BOHR EFFECTS OF THE PARTIALLY-LIGATED (CN-MET) INTERMEDIATES OF HEMOGLOBIN AS PROBED BY QUATERNARY ASSEMBLY

Free energies of quaternary assembly (dimers to tetramers) were determ ined for the 10 ligation species of CN-methemoglobin in the region of the alkaline Bohr effect (pH 7.0-9.5). Analysis of this database yield ed the following principal findings: (1) At each pH, the nine CN-met s pecies exhibit two distinct values of Bohr proton release and Bohr fre e energy. The two Bohr effects are found to distribute in a fashion th at coincides with predictions of a symmetry rule (Ackers et al., 1992) , ie, the first value reflects a ''tertiary Bohr effect'' arising from ligation within the quaternary T tetramer and a second Bohr effect ar ises from the quaternary transition (T --> R) which occurs when both d imeric half-molecules acquire at least one ligated subunit. (2) The Bo hr effects for CN-met ligation are in good agreement with previously-e stablished Bohr effects for stepwise Oz binding under identical condit ions (Chu et al., 1984). (3) In combination with recent studies which show that CN-met species [21] has a quaternary T structure (Daugherty et al., 1991; Doyle and Ackers, 1992; LiCata et al., 1993), the presen t results show that the ''tertiary Bohr effect'' within quaternary T e xceeds the Bohr effect of dissociated dimers, as suggested by Lee and Karplus (1983). (4) The tertiary Bohr effect is found tb account for t he pH dependence of tertiary constraint energy, Delta G(tc), which ''p ays'' for ligand-binding cooperativity prior to the quaternary (T --> R) switchover. Possible origins of the tertiary Bohr effect and its re lationship to the quaternary Bohr effect are considered.