MODULATION OF THE ASSOCIATION REACTION BETWEEN HEMOGLOBIN AND CARBON-MONOXIDE BY PROTON AND CHLORIDE

A cryogenic technique for the isolation of the ligation intermediates in the association reaction between hemoglobin and carbon monoxide at 20 degrees C [Perrella, M., Davids, N., and Rossi-Bernardi, L. (1992) J. Biol. Chem. 267, 8744-8751] was used to study the effects of proton and chloride concentrations on the rates of the stepwise reactions, T he reaction rate was observed to increase continuously in the course o f the ligation process, yet the acceleration of the reaction after the binding of two ligand molecules, observed previously in 100 mM KCl, p H 7, was not observed at other pH values. At pH 6.3, such an accelerat ion occurred after the binding of three ligands, and at pH 8.5, a larg e acceleration was observed after the binding of the first ligand mole cule. Greater CO binding to the beta chains was observed under all con ditions, as in the previous study. The functional heterogeneity of the chains in the first ligation step increased with pH. The chloride con centration did not influence the distribution of the ligand between th e alpha and beta chains at pH 6.3 and 8.5, At pH 7, less binding to th e alpha chains was observed at 7 mM chloride with respect to 100 mM, T he nature of the biliganded component isolated at pH 7 in 100 mM KCl a nd unresolved by the cryogenic technique was studied using a combinati on of cryogenic and noncryogenic isoelectric focusing, This component was a mixture of intermediates (alpha beta)(alpha(CO)beta(CO)), about 65%, and (alpha beta(CO))(alpha(CO)beta), about 35%. The experimental data were compared with the distributions of intermediates calculated according to the Monod kinetic model assuming rapid and concerted tran sitions between two quaternary structures at each ligation step. The m odel provided a qualitative fit of the observed distributions of inter mediates at acidic and neutral pH. A large discrepancy between the exp erimental observations and the predictions of the model was found at a lkaline pH. The mechanism of the association reaction is discussed in the light of the available information on the tertiary/quaternary stru ctures of the intermediates, as obtained from the studies of the deoxy /cyanomet model of ligation.