THE OXYGEN-BINDING INTERMEDIATES OF HUMAN HEMOGLOBIN - EVALUATION OF THEIR CONTRIBUTIONS TO COOPERATIVITY USING ZINC-CONTAINING HYBRIDS

Hemoglobin tetramers [Zn/FeO2] containing oxygenated subunits (FeO2), in combination with unligated subunits containing zinc-substituted hem es (Zn), were analyzed to determine their contributions to the coopera tivity of oxygen binding at the Fe sites. Energetic consequences of po ssible perturbation by zinc substitution were evaluated in all combina tions of unligated Zn/Fe hybrid tetramers, A general thermodynamic str ategy that corrects for the energetic effects of substituting a second metal for Fe showed the perturbations of Zn substitution to be neglig ible, This permitted cooperativity parameters of the native Fe/FeO2 in termediates to be calculated from data on the corresponding Zn/FeO2 mo lecules, These parameters, determined explicitly for all eight oxygen- binding intermediates (Fe/FeO2), were found to be identical to those p redicted earlier from analyzing the O-2 binding data of normal hemoglo bin according to the ''molecular code'' of hemoglobin allostery. The c ooperativity parameters determined for this system showed the same dis tribution pattern found previously for five other oxygen analog system s (Fe/FeCN, Fe/Mn3+, Co/FeCO, Co/FeCN, and Fe/FeCO).