F8 in met-azido derivatives of the isolated chains and intact tetramer of hemoglobin have been identified. Assignment of the two peaks to the individual subunits of the intact tetramer was effected by the basis of the strong similarity of shift of one of the two peaks to that of met-azido semi-hemoglobin, where hemes occupy primarily the alpha subunits, with the heme cavity vacant in the adjacent beta subunits. The magnitudes of the hyperfine shift for both the His-F8 ring NH and the heme methyls reflect the degree of high-spin character in the thermal spin equilibrium between the high-spin, S = 5/2, and low-spin, S = 1/2, states. The changes in these shifts upon tetramer assembly demonstrate that formation of the intersubunit contacts in the R-state met-azido hemoglobin from the isolated chains causes a slight decrease in high-spin character of the alpha (22 to 20%) and a marked increase (5 to 11%) in the high-spin character of the beta subunits. The changes in spin-character are interpreted in terms of slight increase and decrease in the strength of the iron-His F8 bond upon tetramer assembly in the alpha and beta subunits, respectively. These changes in axial bonding upon forming R-state intersubunit contacts are consistent with previous observation on forming the R-state deoxy Hb tetramer from the isolated chains (Nagai, K., La Mar, G.N., Jue, T. and Bunn, H.F. (1982) Biochemistry 21, 842-847).